[CANCER RESEARCH55, 5400-5407. November 15, 19951
`
`ERBB-2 (HER2Ineu) Gene Copy Number, @185HER2Overexpression,
`Intratumor Heterogeneity in Human Breast Cancer'
`
`and
`
`János
`
`Szöllosi,2 Margit
`
`Ba1IZS,2
`
`Burt
`
`G. Feuerstein,
`
`Christopher
`
`C. Benz,
`
`and
`
`Frederic
`
`M. Waldman3
`
`Division of Molecular Cytometry, Department of Laboratory Medicine If. S., B. M., B. G. F., F. M. W.]. Brain Tumor Research institute (B.G.F.], and Cancer Research Institute
`(C. C. B.], University of California, San Francisco, California 94143-0808
`
`@
`
`ABSTRACT
`
`Amplification of the ERBB-2 (HER-2/neu) gene Is accompanied by
`overexpression
`of Its ceH surface
`receptor
`product,
`pl85â€(cid:157)@2. Heteroge
`neity
`has been
`observed
`for both
`the gene
`copy
`number
`and
`the
`level of
`overexpresslon
`ofits proteIn product. To better understand
`their
`relation
`ship, correlation between the level of cellular expression of pf85@@an2and
`ERBB-2 gene amplification was studied in four human breast cancer cell
`lines (BT-474, SK-BR-3, MDA-453,
`and MCF-7)
`and in a primary
`human
`breast
`tumor
`sample.
`The
`relative
`expression
`of pl85@'t2
`was measured
`by hnmunofiuorescence by using flow and/or image cytometry while
`correlated DNA analysis was performed
`on the same cells by fluorescence
`in situ hybridization to determine ERBB-2 gene and chromosome 17 copy
`numbers. Marked heterogeneity was observed in both protein expression
`and ERBB-2 copy number. Despite this heterogeneity, and in accordance
`with previous
`studies,
`the average
`levels of p1ss@R2
`expression
`corre.
`lated well with average ERBB-2 gene copy numbers in the four lines
`examined (r = 0.99). When the relationship between copy number and
`proteIn
`expression was studied
`on a cell-by-cell
`basis, p185â€(cid:157)@2 expres
`sion correlated with both the absolute number ofERBB-2 gene copies/cell
`(r
`0.59—0.63)and chromosome 17 copy number (r
`0.45—0.61).It Is of
`Interest that there was weak or no correlation between p185â€(cid:157)@2protein
`expression
`and the ERBB-2 copy number:chroinosome
`17 copy number
`ratio (r = 0.0-0.25).
`In more than one-half of cells expressing
`a high level
`
`of p185@@E@@2,the chromosome 17 copy number was
`(two or three
`times the average copy number), whereas <2% of an unselected popula
`tion had a high chromosome 17 copy number. Bromodeoxyuridine incor
`poration indicated that
`the S-phase-labeling index was homogeneous
`across various
`p185'@2-expressing
`subpopulations
`In the SK-BR-3
`cell
`line. Analysis of the primary breast tumor sample showed results similar
`to the cell lines, supporting the strong possibility of a mechanistic link
`among p185@2
`overexpresslon,
`ERBB-2 amplification,
`and high chro
`mosome
`17 copy number.
`
`INTRODUCTION
`
`A characteristic feature of cancer cells is the unregulated expression
`of genes involved in cellular growth control. One of these genes is the
`ERBB-2 (HER-2/neu)
`proto-oncogene, which encodes a Mr 185,000
`transmembrane
`glycoprotein
`
`@l85@.@@2)that belongs to a subfamily
`of growth factor
`receptors having intrinsic tyrosine kinase activity,
`including
`the epidermal
`growth factor
`receptor
`and the receptors
`HER-3 and HER-4 (1-3).
`of ERBB-2 is found in 25—30%
`Amplification
`and overexpression
`of primary human breast cancers and is associated with a poor clinical
`outcome (4—6).This suggests that overexpression of pl85}@1t2 plays
`a role in the pathogenesis
`of some human breast cancers
`(5, 6).
`Although overexpression
`of p185HER2 is usually accompanied
`by
`
`tumors overexpress
`rare breast
`amplified ERBB-2 in tumor DNA,
`pl85F@1t2protein or c-ERBB-2 mRNA levels in the absenceof
`detectable gene amplification (7).
`Although amplification of ER.8B-2 is generally considered to be a
`significant
`prognostic
`indicator
`in patients with breast cancer,
`its
`applicability continues
`to be controversial,
`in part because of analyt
`ical discrepancies
`associated with the methods
`traditionally used to
`evaluate
`its amplification
`and/or overexpression.
`These techniques
`include Southern blotting, slot blot analysis, and FISH4 for detection
`of amplification, while ELISA, Western blotting,
`immunohistochem
`istry, and immunofluorescence
`are used to evaluate overexpression
`(8—15).Because FISH allows the observer to distinguish small sub
`populations
`of amplified
`cells,
`it
`is more sensitive
`than blotting
`techniques.
`In addition, FISH allows one to identify particular
`loca
`tions where aberrations
`exist
`in single tumor
`specimens
`(10, 14).
