AM F. R I (EA N
`ASSOCJA‘I'ION FOR THE
`A11\-‘.u\N(;I:rs-1LN‘I‘ (IF
`SCIENCE-L
`
`0
`
`11 MA Y 1989
`
`.‘€~_?..<:.';u
`
`VOL 244 I Run-:5; (ill-"+4-
`
`PFIZER EX. 1027
`
`Page 1
`
`PFIZER EX. 1027
`Page 1
`
`

`

`AMERICAN
`ASSOCIATION FOR THE
`ADVANCEMENT OF
`SCIENCE
`
`SciENCE
`
`ISSN 0036-8075
`12 MAY 1989
`VOLUME 244
`UMBER 4905
`
`Editorial
`
`627 This Week in Science
`
`629 Oil Spills
`
`IH: M. J. PEAK• Climate and Forests: R. A. EDJO •
`631 Budget Cuts at
`Retraction: K. M. MILAM, S. HORN, P. J. TOFILON, D. F. DEEN,
`L. J. MARTON • Educational Reform: D. ELJCJND • Yanomami Indians and
`otilication to Readers:
`Anthropological Ed1ics: B. ALBERT AND A. R. RAMos •
`J. F. FERRER AND P. GUPTA
`
`\t'\\:- 1.."- C o111111t·nt
`
`643 The DingeU Probe FinaUy Goes Public • Credit for Whistle-Blower Vanishes •
`Secret Service Probes Lab Notebooks • "I Am ot a eat Person" • A Question
`of Intent • NIH co Use Forensics
`647 Electrochemists Fail to Heat Up Cold Fusion
`648 Germany Sets Up New Space Agency
`Researchers Irked by Changes to Te timony
`649 Frazier Honored by Psychiauists
`Koop Resigns in a Huff
`Science Artifacts on the Block
`650 Skeleton AUeged in the Stealth Bomber's Closet
`
`M;J$$1i4i4i$(- 652 Two Cultures Find Common Ground
`
`654 Gene Signals Relapse of Breast, Ovarian Cancers
`655
`cw Chip May Speed Genome Analysis
`
`\rtidt':-
`
`Ht·:-t•arch ·\rticle:(cid:173)
`
`Ht·JH1rt-.
`
`659 The Economic Status of the Elderly: M. D. HURD
`664 Observations in Particle Physics from Two Neutrinos to the Standard Model:
`L. M. LEDERMAN
`673 How Old Is the Genetic Code? Statistical Geometry of tRNA Provides an Answer:
`M. EIGEN, B. F. LINDEMANN, M. TIETZE, R. WINKLER-0SWATITSCH, A. DRESS,
`A. VON HAESELER
`
`679 Stereochemisuy of RNA Cleavage by the Tetrahy,nma R.ibozyme and Evidence
`That the Chemical Step Is Not Rate-Linliting: J. A. McSWIGGEN AND T. R. CECH
`
`684 Model Simulation of the Cretaceous Ocean Circulation: E. J. BARRON AND
`W. H . PETERSON
`
`•
`
`•
`
`SCIENCE la publlaMCI ~ on Frldoy, except the lal1- In - . and with an •"'111 I.- In februery
`by the Ameflcan ANoc:lotlon for the Adv1..- of Sc-, 1333 H St...c, NW, Wuhlngton, DC 20005. Sec·
`ond-class postage (pubWcation No. 484460) paid at Washington, DC, and at an additlonal entry. ,_ combined with The
`Sclenttflc Monthly• Copyright O 1989 by the American Association to, lho Advancement of Science. The tiUe SCI·
`ENCE is a registered trademar1< ol the At.AS. Domestic lndMdual membefship and SIJbscription (51 issues): $75. Do(cid:173)
`mestic Institutional subscription (51 issues): $120. Foreign postage extra: Canada $46, other (surlace mail) $46, air
`mail via Amsterdam $85. First class, airmail, school·yaar, and student rates on request. Single copy aalN: Curf9nt Is(cid:173)
`sue. $3.50; back Issues, S5.00; Biotechnology issue, $6.00 (for postage and handling, add per COf7Y so.so U.S., $1 .00 all
`foreign); Guide to Bioteclloology Products and Instruments, $18 (to, postage and handling add per COf7Y $1.00 U.S.,
`
`S1 .SO Cenada, $2.00 other loreign). Bulk rates on request. Change of --= .- 6 -'<s, gMng old and MW ad·
`
`dresses and 1 Hligtt account number. Auth<>rtzation to photocopy material for internal or personal use under circum(cid:173)
`stances not falling witNn the fa~ use provisions of lho Copyright Act Is granted by AAAS 10 ubraries and other usaB reg(cid:173)
`istered with the Copyright Clearance Center (CCC) Transactional Reporting Service, providecl that lho base fff of St per
`COf7Y plus S0.10 per page ls paid directly to CCC, 21 Congress Street, Salem, Massac:llusatts 01970. The ldentlllcation
`COde for Science Is 0036-8075183 $1 + .10. - l e<: Send Form 3579 to $ci,mt;t,, P.O. Box 1722, Riverton, NJ
`oeon. Science Is Indexed In lho Rellde<'s Guide to Periodical UteralUJ'o and In oeveral specieli,ed indexes .
