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`EXHIBIT 11
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`GEOLOGY GEOPHY LIB
`UNIV WISCONSIN
`1215 W DAYTON ST
`MADISON, WI
`53706
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`Case 1:18-cv-01363-CFC Document 82-11 Filed 03/22/19 Page 3 of 11 PageID #:
`9908
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`• CIENCE
`
`ISSN 0036-8075
`12 MAY 1989
`VOLUME 244
`NUMBER 4905
`
`AMERICAN
`ASSOCIATION FOR THE
`ADVANCEMENT OF
`SCIENCE
`
`Letters
`
`News & Comment
`
`Research Articles
`
`Reports
`
`627 This Week in Science
`
`629 Oil Spills
`
`631 Budget Cuts at NIH : M. J. PEAK ■ Climate and Forests: R. A. SEDJO ■
`Retraction: K . M . MILAM, S. HoRN, P . J. TOFILON, D. F. DEEN,
`L. J. MARTON ■ Educational Reform: D . ELKIND ■ Yanomami Indians and
`Anthropological Ethics: B. ALBERT AND A. R . RAMos ■ Notification to Readers:
`J. F. FERRER AND P. GUPTA
`
`643 The Dingell Probe Finally Goes Public ■ Credit for Whistle-Blower Vanishes ■
`Secret Service Probes Lab Notebooks ■ "I Am Not a Neat Person" ■ A Question
`of Intent ■ NIH to Use Forensics
`647 Electrochemists Fail to Heat Up Cold Fusion
`648 Germany Sets Up New Space Agency
`Researchers Irked 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
`654
`655
`
`Two Cultures Find Common Ground
`Gene Signals Relapse of Breast, Ovarian Cancers
`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 . E!GEN, B. F. LINDEMANN, M. TIETZE, R. WINKLER-OSWATITSCH, A. DRESS,
`A. VON HAESELER
`
`679 Stereochemistry of RNA Cleavage by the Tetrahy111e11a Ribozyme and Evidence
`That the Chemical Step Is Not Rate-Limiting: J. A. McSWIGGEN AND T. R. CECH
`
`684 Model Simulation of the Cretaceous Ocean Circulation: E. J. BARRON AND
`W. H . PETERSON
`
`■
`
`■
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`SCIENCE Is published weekly on Friday, except the last week In December, and with an extra Issue In February
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`624
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`SCIENCE, VOL. 244
`
`

`

`Case 1:18-cv-01363-CFC Document 82-11 Filed 03/22/19 Page 4 of 11 PageID #:
`9909
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`;:.-- SciENCE
`
`Color-enhanced tissue section from a human ovanan epithelial
`COVER
`carcinoma. The nunor cells contain >fivefold amplification of the HER-2/11e11
`proto-oncogene. The section is immunostained with antibody to the HER-2/11e11
`protein. The typical cystic nature of the tumor can be seen, and there is intense
`membrane staining (brown color) of the nunor cells lining the cystic structures.
