AM E u [LAN
`
`‘Asscrc1A11()N F()R Tfih
`ANTEMI-LN'I' or
`
`11 MAY 1931)
`
`V0]. 244 -
`
`'5‘ it5'
`
`I
`
`I’Ahl-IS n.2,: - “+-+
`
`';
`r
`l.
`i.
`‘A‘a-J-Zif'r’
`.c” 0115
`l
`txtt:mnxq»ata$:* 5-DIGIT 98195
`r, 0620 00001551“qu 12/15/89 N
`5‘ UNIV OF HASHINGTON
`LIbRARIES
`SERIALS DIVISION
`SEATTLE. HA
`93195
`.
`'
`“v
`“35‘
`‘
`
`-
`
`. v
`
`'
`
`p...
`0» ~ ,
`, T;
`‘l Aw)", ,
`
`.q
`a:
`
`’
`
`*’
`
`.
`:4
`'.
`‘?-~
`
`-
`
`’
`
`‘1
`
`.I
`.k
`3:
`
`5,"
`.D :1
`‘1
`,,
`a “Q
`‘Ql's
`\
`“V,”
`x"
`m
`J
`.
`2‘
`‘ x ,Y
`‘.
`1
`3'
`
`I
`
`:-
`
`,v ,6"
`.
`
`.
`
`‘
`,.
`
`‘1
`I»: N
`
`~
`
`‘
`
`

`

`AMERICAN
`ASSOCIATION FOR THE
`ADVANCEMENT 0::
`SCIENCE
`
`0
`
`
`
`
`
`ISSN 0036-8075
`12 MAY 1989
`VOLUME 744
`NUMBER 4905
`
`627 This Week in Science
`
` 629 on Spills
`
`631 Budget Cuts at NIH: M. I. PEAK I Climate and Forests: R. A. 52930 I
`Retraction: K. M. MILAM, S. HORN, P. I. TOFILON, D. F. DEEN.
`L. 1. Manor: I Educational Reform: D. ELKIND I Yanomami Indians and
`Anthropological Ethics: B. ALBERT AND A. R. RAMOS I Notification to Readers:
`1. F. PERM-3R AND P. Gum
`
` 643 The Dingell Probe Finally Goes Public I Credit for Whistle-Blower Vanishes I
`Secret Service Probes Lab Notebooks I “I Am Not a Neat Person” I A Qusdon
`of Intent I NIH to Use Forensics
`
`647 Electmchemists Fail to Heat Up Cold Fusion
`648 Germany Sets Up New Space Agency
`Researchers Irkcd by Changes to Testimony
`649 Frazier Honored by Psychiatrists
`Koop Resigns in a Huff
`Science Artifacts on the Block
`
`650 Skeleton Alleged in the Stealth Bomber's Closet
`652 Two Cultures Find Common Ground
`654 Gene Signals Relapse of Breast, Ovarian Cancers
`655 New Chip May Speed Genome Analysis
`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. TII-TI'ZE, R. WINKLER-OSWATI’I‘SCH, A. DRESS,
`A. VON HAESELER
`
`679 Stcrcochemisu-y of RNA Cleavage by the Tetrahymma Ribozyme and Evidence
`That the Chemical Step Is Not Rate-Limiting: I. A. MCSW’IGGEN AND T. R. CECH
`m_ 684 Model Simulation of the Cretaceous Ocean Circulation: E. I. BARRON AND
`W. H. PETERSON
`
`I
`
`SCENCEbpuNhMMIymFNdIy.Wmt-atmInn-embanmdwnhenmhunlnhbnm
`wmmunWonmvamc-musam.Imnmwmauammczmss»
`WM(WMWM.W)MMW39HW.DC.andalnnnddiflonnllmry.Nowcambinedwnhm
`mmmwompyrignrc imammmmmwmmmusam.mmsct
`ENCE .nrogmmammomews. Danube Moduli my “dominion [51 Item): $75.01»
`mmmlwbsmmmw1 issuesyflmfimnpodagomfimldlm. mmmnmmnm. air
`mail via Amsterdam $85. First class. airmail. school-year, mdsludenl meson request. ammmmumw
`m. $3.50:me093. $5.00; Biotechnology beam, $8.00 (lor mewm. Iddplrcopy $0.50 U.S.. $1.00 all
`breign); Gumbahlemmbqy Produdsand Inalruments. 518W! Dollie! Irdhurdflngaddpuoovyfl .00 U.$..
`swomszoomumagny.mxmumw.mmmm:umamwmwwmn
`dmmuefignmm www.mmutmhmmmmlbwmmmmmmdm
`mmlallwvmmMoraineWmmmmwhgmmmeblm-mmmm
`isterodwtththeCopyflghtClemCemeHOCC) Trlrtslctlonal Reporting Sonics. pmdadummbmiuofifl pet
`copyplus $0.10pupagnupalddlruouytoCCCJI Congress Stu-I. Siam. Magnet-loam: 01970. WWW
`omeforSduuisOffiG—OO‘IS’BJS! o .10:PomSamFormSSTDloSdamJQBallm.fimnm.m
`oaorr.mulmwlnmmbemomumuwmmwemmm
`I mmmmmmamenmmwummmwmWoodman.new
`mlotunmrmemolsciomisu.mhdlmemwlfimmmgmem.mbsmmnfilnkuManduml-flw.