`The American Association to, lho Advancement of Science was founded In 1848 and lnco<poratad In 1874. Its ol)jects
`are to further the wort< ol scientists, to fecil~ete cooperation among them, to loster scientilic freeclom and responsNily,
`to improve the eflectiveness of science ln the promotion of human wenare. and to Increase public understanding and ap(cid:173)
`praciatlon ol lho Importance and promise of lho methods of science in human progress.
`
`SCIENCE, VOL. 2.44
`
`PFIZER EX. 1027
`Page 2
`
`

`

`Color-enhanced tissue section from a human ovarian epithelial
`COVER
`carcinoma. The rumor ceUs contain > fivefold an1plification of tht HER-2/11e11
`proto-oncogene. The section is immunostained with antibody to the HER-2/11e11
`protein. The typical cy tic narurc of the rumor can be seen, and there is intense
`membrane raining (brown color) of the rumor cells Lining the cystic structures.
`tromal cells and norm1alignant elements arc unstained, indicating the absence of
`the protein ( x 100). ee page 707. (Photograph by D. J. Slamon, Department of
`Medicine, UCLA chool of Medicine, Los Angeles, CA 90024, and M. F. Press,
`Department of Pathology, University of Southern California School of Medicine,
`Los Angeles, CA 90033]
`
`694
`
`686 Hearing in Honey Bees: Detection of Air-Particle Oscillations : W. F. TOWNE AND
`W. H. KIRCHNER
`688 Fibroblast Growth Factor in the Extracellular Matrix of D yscrophic (mdx ) Mouse
`Muscle: J. DIMARIO, N. BUFFINGER, S. YAMADA, R . C. STROHMAN
`690 Corn and Culrure in Central Andean Prehistory: S. JOHANNESSEN AND
`C. A. HAsroRF
`692 Stereochcmical Course of Cataly i by the T etra/1yme1w Ribozyme: J. RAJAGOPAL,
`J. A. DouDNA, J. W. SwsrAK
`Identification of die Fusion Peptide of Primate lmmunodeficiency Viruses:
`M. L. BOSCH, P. L. EARL, K. FARGNOLI, S. Pt
`lAFUOCO, F. GIOMBINI,
`F. WONG-STAAL, G. FRANCHINI
`697 Caliclieamicin 1i' and D A: Molecular Recognition Process Responsible for Site(cid:173)
`Spccificity: N. ZEIN, M. PoNCIN, R. NILAKANTAN, G. A. ELLESTAD
`700 Complementary D A Coding Click Beetle Lucifcrases Can Elicit Biolumincscence
`of Different Colors: K. V . WOOD, Y. A. LAM, H. H. SELIGER, W . D . MCELROY
`702 Reexamination of die Three-Dimensional Structure of the Small Subunit of
`RuBisCo from Higher Plants: S. KN IGHT, I. ANDERSSON, C.-l. BRAND~N
`705 Receptor-Mediated Drug Delivery to Macrophages in Chemotherapy of
`Leishmaniasis: A. MUKHOPADHYAY, G . CHAUDHURI, S. K. ARORA, S. SEHGAL,
`s. K. BASU
`707 Studies of the HER-2/neu Proto-oncogene in Human Breast and Ovarian Cancer:
`D . ]. SIAMON, W . GoDOLPHIN, L.A. JONES, J. A. HOLT,
`. G . WONG,
`D . E. KEITH, w. J. LEVIN, s. G. TUA.RT, J. UDOVE, A. ULLRICH, M. F. PRESS
`713 Activation of 16 T CeUs in the Primary lmmwie Respon e to Mycobaaerim11
`. H . E. KAUFMANN , R. H. CHWARTZ, D . M. PARDOLL
`tuberwlosis: E. M. )ANIS,
`cural Integration of Information Specifying Structure from Sterc:opsis and
`Morion: M. NAWROT AND R. BLAKE
`
`716
`
`Book Review~
`
`Producb & Material!',
`
`719 Ono Folin, reviewed by J. T. EDSALL • Nuts and Bolts of the Past,
`. PUR ELL •
`Evaporite Sedimencology and Evaporitics and Hydrocarbons, P. SONNENFELD •
`Continental Flood Basalts, S. A. MORSE • Books Received
`
`724 Magnetic Cell Sorter • Statistical Experimental De ign Software • In Vivo
`Elcctrochemistry System • Cell and Tissue Adhesive • Population Dynamics
`Software • Mult;variate Data Analysis Software • Literature
`
`Board ot Dlrec:lora
`Watter E. Massey
`Retiring Presk!enr,
`Chairman
`Richard C. Atkinson
`Pros/dent
`Donald N. Langenberg
`President-elect
`
`Mary Ellen Avery
`Francisco J. Ayala
`Floyd E. Bloom
`Mary E. Clutter
`Eugene H. Cota-Robles
`Joseph G. Gavin. Jr.