`Stromal cells and nonmalignant elements are unstained, indicating the absence of
`the protein ( x 100) . See page 707. [Photograph by D. J. Slamon, Department of
`Medicine, UCLA School 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 Dystrophic (mdx) Mouse
`Muscle: J. DIMARIO, N. BUFFINGER, S. YAMADA, R . C. STROHMAN
`690 Corn and Culture in Central Andean Prehistory: S. JOHANNESSEN AND_
`C. A. HASTORF
`692 Stereochemica.l Course of Catalysis by the T etrahy111e11a Ribozyme: J. RAJAGOPAL,
`J. A. DOUDNA, J. W. SZOSTAK
`Identification of the Fusion Peptide of Primate Immtmodeficiency Viruses:
`M. L. BOSCH, P. L. EARL, K. FARGNOLI-, S. PICCIAFUOCO, F . GIOMBINI,
`F. WONG-STAAL, G. FRANCHINI
`1 and DNA: Molecular Recognition Process Responsible for Site(cid:173)
`697 Calicheamicin -y 1
`Specificity: N . ZEIN, M. PoNCIN, R. NILAKANTAN, G . A. ELLESTAD
`700 Complementary DNA Coding Click Beetle Luciferases Can Elicit Bioluminescence
`of Different Colors: K. V. Woon, Y. A. LAM, H. H. SELIG ER, W. D. McELROY
`702 Reexan1ination of tl1e Three-Dimensional Structure of me Small Subunit of
`RuBisCo from Higher Plants: S. KNIGHT, I. ANDERSSON, C.-I. BRANDEN
`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 tl1e HER-2/11e11 Proto-oncogene in Human Breast and Ovarian Cancer:
`D . J. SLAMON, W. GODOLPHIN, L.A. JONES, J. A. HOLT, S. G. WONG,
`D. E. KEITH, W . J. LEVIN, s. G. STUART, J. UDOVE, A. ULLRICH, M . F. PRESS
`713 Activation of-y'& T Cells in the Primary Immw1e Response to Mycobacteri11111
`111/Jerwlosis: E. M. ]ANIS, S. H. E. KAUFMANN, R . H. SCHWARTZ, D. M. PARDOLL
`716 Neural Integration of Information Specifying Structure from Stereopsis and
`Motion: M. NAWROT AND R. BLAKE
`
`719 Otto Folin, reviewed by J. T. EDSALL ■ Nuts and Bolts of the Past, C. PURSELL ■
`Evaporite Sedimentology and Eva.porities and Hydrocarbons, P. SONNENFELD ■
`Continental Flood Basalts, S. A. MORSE ■ Books Received
`
`724 Magnetic Cell Sorter ■ Statistical Experimental Design Software ■ In Vivo
`Electrochemistry System ■ Cell and Tissue Adhesive ■ Population Dyna.mies
`Software ■ Multivariate Data Analysis Software ■ Literature
`
`Book Reviews
`
`Products & Materials
`
`Board of Directors
`
`Walter E. Massey
`Retiring President,
`Chairman
`
`Richard C. Atkinson
`President
`
`Donald N. Langenberg
`President-elect
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`Mary Ellen Avery
`Francisco J . Ayala
`Floyd E. Bloom
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`Richard S. Nicholson
`Executive Officer
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`Editorial Board
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`Elizabeth E. Bailey
`David Baltimore
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`E. Margaret Burbidge
`Philip E. Converse
`Joseph L. Goldstein
`Mary L. Good
`F. Clark Howell
`James D. Idol, Jr.
`Leon Knopoff
`Oliver E. Nelson
`Helen M. Ranney
`David M. Raup
`Howard A. Schneiderman
`Larry L. Smarr
`Robert M. Solow
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`Board of Reviewing
`Editors
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`Oais AI-Awqati
`Don L. Anderson
`Stephen J. Benkovic
`Floyd E. Bloom
`Henry R. Bourne
`James J. Bull
`Kathryn Calame
`Charles R. Cantor
`Ralph J. Cicerone
`John M. Coffin
`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
`Richard M. Held
`Gloria H_eppner
`Eric F. Johnson
`Konrad B. Krauskopf
`Charles S. Levings Ill
`Richard Losick
`Karl L. Magleby
`Philippa Marrack
`Joseph B. Martin
`John C. McGitt
`Mortimer Mishkin
`Carl 0. Pabo
`
`Yeshayau Packer
`Michael I. Posner
`Dennis A. Powers
`Russell Ross
`James E. Rothman
`Erkki Ruoslahti
`Ronald H. Schwartz
`Vernon L. Smith
`Robert T. N. l]ian
`Virginia Trimble
`Emil R. Unanue
`Geerat J. Vermeij
`Bert Vogelstein
`Harold Weintraub
`Irving L. Weissman
`George M. Whitesides
`Owen N. Wille
`William B. Wood
`
`12 MAY 1989
`
`TABLE OF CONTENTS 625
`
`

`

`Case 1:18-cv-01363-CFC Document 82-11 Filed 03/22/19 Page 5 of 11 PageID #:
`9910
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`SciENCE
`
`12 MAY 1989
`VOLUME 244
`NUMBER 4905
`
`American Association for the Advancement of Science
`Science serves its readers as a forum for the presentation
`and discussion of important issues related to the advance·
`ment of science, including the presentation of minority or con~
`llicting points of view, rather than by publishing only material
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`12 MAY 1989
`
`Oil Spills
`
`T he Alaskan oil spill has become the most studied and managed event of its kind.
