throbber
This content downloaded from 173.250.197.161 on Thu, 13 Apr 2017 15:16:32 UTC
`All use subject to http://about.jstor.org/terms
`
`BIOEPIS EX. 1045
`Page 1
`
`

`

`AMERICAN
`AsSOCIATION FOR THE
`ADVANCEMENT OF
`SCIENCE
`
`SciENCE ISSN 0036-8075
`
`9 JANUARY 1987
`VOLUME 235
`NUMBER4785
`
`139
`
`This Week in Science
`
`MptlbiiJQM-
`Letters
`
`141
`
`144
`
`Research News
`
`Articles
`
`Research Articles
`
`151
`153
`154
`155
`156
`158
`159
`
`160
`161
`163
`165
`
`167
`
`172
`
`177
`
`182
`
`•
`
`•
`
`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 Rhythms: R. M. SINCLAIR; C. A. CzEISLER AND J. S. ALLAN •
`Research Practices: W. W. STEWART AND N. FEDER
`
`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
`
`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 Is published weekly on Friday, except the last week In December, and with an extra Issue In February
`by the American Association for the Advancement of Science, 1333 H Street, NW, Washington, DC 20005. Sec(cid:173)
`ond-class postage (publication No. 484460) paid at Washington, DC, and at an additional entry. Now combined with The
`Scientific Monthly® Copyright© 1987 by the American Association for the Advancement of Science. The title SCI(cid:173)
`ENCE is a registered trademark of the AAAS. Domestic individual membership and subscription (51 issues): $65. Do(cid:173)
`mestic institutional subscription (51 issues): $98. Foreign postage extra: Canada $32, other (surface mail) $27, air-sur(cid:173)
`face via Amsterdam $65. First class, alnmail, school-year, and student rates on request. Single copies $2.50 ($3 by mail);
`back issues $4 ($4.50 by mail); Biotechnology issue, $5.50 ($6 by mail); classroom rates on request; Guide to Biotech(cid:173)
`nology Products and Instruments $16 ($17 by mail). Change of address: allow 6 weeks, giving old and new addresses
`and seven-digit account number. Authorization to photocopy material for internal or personal use under circumstances
`not falling within the fair use provisions of the Copyright Act is granted by AAAS to libraries and other users registered
`with the Copyright Clearance Center (CCC) Transactional Reporting Service, provided that the base fee of $1 per copy
`plus $0.10 per page is paid directly to CCC, 21 Congress Street, Salem, Massachusetts 01970. The identification code
`for Science is 0036-8075/83 $1 + .10. Postmaster: Send Form 3579 to Science, 1333 H Street, NW, Washington, DC
`20005. Science is indexed in the Reader's Guide to Periodical Uterature and in several specialized indexes .
`The American Association for the Advancement of Science was founded in 1848 and Incorporated in 1874. Its objects
`are to further the work of scientists, to facilitate cooperation among them, to foster scientific freedom and responsibility,
`to improve the effectiveness of science in the promotion of human welfare, and to increase public understanding and ap(cid:173)
`preciation of the importance and promise of the methods of science in human progress.
`
`SCIENCE, VOL. 235
`
`This content downloaded from 173.250.197.161 on Thu, 13 Apr 2017 15:16:32 UTC
`All use subject to http://about.jstor.org/terms
`
`BIOEPIS EX. 1045
`Page 2
`
`

