fimymmm
`flsmcumgg 702% mg
`mvmmmm m,» g
`
`
`
`
`
`
`
`‘1’
`!
`
`:1; 11' )3: fl:
`"1) ‘2 N
`
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`
`IMMUNOGEN 2117, pg. 1
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2117, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`,
`
`0
`
`AMERICAN
`ASSOCIATION FOR THE
`ADVANCEMENT OF
`SCIENCE
`
`ISSN 0036-8075
`9 JANUARY 1987
`VOLUME 235
`NUMBER 4.785
`
`
`
`139
`
`This week in Science
`
`
` ' EditOriaL ‘ f"
`
`Letters
`.
`g
`‘
`l
`:9 V
`144 Misuse of the Freedom of Information Act: I. R. WILLIAMS I Rtidenberg’s
`Patents: I. L. HUMMER Ii Quality of Biomedical Literature: R. G. MARTIN I
`Quantitative Risk Aspects of the “Woburn Case”: M. T. SMITH I Moonlight and
`Circadian Rhythms: R. M. SINCLAIR; C. A. CZEISLER AND J. S. ALLAN I
`Research Practices: W. W. STEWART AND N. FEDER
`
`Fraud in Science
`
`
`
`NBWS& Comment
`
`.
`
`
`151
`153
`154
`155
`156
`158
`159
`
`
`Science Budget: More of the Same I RSLD and the Deficit
`Peer Review—’oops—Merit Review in for Some Changes at NSF
`Cancer M.D.’s Clash over Interleukin Therapy
`Landsat Commercialization Stumbles Again
`Hazardous Waste: Where to Put It.>
`EEC Research Program in Jeopardy
`Court Rejects Ritkin in Biotech Cases
`Math Papers Called Inaccessiblc
`Briefing: Comings and Goings
`
`
`
`
`
`__
`
`
`
`I
`
`.1,
`
`
`
`'
`
`'
`
`‘
`
`-
`
`'
`
`
`
`
`
`
`‘
`
`'
`
`
`
`
`ReSearch Articles
`
`Oncogenes Give Breast Cancer Prognosis
`160
`161 Materials Scientists Seek a Unified Voice
`163
`Diabetics Should Lose Weight, Avoid Diet Fads I High—Garb Diets Questioned
`165
`Delving into Faults and Earthquake Behavior: HOW to Stop a Quake by logging
`I Earthquakes Are Giving Little Warning I Coastal Ups and Downs Point to a
`
`Big Quake I Cutting the Gordian Knot of the San Andreas
`
`Community Diversity: Relative Roles Of Local and Regional Processes:
`R. E. RICKLEFS
`Band-Gap Engineering: From Physics and Materials to New Semiconductor
`Devices: F. CAPASSO
`
`167
`
`172
`
`177
`
`Human Breast Cancer: Correlation of Relapse and Survival with Amplification of
`the HER-Zineu Oncogene: D. I. SLAMON, G. M. CLARK, S. G. WONG,
`W. I. LEVIN, A. ULLRICH, W. L. MCGUIRE
`
`The Atomic Structure of Mengo Virus at 3.0 A Resolution: M. LUO,
`G. VRIEND, G. KAMER, I. MINOR, E. ARNOLD, M. G. ROSSMANN, U. BOEGE,
`D. G. SCRABA, G. M. DUKE, A. C. PALMENEERO
`
`
`
`
`
`____________.____,__—————-———-
`I
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`SCIENCE, VOL. 235
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`,
`.21
`
`IMMUNOGEN 2117, pg. 2
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2117, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`Human Breast Cancer: Correlation of
`Relapse and Survival with Amplification
`of the HER-Z/neu Oncogene
`
`DENNIS I. SLAMON,* GARY M. CLARK, STEVEN G. WONG, WENDY I. LEVIN,
`AXEL ULLRICH, WILLIAM L. MCGUIRE
`
`
`
`The HER-Zlneu oncogene is a member of the erbB—like
`oncogene family, and is related to, but distinct from, the
`epidermal growth factor receptor. This gene has been
`shown to be amplified in human breast cancer cell lines.
`In the current study, alterations of the gene in 189
`primary human breast cancers were investigated. HER-Z/
`mm was found to be amplified from 2- to greater than 20-
`fold in 30% of the tumors. Correlation of gene amplifica-
`tion with several disease parameters was evaluated. Am-
`plification of the HER-Z/mm gene was a significant pre-
`dictor of both overall survival and time to relapse in
`patients with breast cancer. It retained its significance
`even when adjustments were made for other known
`prognostic factors. Moreover, HER-Z/nm amplification
`had greater prognostic value than most currently used
`prognostic factors, including hormonal-receptor status,
`in lymph node—positive disease. These data indicate that
`this gene may play a role in the biologic behavior and/or
`pathogenesis of human breast cancer.
