Case 1:18-cv-01363-CFC Document 82-6 Filed 03/22/19 Page 1 of 11 PageID #:
`9854
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` RHYU
`EXHIBIT 6
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`Case 1:18-cv-01363-CFC Document 82-6 Filed 03/22/19 Page 2 of 11 PageID #:
`9855
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`Oncogene (1996) 13, 63-72
`© 1996 Stockton Press All rights reserved 0950-9232/96 $1?..00
`
`Detection and quantitation of HER-2/neu gene amplification in human
`breast cancer archival material using fluorescence in situ hybridization
`
`Giovanni Pauletti1, William Godolphin2, Michael F Press3 and Dennis J Slamon1
`
`1Division of Hematology/Oncology, Department of kfedicine, University of California, Los Angeles, School of Medicine, Los
`Angeles, Califomia 90095, USA; 2Departme11t of Clinical Chemistry, Vancouver, Canada, V5Z 1M9; 3Department of Pathology,
`USC School of Medicine, Los Angeles, California 90033, USA
`
`Amplification and overexpression of the HER-2/neu gene
`occurs in 25 - 30% of human breast cancers. This genetic
`alteration is associated with a poor clinical prognosis in
`women with either node negative or node positive breast
`cancers. The initial studies testing this association were
`somewhat controversial and this controversy was due in
`large part
`to significant heterogeneity
`in both the
`methods and/or reagents used in testing archival material
`for
`the presence of the alteration. These methods
`included a number of solid matrix blotting techniques
`for DNA, RNA and protein as well as immunohisto(cid:173)
`chemistry. Fluorescence
`in situ hybridization (FISH)
`represents the newest methodologic approach for testing
`for this genetic alteration. In this study, FISH is
`compared to Southern, Northern and Western blot
`analyses as well as immunohistochemistry in a large
`cohort of archival human breast cancer specimens. FISH
`was found to be superior to all other methodologies
`tested in assessing formalin fixed, paraffin embedded
`material for HER-2/neu amplification. The results from
`this study also confirm that overexpression of HER-2/
`ueu rarely occurs in the absence of gene amplification in
`breast cancer (approximately 3% of cases). This method
`of analysis is rapid, reproducible and extremely reliable
`in detecting presence of HER-2/neu gene amplification
`and should have clinical utility.
`
`Keywords: HER-2/neu; c-erbB-2; gene amplification;
`breast cancer; FISH; interphase cytogenetics
`
`oto-oncogenes and tumor suppressor genes represent
`o classes of genes which play central roles in the
`ulation of cell growth (Bishop, 1991). Alterations in
`embers of either or both of these groups of genes
`pear to be important in the pathogenesis of a
`mber · of human malignancies
`(Hunter, 1991;
`. arshall, 1991). One of the most common genetic
`terations associated with human breast and ovarian
`ncer is amplification of the HER-2/neu (c-erbB-2)
`oto-oncogene (reviewed in Brison, 1993; Hynes and
`. ern, 1994). This gene, located on chromosome 17ql2-
`l.32 (Popescu et al., 1989), encodes a protein which is
`member of the Class I growth factor receptor
`rosine kinase
`family
`(Ullrich and Schlessinger,
`
`rrespondence: Dennis J Slamon
`eived 13 January 1996; revised 3 April 1996; accepted 3 April
`96
`.
`
`1990). Studies done in a number of laboratories have
`demonstrated that 25- 30% of breast and ovarian
`malignancies contain amplification and overexpression
`of the HER-2/neu gene (Slamon et al., 1987, 1989a,b;
`Liu et al., 1992; Press et al., 1993). Moreover, presence
`of this alteration is associated with a poor prognosis
`for those women whose tumors contain it, in that they
`have a shortened disease-free as well as overall survival
`(Slamon et al., 1987, 1989a; Press et al., 1993). A
`number of studies also demonstrate that the degree of
`amplification/overexpression
`is
`important with
`the
`highest
`levels being associated with
`the poorest
`prognoses (Slamon et al., 1987; Press et al., 1993).