`

`Lava flow 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 flow 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]
`
`Reports
`
`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. BoRST, F. C. BAKBR, 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. BAKBR
`207 Direct Activation of Mammalian Attial Muscarinic Potassium Channels by GfP
`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'liALwRAN 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. Coon
`Preferred Microtubules for Vesicle Transport in Lobster Axons: R. H. MILLER,
`R. J. LASEK, M. J. KATZ
`
`217
`
`220
`
`Book Reviews
`
`224
`
`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
`
`Products & Materials
`
`229
`
`Portable Macroscope • Diacylglycerol Kinase • Polyacrylamide Gels for
`Electrophoresis • FORTRAN Language Subroutines • PVC Tubing • Miniature
`Centrifuge • Teflon-Coated Slides • Literature
`
`Board of Dlractors
`Gerard Pie/
`Retiring President,
`Chairman
`Lawrence Bogorad
`President
`Sheila E. Widnall
`President-elect
`
`Robert McC. Adams
`Robert W. Berliner
`Floyd E. Bloom
`Mary E. Clutter
`Mildred S. Dresselhaus
`Donald N. Langenberg
`Dorothy Nelkin
`Linda S. Wilson
`William T. Golden
`Treasurer
`William D. Carey
`Executive Officer
`
`Editorial Board
`Elizabeth E. Bailey
`David Baltimore
`William F. Brinkman
`Philip E. Converse
`Joseph L. Goldstein
`James D. Idol, Jr.
`Leon Knopon
`Seymour Upset
`Walter Massey
`Oliver E. Nelson
`David V. Ragone
`David M. Raup
`Vera C. Rubin
`Larry L. Smarr
`Solomon H. Snyder
`Robert M. Solow
`James D. Watson
`
`Board of Reviewing
`Editors
`John Abelson
`Qais AI·Awqati
`James P. Allison
`Don L. Anderson
`Elizabeth H. Blackburn
`Floyd E. Bloom
`Charles R. Cantor
`James H. Clark
`Bruoe F. Eldridge
`Stanley Falkow
`Theodore H. Geballe
`Roger I. M. Glass
`Stephen P. Goff
`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. McGiff
`AHon Meister
`Mortimer Mishkin
`Peter Olson
`Gordon H. Orians
`John S. Pearse
`
`Yeshayau Pocker
`Jean Paul Revel
`James E. Rothman
`Thomas C. Schelling
`Ronald H. Schwartz
`Stephen M. Schwartz
`Otto T. So/brig
`Robert T. N. Tjian
`Virginia Trimble
`Gee rat 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
`
`This content downloaded from 173.250.197.161 on Thu, 13 Apr 2017 15:16:32 UTC
`All use subject to http://about.jstor.org/terms
`
`BIOEPIS EX. 1045
`Page 3
`
`