`
`F
`
`I
`
`lHE EVIDENCE LINKING PROTO-ONCOGENESTOTHE INDUC-
`
`tion or maintenance of human malignancies is largely cir-
`cumstantial, but has become increasingly compelling. This
`circumstantial evidence is derived from studies of animal models,
`tumor cell lines, and actual human tumors. Data from animal models
`and cell lines include: (i) sequence homology between human proto-
`oncogenes and the viral oncogenes of transforming retroviruses that
`are known to be tumorigcnic in some species (I, 2); (ii) transfcction
`studies showing the transforming potential of prom-oncogenes in
`NIH 3T3 cells and primary embryo fibroblasts (3—5); and (iii) the
`central role of certain proto-oncogenes in tumorigenesis by chronic
`transforming retroviruses such as avian lcukosis Virus (6). Data from
`human tumors include: (i) increased expression of specific proto-
`oncogencs in some human malignancies (7, 8); (ii) localization of
`proto-oncogenes at or near the site of specific, tumor-associated
`chromosomal translocations (9); and (iii) amplification of proto-
`oncogenes in some human tumors (10, 11).
`Additional data linking proto-oncogenes to cell growth is their
`expression in response to certain proliferation signals (12, 13) and
`their expression during embryonic development (14, 15). More
`direct evidence comes from the fact that, of the 20 known proto—
`oncogenes, three are related to a growth factor or a growth factor
`receptor. These genes include c-xir, which is homologous to the
`9 JANUARY 1937
`177
`ARTICLES
`———————————————_——————————-——A
`
`
`
`transforming gene of the simian sarcoma virus and is the B chain of
`platelet—derived growth factor (PDGF) (16, 17); c-fim, which is
`homologous to the transforming gene of the feline sarcoma virus
`and is closely related to the macrophage colony-stimulating factor
`receptor (CSF-lR)
`(18); and c-erliB, which encodes the EGF
`receptor (EGFR) and is highly homologous to the transforming
`gene of the avian erythroblastosis virus (19). The two receptor-
`related prom-oncogenes, c-fim and c-erbB, are members of the
`tyrosine-specific protein kinase family to which many proto-onco-
`genes belong.
`‘ Recently, a novel transforming gene was identified as a result of
`transfection studies with DNA from chemically induced rat neu-
`roglioblastomas (20). This gene, called mu, was shown to be related
`to, but distinct from, the c-erliB proto~oncogene (21 ). By means of
`v-crbB and human EGFR as probes to screen human genomic and
`complementary DNA (cDNA) libraries, two other groups indepen-
`dently isolated human erlIB—related genes that they called HER-2
`(22) and c—m'bB-Z (23). Subsequent sequence analysis and chromo-
`somal mapping studies revealed all three genes (mu, C-erbB-Z, and
`HER-2) to be the same (22, 24, 25). A fourth group, also using v-
`erbB as a probe, identified the same gene in a mammary carcinoma
`cell line, MAC 117, where it was found to be amplified five- to ten-
`fold (26).
`This gene, which we will call HER—201m, encodes a new member
`of the tyrosine kinase family; and is closely related to, but distinct
`from, the EGFR gene (22). HER~2/neu dilfers from EGFR in that it
`is found on hand q21 of chromosome 17 (22, 24, 25), as compared
`to band pll—plS of chromosome 7, Where the EGFR gene is
`located (27). Also,
`the HER-201m gene generates a messenger
`RNA (mRNA) of 4.8 kb (22), which differs from the 5.8- and 10—
`kb transcripts for the EGFR gene (28). Finally, the protein encoded
`by the HER~2/nm gene is 185,000 daltons (21), as compared to the
`170,000-dalton protein encoded by the EGFR gene. Conversely, on
`the basis of sequence data, HER-2mg” is more closely related to the
`EGFR gene than to other members of the tyrosine kinase family
`(22). Like the EGFR protein, IIER-Z/mu has an extracellular
`domain, a transmembrane domain that includes two cysteine-rich
`repeat clusters, and an intracellular kinase domain (21), indicating
`
`
`I. Slamon, S. G. Wong, and W. }. Levin are in the Division of Hematology-
`D.