`Similarly, because immunohistochemically
`stained slides are difficult
`to quantify and because ELISA and Western blotting data do not
`provide information concerning heterogeneity,
`immunofluorescence
`has advantages over these other methods (13, 14).
`Marked heterogeneity has been described in primary breast cancers
`in both the copy number of ERBB-2/cell and in the level of p185@@E1t2
`protein (5—12).Although cell-to-cell differences may be due in part to
`analytical variation, genetic and epigenetic dispersion may also play
`significant
`roles. This heterogeneity provides a potential source for the
`selection of subclones with increased malignant and metastatic poten
`tial, especially
`in the context of
`therapeutic
`targeting
`based on
`ERBB-2 expression.
`Although amplification of ERBB-2 correlates well with overexpres
`sion of pl85@@E1t2protein in cell populations
`(5, 6, 9, 11, 14—16),the
`correlation has not been made on a cell-by-cell
`basis. The present
`communication
`describes our analysis of the extent
`to which ERBB-2
`gene amplification relates to the expression of p185@1t2 on a single
`cell basis. We have found that although pl85@@1t2 expression corre
`lates with the ERBB-2 copy number/cell, p185@@1t2expression cor
`relates poorly with the ERBB-2 copies:ch.romosome
`17 copies ratio.
`Surprisingly,
`there was correlation between p185@1t2 expression and
`chromosome
`17 copy number,
`suggesting that hyperploidy may be
`related to the p185HER2 expression.
`
`MATERIALS
`
`AND METhODS
`
`Cell LInes. Humanbreastcancercell lines, BT-474, SK-BR-3, MDA-453,
`and MCF-7 were obtained from the American Type Culture Collection (Rock
`ville, MD) and grown according to their specifications. The four cell lines were
`characterized
`previously for ERBB-2 gene amplification
`(10). For flow cyto
`metric immunofluorescence measurements, cells were harvested either by
`trypsin
`or 25 mM EDTA in PBS (pH 7.2; Ref. 17). For slide-based
`immuno
`fluorescence measurements,
`cells were cultured in slide chambers
`(Nunc,
`Inc.,
`Naperville, IL). For BrdUrd (Sigma Chemical Co., St. Louis, MO), labeling
`cells were pulsed with 100 @MBrdUrd for 60 mm. Cells were washed three
`times with PBS containing 1 mMCaC12before immunofluorescence labeling.
`Tumor. A biopsy froma node positive,T2tumor,was frozenimmediately
`after resection.
`Imprint preparations were made after thawing by gently touch
`
`4 The
`abbreviations
`used
`are:
`FISH,
`fluorescence
`in
`situ
`modeoxyuridine; chr, chromosome; F!, fluorescence index.
`
`hybridization;
`
`BrdUrd,
`
`bro
`
`Received 6/8/95; accepted 9/14/95.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must
`therefore be hereby marked advertisement
`in accordance with
`18 U.S.C. Section 1734 solely to indicate this fact.
`I This
`research
`was
`supported
`by NIH
`Grants
`CA-49056
`and
`CA-44768
`States-Hungarian
`Joint Fund for Science and Technology (JF292/92B).
`2 Present
`address:
`Department
`of Biophysics,
`Medical
`University
`School
`Nagyerdei krt. 98, H-4012 Debrecen, Hungary.
`of Laboratory
`at Department
`3 To whom
`requests
`for
`reprints
`should
`be addressed,
`Medicine, MCB-230, Box 0808, University of California at San Francisco, San Francisco,
`CA 94143-0808.
`Phone:
`(415)
`476—3821; Fax:
`(415)
`476—8218; E-mail: waldman@
`dmc.ucsf.edu.
`
`and
`
`by United
`
`of Debrecen,
`
`5400
`
`Downloaded from
`
`cancerres.aacrjournals.org
`
`on December 1, 2014. © 1995 American Association for Cancer
`Research.
`
`IMMUNOGEN 2129, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`ERBB.2 EXPRESSION AND AMPLIFICATION
`
`images acquired; they were then hybridized for gene copy number and scored
`after relocating cells analyzed previously. After immunofluorescence analysis,
`slides were
`refixed
`in methanol:acetic
`acid
`(3:1; Carnoys
`solution)
`and air
`dried. FISH was performed as described previously (19) with modifications.
`Briefly, cells on slides were denatured in 70% formamide-2X SSC at 73°Cfor
`3.0 mm, dehydrated in graded ethanols, treated with 0.25 @g/mlproteinase K
`(Sigma) in 20 mMTRIS buffer (pH 7.5) containing 2 mt@iCaCl2 for 7.5 min at
`37°C,and again dehydrated. The hybridization mixture was denatured at 73°C
`for 5 min, reannealed for 30 mm at 37°C,and applied to warmed slides. Ten
`p@lof hybridization mixture contained 6 ng of fluoresceinated chromosome 17
`centromeric probe, 34 ng of rhodaminated ERBB-2 probe, and 10 ng of
`unlabeled, sonicated human placental DNA (Sigma) in 50% formamide, 2X
`SSC, and 10%dextransulfate. Hybridizationwas overnightat 37°C.Slides
`were washed three times for 10 mm each in 55% formamide-2X SSC, once in
`2X SSC at 45°C,and once in 2X SSC at room temperature. Nuclei were
`counterstained
`by using 4',6-diamidino-2-phenylindole
`hydrochloride
`(Molec
`ular Probes, Eugene, OR) at 0.01 g@g/m1in antifade solution (20).