`hiwmflmeflacfivmuuflwmInfluprmubmdhumanwoflm.andlolmrumpubneundammandap-
`medmolmlmnm and pruninufflwmlhodsolmm human progress.
`
`on
`
`sclENCE, VOL. 244
`
`I‘
`‘
`f
`
`
`
`
`
`l
`
`}
`
`

`

`
`
`
`
`
`
`Grier-enhanced tissue section fi-om a human ovarian epithelial
`cover.
`carcinoma. The tumOt cells contain >fivefold arn‘ lification of the HER-21ml;
`prom-oncogene. The section is immunostaincd wi
`' antibody to the HER-Wm
`prorein. The typical cystic nature of the tumor can be seen, and there is intense
`membrane staining (brown color) of the tumor cells lining the cystic structures.
`Stromal cells and nonmalignant elements are unstained, indicating the absence of
`the
`rotein (x100). Sec pa e 707. [Photograph by D. I. Slamon, Department of
`M 'cine, UCLA School 0 Medicine, Los Angelcs, CA 90024, and M. F. Press,
`Department of Pathology. University of Southern California School of Medicine,
`Los Angeles, CA 90033]
`
`686
`
`688
`
`690
`
`692
`
`694
`
`697
`
`700
`
`702
`
`705
`
`707
`
`713
`
`716
`
`719
`
`724
`
`Hearing in Honey Bees: Detection of Air-Particle Oscillations: W. F. TOWNB Anni
`W. H. KIRCHNER
`
`Fibroblast Growth Factor in the Extracellular Matrix of Dystrophic (mdx) Mouse
`Muscle: I. DIMARIO. N. Botswana. 8. YAMADA. R. C. STROHMAN
`
`Corn and Culture in Central Andean Prehistory: S. IOHANNESSEN AND
`C. A. HA5“)!!!
`
`Stereochemical (burst: of Catalysis by the Terrahymena Ribozyme: ]. RAIAGOPAL,
`]. A. Douom, J. W. Szosrrut
`Identification of the Fusion Peptide of Primate Immunodeficiency Vinises:
`M. L. Boson, P. L. EARL, K. FARGNOLI. S. PICCIAFUOCO, F. GIOMBINI,
`F. WONG-STML, G. FRANCHINI
`
`Calicheamicin '1" and DNA: Molecular Recognition Process Responsible for Site-
`Specificity: N. Zsm, M. PONCIN, R. NIIAKAN‘I‘AN, G. A. EILESTAD
`Complementary DNA Coding Click Beetle Luciferases Can Elicit Bioluminesc‘ence
`of Dilfcrent Colors: K. V. WOOD, Y. A. LAM, H. H. Samoan, W. D. MCELROY
`Reexamination of the Three-Dimensional Structure of the Small Subunit of
`
`RuBisCo from Higher Plants: 5. KNIGHT, I. Anosnsson, C.-I. ansn
`Receptor—Mediated Drug Delivery to Macrophages in Chemotherapy of
`Leishmaniasis: A. MUKHOI’ADHYAY, G. CHAUDHUM, S. K. ARORA, S. Seam,
`S. K. anu
`
`Studies ofthc HER'ZIrreu Prom-oncogene in Human Breast and Ovarian Cancer:
`D. I. Sutton, W. GDDOLPHIN, L. A. JONES, I. A. HOLT, S. G. WONG,
`D. B. Ksrru, W. I. LEVIN. S. G. STUART, I. Uoovs, A. ULLRICH, M. F. Puss
`
`Activation of 78 T Cells in the Primary Immune Response to Myrobmm'um
`rubermlasis: E. M. laws, 8. H. E. KAUFMANN, R. H. SCHWARTZ, D. M. PARDOLL
`
`Neural Integration of Information Specifying Structure from Stereopsis and
`Motion: M.’NAWItor AND R. BLAKE
`
`Otto Folio, reviewed by J. T. EDSALL I Nuts and Bolts of the Past, C. PURSELL I
`Evaporite Sedimentology and Evaporines and Hydrocarbons, P. SONNENFBLD I
`Continental Flood Basalts, S. A. Mensa I Books Received
`
`Magnetic Cell Sorter I Statistical Experimental Design Software I In Vivo
`Electrochemistry S
`I Cell and Tissue Adhesive I Population Dynamics
`Software I Multivariate Data Analysis Software I Literature
`
`Mary Ellen Avery
`Francisco .I. Ayala
`Floyd E. Bloom
`Mary E. Chiller
`Eugene H. Cola-Robles
`Jonah G. Gavin. Jr.
`Jam H. Gibbons
`Benn-int A Hamburg
`Willam T. Golden
`from
`moms. Nimol-un
`ExecutiveOlloer
`
`Edml Beard
`Elizabe‘lh E. Bulky
`David Ballroom
`Wilfiam F. Brinlunan
`E. Margaret Bum
`Philip E Converse
`Joseph L, Goldaloln
`Mary L Good
`F. Clark Howell
`Jan-m D. Idol. Jr.