`
`Some 30 teams from various state and federal agencies have, as their goal, assessment
`of damage accruing from it. Each morning at Valdez there have been briefing
`sessions attended by 60 to 70 scientists. The cleanup is being managed by a committee that
`includes representatives from about 12 state and federal agencies.
`Thus far the sequence of events following the spill has been governed by local factors
`such as wind, temperanire, ocean currents, geology, and the composition of the Prudhoe
`Bay crude oil. Nevertheless events have been following a pattern similar to those observed in
`spills of crude oil elsewhere.
`Crude oils difl:er, but in general they contain hundreds, even thousands, of different
`compounds. Some are straight-chain hydrocarbons with carbon numbers ranging from 4 or
`5 to 35 or more. Other hydrocarbons have branched chains with a wide range of carbon
`numbers. Aromatic compounds such as benzene, toluene, and polynuclear hydrocarbons are
`substantially present. Other constinients include waxes and complex, high molecular weight
`asphaltenes. All of these substances and more are present in Prud.hoe Bay crude oil. When
`ingested, most of the compow1ds are nontoxic. Notable exceptions are some of the aromatic
`compounds, including benzene and toluene.
`After a crude oil is spilled in a marine environment, many processes follow. A typical
`crude has a density of about 0.85, more or less, and this factor combined with winds, wave
`action, and currents leads to spreading, which is particularly rapid during the first 24 hours.
`During that period most of the components having boiling points below 200°C volatilize.
`As a result some of the toxic chemicals such as benzene are removed. The composition of the
`floating mixture is further changed immediately and later by photooxidation, biodegrada(cid:173)
`tion, dispersion, and dissolution. About a day after the spill, depending on temperature and
`wave action, an emulsification of oil and sea water occurs, leading to formation of a highly
`viscous material that contains about 70% water. This material is very sticky, and it ad.heres to
`almost all objects that it encow1ters, including birds and sea otters. There has been one
`report of a minor fish kill in Prince William Sound. Observations at other oil spills indicate
`that dispersed oil is not toxic to woplankton when ingested. It is eliminated in the feces. In
`general, oil chemicals are not concentrated in the food chain.
`In Prince William Sow1d local factors have affected the movement and behavior of the
`spill. Part of the time, the sea is relatively calm, but often it is roiled by winds that create
`waves 20 feet high and more. The amplitude of the tide is about 15 feet. The Alaska Current
`enters the Sound on the east and exits on the west. This flow has protected some of the
`Sound from major contamination and has carried part of the spill out of the Sound. As a
`result of various physical , biological, and chemical processes, the inventory of oil in Prince
`William Sow1d (originally 10 million gallons) dropped about 70% during the first 4 weeks
`after the spill. The U.S. Forest Service, one agency active at the site, quotes an Exxon
`estimate as follows: evaporated, 35%; recovered, 17%; burned, 8%; biodegraded, 5%;
`dispersed, 5% . The total in the form ofoil slicks on Prince William Sound amounted to 10%
`of the original spill; that on the shoreline, 18%. A large fleet of vessels was mopping up the
`slicks, and in good weather was capturing about 120,000 gallons a day. Previous experience
`has shown that once the slicks become thin (some micrometers) they are fairly rapidly
`destroyed by photooxidation and microbial action. However, the tarry oil on 300 miles of
`shoreline will only slowly be removed by wave action and by a workforce of about 4000.