`John H. Gibbons
`Beatrix A. Hamburg
`William T. Golden
`Treasurer
`
`Richard S. Nicholson
`Executive 0 6/cer
`
`Editorial Boord
`Eli>abeth E, Balley
`David Baltimore
`WIiiiam F. Brinkman
`E. Margaret Burbidge
`Philip E. Coovem
`Joseph L. Goldstein
`Mary l. Good
`F. Cla'1< Howell
`James 0 . Idol. Jr.
`Leon Knopol
`Oliver E. Nelson
`Helen M. Rannay
`David M. Raup
`Howard A. Schneiderman
`Larry L. Smarr
`Robert M. Solow
`James 0 . Watson
`
`Boord ol Reviewing
`Editors
`
`John Abelson
`OalsAI-Awqati
`Don L. Anderson
`Stephen J. Benl<ovic
`Floyd E. Bk>Om
`HenryR. Bourn<t
`James J. Bull
`Kathryn Calame
`Charles R. Cantor
`Ralph J . Cicerone
`John M. Colin
`Robert Dorfman
`Bruce F. Eldridge
`Paul T. Englund
`Fredric S. Fay
`Theodore H. Geballe
`
`Roger I. M. Glass
`Stephen P. Goff
`Robert B. Goldberg
`Corey S. Goodman
`Jack Gorski
`Stephen J . Gould
`R;ct,a,oM, Held
`Gloria Heppner
`Eric F. Johnson
`Konrad B. Krauskopl
`Chartes S. Levings 111
`Richard Loslck
`Kart L. Magleby
`PhiHppa Marraclc
`Joseph B. Martin
`John C. McGiff
`Mortimer Mishkin
`Carl 0 . Pabo
`
`Yeshayau Pocker
`Michael I. Posner
`Dennis A. Powers
`Russell Ross
`James E. Rothman
`Ell<ki RUOSlahti
`Ronald H. Schwartz
`Vernor> L. Sm<th
`Rober1 T. N. Tjian
`Virginia Trimble
`Emil R. Unanue
`Geerat J. Verrneif
`Be<t Vogelsteln
`HarOld Weinlraub
`Irving L. Weissman
`George M, WMesides
`Owen N. Witte
`William 8 . Wood
`
`12 MAY 1989
`
`TABLE OF CONTENTS 625
`
`PFIZER EX. 1027
`Page 3
`
`

`

`was observed. Finally, the conjugated drug
`caused 90% reduction in the size of the
`lesion 11 days after the initiation of drug
`treatment. The greatest effect on the regres(cid:173)
`sion of the lesions by the conjugated drug
`was observed at a dose of 1 mg/kg per
`footpad. The lack of effect at higher concen(cid:173)
`uations probably reflects saturation of the
`for Mtx(cid:173)
`receptor-mediated uptake proce
`M.BSA. The footpad regressed to nearly
`normal size when Mcx-MBSA was used. In
`contrast, administration of free Moc did not
`significantly affect the footpad lesion. The
`lesions did not reappear even 4 weeks afi:er
`the lase injection of Mtx-MBSA. During the
`the animals re(cid:173)
`experimental period all
`mained healthy with no apparent weight
`loss.
`o antibody against MBSA or Moc(cid:173)
`M.BSA was detectable in these animals after
`3 weeks as determined by the Ouchterlony
`immunodiffusion technique.
`In conclusion, our re ults show that effec(cid:173)
`tive delivery of drug to macrophages can be
`achieved by using the "scavenger" receptor(cid:173)
`mediated endocytic pathway to achieve e(cid:173)
`lective killing of intracellular parasites resid(cid:173)
`ing in macrophages, both in vitro and in
`vivo. A imilar approach may be useful for
`elfcctivc delivery of drugs in the treatment
`of other diseases in which macrophages are
`the primary target, including tuberculosis,
`leprosy, monocytic leukemia, and heavy
`metal storage diseases.
`
`lll!FERENCES AND OTES
`l. M. L. Chance, Br. M,d. J. 283, 1245 ( 1981 ).
`2. J. A. Walsh and K. S. Warren, N . Eng.) . Mtd. 301 ,
`967 ( 1979).
`3. K. P. Chang, Int, Rt•. Cytol ( uppl.) 14 , 267
`( 1983).
`4. J. D. Berman, in uishma11i1UiJ, IC P. Chang and R.
`S. Bray Eds. (Elsevier, Amsterdam, 1985), vol. I,
`pp. 111-138.
`5. ). J. Marr, in Para,itic Dism"', J. M. Mansfield, Ed.