`Loon Knopol
`Oliver E Nelson
`Helen M. RImey
`David M. Raup
`Howard A. Sorrows-man
`Larry L Srnur
`Robert M. Solow
`Jun-es D. Watson
`
`Mum
`
`John Abelson
`on: Al-Aqufl
`Don L Armrsm
`Stephen J. We
`Fbyd E. Bloom
`Henry R. Baum
`James J. Bu!
`Wyn Calms
`Charles R. Cantor
`Pinion J. Cicerone
`John M. Calm
`Hobart Dorlmnn
`Bruno F.
`Paul T. Englund
`Fredric a Fly
`Theodore H. candle
`
`finger I. M. Glass
`aeolian P. Gel
`Robert aW
`Corey S. Goodman
`Jack Gould V
`Stephen J. Gait!
`Richard M. Held
`Gloria Hoppnar
`Eric F. Johnsm
`Kat-rad B. Kramltopl
`Charles S. Loving: lll
`Richard Lnllcil
`Karl L. Maglelry
`PM»! Mutant
`Joesph B. Manln
`John C. MoGr'll
`Mommr M‘ohltln
`Carl 0. Palm
`
`Yashaylu Footer
`Mime-I I. Poanet
`Damls A Power:
`Russell Boos
`James E. Rollin-n
`Erltltl Run-luer
`Ronald H. Scum
`Vernon L. Smllll
`Robert T. N. Tran
`Virginia Td
`Emil Fl. Unlnuo
`Genre! J. Vermeij
`Bert vogMn
`Harold Weinltlm
`Irving L Webernan
`George M. Whileaidu
`Owen N. Willa
`William B. Wood
`
`nous or comm-rs 625
`
`
`
`l’mok Reviva
`
`l’l'Otlllt'lH Oi \lult-rinls
`
`
`
`7: MAY r989
`
`

`

`’¥’
`
`-1??—
`
`observed. Finally. the conjugated drug
`'
`M9096reductioninthesizeofthc
`lesion 11 days after the initiation of drug
`treatment. The greatest eifect on the regim-
`sion of the lesions by the conjugated drug
`Wasobservedatadoseoflmglkgper
`’lbotpad. The lack ofcfl'ect at higher concen-
`trations probably reflects saturation of the
`receptor-mediated uptake process for Mot-
`MBSA. The footpad regressed to nearly
`normal size when Mex-MESA was used. In
`contrast. administration of free Mot did not
`significantly alfect the footpad lesion. The
`lesions did nor reappear even 4 weeks after
`the last injecfion of Mot-MESA. During the
`experimental period all
`the animals
`re-
`mained healthy with no apparent weight
`loss. No antibody against MESA or Mor-
`MBSA was detectable in these animals after
`
`
`
`
`
`
`
`
`3 weeks as determined by the OuchterIOny
`imtnunodilfusion technique.
`In conclusion, our results show that cfi'ec—
`tive delivery of drug to macrophages can be
`achieved by using the “scavenger” receptor
`mediated endoqrtic pathway to achieve se-
`lective killing of intracellular parasites resid-
`ing in macrophages, both in vitro and in
`Vivo. A similar approach may be useful for
`efl'eetive delivery of drugs in the treatment
`of other diseases in which macrophages are
`I the primary target, including tuberculosis,
`leprosy, monocytic leukemia, and heavy
`metal storage diseases.
`
`
`
`
`
`REFERENCES AND NOTES
`
`Int. Rev. Cyml (Suppl) H. 267
`
`4.
`
`9"
`
`.1. M. 1. Chance. Br. Med}. 233. 1245 (1931).
`2. 1. A. Walsh audit. 5. Warren. N. Eng.) Mal 301,
`967 (1979).
`3. K. P. Chang,
`(1983).
`I. D. Batman, in Ln'rhmam'm’r. K. P. Chang and R.
`S. Bray Eds. (Elsevier, Amsterdam. 1985). vol. 1.
`pp. ill—138.
`I. I. Matt. in Puntririr Dunner. I. M. Mansfield, Ed.
`A
`Dehlter. New York. 1984). vol. 2. pp. 201-227.
`P‘ n U
`. V. Black. 6. 1. Watson. 1L 1. Ward, Trans. R.
`. T . Med. Hyg. 71. 550(1971’).
`0r P"
`Ahing. E. A. Suck. W. L. Hanson. P. 5.
`Loirnux, w. 1.. Chapman, lr.. of: Sri. 22. 1021
`.
`C. New. M. L. Chance. 5. C. Thomas, W.
`Peters, NflMe 172. 55 (1978).
`(I. New. M. L. Chance. S. Heath, j. Antimi-
`Cllrmndler. B. 37] [1981).
`Alvin; and G. M. Swat-t2, In, in Uporm
`ogy
`. Gregoriadis. Ed. (CRC Press, Boca
`Raton. FL, 1984). vol. 2. pp. 55-68.
`ll. C. R. Alving er al.. Pm. Natl, fired 50'. 0.5.44. 75.
`2
`s2 g
`...N
`. L. Goldstcin. ‘t'. K. Ho. 5. K. Basil. M. S. Brown.
`2...
`. 7a. 333 (1979).