`Initially there was some controversy surrounding the
`association between this alteration and clinical out(cid:173)
`come (Ali et al., 1988; Gusterson et al., 1988; Zhou et
`al., 1989; Kury et al., 1990; Clark et al., 1991) however,
`the majority of published data now confirm that an
`association between HER-2/neu amplification/overex(cid:173)
`pression and prognosis exists for both node positive
`and node negative breast cancer (Richner et al., 1990;
`Gullick et al., 1991; Seshadri et al., 1993; Press, 1993;
`Hynes and Stern, 1994; Muss et al., 1994). Relevant to
`the current study is the fact that the initial controversy
`regarding this association was multifactorial and due,
`in part, to a number of technical variables in the
`conflicting published studies
`including
`insufficient
`insufficient clinical
`cohort size and/or
`follow-up
`(Slamon et al., 1989a; Gullick et al., 1991; Press et
`al., 1993). The majority of the controversy in the
`literature, however, appears to have been caused by
`technical variables associated with
`the different
`reagents and/or methodologies used in testing for the
`presence of the HER-2/neu alteration (Slamon et al.,
`1989a, b; Press et al., 1994). Solid matrix blotting
`techniques, i.e. Southern, Northern or Western blot
`analyses, all suffer from the problem of variation in
`tumor cell content within a given specimen. Significant
`misinterpretation of data can
`result
`from
`these
`the dilution of malignant cell
`techniques due
`to
`macromolecules (DNA, RNA or protein) by macro(cid:173)
`molecules from normal stromal, vascular or inflamma(cid:173)
`tory cells contained within
`the
`tumor specimen
`(Slamon et al., 1989a). This is a particular problem
`in human breast cancer where non-malignant cells may
`constitute 50% or more of the tissue (Slamon et al.,
`1989b). In a comprehensive analysis of DNA, RNA
`and protein from a large number of tumors, performed
`by Southern, Northern and Western blot as well as
`immunohistochemical analyses on both frozen and
`formalin-fixed, paraffin-embedded
`tissues, all con(cid:173)
`ducted on the same breast cancer specimens, the
`relative strengths and weaknesses of the various
`
`

`

`64
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`Case 1:18-cv-01363-CFC Document 82-6 Filed 03/22/19 Page 3 of 11 PageID #:
`9856
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`FISH analysis of HER-2/neu gene amplification
`G Pauletti et al
`
`approaches used for lcsling for the presence or the
`J-IER-2/11e11 alteraLion were critically evaluated (Slarnon
`et al., 1989a). Among these rnclhods, immunohisto(cid:173)
`chcmical staining or frozen tissue sections was the most
`reliable. This is likely due lo tvvo scparalc reasons: (I)
`it measures
`the allcra tion on a cell by cell has is
`the prohlem or dilutional artifacts
`circumventing
`inherent to extraction of macromolecules f'rom speci(cid:173)
`mens consisting or heterogeneous cell populations :md
`(2) the target protein is not altered in rnven tissue.
`This approach
`is
`limited
`ror general application,
`however, since currently
`tumor specimens are fre(cid:173)
`quently small and/or available only as formalin-iixcd,
`parafJin-embeddcd material making analyses of f'ru!'.en
`tissue either impractical or impossible. Immunohisto(cid:173)
`chcmical analyses or fixed, embedded tissue alleviates
`this problem, although it introduces the new variable
`or l1xation-inducccl changes
`in
`the
`target protein
`(Battifora and Kopinski, 1986; Pcnault-Llorca ct al.,
`1994). Such changes in a target antigen can lead to a
`significant decrease in detection by antibody reagents
`as has been documented for the fIER-2/neu protein in
`fixed tissue (Slamon ct al., 1989a,b). This, compounded
`by the considerable variability in sensitivity or the
`various antibody
`reagents used
`for such
`testing,
`including some of those which are commercially
`for
`to reliability
`marketed
`this purpose, can
`lead
`problems for immunohistochemical analysis of ror(cid:173)
`tissue (Press cl ed.,
`rnalin-f1xed, paraffm-cmbcddcd
`1994).
`Fluorescence in situ hybridization (FISH) (Pinke! et
`al., 1986) offers a new method or testing for the IIER-
`2/neu alteration which has several potential advantages.
`Like immunohistochcmistry, it has the advantage of
`assessing a specimen on a cell specific basis allowing
`direct evaluation of malignant cells (Poddinghe ct a!.,
`1992). Moreover with FISH, adjacent and intermixed
`normal cell populations offer an internal control for
`specificity since they should yield a normal diploid
`signal
`in specimens containing tumor cell specific
`somatic mutations. Again, like immunohistochemistry
`FISH requires very
`little
`tissue since
`it can be
`performed on a single
`tissue section. A potential
`disadvantage of FISH, however, is that it does not
`measure HER-2/neu gene expression which is likely to
`be the important pathogenic factor inherent in this
`alteration. Recent studies, however, demonstrate an
`almost perfect association between amplification and
`overexpression of this gene (Slamon et al., 1989a;
`Naber et al., 1990; Kallioniemi et al., 1992).