`Oncology, Department of Medicine and Jonsson Comprehensive Cancer Center,
`UCLA School of Medicine, Los Angeles, CA 90024. G. M. Clark and W. L. McGuire
`are in the Division of Oncology, Department of Medicine, University of Texas Health
`Science Center at San Antonio, San Antonio, TX 78284. A. Ullrich is
`in the
`Department of Molecular Biology, Genentech, Inc., South San Francisco, CA 94080.
`
`*To Whom correspondence should be addressed.
`
`IMMUNOGEN 2117, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`IMMUNOGEN 2117, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Table 1. Association between HER-Z/neu amplification and disease parame-
`
`ters in 103 breast tumors.
`Number of tumors
`
`ER+
`ER"
`PgR+
`PgR—
`<2
`2:5
`>5
`Unknown
`
`53
`31
`42
`42
`13
`34
`17
`20
`
`21
`52
`11
`
`
`
`
`
`65
`38
`52
`51
`15
`41
`22
`, 25
`
`25
`65
`13
`
`0.99
`
`0'85
`
`0 82
`'
`
`0.83
`
`0‘11
`
`1
`4
`2
`3
`0
`1
`2
`2
`
`1
`4
`0
`
`I,
`'
`
`1:
`_
`L
`_,
`1
`
`1
`,
`-
`
`
`
`3
`
`that it too is likely to be a cellular receptor for an as yet unidentified
`ligand.
`,
`As a result of the published data showing amplification of HER-
`2/mzt in a human mammary carcinoma cell line, and as part of an
`
`
`
` ongoing survey in our laboratory of prom—oncogene abnormalities Factor* Singlc 2 t9 5 5 to?” >29 Total PT
`
`
`in human tumors, we evaluated alterations of the HER-201m gene
`copy
`copies
`copies
`copies
`in a large series of human primary breast cancers. Our results show
`Hormonal rtcepttn‘rmtm
`that amplification of this gene occurs relatively frequently in breast
`2
`9
`cancer, and that it is associated with disease relapse and overall
`l
`2
`patient survival.
`,
`2
`6
`Factors that are lcnown to be im ortant in the ro nosis of breast
`1
`5
`.
`.
`.
`.
`.
`.
`. P
`.
`.P g
`.
`Tumor size (centimeters)
`malignanCies in indwidual patients include: Size of the primary
`1
`1
`tumor, stage of disease at diagnosis, hormonal receptor status, and
`1
`5
`number of axillary lymph nodes involved with disease (positive
`1
`2
`nodes) (29). The current study, which was conducted in two parts,
`0
`3
`involved the evaluation of tissue from 189 separate breast malignan-
`Age at diagnosis (yam)
`550
`cics that were part of a breast cancer study ongoing at the University
`I
`2
`>50
`of Texas, San Antonio. This cohort of tumors was of interest
`2
`7
`-
`because considerable information was available on the majority of Unknown
`0
`, ‘2
`the specimens including size of the primary tumor, estrogen recep-
`Number ofporztmz 13W?[7 nodes
`g:
`1
`tot status, progesterone receptor status, age of patient, disease stage,
`8
`i’
`:8
`(113
`25
`2
`and status of the axillary lymph nodes.
`2
`4
`17
`>3
`
`In the initial survey, tissue from 103 primary breast cancers was
`
`
`
`1 3 117Unknown 22
`
`evaluated for alterations in the HER-2mm gene. DNA from
`,
`*Receptor status was analyzed as described (39). ER, estrogen receptor: + and - refer
`lnleldual tumors was prepared as described (30)., dlge‘qted WIth
`to the presence or absence of 23 fmul of receptor per rmlhgram of protein. PgR,
`ECO R1, and subJCCth to Southern blot anaIySis Wll'h a 321’-labeled
`progesterone receptor: + and — refers to the presence or absence of 25 finol ofreceptor
`_
`_
`-
`-
`,
`g
`-
`'
`'
`per rrrilh rain of protein.
`TStatistical analyses for correlation of HER-2mm muphfi-
`HER 2/118“ 1 prObe’ Wthh IS LIIOVVH t0 dflCCt a 13 kb hybrldlzmg
`cation with disease parameters were performed by die x2 test. P values were computed
`band in human DNA (22). Examples of tumors from the initial
`after combining the cases with 5 to 20 and >20 copies.