`13. M. S. Brown. S. K. BISILC. R. Falk. Y. K. Ho. 1. L.
`GoldminJ. Suprmt‘l. 5m 13. 67 (1930).
`14. O. Stein and Y. Stein. Biathim. Binpkyr. Am 620,
`
`.
`
`D. 5. 515mm. Eds.
`of hutch:
`(North-Holland.
`.
`'
`'
`Armrerdam, 1975). pp. 79-81.
`16. H. I. P. Ryserand W. C. Sheri. Prat. Natl. Ami. Sci.
`USA. 75. 3867 (1978).
`17. A M
`y. G. Gluudhuri. S. K. Basil. un-
`publidiedmflyln some receptor svans chloro-
`quine. especially at relativer high concentrations.
`
`12 MAY 1989
`
`
`
`
`lnwrmaehowever.
`htlubitsuptakeoftlu '
`of ml-
`ehl
`tune W)!
`lamMu-
`A as shown 50111 the following
`observation. Hamster
`were
`’toneal macroph
`incubated at 37°C hips-ludium mnrMnfich-u-
`beled Mot-MESA {10 ug per milliliter of protein).
`The cellular content of radioactivity and that re-
`leased irteo medium (acid-soluble) were manned as
`afianctionoftimemthcpresmceandabmxeof
`chloroquine (3 M0. Cellular content afradimctiv-
`ity continued to increase in chin
`inoueated
`cultures but release of acid-soluble radioacu‘vity in
`the medium was arrested. These results suggmed
`that at this concentration chloroquine inhibited the
`degradation of Mot-MESA and not its uptake.
`
`18. M. Rabinwriuh. V. Zilberfarb, C. WM}.
`Etp. Med. 163. 520 (1986).
`19. s. Sehgal and s. it. Aron. but]. Mal. ea. at. m
`(1985).
`20. 3. Phillips and l. C. Gaaet. New 220. 1140
`(1968).
`21. I. D. Bet-man and o. LWyler. j. l‘ufin. 13.3. 141. as
`(1980).-
`22. Wethankv. K. Kala and G. C. Misha bruitically
`the nunuscript. and the Council ofScicn-
`tific and Industrial Research and Indian Council at"
`Medical Research for award offellowships to AM.
`and G.C.
`
`13 391mm was; accepted 20 February 1939
`
`Studies of the HER-Z/neu Prom-oncogene in Human
`Breast and Ovarian Cancer
`
`DENNIS J. SLAMON.* WILLIAM GODOLPHIN, LOVELL A. Jonas,
`JOHN A. Hour, 8113sz G. WONG, DUANE E. KEITH, WENDY I. LEVIN,
`SUSAN G. STUART, JUDY Uoova, AXEL ULLIUCH, MICHAEL P. Puss
`
`Cardnomaofdtebreastmdovaryacumutforone-dairdofafluncersooumingifl
`women and together are responsible for approximately one-quarter of cancer-related
`deaths in females. The HER-2km: prom-oncogene is amplified in 25 to 30 percent of
`hummpdmarybreasteancersmddfisdterafionisuwdatednddidiseasebehafior.
`In this report, several similarities were found in the bioloy ofHER—Zlneu in breast
`and ovarian cancer, including a similar incidence of amplification, a direct correlation
`between amplification and over-expression. evidence of tumors in which over-expres-
`aiou 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 simultanwusly performed on a large number of both tumor types. This
`analysis identified several potential shortcomings of the various methods used to
`evaluate IRE-2h"! in dies: disuse; (Soudacrn, Northern, and Western blots, and
`immunohismehemistry) and provided information regarding considerations that
`shouldbeaddreseedwhenstudyingageneorgeneproductinhuman tissueThedata
`preamted further support the concept that the HER-21am gene may be involved in the
`pathogenesis of some human cancers.
`
`Rom-oncooauss
`
`REPRESENT
`
`A
`
`family of normal cellular genes that
`were identified on the basis of their
`
`similarity to genetic sequences with known
`tumodgcnic or transforming potential (I).
`Considerable circumstantial evidence now
`exists that alterations in either the structure,
`copy number, or expression ofone or anoth-
`er of these genes may play a role in the
`pathogenesis of some human malignancies
`(2). One such gene. called HER-Zine" or C-
`erb 32. was first identified by transfection
`srudies in which NIH 3T3 cells were trans-
`
`formed with DNA from chemically induced
`rat neuroglioblastomas (3). The gene en-
`codes a protein that has extracellular, trans-
`mcmhrane. and intracellular domains (4)
`which is consisrent with the scruerure of a
`
`growth factor reception.
`Recently, we found a 28% incidence of
`amplification of HER-2mm in 189 primary
`human breast cancers (5). Patients with mul-
`tiple copies of the gene in DNA from their
`tumors had a shorter time to relapse as well
`as a shorter overall survival indicating that
`
`gene amplification was prognostic for disv
`ease behavior in mese individuals. More-
`
`over. multivariatc survival analysis showed
`HERllnru amplification to be more predic-
`tive for clinical outcome than all other-
`
`known prognosticators with die exception
`of positive lymph nodes (5). Since that
`initial report. a number of studies have
`published on the amplification of this gene
`
`D. Slatmnéfs. G. Wong. D. E. KcithI.) W. I. Lev'
`Division
`Hemano
`Oneo
`.