`Insofar as part of the oil leaves Prince William Sound and affects other portions of the
`Alaska coastline, Exxon will have additional problems. However, due to the cleanup, tl1e
`flushing action of tl1e Alaska Current, and the fact that some of Prince William Sound has
`escaped major contan1ination, it is likely that in a few years or less, the fauna of the Sound
`will be substantially restored. But television viewers will not soon forget heartrending scenes
`of oiled birds and sick otters.-PHILIP H . ABELSON
`
`EDITORIAL 629
`
`

`

`Case 1:18-cv-01363-CFC Document 82-11 Filed 03/22/19 Page 6 of 11 PageID #:
`9911
`was observed. Finally, the conjugated drug
`inhibits uptake of the ligand. In our case, however,
`18. M. Rabinowitch, V. Zilbcrfarb, C. Ran1azeilles, J.
`Exp. Med. 163, 520 (1986) .
`chloroquine primarily affected degradation of 1251-
`caused 90% reduction in the size of the
`labeled Mtx-MBSA as shown from the following
`19. S. Sehgal and S. K. Arora, /11d .J. Med. Res. 82, 202
`lesion 11 days after the initiation of drug
`observation . Hamster peritoneal macrophages were
`(1985).
`incubated at 37°C in medium containing 1251-la(cid:173)
`20. B. Phillips and J. C. Gazet, Nature 220, 1140
`treatment. The greatest effect on the regres(cid:173)
`bcled Mtx-MBSA (10 µg per milliliter of protein).
`(1968).
`sion of the lesions by the conjugated drug
`21. J. D. Berman and D. J. Wyler, J. Infect . Dis. 142, 83
`The cellular content of radioactivity and that re(cid:173)
`was observed at a dose of 1 mg/kg per
`leased into medium (acid-soluble) were measured as
`(1980) .
`a function of time in the presence and absence of
`22. We thank V. K. Kalra and G. C. Mishra for critically
`footpad. The lack of effect at higher concen(cid:173)
`chloroquine ( 3 µA1) . Cellular content of radioactiv(cid:173)
`reviewing the manuscript, and the Council of Scien(cid:173)
`trations probably reflects saturation of the
`ity continued to increase in chloroquine-treated
`tific and Industrial Research and Indian Council of
`receptor-mediated uptake process for Mtx(cid:173)
`cultures but release of acid-soluble radioactivity in
`Medical Research for award offellowships to A.M.
`the medium was arrested. These results suggested
`and G.C.
`MBSA. T he footpad regressed to nearly
`that at this concentration chloroquine inhibited the
`normal size when Mtx-MBSA was used. In
`degradation of Mtx-MBSA and not its uptake.
`contrast, administration of free Mtx did not
`significantly affect the footpad lesion. The
`lesions did not reappear even 4 weeks after
`the last injection of Mtx-MBSA. During the
`experimental period all
`the animals re(cid:173)
`mained healthy with no apparent weight
`loss. No antibody against MBSA or Mtx(cid:173)
`MBSA was detectable in these animals after
`3 weeks as determined by the Ouchterlony
`immunodiffusion technique.
`In conclusion, our results 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 se(cid:173)
`lective killing of intracellular parasites resid(cid:173)
`ing in macrophages, both in vitro and in
`vivo. A similar approach may be useful for
`effective delivery of drugs in the treatment
`of other diseases in which macrophages are
`the primary target, including t1,1berculosis,
`leprosy, monocytic leukemia, and heavy
`metal storage diseases .