`Simultaneous detection of BrdUrd incorporation and dual FISH staining
`was performed with three fluorescent dyes (fluorescein- and rhodamine-la
`beled probes and a Cascade Blue-conjugated antibody; Ref. 21). Cells and
`probes were denatured
`and hybridized
`as described
`above. After washing,
`slides were preblocked in 5% Carnation dry milk and 0.1% Triton X-100 in 4X
`SSC for 10 min at room temperature.All staining reactionswere at room
`temperature
`for 30 min. Slides were incubated with IU4 mouse anti-BrdUrd
`(1:400; Caltag, La Jolla, CA), diluted in blocking buffer, washed twice with
`blocking
`buffer,
`and incubated with Cascade Blue-antimouse
`IgG (1:300;
`Molecular Probes), and coverslipped with antifade solution alone.
`Scoring of Interphase Nuclei. Cells analyzed previously for cell surface
`expression of p185HER2 protein were relocated on the basis of their coordi
`nates and scored for chromosome 17 and ERBB-2 signals by using a X100
`NA:1.3 Plan Neofluar oil immersion objective and a computer-controlled
`stage. ERBB-2 doublets were
`counted
`as separate
`signals. Broken,
`torn,
`squashed,
`smeared,
`or overlapping
`nuclei were
`ignored.
`Each
`hybridization
`was accompanied by a control hybridization using normal lymphocytes. The
`scoring results were expressed both as an absolute ERBB-2 copy number/cell
`and as the ERBB-2copy number relative to the 17 centromere copy number.
`Three color images were acquired by using the digital imaging analysis
`system described previously. A triple-band-pass beam splitter and emission
`filters were used (22). Excitation of each fluorochrome was accomplished
`by
`using single-band-pass excitation filters in a computer-controlled filter wheel.
`This made
`it possible
`to collect
`sequential,
`properly
`registered
`images
`of
`the
`three fluorochromes (4',6-diamidino-2-phenylindole hydrochloride or Cascade
`Blue, fluorescein, and rhodamine). The three-color images were processed
`with a Sun IPX workstation using Scil-Image software for pseudocolor display.
`Statistical Analysis.
`Significance
`levels for differences
`in gene copy num
`ber between
`the @185HER.2bright
`and total
`cell populations
`were determined
`by contingency table analysis.
`
`ing the slide surface with tumor material. Slides were then fixed in 1%
`formaldehyde
`for 60 mm at room temperature
`and subsequently
`fixed and
`stored in 70% ethanol. The autofluorescence of air-dried touch imprint prep
`arations was too high for reliable immunofluorescence analysis. Fresh fixation
`of slides in 1% formaldehyde and subsequently in 70% ethanol reduced
`autofluorescence
`significantly.
`Immunolabeling. For flow cytometry,unfixed trypsinizedcells were in
`cubated with 5 ,.tg/ml mAbi (Triton, Alameda, CA) raised against the extra
`cellular domain of p18SHER2,in the presence of 1% BSA on ice for 45 mm,
`washed three times with PBS, and incubated with fluoresceinated rabbit
`antimouse
`IgG (1:100
`dilution;
`Sigma)
`at 0°Cfor 45 mm. After washing with
`PBS, cells were fixed in 1% formaldehyde solution and stored for not more
`than 3 weeks
`at 4°C before
`analysis.
`For image analysis, cells were first fixed in 0.5% formaldehyde solution for
`20 mm at
`room temperature
`and in 70% ethanol
`at 4°C overnight.
`Cells
`on
`slides could be stored in ethanol at 4°Cfor not more than 2 months. Slides were
`then preblocked
`in 5% Carnation
`dry milk, 0.1% Triton X-100
`in 4X SSC (1X
`SSC is 0.15 M NaCl and 0.015 M sodium citrate) for 10 mm at room
`temperature. Staining was at room temperature
`for 45 mm. Samples were first
`incubated with CB1I antibody (BioGenex, San Ramon, CA) specific to the
`intracellular domain of the p185HER.2protein, diluted (1:200) in the blocking
`buffer, washed
`twice with the blocking
`buffer,
`and incubated with fluorescein
`ated rabbit antimouse IgG (1:100; Sigma). After washing, samples were
`refixed in 1% formaldehyde solution in PBS and kept at 4°Cfor not more than
`3 weeks
`before microscopic
`analysis. During
`this
`time,
`no significant
`deteri
`oration of the fluorescence signal was observed.
`To control for nonspecific staining, cells were preincubated with irrelevant
`monoclonal
`antibody of the same isotype before staining with fluorescein
`conjugated
`rabbit antimouse
`IgG. We also compared
`immunofluorescence
`labeling of MDA-453 and SK-BR-3 cells harvested with either trypsin or 25
`mM EDTA in PBS. Trypsinization
`caused
`a 10—15% loss
`in fluorescence
`intensity as compared to cells harvested with 25 mr@iEDTA (data not shown).