`survey are shown in Fig. 1. 0f the 103 samples examined, 19 (18%)
`showed evidence of HER-201m gene amplification. The degree of
`amplification in individual cases was determined by dilution analysis
`(Fig. 2A), as well as soft laser densitometry scanning. To determine
`that the amount of DNA loaded in each lane was equivalent, all
`filters were washed and rehybridized with a 32P-labeled arginase
`gene probe (31). This probe identifies a 15~kb hybridizing band on
`Eco RI—digested human DNA, and was selected as a control
`because it more appropriately assesses the relative amount and
`
`transfer of high molecular weight species than a probe hybridizing
`with low molecular weight species, which transfer more readily on
`Southern blotting. All
`lanes were shown to contain equivalent
`amounts of high molecular weight DNA (Fig. 2B). Individual
`tumors were assigned to groups containing a single copy, 2 to 5
`copies, 5 to 20 copies, and greater than 20 copies of the HER~2/mzu
`gene (Fig. 1). Assignment of tumors to the various groups was done
`
`Fig. 1. Analysis of alterations of the HER-201m
`gene in human breast cancer. Shown are 79 of the
`189 breast tumors used in this analysis. Tumors
`with a single copy of HER-201m: 3, 4, 10 to 15,
`20, 23 to 25, 27 to 29, 31, 38, 42 to 46, 48, 49,
`52, 55, 61, 65, 66, 71, 72, and 74. Tumors with
`two to five copies ofHER-Z/ueu: 1, 2, 5, 7, 9, 16,
`17, i9, 21, 22, 32, 35, 36, 47, 50, 54, 56 :6 58,
`60, 62, 70, and 75 to 77. Tumors with 5 to 20
`copies of HER<2/m:u: 6, 8, 26, 34, 37, 39 to 41,
`51, 53, 63, 64, 67, 69, 73, and 79. Tumors with
`more than 20 copies of HER—201m: 18, 30, 33,
`59, 68, and 78. Examples oftumors 77 to 79 have
`rearrangements in the HER-Z/nm gene. DNA
`was extracted from tissues and digested with Eco
`RI as described (30). A total of 12 ug ofEco RI—
`digested DNA was loaded onto 0.8% agarose
`gels, separated by electrophoresis, and transferred
`onto nylon filter papers (Biodyne) (30). All filters
`were baked in a vacuum oven for 3 hours at 80°C,
`prehybridized in 5X SSC (standard saline citrate)
`containing 50% formamidc, 10% dextran sulfate,
`0.1% SDS, denatured salmon sperm DNA (1 mg/
`ml), and 4X Denhardts solution for 12 hours,
`then hybridized in the same solution containing
`32P-labeled nick-translated HER-2 probe (21)
`specific activity of 1 X 108 cpm per microgram of
`DNA; 2 X 106 cpm/ml. Hybridization occurred
`at 42°C for 48 hours, followed by washing of
`filters under the following conditions in succes-
`
`178
`2W
`
`i
`
`2 34 5 «
`
`6
`
`7 8 9
`
`.1011121314
`
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`3031323334
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`_
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`L
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`151617181920 ' 212223242526272829
`
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`41424344454647484950
`l
`
`
`
`51 52 53 54 55
`‘
`‘
`
` mmmmw
`
`
`SCIENCE, VOL. 235
`
`-
`-
`‘
`sion: 2X SSC for 20 minutes at room tempera-
`ture; two washes of 30 minutes each in 2X SSC,
`0.1% SDS at 65°C, one wash of 30 minutes in
`
`2
`0,5X SSC, 0.1% SDS at 65°C. Filters were their
`exposed to XAR—S X-ray film (Kodak) for autora—
`diogi'aphy.
`
`IMMUNOGEN 2117, pg. 4
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2117, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Fig. 2. (A) Example of dilutional analysis to assess degree of HER-Zlmu
`gene amplification. Lanes a, g, k, and p were loaded with 12 pg of Eco RI—
`digested breast tumor DNA. Lane a is DNA from tumor 31 (Fig, 1), which
`represents a tumor with a single copy of the HER/24ml gene. Lane g is
`DNA from tumor 33, which represents a tumor with >20 copies of the
`HER—Z/neu gene. Lanes b to fare serial dilutions (1:100, 1:20, 1:10, 1:5,
`and 1:2, respectively) of the DNA sample in lane g. Lane k is DNA from
`tumor 35 (Fig. 1), which represents a tumor containing two to five copies of
`the HER-2km; gene. Lanes h toj are serial dilutions (1:10, 1:5, and 1:2,
`respectively) of the DNA sample in lane k. Lane p is DNA from tumor 34
`(Fig. 1), which represents a tumor with 5 to 20 copies of the HER-Z/neu
`gene. Lanes l to 0 are serial dilutions (1:20, 1: 10, 1: 5, and 1:2, respective-
`ly) ofthc DNA sample in lane p. The filter was prepared and hybridized with
`a 32P~labeled HER-2 probe as in Fig. 1. (B) Example of arginase probe
`hybridization to demonstrate that equivalent amounts of tumor DNA were
`loaded into each lane. Rehybridization of filter containing lanes 30 to 40
`(Fig. 1). The filter was first stripped of label by washing in a buffer made up
`of 50% formamidc, 3X SSC, and 0.1% SDS at 65°C for 20 minutes,
`following by three successive washes of 5 minutes each in 0.1 X SSC at room
`temperature. Filters were exposed overnight on XAR‘S film (Kodak) to
`ensure removal of all radioactive probe, then rehybridi7ed as in Fig. 1 with a
`32P-labelcd human arginase gene probe (31).