`(Dckk<r,
`'cw York, 1984), ,'DI . 2, pp. 201- 227.
`6. C. D . V. Black, G. J. Wats0n, R. ) . Ward, Tranr. R.
`Soc. Trop . Mtd. Hyg. 71 , 550 ( 1977).
`7. C. R. Alving, E. A. Steck, W. L. Hanson, P. S.
`Loiz.c:aw:, W. L. Chapman, Jr., Life Sri. 22, 1021
`(1978).
`8. R. R. C. New, M . L. Chance, S. C. Thomas, W.
`Peters, aoir, 272, 55 ( 1978).
`cw, M . L. Chance, S. Heath, ) . A111imi-
`9. R. R. C.
`1Tob. Chrmotha. 8, 371 (1981 ).
`10. C. R. AJving and G. M. S-=-, Jr., in Liposomt
`T«hnalogy, G. Gregoriodis, Ed. (CRC Press, Boca
`Raron, FL, 1984), ,'OL 2, pp. 55- 68.
`11. C. R. AJving ti al., Prot.
`01/. A cad
`ri. U. .A . 75,
`2959 (1978).
`12. J. L. Goldstein, Y. K. Ho, S. K. Basu, M. S. Brown,
`ibid. 76 , 333 ( 1979).
`13. M. S. Brown, S. K. Basu, C.R. Falk, Y. K. Ho, ). L.
`tnut. 13, 67 ( 1980).
`Goldstein, ) . upramol.
`14. 0. Srein and Y .
`rein, Bi« him. Biophy,. Acta 620,
`631 ( 1980).
`15. A. N . Gl=r, R. S. Delange, D. S. Sigman, Eds.,
`Ch<r0ical Modifaation of Prottim ( orth-HoUand,
`Amsterdam, 1975), pp. 79-81.
`16. H . ). P. Ryser and W,
`. Shen, Pro<. at/. Acad. Sri.
`U.S.A . 75 , 3867 (1978).
`17. A. Mukhopadhyay, G. O,audhuri,
`. K. Basu, un,
`published dara. In some rettpror systems chloro(cid:173)
`quine, especially at relatively high concentrations,
`
`inhibia uptake of the ligand. In our case, however,
`chloroquine primarily alf'ccted degradation of '"I·
`labeled Mtx·MBSA as shown from the foUowing
`observation. Hamsrer pcrironcal macrophages were
`incubated at 37°C in medium containing ""I· la·
`bcled Mtx· MBSA (10 µg per milliliter of protein).
`The ccUub.r conccnr of radioactiviry and that re·
`leased inro medium (ocid-solublc) were mca.mred ..
`a function of rime in the presence and absence of
`chloroquine (3 µM) . CcUub.r content of radioactiv(cid:173)
`iry continued ro increase in chloroquine-trcared
`cultures but release of acid-soluble radioactiviry in
`the medium was arrested. These rcsula suggcsrcd
`that at this concentration chloroquine inhibited the
`degradation of Mex-MB A and nor ia uptake.
`
`18. M. Rabinowirch, V. Zilbcrfarb, C. Rama,.cillcs, J.
`Exp. Md. 163, 520 ( 1986).
`19. S. Sehgal and S. K. Arora, Ind. ). Mtd. R<J. 82,202
`( 1985).
`20. B. PhilliP5 and J. C. Gazct,
`( 1968).
`21. J. D. Berman and D. J. Wyler, J. l•f«t. Dis. 142, 83
`(1980).
`22. We thank V. K. Kalra and G. C. Mishra for critically
`reviewing the manuscript, and the C.ouncil of Scien (cid:173)
`tific and lndusuial Rcscan:h and Indian Council of
`Medical Rcscarch for award offc!JowshiP5 to A.M.
`and G.C.
`
`atun 2 20, 1140
`
`13 September 1988; accepted 20 February 1989
`
`Studies of the HER-2/neu Proto-oncogene in Human
`Breast and Ovarian Cancer
`
`DENNIS J . S I.AMON,* W ILLIAM GoooLPHIN, L OVE LL A. JONES,
`]OHN A. HOLT, Sulv:EN G. W ONG, D UANE E. l<Em-I, W ENDY J. LEvlN,
`SUSAN G . STUART, J U DY UDOVE, Ax.EL ULLRI CH , MICHAEL F. PRESS
`
`Carcinoma of the breast and ovary account for one-third of all cancers occurring in
`women and together arc responsible for approximately one-quarter of cancer-related
`deaths in females. The HER-2/neu proto-oncogcne is amplified in 25 to 30 percent of
`human primary breast cancers and this alteration is associated with disease behavior.