`Because this loss was not significant,
`and the two other cell lines could not be
`harvested with 25 mM EDTA, we used trypsin to harvest
`cells
`for
`flow
`cytometric analysis. Results from monoclonal antibody (mAbi) raised against
`the
`extracellular
`domain
`of @l85H@.2 protein were
`similar
`to those
`from
`monoclonal
`antibody
`(CB11)
`raised
`against
`the
`intracellular
`domain
`of
`the
`protein. With mAbI
`, prefixation
`was
`unnecessary,
`resulting
`in lower
`non
`specific binding.
`Flow Cytometry. Cell suspensions were filtered through a 35-g.@mnylon
`mesh to remove aggregates before flow cytometric analysis. Analysis was
`performed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA)
`equipped with a 15 mW argon laser
`(488 nm) and pulse-width
`doublet
`discrimination. A total of 10,000 events were recorded
`in list mode after
`logarithmic
`amplification
`of the fluorescence
`signal.
`Digital Image Analysis. The fluorescence of cells stained on slides was
`analyzed by using a digital image analysis system based on a Zeiss Axioplan
`microscope equipped with the Microlmager 1400 Digital camera (Xillix Tech
`nologies Corp., Vancouver, British Columbia, Canada). Images were captured
`through a fluorescein excitation filter, beam splitter, and emission filter by
`using
`a X 20, NA: 0.5 Plan Neofluar
`objective.
`Images were
`processed
`and
`and Expression in Breast Cancer Cell
`ERBB-2 Amplification
`quantitatively
`analyzed with a Sun IPX workstation
`using Scil-Image
`software
`Lines
`Four breast cancer cell
`lines, MCF-7, MDA-453, SK-BR-3,
`(National Research Institute, Delft, The Netherlands). Local background
`flu
`and BT-474, known to have various
`levels of amplification
`of the
`orescence was determined for each image, and the average autofluorescence
`of
`ERBB-2 gene (10) were studied for distribution of ERBB-2 gene copy
`the isotypic control cells was subtracted from the total fluorescence intensity of
`number and chromosome
`17 centromere copy number
`(Fig. 1). Am
`labeled cells. The Fl was defined as a ratio of the corrected total fluorescence
`plification of the ERBB-2 gene can be expressed as copy number/cell
`intensity of labeled cells to the mean autofluorescence
`of the isotypic control
`or as copy number relative to chromosome 17 copy number. Using a
`cells.
`DNA Probes and Probe Labeling. Two contiguous ERBB-2 cosmid
`relative measure is especially important
`for those cell
`lines that are
`clones (cRCNeul and cRCNeu4), together spanning 55 kb of genomic DNA
`aneusomic for chromosome
`17. Amplification
`of ERBB-2 gene was
`(10), were used in combination
`with a probe
`specific
`for
`the chromosome
`17
`observed in MDA-453, SK-BR-3, and BT-474 cell lines, using either
`pericentromeric
`sequence
`(p17H8; Ref. 18)
`for
`two-color
`FISH analysis.
`the definition of amplification as total ERBB-2 copies/cell or the ratio
`Probes were directly labeled with fluorescein-11-dUTP or tetramethylrhodam
`of ERBB-2 copy number to chromosome 17 copy number. There was
`inc-I 1-dUTP
`(Boehringer Mannheim,
`Indianapolis,
`IN) by nick translation
`by
`marked heterogeneity for ERBB-2 copy number, chromosome 17 copy
`using commercially available kits (Bethesda Research Laboratories, Gaithers
`number, and their ratios in the three cell lines with ERBB-2 amplifi
`burg, MD).
`cation. In MCF-7,
`the ERBB-2 gene was deleted (ERBB-2 gene copy
`in Situ Hybridization and Staining for BrdUrd. Dual
`fluorescence
`number was less than the chromosome 17 copy number/cell) and there
`analysis of gene copy number and protein expression was done as a two-stage
`procedure. Slides were first stained for protein expression and fluorescence
`was less heterogeneity in ERBB-2 gene copy number/cell
`and in the
`5401
`
`RESULTS
`
`Downloaded from
`
`cancerres.aacrjournals.org
`
`on December 1, 2014. © 1995 American Association for Cancer
`Research.
`
`IMMUNOGEN 2129, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`@@@@.
`
`@
`
`Lu1
`
`ERBB.2 EXPRESSION AND AMPLIFICATION
`
`.@
`
`I-..
`
`‘,
`
`@
`
`@
`
`@
`@@
`@
`
`
`
`
`
`@@‘“..
`
`17 ratio. The mean values and the SDs of the
`ERBB-2:chromosome
`copy number distributions
`are summarized in Table 1.
`We next characterized the expression levels of the ERBB-2 gene
`product p185HER2 by flow cytometry. Fig. 2 shows the fluorescence
`intensity histograms of the four cell lines labeled with mAbi against
`p185@@E@2. Heterogeneity
`of expression
`of p185'@@2 was similar
`in
`the four cell
`lines. The MCF-7 cell
`line was the least positive, only
`twice background, whereas the BT-474 cells were the most positive.