`
`in a blinded fashion, in that they were made without knowledge of
`disease parameters. Analysis of the data for association between gene
`amplification and a number of disease parameters was then per—
`formed.
`
`Of 103 tumors evaluated in the initial survey, there was essentially
`no correlation between gene amplification and estrogen receptor
`status, progesterone receptor status, size of tumors, or age at
`diagnosis (Table 1). However, when analysis was performed for
`association between HER-2mg“. amplification and number of posi-
`tive lymph nodes, a trend was noted. This analysis showed that 4/34
`(11%) of patients with no involved nodes, 2/20 (10%) with l to 3
`involved nodes, and 8/25 (32%) with >3 involved nodes had gene
`amplification (P = 0.11). If these data were examined by comparing
`0 to 3 positive nodes versus >3 positive nodes, the correlation with
`gene amplification became more significant (P < 0.05). Thus, there
`was a significant increase in incidence of HER-Z/neu gene amplifica-
`tion in patients with >3 axillary lymph nodes involved with disease.
`A multivariate regression analysis to correlate HER—Z/m’u amplifica-
`tion with various disease parameters identified the number of
`positive nodes as the only significant factor, either alone or in
`combination, to correlate with amplification.
`This initial study indicated that it might be possible to discrimi-
`nate among node— positive patients on the basis of HER-Z/nm gene
`
`1.0
`
`71.:
`
`
`
`
`amplification. It is well known that die number of positive nodes is
`the best prognostic factor for disease recurrence and survival in
`patients with breast cancer (29). Given the correlation between
`number of nodes positive and HER-2mm amplification, one might
`predict
`that amplification of this gene might also have some
`prognostic value. No long—term follow-up data, however, were
`available on the 103 patients analyzed in the initial study. For this
`reason, a second study was conducted on 100 breast cancer samples
`from patients with positive axillary lymph nodes. All of the informa»
`tion available for the first group of 103 patients was available for
`these patients. In addition, relapse and survival information was
`available, since these cases had a median follow—up of 46 months
`(range 24 to 86 months). Of these 100 samples, 86 yielded suflicient
`DNA for study. Amplification of the HER-201314 gene was mea-
`sured as in the initial survey, and examples of tumors from mis study
`are shown (Fig. 1). Amplification was found in 34/86 (40%) of
`these patients. For this larger sample of node-positive patients,
`several statistically significant or nearly significant relationships were
`observed. In agreement with the preliminary survey, there was an
`association between number of involved lymph nodes and HER-2/
`new amplification (Table 2). In addition,
`the presence of gene
`amplification was correlated with estrogen receptor status and size
`of primary tumor (Table 2). Together, these two surveys yielded
`data on 189 patients and the association of HER-Z/ncu amplifica—
`tion with various disease parameters in the combined group is
`shown in Table 3.
`
`While these correlations were of interest, the strong relationship
`
`,_l
`
`Not amplified (11:52)
`
`
`
`
`Neil amplified (n:52)
`
`
`Amplified (11:34)
`
`1‘
`;
`a
`I
`
`—
`
`3 0.8 A
`L N
`3%
`~T a 0.6 a
`m 9
`g a
`$73“ 0.4 a
`0;
`in 0.2 ~
`
`A
`1] ,‘r W ,1 7
`
`I
`
`I
`
`I
`
`, ,1
`
`I
`
`
`
`Amplified (17:11)
`~
`>5 copies
`
`C
`I
`I
`I
`I
`I
`I
`_I
`
`
`
`
`
`~
`'
`'
`Flg. 3. Actuarial cun’e for relapse in (A) node—
`positive patients with no amplification versus
`node-positive patients with any amplification (>2
`copies) of HER»2/m’u and (C) node»positive pa-
`tients with no amplification versus node—positive
`atients with rcater than 5 co ies of HER»2/fleu.