`
`13 September 1988; accepted 20 February 1989
`
`Studies of the HER-2/neu Proto-oncogene in Human
`Breast and Ovarian Cancer
`
`DENNIS J. SLAMON,* WILLIAM GODOLPHIN, LOVELL A. JONES,
`JOHN A. HOLT, STEVEN G. WONG, DUANE E. KEITH, WENDY J. LEVIN,
`SUSAN G. STUART, JUDY UDOVE, AxEL ULLRICH, MICHAEL F. PRESS
`
`Carcinoma of the breast and ovary account for one-third of all cancers occurring in
`women and together are responsible for approximately one-quarter of cancer-related
`deaths in females. The HER-2/ne11 proto-oncogene 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 of HER-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 overexpres(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 used to
`evaluate HER-2/nrn in these diseases (Southern, Northern, and Western blots, and
`immunohistochemistry) 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-2/11e11 gene may be involved in the
`pathogenesis of some human cancers.
`
`P ROTO-ONCOGENES REPRESENT A
`
`family of normal cellular genes that
`were identified on the basis of their
`similarity to genetic sequences with known
`tumorigenic or transforming potential (1).
`Considerable circun1stantial evidence now
`exists tl1at alterations in either the structure,
`copy nwnber, or expression of one or anoth(cid:173)
`er of these genes may play a role in the
`pathogenesis of some human malignancies
`(2) . One such gene, called HER-2/11e11 or c(cid:173)
`erb B2, was first identified by transfection
`studies in which NIH 3T3 cells were trans(cid:173)
`formed with DNA from chemically induced
`rat neuroglioblastomas (3). The gene en(cid:173)
`codes a protein that has extracellular, trans(cid:173)
`membrane, and intracellular domains ( 4)
`which is consistent with tl1e structure of a
`growtl1 factor reception.
`Recently, we found a 28% incidence of
`amplification of HER-2/11e11 in 189 primary
`hwnan breast cancers ( 5). Patients with mul(cid:173)
`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 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 outcome than all other
`known prognosticators with the exception
`of positive lymph nodes (5) . Since that
`initial report, a number of studies have been
`published on the amplification of tl1is gene
`
`D. Slan1on, S. G. Wong, D. E. Keith, W. J. Levin,
`Division of Hematology-Oncology, Department of
`Medicine, and the Jonsson Comprehensive Cancer Cen(cid:173)
`ter, U.C.L.A. School of Medicine, Los Angeles, CA
`90024.
`W. Godolphin, Department of Clinical Chemistry, Van(cid:173)
`couver General Hospital, Vancouver, Canada V5ZlM9.
`L. A. Jones, Department of Gynecology, M. D. Ander(cid:173)
`son Hospital, Houston, TX 77030.
`J. A. Holt, Department of Obstetrics and Gynecology,
`Un iversity of Chicago Medical Center, Chicago, IL
`60637.
`S. G. Stuart, Triton Biosciences, Inc.; Alameda, CA
`94501.
`J. Udove and M. F. Press, Department of Pathology,
`University of Southern California, School of Medicine,
`Los Angeles, CA 90033.
`A. Ullrich, Department of Molecular Biology, Genen(cid:173)
`tech, Inc., South San Francisco, CA 94080.
`
`*To whom correspondence should be addressed.
`
`REPORTS 707
`
`REFERENCES AND NOTES
`
`l. M. L. Chance, Br. 1\!led.J. 283, 1245 (1981 ).
`2. J. A. Walsh and K. S. Warren, N. Ellg. J. Ivied. 301 ,
`967 (1979).
`3. K. P. Chang, /111 . Rei,. C ytol (Suppl. ) 14, 267
`(1983) .
`4. J. D. Berman, in Leislnnaniasis, K. P. Chang and R.
`S. Bray Eds. (Elsevier, Amsterdam, 1985), vol. l ,
`pp. lll-138.
`5. J. J. Marr, in Parasitic Diseases, J.M. Mansfield, Ed.
`(Dekker, New York, 1984), vol. 2, pp. 201- 227.
`6. C. D. V. Black, G. J. Watson, R. J. Ward, TrallS . R.
`Soc. Trap. Med. Hyg. 71, 550 (1977).
`7. C. R. Alving, E. A. Steck, W. L. Hanson, P. S.