`
`,n © an © ,,@
`——e@ ri
`
`In © $fl 0
`
`@‘i‘@‘‘@‘Siin
`ERBB-2 Signals/Cell
`
`V@
`
`I
`
`I
`
`c)
`
`0
`
`:
`
`40
`
`20
`
`U
`
`80
`
`60
`
`.
`
`.—--
`•1•2
`
`0
`
`1
`
`2
`
`3
`
`r—
`
`r
`
`;-@-
`
`4
`5
`6
`7
`8
`Chr 17 Signals/Cell
`
`9
`
`10
`
`11
`
`12
`
`C
`
`Ez —
`
`L)
`
`100
`10
`Intensity
`Fluorescence
`Fig. 2. Frequency distribution of fluorescence intensity after immunofluorescence
`staining for p1@5HER.2.Trypsinized
`cells were labeled with mAbi
`raised against
`the
`extracellular domain of @185UER.2and then with fluorescein-conjugated rabbit antimouse
`IgG.Anirrelevantprimaryantibodyofthesameisotype,followedbyfluorescein
`conjugated rabbit antimouse
`IgG, was used for the blank control
`(SK-BR-3 cells). The
`mean values of these distribution curves are summarized in Table 1. Note that the level
`of heterogeneity (width of the intensity profiles on this log intensity scale) is similar in the
`four cell lines, although the absolute amount of p185'@'@2 varies greatly from line to line.
`
`A
`
`U BT-474
`U MDA-453
`
`0
`
`SK-BR-3
`
`MCF-7
`
`N t'@
`in m in
`
`B
`
`The mean values and the SDs of the fluorescence intensity histograms
`are summarized
`in Table 1. The mean fluorescence
`intensity was
`strongly
`correlated with
`the mean ERBB-2
`copy
`number/cell
`(r
`0.99; Table 1). A strong correlation was also observed between
`the mean protein expression and mean ERBB-2:chromosome
`17 ratio
`(r
`0.99), whereas
`there was a weaker correlation with average
`chromosome
`17 copy number
`(r = 0.75).
`on a Single Cell
`ERBB-2 Gene Expression and Amplification
`Basis. Protein expression
`and copy number were measured in the
`same individual cells to study their correlation on a single cell basis.
`This was especially
`relevant given the wide range in both copy
`number and immunofluorescence
`observed (Figs. 1 and 2). Immuno
`fluorescence intensity of individual SK-BR-3 and MDA-453 cells was
`studied by image microscopy,
`and the same cells were identified and
`scored for ERBB-2 gene and chromosome
`17 copy number after dual
`FISH labeling. The fluorescence intensity was too low in MCF-7 to
`perform quantitative image cytometry, and BT-474 cells could not be
`separated from each other during image analysis because of their
`piled-up growth pattern.
`and ERBB-2
`of @185Han@2expression
`Correlated measurement
`copy number was performed in the same cells by consecutive analysis
`(Fig. 3). The fluorescence
`images of cells displayed in Fig. 3B are
`shown after double-target hybridization in Fig. 3C. The green signals
`correspond
`to chromosome
`17 centromere,
`and the red signals
`to
`ERBB-2 signals. The heterogeneity
`of pl85I@@t2 expression in 5K-
`BR-3 cells by image microscopy (Fig. 3A) was similar to that found
`by flow cytometry (Fig. 2).
`and ERBB-2 gene
`expression
`The linked analysis of p185H@2
`in Figs. 4 and 5. Note the use
`amplification
`in SK-BR-3
`cells
`is shown
`of a Fl for these measurements,
`rather than absolute intensity (as was
`used for the flow measurements),
`in order to control for the increased
`levels of autofluorescence
`in these fixed samples. ERBB-2 copy
`number showed a significant correlation with protein expression on a
`cell-by-cell
`basis. The
`correlation was
`stronger
`using
`absolute
`ERBB-2 copy number/cell
`(Fig. 4A)
`than when using a relative
`5402
`
`0 0.5 1
`
`2
`3
`4
`5
`6
`7
`ERBB-2 Signals/Chr
`
`8
`9 10 11 12 13
`17 Signals
`
`Fig. 1. Number of ERBB-2and chromosome 17 centromere copies in four breast cancer
`lines. A, frequency distribution of ERBB-2 signals/cell; B, chromosome
`17 signals]
`cell
`cell; C, ERBB-2:chromosome
`(Chr) 17 ratio. The values along the abscissa represent
`the
`lower limits of the range of values for each category. At least 100 cells were scored to
`create the distribution histograms. The mean values and their SDs are summarized in
`Table 1. Note the wide heterogeneity
`present
`in all but the MCF-7 distributions.
`
`linesBreast
`Table 1ERBB-2
`
`amplification and expression in breast cancer celi
`
`cancer
`17FPMCF-72.2
`linesERBB-2'@Chr
`cell
`
`17bERBB-2/Chr
`
`±±1.00.60.220±9MDA-45311.0±3.94.1±1.62.8±1.0186±75SK-BR-331.0
`±05d3.8
`
`
`
`
`
`114BT-47452.0
`
`±9.06.9
`
`±1.04.5
`
`±11.36.0
`
`±1.19.0
`
`±1.2326
`
`±2.3549
`
`±
`
`±165
`
`number/cell.