`P
`-
`g
`P-
`-
`Actuarial curve for overall survrval in (B) node?
`positive patients with no amplification versus
`node-positive patients with any amplification (>2
`copies) ofHER-Z/Hrn and (D) node~positive pa-
`tients with no amplification versus node~positive
`patients with greater than 5 copies of HER-Z/m’u.
`Actuarial curves for both relapse and overall sur—
`vival were computed by the method of Kaplan
`and Meier (44) and compared by the log rank test
`(424.14).
`
`
`
`
`
`
`"0—%
`
`
`
`
`
`Not amplified 01:52)
`5 0,3 a
`_H
`
`:5
`
`:E 0 0 7
`
`g g '
`5; 0.4 A
`E
`”’5, 0,2
`
`“"‘P'me'j l" =34)
`
`B
`
`o
`
`0
`
`I
`12
`
`I
`24
`
`I
`36
`
`T
`48
`
`I
`60 .
`
`I
`72
`
`-
`
`_
`
`_
`
`D
`
`
`
`;
`'
`'
`N°‘a"‘p""ed (’7 52)
`
`i
`
`Amplified ("=10
`>5 °°p‘°5
`
`I
`12
`
`I
`24
`
`r
`as
`
`I
`as
`
`I
`so
`
`I
`72
`
`j
`84
`
`o
`84
`Time (months)
`
`9 JANUARY 1987
`
`lib—“.—
`
`ARTICLES
`
`179
`
`IMMUNOGEN 2117, pg. 5
`Phigenix v. Immunogen
`|PR2014-00676
`
`IMMUNOGEN 2117, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`between HER—Z/nen amplification and nodal status (P = 0002)
`indicated that
`information on amplification of this gene may
`correlate with disease behavior; that is, recurrences and survival. To
`test
`this, univariate survival analyses were performed in which
`amplification was compared to relapse and survival in this patient
`group. A total of 35 patients had a recurrence of the disease, and 29
`had died at the time of the analyses. Median times to relapse and
`death were 62 months and 69 months, respectively. The median
`follow-up time for patients still alive was 47 months, ranging from
`24 to 86 months. A total of 71 of the 86 patients (83%) received
`some form of therapy after mastectomy: adjuvant systemic therapy
`alone, 47%; adjuvant systemic therapy plus local radiation, 19%;
`and local radiation alone, 17%. A strong and highly statistically
`significant correlation was found between the degree of gene
`amplification and both time to disease relapse (P = <0.0001) and
`survival (P = 0.0011) (Table 4). Moreover, when compared in
`univariate analyses to other parameters, amplification ofHER-2/nen
`was found to be superior to all other prognostic factors, with the
`exception of the number of positive nodes (which it equaled) in
`predicting time to relapse and overall survival in human breast
`cancer (Table 4). The association between HER—Z/nen amplification
`and relapse and survival can be illustrated graphically in actuarial
`survival curves (Fig. 3, A to D). While there was a somewhat
`shortened time to relapse and shorter overall survival in patients
`having any amplification of the HER-Z/nen gene in their tumors
`(Fig. 3, A and B),
`the greatest differences were found when
`comparing patients with >5 copies of the gene to those without
`amplification (single copy) (Fig. 3, C and D). Patients with greater
`than five copies of HER-2/nen had even shorter disease-free survival
`times (P : 0.015) and overall survival
`times (P = 0.06) when
`compared to patients with no amplification. The phenomenon of
`greater gene copy number correlating with a worse prognosis has
`also been seen in evaluations of N—myr gene amplification in human
`neuroblastomas (32).
`To determine if amplification of HER-2/nen was independent of
`other known prognostic factors in predicting disease behavior,
`multivariate survival analyses were performed on the 86 trade
`positive cases. Amplification of the gene continued to be a strong
`prognostic factor, providing additional and independent predictive
`information on both time to relapse and overall survival in these
`
`51
`
`52
`
`53
`
`54
`
`55
`
`56
`
`57
`
`58
`
`59
`
`60
`
`61
`
`62
`
`53
`
`64
`
`65
`
`66
`
`
`
`Fig. 4. Example of rehybridization offilter with human EGFR probe. Filters
`were stripped as in Fig. 2B, and hybridized with 32P-labeled human EGFR
`probe (28), as in Fig. 1. Shown are the lower molecular weight bands
`hybridized with 32P—labeled EGFR probe in filter-containing lanes 51 to 66
`(Fig. 1). The bands from top to bottom are 2.8, 2.2, and 1.8 kb, respectively.