`In this report, several similarities were found in the biology ofHER-2/neu in breast
`and ovarian cancer, including a similar incidence of amplification, a direct correlation
`between amplification and over-expression, evidence of tumors in which overcxprcs(cid:173)
`sion occurs without amplification, and the association between gene alteration and
`clinical outcome. A comprehensive study of the gene and its products (RNA and
`protein) was simultaneously performed on a large number of both tumor types. This
`analysis identified several potential shortcomings of the various methods IUed to
`evaluate HER-2/neu in these diseases (Southern, Northern, and Western blots, and
`immuoohistochcmistry) and provided information regarding considerations that
`should be addressed when studying a gene or gene product in human tissue. The data
`presented further support the concept that the HER-2/neu gene may be involved in the
`pathogenesis of some human cancers.
`
`P ROTO·ONCOGBNBS REPRESENT A
`
`f.uni ly of normal cellular genes that
`were identified on the basis of their
`sin1ilarity to genetic sequences with known
`rumorigcnic or transforming potential (1).
`C.onsiderable circumstantial evidence now
`exists that alterations in either the srrueture,
`copy number, or expression of one or anoth(cid:173)
`er of these genes may play a role in the
`pathogenesi of some human malignancies
`(2). One such gene, called HER-2/11e11 or c(cid:173)
`erb B2, wa
`first identified by transfection
`studies in which
`lH 3T3 cells were trans(cid:173)
`formed with DNA from chemically induced
`rac ncuroglioblastomas (J). The gene en(cid:173)
`codes a protein that has extracellular, trans(cid:173)
`membrane, and intracellular domains ( 4)
`which is consistent with the structure of a
`growth factor reception.
`Recently, we found a 28% incidence of
`amplification of HER-2/neu in 189 prim.ary
`human breast cancer (5). Patients with mul(cid:173)
`tiple copies of the gene in D A from their
`rumors had a shorter time to relapse as well
`as a shorter overall survival indicating that
`
`gene amplification was prognostic for dis(cid:173)
`ease behavior in these individuals. More(cid:173)
`over, multivariate survival analysis showed
`HER-2/11e11 amplification to be more predic(cid:173)
`tive for clinical o utcome than all other
`known prognosticators with the exception
`of positive lymph nodes (5). Since that
`initial report, a number of studies have been
`publi hed on the amplification of this gene
`
`D. Slamon, S. G. Wong, D. E. Keith, W. J. Levin,
`Division of Hematology-Oncology, Department of
`Medicine, •nd the Jonsson Comprehensive Cancer Ccn·
`ter, U.C.L.A. School of Medicine, Los Angeles, CA
`90024.
`W. Godolphin, Dcputment of Clinical Chemistry, Van(cid:173)
`couver General Hospital, Vancouver, Canada V5Zl M9 .
`L. A. Jones, Department of Gynecology, M. D. Ander·
`son Hospital, Houston, TX 77030.
`J. A. Holt, Department of Obstetrics and Gynccok,ji;y,
`nivcrsiry of Chicago Medical Center, O,icago, TL
`60637.
`S. G. Sruan, Triton Biosciences, Inc., Alameda, CA
`94501.
`J. Udovc and M. F. Press, ~
`t of P•thology,
`Unil'ersiry of Southern Califom,a, School of Medicine,
`Los Ant;clcs, CA 90033.
`A. Ullrich, Ocpanmcnr of Molecular Biology, Gcncn·
`tech, Inc., South an Franci<co, CA 94080.
`
`•To whom correspondence should be addressed.
`
`REPOlj.TS 707
`
`PFIZER EX. 1027
`Page 4
`
`

`

`in human breast cancer and the association
`of gene amplification with clinical behavior
`( 6-8). There is considerable variability in
`both the reponed incidence of amplification
`and the correlation of gene amplification
`with patient outcome (5-10). Some groups
`have found amplification rates as low as
`10% and no correlation to outcome data
`while others have found rates as high as
`33% and a strong association with outcome
`(7, 10). Given the variable narural history
`and heterogeneity of human breast cancer,
`all studies published to date suffer from a
`similar problem, which is small numbers of
`
`evaluated cases (5-10) . Perhaps a more sig(cid:173)
`nificant shoncoming of most prior studies
`of oncogenes in human tumors including
`our own is that only one aspect of the gene
`in question (DNA, RNA, or protein starus)
`is evaluated (5, 11 , 12). The potential errors
`inrroduced by dilution of tumor cell macro(cid:173)
`molecules with macromolecules from sur(cid:173)
`rounding normal vascular, stromal, or in(cid:173)
`flammatory cells is a general problem in
`human tumor tissue and a panicular prob(cid:173)
`lem in breast cancer where these non-malig(cid:173)
`nant cells can account for more than 50% of
`the tissue. All solid matrix-blotting tech-
`
`niques (Southern, Western, or onhem)
`are susceptible to these errors. Similarly,
`these techniques cannot determine whether
`an observed alteration is specific for only the
`malignant cells in the tissue.