`The objectives of this study are twofold: (1) to
`develop and assess a FISH based assay for use in
`formalin-fixed/paraffin-embedded tissue with the requi(cid:173)
`sites that it be technically simple, reproducible, and
`relatively rapid; and (2) to evaluate the sensitivity and
`specificity of this assay compared to other techniques
`used in testing human archival specimens for the
`presence of the HER-2/neu alteration. To achieve these
`objectives we tested two FISH probes labeled with
`different technologies, to study a cohort of specimens
`which had previously been characterized as to the
`presence or absence of HER-2/neu amplification and
`overexpression (Slamon et al., 1989a), and compared
`FISH analysis to Southern, Northern and Western
`blotting as well as immunohistochemistry in frozen
`tissue sections.
`
`Results
`
`Anafrsis of f/ER-2/neu gene u111plificotio11 hy
`j!11ore.1·cc11ce in situ hyhridizotio11 in thin tissue section.\
`ji·o111 {/ l!IO(crn!orly c/1{{/'il('/CJ'IZCd cohort of h1111w11 hl'C(/,\/
`C//!7('e/' S/!CC/1!/CIIS
`
`;\ comprehensive analysis or the H ER-2/ncu gene
`alteration was previously conc!uclcd on 143 infiltratin~
`ductal carcinoma of the brc,1sl. In this cohort, HER~
`2/11c11 gene copy number was determined by Southern
`blot analysis using a HER-2/11e11 probe f'ollowcd hv
`rebyhridi!'.ation with a rnycluperoxidasc probe as ;1
`control for chromosome 17 copy number, this was
`followed by scanning densitometry comparing
`the
`HER-2/11e11 band Lo
`the control probe band with
`samples being grouped into one of four categories as
`l, 2 - 5, 5- 20 or
`20
`follows:
`tumors containing
`copies of lhc HER-2/neu gene (Slarnon and Clark.
`cl al.,
`1988; Shimon
`1989a). HER-2/neu gene
`expression was determined at
`the
`transcript and
`these same samples by Northern
`protein
`levels
`in
`blot analysis and scanning densitometry
`to assess
`levels and Western blot
`relative messenger RNA
`analysis followed by scanning densitometry to assess
`relative protein
`levels. These same specimens were
`then studied by immunohistochcmical analysis per(cid:173)
`formed on fro!'.en sections of the tumor tissue. Use of
`frcven sections circumvented the problem of hetero(cid:173)
`geneous immunostaining which is sometimes seen in
`fixed and embedded tissue, and demonstrated uniform
`staining
`throughout
`the malignant cell population
`(Slarnon ct al., 1989a, h). Relative levels of HER-2
`ncu
`irnnrnnostaining were determined
`by direct
`microscopic examination of the tissue and grouped
`into one of four staining categories (negative/weak,
`1 +, 2 + or 3 + ). Evaluation of
`the association
`between HER-2/neu gene amplification as determined
`by Southern biol analysis and HER-2/ncu gene
`expression as determined by Northern and Western
`blot
`analysis
`as well
`as
`immunohistochemistry
`demonstrated a direct correlation between HER-2
`ncu DNA copy number and gene expression. Further
`analysis of the data demonstrated
`that Western
`blotting was the least sensitive of these techniques
`for determining levels of expression while immunohis(cid:173)
`tochemical analysis of frozen tissue sections proved to
`be the most reliable method (Slamon et al., 1989a, b).
`Using this same cohort of specimens we assessed the
`specificity and sensitivity of FISH analysis in detecting
`HER-2/neu gene amplification in human breast cancer.