`epartn'tent
`Medicine. and the long Comp
`ive Cancer Cen-
`ter, U.C.L.A. School of Medicine. has Angeles. CA
`90024.
`
`. Van.-
`w. Godnlplun.‘ Department of Clinical
`cottvcr General Hospital. Vancouver. Canada V5 1M9.
`L.A.Ionca.De
`tof7G’vnecology. M. Blinder-
`son Hospital.
`ouston. TX
`030.
`l. A. Hair. Department of Obstetrics and
`analog.
`Univci’rsity of Chicago Medical
`(Butter. OGiiHcagot
`s. 5%. .Sruart. Triton a'm'noes. 1m. annual. CA
`94
`l.
`I.UdoveandM.P.Prcs.Dcpamtet1tofPathology
`University ofSouthern California. saloon of Medicine:
`Los Angela, CA 90033.
`A Ullnch. De
`tuanlecularBiology.Genen-
`tedi. Inc. Sou
`San Francisco. CA 94030.
`
`‘To Idiom correspondence should be addressed.
`
`REPORTS 707
`
`

`

`
`
` ?
`
`in human breast cancer and the association
`
`of gene amplification with clinical behavior
`(6—8). There is considerable variability in
`both the reported 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 natural history
`and heterogeneity of human breast cancer,
`all studies published to date sufl‘er from a
`similar problem, which is small numbers of
`
`evaluated cases (5—10). Perhaps a more sig-
`nificant shortcoming of most prior studies
`of oncogenes in human tumors including
`our own is that only one aspect ofthe gene
`in question (DNA, RNA, or protein status)
`is evaluated (5, 11, 1‘2). 'lhc potential errors
`introduced by dilution of tumor cell macro—
`molecules with macromolecules fi'om sur-
`rounding normal vascular, stromal, or in-
`flammatorycellsisageneralproblemin
`human tumor tissue and a particular prob-
`lem in breast ancer where these non-malig-
`nant cells can account for more than 50% of
`
`the tissue. All solid matrix—blotting tech-
`
`.
`
`niqnes (Southern, Western, or Northern)
`are susceptible to these errors. Similarly,
`these tedutiques cannot determine Whether
`an observed alteration is specific [be only the
`malignant cells in the tissue.
`Studies of the rat new gene isolated from
`the diunically induced neuroglioblasmrnas
`revealed it to contain a single mutation in
`the transmembranc domain that difl'crentiar-
`ed it from the neutransfbnning neu gene
`[bum in normal rattissues. This mutation is
`
`criticalintheoonversionofthe normalgcne
`into a transforming gene (13). To address
`
`ll.§tl'.. - u
`
`IDUTH INN
`
`""ll' '~'.l m...
`
`
`
`
`
`
`’ I
`
`
`
`
`
`
`MM HIS?
`
`Fig. 1. Examples of the correlation between HER-rm
`e amplification and expression. Southern
`blotan
`alyses show the [2.5-kb HER—2mm band seen wi
`Eco RI cut DNA. All DNA samples were
`ghr species and samples
`showing evidence of DNA
`checked for integrity of high molecular wci
`number dctertninalions were
`degradation were not evaluated (9). Southern blots and HER-Zincu copy
`as described (5, 9). Hybridizations were done with a 1.4-kb, 3' Eco RI fr
`agment of the human HERA
`Zine" cDNA clone. Northern blot
`shmv the 4.5-kb HER-21m transcri
`pt. All RNA samples
`Were checked for integrity ofthc 283 and 185 ribosomal RNA 5
`pecies. Total RNA (20 FE) from
`aampleswith intact RNAwererunonana
`gel, transferred to a nylon
`liltcr, and hybridized with a
`(11). Samples with intact DNA but showing some
`32P-labeled HER—21mm probe as described
`degradation of the RNA were evaluated
`IoadinngugofrotalRNAonthefilter
`by slot-blot analysis by
`'
`'onol'bothDNAandRNAwerenotusedbr
`and hybridizing as above. Samples showing
`density (0.D.) ofbands was determined
`RNA analysis. The relative optical
`bysolilaserdensitomerry
`maturing and ranged from a low of0.1 O.D. units to a high
`oils 0.1). units. Twnotswercgmuped
`into RNA expression ca
`tegories as follows: 0.1 m 0.5 CD. units, 1+; >05 to 1.0 0D. units, 2+;
`>l.0 to 1.5 OD. units, 3+; and >l.5 OD. units, 4+. Western blot analyses show the 185<|LD HER-
`2lneu protein band. The relative OD. ofbands ranged from a low of0.l OD. units In a high 4.5 CD.
`units. Tumors were
`pcd into protein expresswn ategories as follows; 0.1 to 0.5
`units, 1+;
`>05 to 1.0 0D. units, 2+; >10 to 1.5 OD. units, 3+; >l.5 0D. units, 4+. lnununohrstocbmucal
`analysis was done as described (20) with the anti-HER-Z/mu specific antibody and frozen sections (14).