`copy
`a ERBB-2
`b Chr, chromosome 17 copy number/cell.
`C Mean
`fluorescence
`intensity
`determined
`
`d Data
`
`expressed
`
`as mean,
`
`±SD.
`
`from
`
`flow
`
`cytometric
`
`histograms.
`
`Downloaded from
`
`cancerres.aacrjournals.org
`
`on December 1, 2014. © 1995 American Association for Cancer
`Research.
`
`IMMUNOGEN 2129, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`A
`
`4/
`
`a?
`
`I
`
`C
`
`I
`
`ERBB.2 EXPRESSION AND AMPLIFICATION
`
`B
`
`C
`
`4,
`
`,@
`
`..,
`
`0
`
`‘4
`
`7
`
`..
`
`Fig. 3. Linked detection of pl85@@ER2expression and gene amplification in individual cells. A, SK-BR-3 cells display immunofluorescence staining for pi85@R2 expression (X20
`objective) after staining with mAbl. B, computer magnification (X5) of the rectangle in A. C, FISH detection of ERBB-2 (red) and chromosome
`17 centromeres
`(green)
`in identical
`cells shown in B (X 100 objective). Cells were refixed after immunofluorescence labeling and denatured and hybridized with directly labeled ERBB-2 and chromosome 17
`centromere-specific
`probes. Not all signals are visible in this image because the plane of focus is thinner
`than the specimen. Anti-BrdUrd labeling (blue)
`is positive in the top cells.
`These are pseudocolor,
`contrast-enhanced
`digital
`images.
`
`17 copy
`(ERBB-2:chromosome
`measure of ERBB-2 amplification
`number
`ratio; Fig. 4B). There was also a correlation seen between
`p185H@@@2expression and copy number of chromosome 17 (Fig. 4C),
`perhaps due to an second association between aneuploidy and ERBB-2
`amplification.
`A subpopulation of cells was seen, which stained especially brightly
`for p185H@2•To test whether this was due to genetic heterogeneity or
`
`the genetic composition of “brightâ€(cid:157)cells (with
`
`to phenotypic dispersion,
`more than four times more fluorescence intensity than the nonspecific
`staining of isotypic control cells) was analyzed as a separate group. We
`compared the distribution of ERBB-2 copy number (Fig. 5A), ERBB-2:
`chromosome 17 copy number ratio (Fig. SB) and the chromosome 17
`copy number (Fig. SC) of bright cells to an unselected population and
`found that these differences were all highly significant.
`5403
`
`Downloaded from
`
`cancerres.aacrjournals.org
`
`on December 1, 2014. © 1995 American Association for Cancer
`Research.
`
`IMMUNOGEN 2129, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`ERBB-2 EXPRESSION AND AMPLIFICATION
`
`SK-BR-3
`
`0
`
`0
`
`°
`
`o@@d?
`
`0
`
`oo
`c@0
`o@°°8o@
`
`100
`
`10
`
`1
`
`A
`
`0
`
`significant difference (P = 0.29) in the average fluorescence intensity.
`S-phasecellshada higheraverageERBB-2genecopynumberand
`
`non-S-phase
`did
`than
`ratio
`number
`copy
`17
`ERBB-2:chromosome
`respectively),
`perhaps
`cells (43.7 versus 38.7 and 6.3 versus 5.0,
`because doublets forming during DNA synthesis were scored as two
`separate gene copy numbers as described in “Materialsand Methods.â€(cid:157)
`The labeling index of the whole cell population (39.7% of 224 cells)
`.
`.
`and for cells with >10 chromosome 17 copies (38.3% of 60 cells) did
`
`0
`
`r=0.60
`
`0
`
`20
`
`40
`
`SO
`60
`ERBB-2 Signals/Cell
`
`100
`
`‘
`
`120
`
`.
`
`1'@0
`
`not
`
`differ.
`
`@
`
`@
`
`@@
`
`100
`
`10
`
`1
`
`‘0
`
`‘I
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0
`
`0@6'@0@
`
`000
`
`°
`
`i@'6'@o@t'@ 0
`
`0
`
`0
`
`0
`
`B
`
`0
`
`F = 0.25
`
`0
`
`2
`
`8
`6
`4
`ERBB-2 Signals/Chr
`
`10
`17 Signals
`
`I'2
`
`1'4
`
`on a Primary
`and Amplification
`ERBB-2 Gene Expression
`Tumor
`Sample. The results of consecutive
`analysis of pl85H@@2
`expression
`and ERBB-2 gene amplification
`in primary tumor cells
`(caseno.B372)areshowninFigs.8and9.Positivecorrelationswere
`found between p185HER2 expression and ERBB-2 gene copy number
`(Fig. 8A), the ERBB-2:chromosome
`17 ratio (Fig. 8B), and the chro
`mosome 17 copy number
`(Fig. 8C). There were significant differ
`ences in the distribution of the ERBB-2 copy number
`(Fig. 9A),
`the
`ratio of ERBB-2:chromosome
`copy number
`(Fig. 9B), and the chro
`mosome 17 copy number (Fig. 9C) when bright cells were compared
`to
`the
`unselected
`population.