`Representative FISH .photomicrographs are shown in
`Figure la to f. The spectrum of HER-2/neu gene
`amplification in this cohort as determined by FISH is
`demonstrated by ordeting
`tumors according
`to
`decreasing levels of gene copy number using the
`scoring criteria described in Materials and methods
`(Figure 2a). When Northern and Western blot as well
`as
`immunohistochemistry data are plotted in
`the
`identical saniple order, a clear correlation between
`overexpression and gene amplification as determined
`by FISH is apparent (Figure 2b and c). These data also
`demonstrate that the correlation between expression
`and amplification detected by FISH assay is superior to
`the correlation with amplification as determined by
`Southern blot analysis (Table 1). Immunohi:;;tochemical
`
`

`

`Case 1:18-cv-01363-CFC Document 82-6 Filed 03/22/19 Page 4 of 11 PageID #:
`9857
`
`FISH analysis of HER-2/neu gene amplification
`G Pauletti et al
`
`analysis of the corresponding frozen sections detected
`ovcrexprcssion
`in essentially all
`forty (28'%) cases
`which contained greater than 10 IIER-2/neu signals
`per cell (Figure 2b and c). In addition Northern and
`Western blot analyses demonstrate higher relative
`levels or mRNA and protein for this group or tumors
`(Figure 2b, c and Table 2). Only four tumors (2.8% of
`the total cohort) classified as single copy by FISH
`showed evidence or ovcrcxpression by immunohisto(cid:173)
`chemistry. Taken together,
`these data indicate that
`J-IER-2/neu gene amplification as defined by > IO
`signals per nucleus in
`!'our micron
`tissue sections
`
`(uncorrected f'or chromosome 17 polysorny) is uni(cid:173)
`formly associated with HER-2/neu ovcrcxpression at
`both the transcript and protein levels.
`Aneuploidy is frequently found in human breast
`malignancies (Dcvilee and Cornelissc, 1994). Moreover
`aneusorny or chromosome 17 has specifically been
`reported in human breast cancer specimens (Dhingra et
`al., 1992; Matsumura et al., 1992; Micale ct al., 1994;
`Hyytincn ct al., 1994; Visscher et al., 1995; Murphy ct
`al., 1995). As a result, accurate determination of HER-
`2/neu gene
`amplification
`requires assessment or
`chromosome 17 copy number
`to circumvent
`the
`
`65
`
`a
`
`C
`
`e
`
`b
`
`d
`
`f
`
`Figure 1 Representative photomicrographs of breast tumor tissue sections after fluorescence in situ hybridization (FISH). A single(cid:173)
`band pass filter combination has been used in all photomicrographs. Magnification is x 630. (a) HER-2/neu gene single copy status
`(after prolonged exposure demonstrates the absence of nonspecific hybridization. (b) Visualization of DAPI counterstaining in the
`same field. (c, d and e) examples of tumors with increasing levels of HER-2/neu gene amplification. (f) FISH with the chromosome
`17 centromere probe
`
`

`

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`9858
`
`FISH analysis of HER-2/neu gene amplification
`G Pauletti et a/
`
`66
`
`possibility that increases in HER-2/neu signal are due
`to increases in chromosome 17 number rather than true
`gene amplification. This is particularly critical for cases
`
`60--r-------------------,
`a
`
`_ 50
`ai
`u
`~40
`'ii
`0
`~ 30
`
`1/)
`(1)
`
`(1)
`C:
`(::;J
`
`ffi 20
`
`I
`I
`~ 10
`
`0 J_ ___ ~~m!J~lnltllJWJM~L,__j
`5.0 ~ - - - - - - - - - - - - - - - - - - -~
`•
`• ti
`b
`•
`
`~
`
`(1)
`
`•
`•
`
`C
`::,
`f;
`E
`2
`'iii
`C
`(1) :s
`
`••
`
`•
`•
`•
`•
`=
`• •
`11 5- • •
`t
`.
`0
`z1.o-
`0
`0
`0
`•
`ti'
`0.5-
`0
`0
`0
`00
`'t5' CO O O
`,:om\' 4:, c<JD'?n d3 0
`e o
`O. O -'----•---..._o,.,_o _<D_•rr-'o0"-<oo-co-'. ''<>-<'-o-'.u.n'o__,~R-oe--<:><D..M--'°ro-ant. _ _ __,
`
`~
`0
`(0
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`•
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`\ • •
`
`..