`Lane 52 is an example of a tumor showing marked amplification (>50
`copies) of the EGFR gene.
`
`patients, even when other prognostic factors were taken into
`account (Table 4).
`Rearrangement of the HER—Z/ncn gene was rare. Of the total 189
`tumors evaluated, three showed evidence of rearrangement, and in
`two of the three cases, the rearrangement was identical (Fig. 1, cases
`77 to 79). Also,
`two of the rearranged HER-Z/nzn loci were
`amplified (Fig. 1, cases 78 and 79). The incidence of HER-Z/nen
`rearrangement as determined by Eco RI digestion was too small to
`attempt statistical correlations.
`To determine whether the phenomenon of amplification of HER-
`2/nen in breast cancer extended to related growth factor receptors,
`all filters were analyzed with the EGFR probe (Fig. 4). Amplifica-
`tion of the EGFR gene was found in 4/189 (2%) of the cases, and
`rearrangement of the EGFR gene was found in one of those four
`cases. The incidence of EGFR amplification and rearrangement was
`too small to attempt statistical correlation. Comparison of HER—2/
`nen amplification (53/189 or 28%) with that of the EGFR gene
`reveals the incidence of the former to be 14 times greater than that
`of the latter, indicating that the phenomenon of gene amplification
`is not a general one for a related tyrosine kinase—specific receptor in
`human breast cancer. Moreover, studies examining alterations of
`two other tyrosine kinase—specific prom—oncogenes, ab! and fizs, in
`breast cancer did not show amplification of these genes (33).
`Alterations of non—tyrosine lcinase—rclated proto-oncogenes in these
`
`Table 3. Association between HER-Z/nen amplification and disease parame-
`ters in combined surveys (189 patients).
`————.___
`Single
`2 to 5
`5 to 20
`>20
`_
`.
`.
`Factor*
`Total
`
`COPY
`C013163
`“717'“
`”Pics
`
`PT
`
`Harmorial receptor status
`23
`14
`3
`6
`20
`10
`Hormmml receptor status
`6
`10
`21
`5
`Tumor size (centimeters)
`2
`4
`9
`4
`18
`4
`1s
`7
`5
`5
`4
`6
`Tumor size (centimeters)
`0
`3
`g
`3
`Age at diagnosis (years)
`12
`2
`2
`13
`8
`3
`4
`5
`13
`10
`Age at diagnosis (years)
`0
`0
`2
`1
`12
`6
`Ntimber ofpositive lymph nodes
`1
`,
`11
`3
`34
`0
`3
`1
`30
`0
`Number afpositinz lymph nodes
`65
`7
`6
`1
`51
`1—3
`43
`7
`5
`0
`31
`1-3
`68
`18
`8
`4
`38
`>3
`43
`16
`4
`2
`21
`>3
`22
`l
`17
`W UflkHOVVl'l
`3
`l
`——-m-———-—-——-———___._
`*ER and PgR are as described in Table 1.
`TStatistical analyses for correlation of
`HER-Z/mm amplification with various disease
`arameters were performed by the x2
`test. P values were computed after combining the 5 to 20 and >20 cases, since there
`were so few samples in the >20 group.
`180
`
`91
`45
`73
`63
`
`31
`62
`23
`20
`
`37
`88
`11
`
`2
`5
`3
`4
`
`0
`2
`3
`2 -
`
`130
`59
`106
`83
`
`44
`84
`36
`25
`
`60
`116
`13
`
`005
`
`0.06
`
`0‘19
`
`0.11
`
`0.002
`
`0.05
`
`0.14
`
`009
`
`52
`2—5
`>5
`Unknown
`
`550
`>50
`Unknown
`
`005
`
`0.06
`
`'l Statistical analyses for correlation of
`*ER and PgR are as described in Table 1.
`HER-Zine“ amplification with various disease parameters were performed by the x2
`test. P values were computed after combining the cases with 5 to 20 and >20 copies.
`
`SCIENCE, VOL. 235
`
`IMMUNOGEN 2117, pg. 6
`Phigenix v. Immunogen
`|PR2014-00676
`
`Table 2. Association between HER-Zlnen amplification and disease parame—
`ters in 86 breast tumors from node-positive patients.
`————_—_—
`.
`Single
`2 to 5
`5 to 20
`>20
`.