`

`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 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 tmnors (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 [3 chain of
`platelet-derived growth factor (PDGF) (16, 11); c-fins, which is
`homologous to the transforming gene of the feline sarcoma virus
`and is closely related to the macrophage colony-stimulating factor
`receptor (CSF-1R) (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-fins 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 (eDNA) 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 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 transmembrane domain that includes two cysteine-rich
`repeat clusters, and an intracellular kinase domain (21), indicating
`
`D. J. Slamon, S. G. Wong, and W. J. Levin are in the Division of Hematology·
`Oncology, Department of 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
`
`This content downloaded from 173.250.197.161 on Thu, 13 Apr 2017 15:17:17 UTC
`All use subject to http://about.jstor.org/terms
`
`BIOEPIS EX. 1045
`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 rumors 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-1 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-Iabeled 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
`
`Table1. Association between HER-2/neu amplification and disease parame-
`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
`
`Total
`
`53
`31
`42
`42
`
`13
`34
`17
`20
`
`21
`52
`11
`
`30
`20
`17
`17
`
`1
`4
`2
`3
`
`H~ recept~~r sti#US
`9
`2
`1
`2
`2
`6
`1
`5
`Tum~~r size (centimeters)
`1
`1
`1
`5
`1
`2
`0
`3
`4!Je at dillgmJSis (years)
`1
`2
`1
`4
`2
`7
`0
`0
`2
`Number of positive lymph nctles
`0
`3
`1
`0
`1
`1
`2
`4
`2
`3
`1
`1
`
`0
`1
`2
`2
`
`65
`38
`52
`51
`
`15
`41
`22
`25
`
`25
`65
`13
`
`34
`22
`25
`22
`
`Pt
`
`0.99
`
`0.85
`
`0.82
`
`0.83
`
`0.11
`
`*Receptor srarus was anaiyud as described (39). ER, estrogen receptor: + and - refer
`to the presence or absence of ;o3 finol o( receptor per milligram of protein. PgR,
`ptogestcrone receptor: + and - refers to the presence or absence of ;oS finol of receptor
`per ~ of protein.
`tSratistical analyses for correlation of HER -2/,.,. amplifi(cid:173)
`cation With diseaSe parameters were perfonncd by the X: 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 are 79 of the
`189 breast tumors used in this analysis. Tumors
`with a single copy ofHER-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 ofHER-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 of HER-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
`RI as described (30) . A total of 12 f.l.g ofEco RI(cid:173)
`digested DNA was loaded onto 0.8% agarose
`gels, separated by electrophoresis, and transferred
`onto nylon filter papers (Biodyne) (30) . All filters
`were baked in a vacuum oven for 3 hours at 80°e,
`prehybridized in 5x sse (standard saline citrate)
`containing 50% formamide, 10% dextran sulfate,
`0.1% SDS, denatured salmon sperm DNA ( 1 mgl
`ml), and 4 x Denhardts solution for 12 hours,
`then hybridized in the same solution containing
`32P-labeled nick-translated HER-2 probe (21)
`specific activiry of 1 x 108 cpm per microgram of
`DNA; 2 x 106 cprnlml. Hybridization occurred
`at 42"e for 48 hours, followed by washing of
`filters under the following conditions in succes-
`
`178
`
`1 2 3 4 5
`
`• 6 7 8 9
`
`• 10 11 12 13 14
`
`mwvfflm~ ~~~~~~v~~
`
`'
`
`41 42 43 44 45 46 47 48 49 50
`
`•
`
`51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
`
`67 68 69 70 71 72 73 74 75 76
`
`n 78 79
`
`kb
`-12
`
`- 4
`-3.1
`
`-18
`
`sion: 2X SSe for 20 minutes at room tempera(cid:173)
`ture; two washes of 30 minutes each in 2 X SSe,
`0.1% SDS at 65oe; one wash of 30 minutes in
`
`0.5x sse, 0.1% SDS at 65°e. Filters were then
`exposed to XAR-5 x-ray film (Kodak) for autora(cid:173)
`diography.
`
`SCIENCE, VOL. 235
`
`This content downloaded from 173.250.197.161 on Thu, 13 Apr 2017 15:17:17 UTC
`All use subject to http://about.jstor.org/terms
`
`BIOEPIS EX. 1045
`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 ~J.g of Eco Rl(cid:173)
`digested breast rumor DNA. Lane a is DNA from nunor 31 (Fig. 1), which
`represents a rumor with a single copy of the HER/2-neu gene. Lane g is
`DNA from rumor 33, which represents a rumor 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 rumor 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 lane k. Lane p is DNA from nunor 34
`(Fig. 1), which represents a rumor 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-labcled HER-2 probe as in Fig. l. (B) Example of arginase probe
`hybridization to demonstrate that equivalent amounts of rumor DNA were
`loaded into each lane. Rehybridization of filter containing lanes 30 to 40
`(Fig. 1). The filter was first stripped oflabel by washing in a buffer made up
`of 50% forrnaruide, 3x SSe, and 0.1% SDS at 65oe for 20 minutes,
`foUowing by three successive washes of 5 minutes each in 0.1 x sse at room
`temperarure. Filters were exposed overuight on XAR-5 film (Kodak) to
`ensure removal of aU 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 1 to 3
`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
`
`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
`
`1.0
`
`~ 0.8
`:c
`~ 0.6
`e c.
`
`~ 0.4
`·;;
`~ 0.2
`
`1.0
`
`~ 0.8
`
`:c = -g 0.6
`a.
`~ 0.4
`~
`; 0.2
`
`A
`
`B
`
`Amplified (n : 34)
`
`Amplified (n : 34)
`
`c
`
`D
`
`Amplified (n =11)
`>5 copies
`
`Amplified (n :11)
`>5 copies
`
`12
`
`24
`
`36
`
`48
`
`60
`
`72
`
`84
`Time (monlhs)
`
`12
`
`24
`
`36
`
`48
`
`60
`
`72
`
`84
`
`ARTICLES 179
`
`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) ofHER-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 overaU survival in (B) node(cid:173)
`positive patients with no amplification versus
`node-positive patients with any amplification ( > 2
`copies) ofHER-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 overaU 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
`
`This content downloaded from 173.250.197.161 on Thu, 13 Apr 2017 15:17:17 UTC
`All use subject to http://about.jstor.org/terms
`
`BIOEPIS EX. 1045
`Page 6
`
`