`In
`general,
`the
`correlation
`patterns
`observed
`in this touch imprint preparation were similar
`to those
`observed in tumor cell
`lines.
`
`SK-BR-3
`
`C
`
`40
`
`0 0
`
`0
`
`o
`
`00
`
`100
`
`10
`
`20
`
`30
`
`= 0.45
`
`0 r
`
`00
`8°@°
`00
`
`0
`0
`
`A
`
`0
`
`900
`
`10
`
`20
`Chr 17 Signals/Cell
`
`I
`
`0
`
`0
`
`@@@@@
`
`
`
`
`
`
`
`@@@@@@@@@@@
`
`@
`
`@
`
`@
`@@
`
`@@@@@@
`
`
`
`
`
`
`
`
`@@@@@@@@
`
`
`
`
`
`@@@@@
`
`Fig. 4. ERBB@2gene expression and amplification in single SK-BR-3 cells. Expression
`level of p185@@E@2protein
`plotted against: A, ERBB-2 copy number; B, ERBB-2:
`chromosome
`17 ratio; and C, chromosome
`17 copy number. Cells were labeled with
`antibody (CB11) against the intracellular domain of p18SHER2protein. Data from 184
`cells are shown. There is a good correlation between p185â€(cid:157)@2 expression and copy
`number of either ERBB.2 or chromosome
`17 centromere
`(A and C). The correlation
`between @185HER.2expression
`and ERBB.2:chromosome
`17 copy number
`ratio was
`weaker(B).
`
`The results in MDA-453 cells were similar
`to SK-BR-3 cells for
`ERBB-2 gene amplification
`and protein expression (Figs. 6 and 7).
`However,
`there was no correlation between protein expression and
`ERBB-2:chromosome
`17 copy number
`ratio (Fig. 6B), and the distri-
`bution of ERBB-2:chromosome
`17 ratio of bright cells did not differ
`significantly
`from the unselected
`population
`(Fig.
`7B).
`In both cell
`lines, the centromere 17 copy number was high (two or three times the
`average copy number)
`in >50% of the bright cells (expressing a high
`level of p185HER2), whereas <2% of the unselected population had a
`high chromosome
`17 copy number.
`Relationship
`between DNA Synthesis and ERBB-2 Gene Ex
`pression
`and AmplificatiOn. We next addressed the issue of
`whether
`the bright cells having high pl85'@@2 expression and high
`chromosome
`17 copy number were proliferatively
`active. SKBR-3
`cells were pulse labeled with BrdUrd, p185H@@@@2expression was
`determined before fixation, and then BrdUrd incorporation and dual
`color FISH were detected simultaneously
`(for demonstration see Fig.
`3C) Correlation between ERBB-2 gene amplification
`and protein
`expression in these cells (data not shown) was similar to that found in
`
`prefixed cells (Figs. 4 and 5). When BrdUrd-positive
`phase) were compared with BrdUrd-negative
`cells,
`
`cells (cells in S
`there was no
`5404
`
`,.@
`c)
`
`0
`
`b@
`
`I
`I
`20@
`
`I
`@o-I
`]
`I
`01
`
`©
`
`60
`
`40
`
`20
`
`in Q in Q in ©in
`,,
`@,
`,,@
`
`in ©in
`.@
`ERBB-2
`
`Signals/Cell
`
`A
`
`U Average
`D Bright(Fl>4)
`
`p < 0.0001
`
`in
`
`in ©in
`
`in
`
`in ©in
`
`V
`
`B
`
`p<0.002
`
`I LI
`
`LL@m@
`
`in ©@I(@
`if@©@lf@©@in@
`,-
`— r-@ e@ e@ r')
`in
`
`in@
`in
`
`in@
`@cr-.
`
`V@Q@in@©
`©
`
`ERBB.2 Signals/Chr
`
`17 Signals
`
`C
`
`p < 0.0001
`
`© e@r.@
`
`in @cr..
`
`o _ ei
`
`an @ar. ao
`
`@‘17 SignalS/Cell
`Fig.5. ERBB.2copy number(A), ERBB.2:chromosome(Chr) 17 ratio(B), and
`chromosomei7 copy number(C) in unselectedand in highlyp185@52-expressing
`(Fl > 4) SK-BR-3 cells. Distributions of bright cells were determined from cells plotted
`in Fig.4. Cellsthatexpressedp185HER2at a highlevelhadsignificantlymorecopiesof
`ERBB-2andchromosome17,anda higherratioofthetwo,thananunselectedpopulation.
`
`Downloaded from
`
`cancerres.aacrjournals.org
`
`on December 1, 2014. © 1995 American Association for Cancer
`Research.
`
`IMMUNOGEN 2129, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`ERBB.2 EXPRESSION AND AMPLIFICATION
`
`linkage between gene copy number and protein expression was still
`present within each cell
`line (r = 0.59—0.72) but was weaker
`than that
`
`A
`
`MDA-453
`
`0
`
`0
`
`0
`
`@0
`
`.
`
`‘
`10
`
`.
`
`,
`,
`30
`20
`ERBB-2 Signals/Cell
`
`.