`Loizcaux, W. L. Chapman, Jr. , Life Sci. 22, 1021
`(1978).
`8. R. R. C. New, M. L. Chance, S. C. Thomas, W.
`Peters, Nature 272, 55 (1978).
`9. R. R. C. New, M. L. Chance, S. Heath, J. A11ti111i(cid:173)
`crob . Che111otlier. 8, 371 (1981).
`10. C. R. Alving and G. M. Swartz, Jr. , in Liposo111e
`Tecln10/ogy, G. Gregoriadis, Ed. (CRC Press, Boca
`Raton, FL, 1984), vol. 2, pp. 55-68.
`11. C. R. Alving et al., Proc. Natl. A cad Sci. U. S.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, J. L.
`Goldstein,]. Supra111ol. Stnut . 13, 67 (1980).
`14. 0. Stein and Y. Stein, Biochi111 . Biophys. Acta 620,
`631 (1980) .
`15. A. N. Glazer, R. S. Delange, D. S. Sigman, Eds.,
`Chemical Modification of Proteins (North-Holland,
`Amsterdam, 1975), pp. 79- 81.
`16. H.J. P. Ryser and W. C. Shen, Proc. Natl . A cad. Sci.
`U.S .A . 75, 3867 (1978).
`17. A. Mukhopadhyay, G. Chaudhuri, S. K. Basu, un(cid:173)
`published data. In some receptor systems chloro(cid:173)
`quine, especially at relatively high concentrations,
`
`12 MAY 1989
`
`

`

`Case 1:18-cv-01363-CFC Document 82-11 Filed 03/22/19 Page 7 of 11 PageID #:
`9912
`evaluated cases (5- 10). Perhaps a more sig(cid:173)
`in human breast cancer and the association
`niques (So~thern, Western, or Northern)
`nificant shortcoming of most prior studies
`of gene amplification with clinical behavior
`are susceptible to these errors_- Similarly,
`( 6-8). There is considerable variability in
`of oncogenes in human tumors including
`these techniques cannot determme whether
`our own is that only one aspect of the gene
`both the reported incidence of amplification
`an observed alteration is specific for only the
`in question (DNA, RNA, or protein status)
`and the correlation of gene amplification
`malignant cells in the tissue.
`with patient outcome (5-10). Some groups
`is evaluated (5, 11, 12). The potential errors
`Studies of the rat neu gene isolated from
`introduced by dilution of tumor cell macro(cid:173)
`have found amplification rates as low as
`the chemically induced neuroglioblastomas
`molecules with macromolecules from sur(cid:173)
`10% and no correlation to outcome data
`revealed it to contain a single mutation in
`rounding normal vascular, stromal, or in(cid:173)
`while others have found rates as high as
`the transmembrane domain that differentiat(cid:173)
`33% and a strong association with outcome
`flammatory cells is a general problem in
`ed it from the nontransforming neu gene
`( 7, 10) . Given the variable natural history
`human tumor tissue and a particular prob(cid:173)
`found in normal rat tissues. This mutation is
`and heterogeneity of human breast cancer,
`lem in breast cancer where these non-malig(cid:173)
`critical in the conversion of the normal gene
`all studies published to date suffer from a
`nant cells can account for more than 50% of
`into a transforming gene (13). To address
`similar problem, which is small numbers of
`the tissue. All solid matrix- blotting tech-
`
`12. 5 kb
`
`4.4 kb
`
`SOUTHERN
`
`N O RTHCAN
`
`pl85
`
`"'
`
`WESTERN
`
`I
`
`IMM HIST
`
`Fig. 1. Examples of the correlation between HER-2/neu gene amplification and expression. Southern
`blot analyses show the 12.5-kb HER-2/neu band seen with Eco Rl cut DNA. All DNA 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/neu copy number determinations were
`as described (5, 9). Hybridizations were done with a 1.4-kb, 3' Eco Rl fragment of the human HER-
`2/neu cDNA clone. Northern blot analyses show the 4.5-kb HER-2/neu transcript. All RNA samples