`Tissues were scored and placed in one ofthc flour staining categories shown on the basis ofthe relative
`levdof
`egative to weak, 1+, 2+,
`specific staining as judged by microsco ic exarmnation as follows: n
`CRIN
`inidenticalordcrfromleftroti
`3+.Thefivesamplea
`d h
`ght in each
`The
`HER-201m m
`copynumbers(frornlefitoright)were >20,5no20, 2 r05, l,and l,res
`pectively. The
`from the Northern blots were 4.3, 1.4, 0.9. 0.2. and 2.3, res
`pectively. The
`corresponding (1D. readings
`mnetponding 0.1). readings from the Western blots were 2.0. 1.1, 0.6, 0.12, and 2.1, respectiVely.
`The corresponding inununohistochcmistry readings were 3+. 2+, l+1 weak, and 3+, respectively.
`
`
`
`Flu. 2. (blnparison
`of immunoperomidasc stain»
`ing with Western blot on aroma-rich breast
`cancer. The inset (upper left) is a hematortylin-
`eosinstainofabreasttiunotrichin
`tissue.
`Note the absence ofsignificanr numbers oftumor
`cells. Large middle panel
`is the inununoper-
`oxidasesrainingol‘rumotcellsCTCHoundindiu
`tissue.'I'licsniningisS+,placingtl1istumoriri
`thchlghestcategory of HER-21m expressionas
`‘
`byirnmmosraining.80urlmanalysisre~
`rworolivempiesofthcgeneindieDNA
`aadede blot analysis gaveanOD. reading
`of 0.6 (2+). Western blot of protein from this
`sample isshown in lane A ofthe lowerright inset.
`The 0.D.teadingforthissamplewas0.18(l+).
`while the Western blot of protein fiom another
`specimen with amplified HER~Zlm and greater
`numbetsoftumoreellsisshowninlaneB.The
`0.1). reading for this tumor (lane B) was 3.2
`(4+). Eight of the 11 tumors found to have
`inappropriately lowWestem blotdatainoompar—
`irontoodierdataweresimilarinthatdreywere
`
`. B
`
`Fly. 3. Comparison of
`stainingofHER-Wneu proteininthesamebreast
`cancerspecimenevalmtedwidifroomtismeand
`fomialin-fixed, panfin‘embedded tissue. Tissue
`showninpanelsAandBar-eftomthesametumor
`whichwasfoundtohaveSroncopiesofdie
`geneanda2+expressionlcvelbyNortliemand
`Western blot analyses. Panel A is the frozen
`scctionarrdshows2+irnrn
`‘
`'
`.l’anelBis
`theformalin-limed. paralfin-em
`sectional"
`memenmiorandslwwsnegativeimmuno-
`staining.
`
`SCIENCE, VOL 244-
`
`

`

`
`
`
`; 'r' v. H
`.
`.‘
`a
`.
`
`
`f
`P.
`a‘.
`
`
`Table 1. Umvxfiatgjnd”
`-
`survival
`1‘
`analyses can-pm" " airmen {relapse-her r, ’
`
`
`survival to
`'
`factors“
`345 nude-positive breast canon patients. Statistical
`x1 test
`performed
`by Cox’s
`nonparametric regression analysis to evaluate
`'l'actorsinamulxiv ‘
`I
`predktivrpowaofvarious combinatioosand inmactiomofprognmuc
`..
`'
`'
`parameters evaluated include number of nodes (Nodes). HER-1i
`manner as described Prognostrc'
`receptor (ER).
`terone receptor (PGR), size of .‘
`um gene amplification (HER-21m). estrogen
`primary l:urnovr(Size)1 and ageof patient at diagnosis (Age). The median follow up was 57 months (60
`months For those still alive).
`
`
` l
`
`
`
`I
`
`I
`I
`.
`1
`-
`
`'
`
`.
`
`)
`‘
`
`1
`l
`
`'
`
`I
`‘
`
`Afi&es
`
`(l
`
`'on of whether or not a similar
`
`
`. occurred in an amplified HER-
`genc found in human breast cancer.
`
`1, Mloncs from tumor tissue rather than
`
`‘M were generated by means of the
`.r -Betg vector (14, 15). Tissue was
`{1 to circumvent acquired genetic changes
`
`'
`r can occur in vitro. Analysis of the
`y ‘ ‘ ~
`an brane domain of eight clones from
`
`- ' separate tumors showed the identical
`'5- a
`c. There was no glutamic acid for
`
`substitution as reported in the trans-
`
`H2“! lug mu gene from the chemically in-
`
`.51.: rat tumors (13). There was, however,
`a a tral change of isoleucinc For valine at
`
`- " 'on 655 in the transmernhranc domain,
`ch is similar to the sequence found in
`‘
`
`y» . mnccr cell lines (16). Analysis of the
`
`coding sequence of full-length clone
`
`, comparison with the published platen.
`"* sequence (3) showed no other significant
`
`»
`; a (14). These data are consistent with
`'
`
`a concept that overexpression of a normal
`0‘ R—Zimrr gene product rather than muta-
`
`__ to an abnormal gene may be an impor-
`_ ', pathogenic event for some nunors.