`

`between HER-2/neu amplification and nodal starus (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 7l of the 86 patients (83%) received
`some form of therapy afrer 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 foWld 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
`Wlivariate analyses to other parameters, amplification ofHER-2/neu
`was foWld 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 berween 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 rumors
`(Fig. 3, A and B), the greatest differences were foWld 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
`
`Fig. 4- Example of rehybridization of filter with human EGFR probe. Filters
`were stripped as in Fig. 2B, and hybridized with 32P-Iabeled human EGFR
`probe (28), as in Fig. 1. Shown are the lower molecular weight bands
`hybridized with 32P-Iabeled EGFR probe in filter-containing lanes 51 to 66
`(Fig. 1). The bands from top to bonom are 2.8, 2.2, and 1.8 kb, respectively.
`Lane 52 is an example of a tumor showing marked amplification (>50
`copies) of the EGFR gene.
`
`patients, even when other prognostic factors were taken into
`accoWlt (Table 4).
`Rearrangement of the HER-2/neu gene was rare. Of the total189
`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 of HER-
`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 foWld in 4/189 (2%) of the cases, and
`rearrangement of the EGFR gene was foWld in one of those four
`cases. The incidence of EGFR amplification and rearrangement was
`too small to attempt statistical correlation. Comparison of HER-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 fes, 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. AssQciation 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
`
`s50
`>50
`
`1-3
`>3
`
`38
`14
`31
`21
`
`18
`28
`6
`
`16
`36
`
`31
`21
`
`1
`1
`
`Hl1mi01UII receptor st/ltus
`21
`5
`2
`4
`18
`4
`5
`5
`Tunwr size ( centimetm)
`3
`8
`12
`2
`3
`4
`Age M diagtuJSis (ye/U'S)
`12
`6
`11
`3
`Number of positive lymph not/a
`7
`0
`5
`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 and PgR are as described in Table l.
`tStatistical analyses for correlation of
`HER-2/neu amplification with various disease J:aramcters were perfOrmed by the x2
`test. P values were computed after combining e S to 20 and > 20 cases, since there
`were so few samples in the > 20 group.
`
`ER+
`ER-
`PgR+
`PgR-
`
`s2
`2-5

This document is available on Docket Alarm but you must sign up to view it.


Or .

Accessing this document will incur an additional charge of $.

After purchase, you can access this document again without charge.

Accept $ Charge
throbber

Still Working On It

This document is taking longer than usual to download. This can happen if we need to contact the court directly to obtain the document and their servers are running slowly.

Give it another minute or two to complete, and then try the refresh button.

throbber

A few More Minutes ... Still Working

It can take up to 5 minutes for us to download a document if the court servers are running slowly.

Thank you for your continued patience.

This document could not be displayed.

We could not find this document within its docket. Please go back to the docket page and check the link. If that does not work, go back to the docket and refresh it to pull the newest information.

Your account does not support viewing this document.

You need a Paid Account to view this document. Click here to change your account type.

Your account does not support viewing this document.

Set your membership status to view this document.

With a Docket Alarm membership, you'll get a whole lot more, including:

  • Up-to-date information for this case.
  • Email alerts whenever there is an update.
  • Full text search for other cases.
  • Get email alerts whenever a new case matches your search.

Become a Member

One Moment Please

The filing “” is large (MB) and is being downloaded.

Please refresh this page in a few minutes to see if the filing has been downloaded. The filing will also be emailed to you when the download completes.

Your document is on its way!

If you do not receive the document in five minutes, contact support at support@docketalarm.com.

Sealed Document

We are unable to display this document, it may be under a court ordered seal.

If you have proper credentials to access the file, you may proceed directly to the court's system using your government issued username and password.


Access Government Site

We are redirecting you
to a mobile optimized page.





Document Unreadable or Corrupt

Refresh this Document
Go to the Docket

We are unable to display this document.

Refresh this Document
Go to the Docket