`
`••• 0
`
`<D
`
`0
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`•
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`5.0 ~ - - - - - - - - - - - - - - - - - -~
`C
`•
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`•
`• •
`••••
`•• •
`••
`• •
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`
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`
`Tumor samples
`
`160
`
`Figure 2 Comparison of HER-2/neu amplification, as deter(cid:173)
`mined by FISH analysis in four micron tissue sections, with
`HER-2/neu overexpression, as determined by Northern, Western
`and immunohistochemistry analyses. (a) Spectrum of HER-2/neu
`amplification in the tumor cohort, tumors are ordered from left to
`right according to decreasing level of HER-2/neu gene copy
`number. For copy number determination see Materials and
`methods. Each bar represents the range of signal measurements in
`a given sample. Tumors with the identical copy number range
`have been randomly ordered. (b) mRNA levels as determined by
`Northern blot analysis (order is the same as in a). (c) HER-2/neu
`protein levels as determined by Western blot analysis, tumor
`(order is the same as in a). Positive, ;;;,2+ (e, •) and negative
`(0, O) immunohistochemistry is indicated for each tumor both in
`b and c. 11 indicates that immunohistochemistry data were not
`available
`
`containing low level gene amplification. In this study a
`ratio of HER-2/neu
`signal
`to chromosome
`17
`centromere signal of greater than two was considered
`indicative of HER-2/neu gene amplification. These
`corrected data demonstrate that 42 of the 143' tumor
`samples (29.4%) show evidence of true HER-2/neu
`gene amplification. Of the remaining samples, 84
`(58.7%) show no gene amplification and 17 (11.9%)
`fall into the borderline category with a ratio between
`two HER-2/neu genes/cell
`one and
`relative
`to
`chromosome 17 centromeres (Figure 3). Of note is
`the fact that no evidence of chromosome 17 polysomy
`was found in any cases scored as single copy with the
`HER-2/neu probe. The overall correlation between
`ampiification and overexpression was improved by
`defining HER-2/neu copy number based on a ratio of
`HER-2/neu signals per chromosome 17 centromcrc
`signals per cell, i.e. a corrected analysis (Table l ).
`Improvement of the correlation is likely clue to the
`greater accuracy or this scoring criteria. Tumors
`containing true amplification of the HER-2/neu gene
`as determined by I•ISH, uniformly show evidence of
`RNA and protein overexpression. While data from the
`uncorrected analysis, also demonstrated a correla lion
`between overexpression and samples containing > IO
`IIER-2/neu signals per nucleus,
`there were eight
`samples from this analysis which showed evidence of
`overcxpression 111 tumors with < 10 signals/cell (Figure
`2). After correction for chromosome 17, however, three
`of these
`tumors were found
`to contain low
`level
`amplification and only five remained in the single copy
`or borderline categories (Figure 3). While there is no
`significant expression difference between the corrected
`single copy and borderline categories, a significant
`increase is apparent when either corrected group (single
`or borderline) is compared to any of the corrected
`amplified categories (Table 2). Since only 5/143 (3.5%)
`tumors (four
`in
`the single copy and one
`in
`the
`borderline category)
`showed
`imnrnnohistochernical
`evidence of overexpression, the concept that HER-2/
`neu overexpression is almost always associated with
`actual locus specific gene amplification rather than
`phenomena associated with single gene copy over(cid:173)
`expression or chromosome 17 duplication is supported
`by these data .
`relatively
`tumors show a
`All highly amplified
`constant spatial distribution of FISH signals within
`the nuclei and these signals are grouped in clusters of
`variable size. Usually one to four clusters are seen,
`though rarely, more are visible. Only one of 18 of the
`
`Table 1 Correlation analysis of HER-2/neu gene copy number,
`both absolute and relative to chromosome 17 centromere, as
`detected by FISH and gene copy number as determined by
`Southern blot with the level of overexpression as determined by