`
`Having obtained and sequenced a full-
`
`);a cDNA clone from a human breast
`or, we next wanted to generate antisera
`u e human gene product. The generation
`.. . characterization of this antiserum is
`
`_-“--w 'bcd elsewhere (14) and the antibody is
`_-
`.- e blc of identifying the gene product both
`‘ Western blot analysis of tissue homoge-
`‘j w «- and immunohistochcmically in tissue
`~
`ons.
`
`In the current study. we collected a total
`23 668 human breast cancer specimens. Of
`-< . 526 had suflicient clinical follow-up
`‘;~ allow for evaluation of an association
`'-
`ecn gene amplification and charm: out~
`u.
`. As in our initial study (5), we per-
`3-
`ed Southern analysis on samples with-
`1'-
`I knowledge of the clinical outcome. All
`1" NA blots were stripped and reprobed with
`«lit
`p53 and myeloperoxidase probes to
`.-
`~ uare the relative loading ol’DNA in each
`‘ w
`and to exclude the possibility that “am-
`ification” was caused by partial or com-
`l-
`e duplication of chromosome 17 (9).
`lots were scanned by soft laser densitome-
`1'
`and the level of HER-21mm amplification
`‘9‘ determined by the ratio of the HER-
`‘1 mu signal relative to the single-copy p53
`flgnal.
`We evaluated 345 patients with node-
`‘- n 'lin: disease in a blinded Fashion (Table
`). 0f drcsc, 101 (27%) had evidence of
`' -2/m'u amplification. Univariate surviv-
`analysis showed amplification of the
`‘t
`m-Z/Hfll gene to be a significant predic-
`rr of both diseasevl'ree survival and overall
`
`'
`
`mival for these patients (Table 1). Tumor
`m was slightly better than HER—211ml
`* row was
`
`Disease free survival
`Multi-
`variant (P)'
`
`Uni»
`variate (P)
`
`Uni-
`variare (P)
`
`Overall survival
`Multi-
`variate (P)'
`
`(0.0001 [0.0912 3 0.0346]
`(0.0001
`<0.0001 [0.0818 1': 0.0214]
`(0.0001
`Nodes
`0.045 [0.0864 2 0.0288]
`0.041
`0.006 [0.1142 x 00113]
`0.01
`HER-2mm
`0.157
`0.091
`0.60
`0.235
`ER
`0.221-
`0.20
`0.07
`0.045
`PGR
`Sizc
`0.003
`0.15
`0.006
`0.16
`Age
`0.92
`0.96
`0.20
`0.1 l
`
`
`'Regrrssimeoelfidenuxsflaretlwnmsquarebnclrcu.
`ic to tumor cells in these specimens. When
`blotting
`alone are used, there is
`the risk of dilutional ell‘ects. which make it
`impossible to distinguish signals from tu-
`mor cells versus those from normal cells.
`Third, this approach should give some indi-
`cation of the relative strengths and wealt-
`nesses of the various techniques used in
`assessing the status of the HER-2km gene
`audits products il'ltl'lesan'leprimaryhurnao
`tissue. Studies at both the RNA and protein
`
`amplification in the univariate analysis but
`lost its significance on multivariate analysis,
`which indicates limit was not independent
`ofnodal status (Table 1). Multivariate analy-
`sis showed HER-Elam amplification to be
`an independent
`of both disease
`relapse and overall survival (P = 0.006 and
`l’ = 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 DNAli'om tumors of 181
`node-negative patients with a median ibi-
`low-up of 59 months (62 months for those
`still alive). Ofthese. 45 (2596) had amplifi-
`cation of the PIER-211w gene. Univariau:
`and multivariate analysis did no: show an
`association
`gene amplification and
`disease oust-tune lit this group of patients.
`There were 187 tumor samples of sulfi-
`cient sire and integrity to allow for multiple
`studies in the Same specimen. This group of
`specimens was representative of the overall
`group in that
`fi'om both node-nega-
`tive as well as node~posicive patients were
`included as well as tumors of varying Sim
`(<1 cm to >15 on). The availability of a
`cDNA clone for HER~2lnru from a human
`
`tumor as well asantisera that could identify
`the protein in both Western blots and tissue
`sections allowed lbr a comprehensive evalu-
`ation ofthe gen: and its products (RNA and
`protein) in these tissues. Such a study ad-
`dresses
`several
`critical
`issues
`regarding
`HER-21hr“ 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 that are amplified in breast cancer,
`such as rrb A (6)1 are nor expressed: these
`genes may serve as useful markets but are
`unlikely to be involved in the pathogenesis
`of the disease. Second. it should be possible
`to address the issue of whether amplification
`and mervexpmion of HER-2km: is specif-
`
`levels are important since there are examples
`
`of human tumors in which transcripts of a
`particular gene are present but no protein
`can be detected (17)- Fourth. ifgene expres~
`sion correlates closely with amplification, a
`separate assessment of the incidence of am-
`plification can be made.