`Studies of the rat neu gene isolated from
`the chemically induced neuroglioblastomas
`revealed it to contain a single mutation in
`the transmembrane domain that differentiat(cid:173)
`ed it from the nontransforming neu gene
`found in normal rat tissues. This mutation is
`critical in the conversion of the normal gene
`into a transforming gene (13) . To address
`
`12.5 1•
`
`Hkb
`
`SOUTHERN
`
`NCRTMCRN
`
`IMM HIST
`
`Ag. 1. Examples of the correlation between HER-2/11eu gene amplification and expression. Southern
`blot analyses show the 12.5-kb HER-2/11eu band seen with Eco Rl cut D A. All D A samples were
`checked for integrity of high molecular weight species and samples showing evidence of DNA
`degradation were not evaluated (9). Southern blots and HER-2/11eu copy number detenninations were
`as described (5, 9). Hybridizations were done with a 1.4-kb, 3' Eco Rl fragment of the human HER-
`2/11n1 cD A clone. Northern blot analyses show the 4 .5-kb HER-2/11eu transcript. All RNA samples
`were checked for integrity of the 28 and 18
`ribosomal RNA species. Total RNA (20 µ.g) from
`samples with intact RNA were run on an agarose gel, transferred co a nylon filter, and hybridized with a
`32P-labeled HER-2/11eu probe as described ( 11 ). Samples with intact D A but showing some
`degradation of the RNA were evaluated by slot-blot analysis by loading 12 µ.g of total RNA on the filter
`and hybridizing as above. Samples showing degradation of both DNA and RNA were not used for
`RNA analysis. The relative optical density (0 .0 .) of bands was determined by soft laser densitometry
`scanning and ranged from a low ofO.l 0 .0 . units to a high of 3.8 0 .0 . units. Tumors were grouped
`into RNA expression categories as foUows : 0.1 to 0.5 0 .0. units, l +; > 0.5 to 1.0 0 .0 . units, 2 +;
`> 1.0 to 1.5 0.0. units, 3+; and > 1.5 0.0. units, 4+. Western bloc analyses show the 185-kD HER-
`2/ne,, protc.in band. The relative 0.0. of bands ranged from a low ofO. I 0 .D. units to a high 4.5 O .D.
`units. Tumors were grouped into protein expression categories as follows: 0.1 to 0.5 O.D. units, l + ;
`> 0.5 to 1.0 0 .D . units, 2 +; > 1.0 to 1.5 0 .0. units, 3+ ; > 1.5 0 .0. units, 4+. lmmunohiscochcmical
`analysis was done as described (20) with the anti-HER-2/r1eu specific antibody and frozen sections (14).
`Tissues were scored and placed in one of the four staining categories shown on the basis of the relative
`level of specific staining as judged by microscopic examination as follows: negative to weak, l +, 2+,
`3+. The five samples analyzed here arc arranged in identical order from left to right in each panel. The
`HER-2/,reu copy numbers (from left to right) were >20, 5 to 20, 2 to 5, I, and 1, respectively. The
`corresponding 0 .0 . readings from the Northern blocs were 4.3, 1.4, 0.9, 0.2, and 2.8, respectively. The
`corresponding 0 .0 . readings from the Western blots were 2.0, I.I, 0.6, 0.12, and 2. 1, respectively.
`The corresponding immunohiscochcmi cry readings were 3+, 2+, I + , weak, and 3+, respectively.
`
`7o8
`
`-o••a
`
`Fig. 2. Comparison of immunoperoxidase stain(cid:173)
`ing with Western bloc on stroma-rich breast
`cancer. The inset (upper left) is a hcmatoxylin(cid:173)
`cosin stain of a breast rumor rich in srromal tissue.
`Note the absence of significant numbers of rumor
`cells. Large middle panel is the immunoper(cid:173)
`oxidase staining of rumor cells (TC) found in this
`tissue. The staining is 3 + , placing this rumor in
`the highest category of HER-2/11n1 expression as
`judged by immunostain.ing. Southern analysis re(cid:173)
`vealed two to five copies of the gene in the DNA
`and Northern blot analysis gave an 0 .0 . reading
`of 0.6 (2+ ). Western blot of protein from this
`sample is shown in lane A of the lower right inset.
`The 0.D. reading for this sample was 0.18 (I+ ),
`while the Western blot of protein from another
`specimen with amplified HER-2/neu and greater
`numbers of rumor cells is shown in lane B. The
`0 .0 . reading for this rumor (lane B) was 3.2
`(4+ ). Eight of the II rumors found to have
`inappropriately low Western blot <Uta in compar(cid:173)
`ison to other data were similar in that they were
`stromal-rich rumors.
`
`A
`
`Fig. 3. Comparison of immunohistochcmical
`staining of HER-2111eu protein in the same breast
`cancer specimen evaluated with frozen tissue and
`formalin-fixed, paraffin-embedded tissue. Tissue
`shown in panels A and B arc from the same rumor
`which was found co have 5 to 20 copies of the
`gene and a 2 + expression level by Northern and
`Western blot analyses. Panel A is the frozen
`section and shows 2+ immunoscaining. Panel Bis
`the formalin-fixed, paraffin-embedded section of
`the same tumor and shows negative immuno(cid:173)
`staining.