`
`Factor"
`copy
`COPE,
`copies
`copies
`Fowl
`PT
`1311+
`BR
`PgR+
`PgR—
`
`ER+
`ER—
`PgR+
`PgR-
`
`38
`14
`31
`21
`
`1
`1
`1
`1
`
`65
`21
`54
`32
`
`52
`2—5
`>5
`
`$50
`>50
`
`18
`28
`6
`
`15
`36
`
`0
`1
`1
`
`29
`43
`14
`
`35
`51
`
`
`
`
`
`
`
`IMMUNOGEN 2117, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`
`
` es comparing disease-free survival (relapse) and overall survival to prognostic factors in node-positive patients. Table 4. Univariate and multivariate analys
`Multivariate*
`Univariate (P)
`Factor
`
`Survival
`Relapse
`Survival
`
`Relapse
`
`Number of positive nodes
`HER-201m
`Log (PgR)
`Tumor size
`Log (ER)
`Age
`
`
`0.0001
`0.0011
`0.05
`0.06
`0.15
`0.22
`
`0.0002
`<0.0001
`0.05
`0.06
`0.10
`0.61
`
`0.0003
`0.02
`
`(0.0938 i 0.0256)
`(0.0872 i 0.0388)
`
`.
`
`0.001 (0.0849 : 0.0266)
`0.001 (0.1378 : 0.0425)
`
`0.03
`
`(—0.5153 x 0.2414)
`
`*Cox’s partially nonparametric regression model was used to evaluate the predictive power ofvarious combinations and interactions of prognostic factors in a multivariate manner
`(42%). Results are shown as P (regression cocflicient : SE).
`
`receptors in the biology of breast cancer is well established (29, 39,
`40). It is easy to speculate that a gene encoding a putative growth
`factor receptor, when expressed in inappropriate amounts, may give
`a growth advantage to the cells expressing it. Alternatively, altet~
`ation in the gene product itself may lead to a critical change in the
`receptor protein. A single point mutation in the transmembrane
`domain of the protein encoded by the rat nm oncogene appears to
`be all that is necessary for the gene to gain transforming ability (41).
`Whether this or a similar alteration is found in the amplified HER-
`2/mm gene in human breast cancer will require sequence analysis of
`the homologous region in the amplified human gene. In addition,
`studies evaluating the expression of this gene at the RNA and/or
`protein level will prove important in determining if HER-201cm
`amplification results in an expected increased gene expression. The
`question of amplification of HER~2/nzu in metastatic as compared
`to primary lesions in a given patient is important. The current study
`utilized only primary breast tumors for analyses. It would be of
`interest to determine if HER-202m copy number is altered as the
`tumor metastasizes. A recent study evaluating N-myc copy number
`in human small cell carcinoma of the hing showed no difference
`between primary and metastatic lesions (11).
`The initial survey from the current study showed that 15% of
`breast cancer patients with stage 1 disease (node-negative) have
`HER-20m; amplification. Unfortunately, no long-term follow~up
`data were available for these patients. This stage I setting may be an
`additional group in which HER-2/nzu measurements will have an
`impact in predicting biologic behavior of the tumor, and as a result,
`in design of treatment strategy. Finally,
`if the HER-Z/ncu gene
`product functions as a growth factor receptor that plays a role in the
`pathogenesis of breast cancer,
`identification of its ligand and
`development of specific antagonists could have important therapeu-
`tic implications.
`
`
`REFERENCES AND NOTES
`
`9pwfiomnmwr¢pwwow+w-
`
`Nu—IHh‘r-H-n—n—u—u—w—I
`
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`H’ Land, 1.. F. Parada, R. A. Weinberg, Nrmm? (Landau) 304, 596 (1983).
`H E. Ruley, [111%, p. 602.
`M Schwab, H. E. Varmus, J. M. Bishop, illid. 316, 160 (1985).
`W.
`s. Hayward, 11. (j. Neel, s. M. Astrin, ibid. 290, 475 (1981).
`D. I. Slamon st 111., Srimrr 224, 256 (1984).
`M D. Erisman of (11.,Mal. Cal]. Biol. 5. 1969 (1985).
`C M. Croce and G. Klein, Scizlm. 252, 54 (March 1985).
`,G M. Brodeur at £11., Srima: 224, 1121 (1984).
`A I. Wong at fl/.,
`illin’. 233, 461 (1986).
`Campisi It 111., Cell 33, 357 (1983).
`.M
`E. Gteenberg and E. B. Zilf, Natlm: (London) 311, 433 (1984).
`R
`Muller at 111., ibid. 299, 640 (1982).
`.D
`J. Slamon and M. I. Cline, Prop. Natl. Ara/i. Sci. U.S.A. 81, 714