`were checked for integrity of the 28S and 18S ribosomal RNA species. Total RNA (20 µg) from
`samples with intact RNA were run on an agarose gel, transferred to a nylon filter, and hybridized with a
`32P-labeled HER-2/neu probe as described (11). Samples with intact DNA but showing some
`degradation of the RNA were evaluated by slot-blot analysis by loading 12 µg 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 (O.D.) of bands was determined by soft laser densitometry
`scanning and ranged from a low of 0.1 O.D. units to a high of 3.8 O.D. units. Tumors were grouped
`into RNA expression categories as follows: 0.1 to 0.5 O.D . units, l+; >0.5 to 1.0 O.D. units, 2+;
`> 1.0 to 1.5 O .D . units, 3+; and > 1.5 O.D. units, 4+. Western blot analyses show the 185-kD HER-
`2/neu protein band. The relative O.D . of bands ranged from a low ofO. l O.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, 1 +;
`>0.5 to 1.0 O.D. units, 2+; > 1.0 to 1.5 O.D. units, 3+; > 1.5 O.D. units, 4+ . Immunohistochemical
`analysis was done as described (20) with the anti-HER-2/neu 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, 1 +, 2 +,
`3 +. The five samples analyzed here are arranged in identical order from left to right in each panel. The
`HER-2/neu copy numbers (from left to right) were >20, 5 to 20, 2 to 5, 1, and 1, respectively. The
`corresponding O.D. readings from the Northern blots were 4.3, 1.4, 0.9, 0.2, and 2.8, respectively. The
`corresponding O .D . readings from the Western blots were 2.0, 1.1, 0.6, 0.12, and 2.1, respectively.
`The corresponding imrnunohistochemistry readings were 3+, 2+, 1 +, weak, and 3+, respectively.
`
`708
`
`A B
`
`- p1B5
`
`STIIOMA
`
`•
`
`Fig. 2. Comparison of imrnunoperoxidase stain(cid:173)
`ing with Western blot on stroma-rich breast
`cancer. The inset (upper left) is a hematoxylin(cid:173)
`eosin stain of a breast tumor rich in stromal tissue.
`Note the absence of significant numbers of tumor
`cells. Large middle panel is the imrnunoper(cid:173)
`oxidase staining of tumor cells (TC) found in this
`tissue. The staining is 3 +, placing this tumor in
`the highest category of HER-2/neu expression as
`judged by imrnunostaining. Southern analysis re(cid:173)
`vealed two to five copies of the gene in the DNA
`and Northern blot analysis gave an O.D. reading
`of 0.6 (2+ ). Western blot of protein from this
`sample is shown in lane A of the lower right inset.
`The O.D. reading for this sample was 0.18 (1 + ),
`while the Western blot of protein from another
`specimen with amplified HER-2/neu and greater
`numbers of tumor cells is shown in lane B. The
`O.D. reading for this tumor (lane B) was 3.2
`( 4 +) . Eight of the 11 tumors found to have
`inappropriately low Western blot data in compar(cid:173)
`ison to other data were similar in that they were
`stromal-rich tumors.
`
`A
`
`·-
`
`B
`
`P.t.11,VPIN
`
`Fig. 3. Comparison of imrnunohistochemical
`staining of HER-2/neu protein in the same breast
`cancer specimen evaluated with frozen tissue and
`formalin-fixed, paraffin-embedded tissue. Tissue
`shown in panels A and B are from the same rumor
`which was found to 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+ imrnunostaining. Panel Bis
`the formalin-fixed, paraffin-embedded section of
`the same tumor and shows negative imrnuno(cid:173)
`staining.
`
`SCIENCE, VOL. 244
`
`

`

`Case 1:18-cv-01363-CFC Document 82-11 Filed 03/22/19 Page 8 of 11 PageID #:
`9913
`Table 1. Univariate and multivariate