`
`SCIENCE, VOL. 244
`
`PFIZER EX. 1027
`Page 5
`
`

`

`c que tion of whether or noc a similar
`change had occurred in an amplified HER-
`2/11n1 gene found in human breast cancer,
`cDNA clones from rumor tissue rarher rhan
`ccU lines were generated by mean of rhe
`Okayama-Berg vecror (14, 15). Tissue was
`used to circumvent acquired genetic changes
`which can occur in vitro. Analysis of rhc
`tran5membrane domain of eight clones from
`rwo separate rumors showed rhe identical
`sequence. There was no glutamic acid for
`valine ubstirucion as reported in the trans(cid:173)
`forming ,w, gene from the chemically in(cid:173)
`duced rat tumors (13). There was, however,
`a neutral change of isoleucine for valinc at
`position 655 in rhe transmembrane domain,
`which i
`imilar to the sequence found in
`breast cancer cell line (16). Analysis of the
`entire coding sequence of full-length clone
`and comparison wirh the published placen(cid:173)
`tal sequence (J) showed no other significant
`changes (14). These data are consi tent with
`the concept that overexpression of a normal
`HER-2/neu gene product rather than muta(cid:173)
`tion to an abnormal gene may be an impor(cid:173)
`tant pathogenic event for some rumors.
`Having obtained and sequenced a foll(cid:173)
`length cDNA clone from a human breast
`rumor, we next wanted to generate antisera
`to the human gene product. The generation
`and characterization of chis antiserum is
`described el cwhere (14 ) and the antibody is
`capable of identifying the gene producr both
`by Western bloc analy is of ti ue homoge(cid:173)
`nates and immunohistochemically in tissue
`sections.
`In the current rudy, we coUected a total
`of 668 human breast cancer specimens. Of
`th e, 526 had ufficient clinical foUow-up
`to allow for evaluation of an association
`between gene amplification and di ease out(cid:173)
`come. As in our initial study (5), we per(cid:173)
`formed outhcm analysis on samples with(cid:173)
`out knowledge of the clinical outcome. All
`D A blots were stripped and reprobed with
`both p53 and myeloperoxidase probes ro
`evaluate the relative loading of D A in each
`lane and ro exclude the po ibiliry that "am(cid:173)
`plification" wa cau ed by partial or com(cid:173)
`plete duplication of chromosome 17 (9).
`Blocs were scanned by soft laser densitome(cid:173)
`try and the level ofHER-2/11e11 amplification
`was determined by d1e ratio of the HER-
`2/ueu
`ignal relative to the single-copy p53
`signal.
`We evaluated 345 patients with node(cid:173)
`positive disease in a blinded fashion (Table
`1). Of d1esc, 101 (27%) had evidence of
`HER-2/11e11 amplification. Univariate surviv(cid:173)
`al analysis showed amplification of the
`HER-2/neu gene co be a significant predic(cid:173)
`tor of both disease-free survival and overall
`survival for these patients (Table l ). Tumor
`size was slightly better than HER-2/11eu
`
`12 MAY 1989
`
`Table 1. Univariate and multivariate survival analyses comparing disease-free {relapse) and overall
`survi al to prognostic factors in 345 node-positive breast cancer patients. Statistical analyses were
`performed by the )(2 test and by Cox's partially nonparametric regression analysis to evaluate the
`predictive power of various combinations and interactions of prognostic factors in a multivariate
`manner as described (5). Prognostic parameters evaluated include number of nodes {Nodes), HER-2/
`""" gene amplification (HER-2/11ru), estrogen receptor (ER), progesterone receptor {PGR), size of
`primary rumor (Size), and age of patient at diagnosis (Age). The median follow up was 57 months (60
`months for those still alive).
`
`Disease free survival
`
`Overall urvival
`
`Uni-
`variate (P)
`
`< 0.0001
`0.01
`0.235
`0.045
`0.003
`0.92
`
`odes
`HER-2/11,"
`ER
`PGR
`Size
`Age
`
`Multi-
`variate (P)•
`
`Uni -
`variate {P)
`
`Multi-
`,•ariate { P) •
`
`<0.0001 (0.0818 ± 0.0214)
`0.006 [0.1142 ± 0.0413)
`0.60
`0.07
`0.15
`0.96
`
`< 0.0001 <0.0001 [0.0912 ± 0.0346)
`0.041
`0.045 [0.0864 ± 0.0288)
`0.091
`0.157
`0.20
`0.24
`0.16
`0.006
`0.ll
`0.20
`
`• Regression codli icnts :!: SE arc shown in square brackets.