`Northern (N.) blot, Western (W.) blot and Immunohistochemistry
`(IHC)
`
`N. blot
`
`W. blot
`
`JHC
`
`Southern blot
`
`FISH (HER-2/neu per cell)
`
`0.421
`0.497
`97
`112
`0.627
`0.554
`115
`132
`0.673
`FISH (HER-2/neu per chromosome
`0.554
`I 7 cen tromere)
`115
`132
`Spearman's correlation coefficients/number of observations
`
`0.452
`107
`0.568
`125
`0.615
`[25
`
`

`

`Case 1:18-cv-01363-CFC Document 82-6 Filed 03/22/19 Page 6 of 11 PageID #:
`9859
`
`Table 2 HER-2/11e11 gene copy number group assignment upon FISH analysis: multiple comparisons of
`the ovcrcxpression level of each group as determined by Northern (N.) blot, Western (W.) blot and
`immunohistochemistry (II-IC)
`
`- - - -
`
`67
`
`FISll analysis of HER-2/neu gene amplification
`G Pauletti et al
`
`FIS!/ groups
`
`FISH~ 11 FR-2/neu gei1es per nucleus
`FISII < 4 vs 4<FISH<IO
`
`4<1'1SH< 10 vs 111SJI > 10
`
`FISH< 4 vs FISH > 10
`
`FIS// - I/ /\'R-2/ncu genes per c/1ro111osm11e
`17 ee11/ro111ere (Categories)
`!ilSII ~ I I'S Ji[SII -
`I
`2
`
`FJSll~I 1•s JiJSll~2-5
`
`FISH~ I vs FISH ~5 20
`
`FISH-I
`
`2 vs 111SJl~2-5
`
`!'ISi-i- I
`
`2 1•s !1JSII ~ 5 - 20
`
`N. h/01
`
`NS
`54 m 29
`0.0001
`29 \'S 32
`0.0001
`54 I'S 32
`
`NS
`6(, I'S I(,
`(l.001
`(J{) I'S<)
`0.0001
`66 I',\' 24
`0.001
`](, \',\' 9
`0.0001
`](, \',\' 24
`(J.01
`9 I'S 24
`
`W. h/01
`
`NS
`65 1·s 30
`0.0001
`30 \'S 37
`0.0001
`65 J'S J7
`
`NS
`78 I'.\' I(,
`NS
`78 rs 12
`0.0001
`78 I'S 26
`NS
`J(J \'S \2
`0.0001
`16 rs 2(i
`0.003
`12 J'S 26
`
`fI/C
`
`NS
`59 \',\ 31
`O.OIJIJ I
`.11
`\'\ .15
`0.0001
`:'.?) 1'.I' 35
`
`NS
`73 ,•s I/,
`0.0007
`T!> n II
`0.0001
`73 \'.I' 2.-;
`0.02(,5
`!(1 I'S I I
`0.0001
`16 1'.I' 2)
`0.0014
`I I i·s 2'
`
`Pair-wise comparisons among groups were performed using l:lonfcrroni test. IHC: data were compared
`with chi-square test. /' values arc indicated along with the number of observations in each group. NS,
`not significant
`
`highly amplified samples demonstrate a scattered signal
`pattern throughout tumor cell nuclei within the section.
`represent a case or extrachromosomal
`This may
`amplification.
`In
`samples with
`low
`level gene
`amplification, however, we did observe a scallered
`pallcrn of signal distribution.
`The relative sensitivities or FJSH and Southern blot
`analysis for detecting the HER-2/neu alteration was
`compared in this cohort. Using HER-2/neu immuno(cid:173)
`histochemical staining
`in
`frozen
`sections as
`the
`'standard' ror detecting the presence or the alteration
`(Slamon el al., 1989a) FISH was found to have a
`sensitivity of 96.5°/.1 while Southern blot analysis had
`a sensitivity or 92.4'½,. or interest is the fact that one
`case detcnnined to be amplified by Southern blot but
`not amplified by FISH, showed no evidence of
`overexpression. Conversely, six specimens considered
`single copy by Southern blot analysis were found to
`be amplified by FISH and five of these showed
`evidence of HER-2/neu overexpression (Figure 3).
`Moreover, assessment of the methodologic success
`rate of FISH (i.e. the ability to successfully complete
`the analysis on the first attempt) demonstrated that it
`could be successfully performed on 142 out of 143
`tumors (99%) with only one case requiring repeat
`analysis due to the loss of the tissue section from the
`slide. Conversely Southern blot analysis could be
`conducted in 119/143 (83%), Northern blot analysis in
`117/143 (82%) and Western blot analysis in 132/143
`(92 % ) of the samples due to partial or complete
`degradation of the respective macromolecules being
`examined.
`Having assessed the relative methodologic success
`rates of the various techniques used for testing a
`sample for the HER-2/neu alteration and demonstrat(cid:173)
`ing
`the superiority of FISH, we next wanted to
`compare a direct-labeled versus an indirectly labeled
`FISH probe. This was done using the SpectrumOr-
`
`ange-labeled and biotin-labeled probes respectively. As
`mentioned above, using Lhe direct labeled probe we
`had a methodologic success rate or 99'¾, (142/143). The
`indirect labeled FISH probe however gave a methodo(cid:173)
`logic success
`rate or only 79.7%
`(114/143) with
`unsuccessful hybridization evidenced by either cases
`in which no signal (single copy or otherwise) was seen.