`The samples used for the comprehensive
`analysis were between 0.5 and 1 g in size
`and had been stored for various periods of
`time at —70“ to - 140°C. Tumor tissue was
`li‘acruredinliquidnitrogentoobtaina
`representative piece suitable for cryostath
`sectioning and inununohistochemical analy-
`sis. The remainder of the entire specimen
`was ground to powder in liquid nitrogen
`with a mortar and pestle. This process al-
`lowed for an even distribution oftumor cells
`
`in all subsequent analyses. A portion of the
`tissue powder (50 mg) was stored for West-
`ern blot analysis and the remainder was
`attracted for DNA and RNA simultaneous-
`
`ly. As in our clinical correlation studies. each
`procedure was conducted separately and
`without knowledge of the results obtained
`by Other modalities of evaluation. Southern
`blots with appropriate p53 and mycloperox-
`idase controls were perfiimrcd and HER?
`Zhrerr copy number deten'nined as described
`(Fig. l). Northem blots were performed on
`zougoftotal RNAcxtracted {tomthe
`sample and were analyzed for the presence
`and relative intensity of the 4.5-kb HER-
`Jim-u messenger RNA using soft laser dcnsi~
`carom-s 709
`
`

`

`
`
`tomctryandgroupedinmoneoffourRNA
`expression categories depending on the den-
`sitomctry results (Fig. 1). Western blots
`werepcrfonnod on lODugoftotal protein
`extracted from each sample and were ana-
`lyzed for the presence and relative intensity
`of the lSS-kD HERoZB‘ueu gene product by
`soft laser densitotnctry and grouped into
`one of four protein expression categories
`(Fig. l). lnununohistochemical analysis was
`performed by an lmmunoperoxidase stair»
`ing technique on frozen sections of the
`tumor
`tissue. The immunohistochemical
`
`specificity of the antibody was previously
`shown (14).
`After completion of testing of all samples
`by each modality, the study was unblindcd
`and a direct correlation analysis between
`gene amplification and expression was per-
`formed. Ofthe 187 samples evaluated in this
`manner there was almost complete concor-
`‘dancc ofthc data, with a few notable excep-
`Fifty-one samples (27%) of the cases
`were called amplified by the Southern blot
`analyses, and in every instance the amplified
`samples were shown to over-express the
`HER-2mm gene product relative to nonam-
`plified cases.
`In 46 of the amplification-
`positive samples (90%} there was complete
`concordance of all tests, that is, amplifica-
`tion correlated with overexprcssion as deter-
`mined by Northern, Western and immuno-
`In all 51 cases with ampli-
`fication, two of the three measures of ex-
`pression Were concordant in showing over-
`expression.
`The Western blot analysis was most dis-
`cordant, being inconsistent with data ob~
`rained by Southern blot, Northern blot, and
`immunohistochcmistry in 11 of the 187
`eases (6%). A possible explanation for this
`phenomenon is that samples showing a
`weak signal by Westem blot may do so
`because of a relatively large amount of mo
`mainthetissue.ln80fthcllcasesin
`Which the Western was distordant, histolog-
`ic examination confirmed the presence of
`excessive snomal elements (Fig. 2). DNA
`and RNA analyses were less sensitive to this
`problem since there was evidence ofamplifi-
`cation and increased transcript levels in these
`tumors. The increased
`of the
`Western blot to dilutional e’li'ocrs is
`due to the fact that large amounrs ofnoncel-
`lular connective tissue can substantially oonv
`tribute to the total protein in a sample by
`adding significant amounts of extracellular
`matrix proteins such as collagen to the ly-
`satt. The stroma, however, is relatively poor
`in cellular-icy and will make only minimal
`contributions to the total DNA or RNA
`crunched floor the same specimen.
`The Northern blot analysis was discor-
`dent in only four cases (2%) judged ampli-
`
`710
`
`mchcmical data were inconsistent in only
`mocases(l%).lneachofthesecases,thc
`other two modalities used shower! over-
`
`orpression. The correlation between amplifi-
`cation and overexpression (P < 0.0001)
`shown in the comprehensive analysis confimns
`previously published amplification rates of 25
`to 30% for HER-Zinc“ in human breast
`cancer. There were 18 cases (10%) whidi
`were judged as single copy by DNA analysis
`but which showed clear over-expression at the
`RNA and protein levels (Fig. 1, lane 5). These
`cases may represent examples of alterations
`that occur in control mechanisms for gene
`expression, rather than increases in gene copy
`number. Alternatively, these cases may repre-
`sent instances of true gene amplification at
`low levels {Mo- to fivefold) which are missed
`becameofdilutionofthctumorcellDNA
`
`with DNA from norunalignant tissue.
`Some studies have used only immuno-
`staining of formalin-fixed, parafiin-embcd-
`dcdtissue massessmestams ofthc HER-
`
`IS). This approach
`Zlneu product (8, 10,
`presents some problems, since fixation and
`embedding of tissue frequently decreases or
`totally destroys reactivity for many antigens
`(19). Furthermore, the degree of lass can
`vary considerably depending on the dura-
`tion of fixation with the same fixative (19,
`20). For42 ofrhc 187cases in the compre-
`hensive analysis,
`fonnalin-fixcd, paraflim
`embedded tissue was available. This allowed
`for a comparison of immunohistochemical
`staining of the HER-Ilium protein in the
`same specimen prepared in different ways.
`Evaluation of HER-Zlnm immuno-reactiv-
`
`'
`4+9, the protein was visible by '
`in tissue prepared by