`
`I
`40
`
`r = 0.63
`
`•
`50
`
`B
`
`level
`
`cell
`
`lines
`
`were
`
`compared
`
`at
`
`the
`
`population
`
`when
`observed
`(r
`0.99).
`Several reasons may account for the observed dissociation between
`ERBB-2 copy number and protein expression. A normal dispersion in
`ERBB-2
`transcription
`and
`translation
`rates,
`or
`in the
`half-lives
`of RNA
`transcripts and protein products, might lead to a weakened linkage
`between
`genotype
`and
`phenotype.
`ERBB-2
`transcript
`levels
`in ampli
`fled SK-BR-3 and BT-474 cells are 20—40times that of immortalized
`but
`nontumorigenic
`HBL-100
`breast
`epithelial
`cells,
`although
`the
`average level of ERBB-2 gene copy number in the amplified cell lines
`was
`only
`8-fold
`that
`of
`the HBL-100
`cells
`(determined
`by Southern
`
`0
`
`0
`
`0
`
`2
`
`8I'@
`
`gc@
`
`°
`
`%oo
`@c@cP6oo0
`0
`,@°o
`
`o
`
`0
`
`9
`
`0
`
`o
`
`0
`
`8
`
`:
`
`8
`
`blotting)
`
`(26).
`
`Another
`
`cause
`
`of
`
`the
`
`lower
`
`association
`
`on a cell-by-cell
`
`immu
`or
`number
`copy
`gene
`either
`for
`variation,
`analytical
`is
`basis
`nofluorescence intensity, which is much less of a factor when the
`.
`.
`.
`.
`.
`.
`.
`HER-2
`entire
`population
`is measured.
`Asymmetric
`distribution
`of p185
`
`protein
`
`occurring
`
`during
`
`mitosis,
`
`which
`
`occurs
`
`in exponentially
`
`grow
`
`r = 0.04
`
`ing cellpopulations (27),might also lead to lesslinkage between gene
`
`copy
`
`number
`
`and
`
`expression.
`
`100
`
`10
`
`I
`
`.1
`
`0
`
`100
`
`‘0
`
`-
`
`0
`
`0
`
`0
`
`8
`
`10
`
`I
`
`I
`
`@
`
`@
`
`@
`
`@@
`@
`@@@
`
`
`
`
`
`
`
`@@@@@
`
`@@@@
`
`@
`
`@
`
`@@@@@@
`
`
`
`@@@@
`
`
`
`@@
`
`
`
`
`
`
`
`@@@@@@@@
`
`
`
`
`
`
`
`I
`
`.
`
`)
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`ERBB-2 Signals/Chr
`
`17 Signals
`
`interest
`is of
`It
`number:chromosome
`
`that
`
`copy
`ERBB-2
`the
`and
`expression
`p185'@@2
`17 copy number
`ratio were not closely linked
`
`100
`
`10
`
`0
`
`C
`
`0
`
`0
`
`8 °
`0i0@0@ 00
`
`0
`
`0
`
`°
`
`0
`e@
`
`o
`
`-
`
`0
`
`0
`
`.
`
`-
`
`10
`Chr 17 Signal/Cell
`
`20
`
`40@
`
`30
`
`20
`
`10
`
`MDA-453
`
`A
`
`. Average
`Bright(Fb4)
`
`1
`
`e@ ‘@‘000
`
`c@ ‘@‘C
`e
`—————e@ e@ r-4e@ e-1r'@
`
`p < 0.004
`
`© ei
`
`‘@‘@C
`
`F]
`
`‘@
`
`B
`
`p = 0.72
`
`Fig. 6. ERBB.2 gene expression and amplification in individual MDA-453 cells. Fl
`plotted against: A, ERBB-2 copy number; B, ERBB-2:chromosome
`17 ratio; and C,
`chromosome
`i7 copy number. Cells were labeled with antibody (CB1 1) against
`the
`intracellular domain of pi8S'@'@2protein. Data from 239 cells are shown. There is a good
`correlation between p185HER2 expression and copy number of either ERBB-2 or chro
`mosome 17 centromere
`(A and C) but no correlation between p185@@2 expression and
`ERBB.2 to chromosome (Chr) 17 ratio (B).
`
`DISCUSSION
`
`ERBB-2 Signals/Cell
`
`in
`0
`— — 1'1 e@
`
`in
`
`0
`in
`in
`0
`@e e'i ‘@‘@in in
`
`in
`
`ERBB-2 Signals/Chr
`
`17 Signals
`
`of p185â€(cid:157)@2 can occur as a result of either DNA
`Overexpression
`amplification or by increased levels of RNA transcription. Concordant
`ERBB-2 gene amplification
`and p185HER2 overexpression
`has been
`found in both human mammary cancers and cell
`lines, with good
`correlation between the level of ERBB-2 gene amplification and the
`average p185HER2 protein overexpression (5, 6, 9, 11, 14—16,23—25).
`However,
`there has been no prior analysis of this genotype-phenotype
`association on a single-cell basis.
`To investigate the cell-by-cell basis for the correlation between
`ERBB-2 amplification
`and overexpression,
`several well-established
`breast cancer cell lines having a wide range of gene am