`5.6% (8/143) or cases
`in which high background
`lluorcscence was seen, i.e. 14.7% (21 143), making it
`difficult lo unambiguously recognize a clear H ER-2/11c11
`signal (Figure 4a, b). Single-band pass filter combina(cid:173)
`tions were significantly superior lo double or triple(cid:173)
`band pass 1iltcrs for visualization of the l1uoruehromes
`the FIS! I probes as well as
`used
`in
`the DAPI
`counterstain.
`In addition.
`single-band pass
`filter
`combinations maximized signal intensity while mini(cid:173)
`mizing cellular autofluorescence and eliminating signal
`reduction due to superimposing · color of nuclear
`staining. The most informative filter combination for
`DAPI was
`the Zeiss 02
`filter which
`facilitated
`delineation of nuclear boundaries in compact cell
`populations.
`
`Discussion
`
`Amplification of the HER-2/neu gene is an important
`alteration in human breast cancer occurring in 25 -
`30% of infiltrating ductal carcinomas (Slamon, 1987;
`Slamon et al., 1989a). Identification of the HER-2/neu
`alteration
`in
`these. malignancies carries
`important
`prognostic information for node positive as well as
`node negative breast cancers (Slamon et al., 1989a;
`Gullick et al., 1991; Press et al., 1994; Silverstein et al.,
`1995). To date detection of its presence has been
`accomplished by either solid matrix blotting or
`immunohistochemical methodologies. Fluorescence in
`situ hybridization (FISH) represents the newest method
`
`

`

`Case 1:18-cv-01363-CFC Document 82-6 Filed 03/22/19 Page 7 of 11 PageID #:
`9860
`
`68
`
`FISH analysis of HER-2/neu gene amplification
`G Pauletti et a/
`
`1
`
`1-2
`2-5
`5-10
`FISH (HER/neu per chr. 17 cen)
`
`>10
`
`1-2
`2-5
`5-10
`FISH (HER/neu per chr. 17 cen)
`
`>10
`
`a,20DDDCJD
`i'·"DDDDD
`1,~00000
`,[IJDlJll
`b [JD[~IDD
`~IS.OD
`
`.!!E
`:3_.g 1.5
`~ i J:~
`~M
`~
`~~~~
`Northern Blot
`(densitomatr!c units)
`
`(a) Comparison of HER-2/neu copy number as
`Figure 3
`determined by FISH with copy number as determined by
`into categories
`Southern blot analysis. Data are grouped
`represented by the small squares. (b) Tumors in which Southern
`blot analysis was unsuccessful due to DNA degradation, are
`sorted into categories by FISH as in (a). As indicated in (c), the x(cid:173)
`axis in these squares represents expression levels as determined by
`Northern blot analysis, the y-axis represents protein levels as
`determined by Western blot analysis. The scale is the same as in
`Figure 2b and c. Tumors with positive ;;,2 (e) and negative (0)
`are indicated. /1 immunohistochemistry not determined
`
`of analysing human breast cancer specimens for the
`presence of the HER-2/neu alteration and offers several
`advantages over other approaches. Like immunohis(cid:173)
`is superior
`to methods requiring
`tochemistry,
`it
`measurement of macromolecules by solid matrix
`blotting techniques. This is likely due to the fact that
`FISH avoids dilutional artifacts inherent in extraction
`of these molecules from tumor cells contained in
`heterogeneous tissue specimens. Again, like immuno(cid:173)
`histochemistry, it offers the advantage of requiring only
`a small amount of tissue, since it can be performed on
`a single four micron tissue section. An additional
`advantage of FISH over
`immunohistochemistry,
`however,
`is
`the
`relative stability of DNA
`for
`hybridization based technologies (Greer et al., 1991;
`Nuovo et al., 1989). The methodologic liability of
`protein is likely secondary to the progressive cross
`linking effects of formalin based fixatives as well as the
`high temperatures used in embedding tissue since both
`of these phenomena can result in antigenic alteration of
`target proteins (Battifora and Kopinski, 1986).
`Using a cohort of breast cancer specimens, all of
`which had been analysed by Southern, Northern and
`Western blot methodologies as well as immunohisto-
`
`a
`
`b
`
`Figure 4 Comparison of probe labeling technologies for FISH in
`tissue sections. Magnification is x 1000. (a) Direct detection of a
`SpectrumOrange labeled HER-2/11e11 probe hybridized to tumor
`cell nuclei which display a single copy status. (b) the same tumor
`hybridized with a HER-2/neu probe labeled with biotin and
`detected via avidin-FITC followed by one cycle of amplification.