`
`amplification in the univariate analysis but
`lost its significance on multivariate analysis,
`which indicates mat it was not independent
`ofnodal status (Table l). Multivariate analy(cid:173)
`sis showed HER-2/11eu amplification to be
`an independent predictor of both disease
`relapse and overall survival (P = 0.006 and
`P = 0.045, respectively) and superior to all
`other known prognostic factors with the
`exception of a number of positive lymph
`nodes for this group of patients (Table 1 ).
`We also evaluated DNA from rumors of 181
`node-negative patients with a median fol(cid:173)
`low-up of 59 monrhs (62 months for chose
`still alive). Of these, 45 (25%) had amplifi(cid:173)
`cation of the HER-2/11e11 gene. Univariate
`and multivariate analysis did not how an
`association between gene amplification and
`disease outcome in chis group of patients.
`There were 187 rumor amples of suffi(cid:173)
`cient size and integrity to aUow for multiple
`studies in the same specimen. This group of
`specimen was representative of the overaJJ
`group in that lesions from both node-nega(cid:173)
`tive as welJ as node-positive patients were
`included as welJ as rumors of varying sizes
`(< I cm to >2.5 cm). The availability of a
`cDNA done for HER-2/r1e11 from a human
`rumor as well as antisera that could identify
`die protein in both Western blots and ti ue
`ections aUowed for a comprehensive evalu(cid:173)
`ation of the gene and its products (RNA and
`protein) in these tissues. uch a study ad(cid:173)
`dresses several critical
`issues
`regarding
`HER-2/11eu in human breast cancer. First,
`the correlation between a given level of
`amplification and relative expression for
`both RNA and protein is important. Some
`genes chat are amplified in breast cancer,
`such as erb A ( 6), are not expressed; these
`genes may crve as useful markers bur are
`unlikely to be involved in die pathogenesis
`of the disease. ccond, it should be possible
`to address the issue of whether amplification
`and over-expression of HER-2/neu is pecif-
`
`ic to rumor cells in the e specimens. When
`blotting techniques alone are used, there is
`the risk of dilutional effects, which make it
`impossible to distinguish signals from ru(cid:173)
`mor cells versus those from normal cells.
`Third, chis approach should give some indi(cid:173)
`cation of the relative strengths and weak(cid:173)
`nesses of the various techniques used in
`assessing the status of the HER-2/11eu gene
`and its produces in the same primary human
`tissue. Studies at both the RNA and protein
`levels are important since there are examples
`of human rumors in which transcripts of a
`panicular gene are present but no protein
`can be detected ( 17). Fourth, if gene expre ·
`sion correlates dosely with amplification, a
`separate assessment of the incidence of am(cid:173)
`plification can be made.
`The samples used for the comprehensive
`analysis were between 0.5 and l g in size
`and had been cored for various periods of
`time at - 70° co - l 40°C. Tumor tissue was
`fracrured in I iquid nitrogen to obtain a
`representative piece uitable for cryoscatic
`sectioning and imrnunonistochemical analy(cid:173)
`si . The remainder of the entire specimen
`was ground to powder in liquid nitrogen
`with a mortar and pestle. This process al(cid:173)
`lowed for an even di tribution of rumor cells
`in all ubsequcnt analyses. A portion of the
`tissue powder (50 mg) was rored for West(cid:173)
`ern blot analysis and the remainder was
`extracted for D A and RNA simultaneous(cid:173)
`ly. As in our clinical correlation studies, each
`procedure was conducted separately and
`without knowledge of the results obtained
`by other modalitie of evaluation. outhem
`blocs with appropriate p53 and myeloperox(cid:173)
`idase controls were performed and HER-
`2/11e11 copy number determined as described
`(Fig. 1). Northern blots were performed on
`20 µ.g of rotal RNA extracted from the
`sample and were analyzed for the presence
`and relative intensiry of the 4.5-kb HER-
`2/ueu me
`··ngcr RNA using soft laser dcnsi-
`
`R.EPORTS 709
`
`PFIZER EX. 1027
`Page 6
`
`

`

`tometry and grouped into one of four RNA
`expression categories depending on the den(cid:173)
`sitometry results (Fig. I). Western blots
`were performed on I 00 µg of total protein
`exrracted from each sample and were ana(cid:173)
`lyzed for the presence and relative intensity
`of the 185-kD HER-2/neu gene product by
`soft laser densitometry and grouped into
`one of four protein expression categories
`(Fig. 1). Immunohisrochemical analysis was
`performed by an immunoperoxidase rain(cid:173)
`ing technique on frozen sections of the
`tumor tissue. The immunohistochemical
`speci.ficity of the antibody was previously
`hown (14).
`After completion of testing of all amples
`by each modality, the study was unblinded
`and a direct correlation analysis between
`gene amplification and expression was per(cid:173)
`formed. Of the 187 amples evaluated in this
`manner there was almost complete concor(cid:173)
`dance o