`Note the high background
`
`chemical staining of frozen and fixed/embedded tissue,
`we demonstrate that, compared to the best of these
`approaches, (immunohistochemistry in frozen
`tissue
`sections), the sensitivity of FISH is 96.5% while its
`specificity is 100%. The only potential disadvantage to
`FISH is represented by its 3.5% failure in sensitivity
`related to the fact that FISH does not assess HER-2/
`neu expression and therefore cannot detect those few
`cases which overexpress the gene product in absence of
`gene amplification. The current data, however, confirm
`that ,HER-2/neu overexpression is rarely found in
`· tumors with a single copy 'of the gene. Previous
`studies of this cohort estimated that this phenomenon
`may occur in up to 10% of cases of invasive carcinoma
`(Slamon et al., 1989a). This estimate, however, was
`made with the caveat that cases showing overexpres(cid:173)
`sion by immunohistochemistry but assigned to the
`single gene category using Southern blot analysis,
`might be so assigned based on dilutional artifacts
`inherent in the methodology rather than true single
`copy status (Slamon et al., 1989a). FISH analysis
`circumvents this problem and evaluation of the cohort
`using FISH demonstrates a true single copy-over(cid:173)
`expression rate of approximately 3% confirming data
`from two smaller studies of invasive breast cancers
`(Naber et al., 1990; Kallioniemi et al., 1992). In
`addition, discrepancies between FISH and Southern
`blot analyses in detecting HER-2/neu amplification in
`
`

`

`69
`
`Case 1:18-cv-01363-CFC Document 82-6 Filed 03/22/19 Page 8 of 11 PageID #:
`9861
`
`some lumors may be related lo !he probes traditionally
`used as a baseline ror determining gene arnplilic,1lion
`level by Southern. This is especially true in studies
`where p53 is used as a rclcrcnce marker for Southern
`biol analysis since loss or hcterozygosity on the short
`arm or chromosome 17 is a common genetic event in
`breast cancer (Dcvilcc and Cornelisse, 1994) making its
`use as a reference marker questionable. Indeed, use or
`p53 as a reference gene may lead to overestimation of
`HER-2/neu gene amplification. This fact coulcl account
`for the higher percentage or amplification (33 40'1/ti)
`reported in some studies which in !urn may account for
`the lack of correlation seen in those studies (Clark and
`McGuire, 1991 ).
`Results from the FISH analyses pcrf'ormed in this
`study demonstrate that specimens containing more
`than 10 HER-2/neu signals per cell unirormly contain
`ovcn:xprcssion or the gene product, indicating a level
`of gene amplification, uncorrected for chromosome 17
`polysomy, which
`is
`consistently associated with
`detectable ovcrcxprcssion. The advantage of such a
`threshold based association is that it can be done in a
`single step and
`is
`independent of more subjective
`measures of the alteration such as staining intensity as
`used in immunohistochcrnistry. The disadvantage of
`simply using HER-2/neu signal, however,
`is
`the
`possibility that chromosome 17 polysomy could result
`in an increase in signal number without true gene
`amplification. Tn critically assessing tissue for presence
`of IIER-2/neu amplification as well as assessing the
`association between increased DNJ\ copy number and
`ovcrexpression,
`correction of HER-2/nrn
`signal
`number per chromosome 17 ccntromcrc signal
`is
`necessary to obtain a true read or HER-2/neu gene
`copy number (Figure 3). Using this approach, the
`association between HER-2 amplification and over(cid:173)
`expression was consistently found
`in tumors which
`contain more than 2 HER-2/neu genes per chromo(cid:173)
`some 17 centromere. HER-2/neu overexpression in the
`borderline group (> 1, ~2 HER-2/neu genes/chromo(cid:173)
`some 17) occurs in only 1/14 (7%) of these cases and
`it is unclear whether this is on the basis of an increase
`in HER-2/neu DNA copy number secondary
`to
`chromosome 17 polysomy or due to true low level
`gene amplification. However, while it is assumed that
`chromosome 17 centromere signal corresponds to
`copies of intact chromosome 17 in tumor cells as it
`·· does in normal cells, it is possible that loss of physical
`>linkage between the centromere and a gene of interest
`tnay occur very early i