The Combination of In Situ Hybridization and
`lmmunohistochemical Analysis: An Evaluation of
`Her2/ neu Expression in Paraffin-Embedded Breast
`Carcinomas and Adjacent Normal-Appearing
`Breast Epithelium
`Nora Ratcliffe, M.D., Wendy Wells, M.D., Karlya Wheeler, H.T. (A.S.C.P.), H.T.L., l.H.C,
`Vincent Memoli, M.D.
`Department of Pathology, Dartmouth Medical School, Dartmouth Hitchcock Medical Center, Lebanon,
`New Hampshire
`
`The combination of in situ hybridization and im(cid:173)
`munohistochemical techniques can successfully
`identify viral DNA/RNA in specific subsets of cellular
`populations. We recently modified this method to
`evaluate amplification of the oncogene Her2/neu
`and overe:xpression of its protein c-erbB-2 in a se(cid:173)
`ries of 15 breast carcinomas. This combination al(cid:173)
`lows the simultaneous evaluation of the oncogene
`and its corresponding protein expression in single
`cells and specific cellular populations in histologic
`tissue sections. Double staining demonstrated het(cid:173)
`erogeneity within breast carcinomas. In addition,
`both nuclear and cytoplasmic signals were often
`detected in morphologically normal-appearing ad(cid:173)
`jacent breast epithelium. The ability to view both
`the oncogene and its corresponding protein in sin(cid:173)
`gle cells offers a unique look at the biology of c(cid:173)
`erbB-2.
`
`KEYWORDS: Breast cancer, c-erbB-2, Double stain(cid:173)
`lmmunohistochemistry, In situ
`ing, Her2/neu,
`hybridization.
`
`Mod Pathol 1997;10(12):1247-1252
`
`Overexpression of the Her2/ neu oncogene's protein
`product c-erbB-2 as an indication of poor prognosis
`in women with breast carcinoma was first de(cid:173)
`scribed in 1987 by Slamon et al. (1). During the next
`
`0893-3952/97 /0100 12-1247$3.00/0 MODERN PATHOLOGY
`Copyright© 1997 by The United States and Canadian Academy of
`Pathology, Inc.
`VOL. 10, NO. 12, P. 1247, 1997 Printed in the U.S.A.
`Date of acceptance: September 10, 1997.
`NR was supported by Grant T32 ARO 75760 - 02 from the National Insti(cid:173)
`tutes of Health Training Grant in Autoimmunity and Connective Tissue
`Biology.
`Address reprint requests to: Wendy Wells, M.D., Department of Pathology,
`Dartmouth Med ical School, Dartmouth llitchcock Medical Center, One
`Medical Center Drive, Lebanon, NH 03756; fax: 603-650-4845.
`
`10 years, numerous investigators found protein
`overexpression in approximately 25 to 30% of
`breast cancers. An increased incidence is seen in
`both intraductal carcinomas, especially of the
`comedo type, and in Paget's disease of the breast,
`thus generating the hypothesis that amplification/
`overexpression of c-erbB-2 is an early event (2- 7).
`The role of this protein, however, which is homol(cid:173)
`ogous to the epidermal growth factor receptor, in
`the etiology and pathogenesis of breast carcinoma
`remains obscure.
`Historically, the evaluation of tissue for protein
`and gene expression involved serial sections and
`employed the classic techniques of Southern,
`Northern, and Western blotting. Problems with
`sample dilution and sample size often limited the
`usefulness of these approaches. Slamon et al. (8), in
`a comparative study, reported that immunohisto(cid:173)
`chemical analysis of the Her-2/ neu in frozen sec(cid:173)
`tions offered the best correlation with all other an(cid:173)
`alytic data. After these earlier studies, antibodies
`effective in paraffin-embedded tissues (9, 10) were
`developed, eliminating the requirement for frozen
`samples. In addition, fluorescent in situ hybridiza(cid:173)
`tion (FISH), which allowed evaluation of specific
`tumor genetics also using paraffin-embedded tis(cid:173)
`sues, has been refined.
`The combination of ISH and immunohistochem(cid:173)
`ical techniques has been successful in characteriz(cid:173)
`ing viral infections in specific cellular populations
`(11-13). We describe here a technique combining
`conventional automated protocols for ISH of the
`amplified oncogene Her2 / neu and immunohisto(cid:173)
`chemical techniques for its protein product c(cid:173)
`erbB -2 in formalin-fixed, paraffin-embedded breast
`carcinomas. The combination of these two meth(cid:173)
`ods enhances the evaluation of tumor genetics at
`both the gene and protein level, thus allowing the
`
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`study of oncogene induction and expression in tu(cid:173)
`mor and other related cell populations.
`
`MATERIALS AND METHODS
`
`Case Selection and Scoring
`We selected 12 consecutive cases of breast carci(cid:173)
`noma from April 1995 through August 1995 that had
`been routinely imrnunostained for c-erbB-2 (CBll;
`BioGenex, San Ramon, CA) and 3 additional cases
`from 1992 and 1993 under consideration for inclu(cid:173)
`sion in an immune therapy protocol using c-erbB-2
`as the target antigen. All of the tissues were fixed in
`10% buffered
`formalin
`(Biochemical Science,
`Swedesboro, NJ) and embedded in paraffin, per our
`laboratory routine. Standard fixation times ranged
`from 12 to 56 hours. Sections were cut at 4 µm and
`mounted on ChemMate Capillary Gap Plus slides
`(Ventana BioTek, Tucson, AZ) . Immunohistochem(cid:173)
`ical results from all of the cases was reviewed by the
`authors and scored as either positive or negative for
`c-erbB-2. A negative score (for immunohistochemi(cid:173)
`cal results) meant no staining, weak membrane
`staining, or weak cytoplasmic staining. A positive
`score meant that the tumors displayed typical
`strong membrane staining in at least 70% of the
`tumor cells. The tumors were also scored indepen(cid:173)
`dently by ISH for amplification of Her2/ neu. Tu(cid:173)
`mors were considered positive by ISH if a discrete
`nuclear signal was identified in greater then 70% of
`the tumor cells.
`
`FI SH
`FISH using Her2/ neu, a single-stranded cosmid
`probe, (Oncor, Gaithersburg, MD) was performed
`per protocol for tissue ISH from Oncor. In brief,
`protein digestion was done at 37° C with Oncor
`protein digestion enzyme, 400 µ L in 40 mL of 2 X
`standard saline citrate (SSC) (Proteinase K, 25 mg /
`mL) for 20 minutes. Slides were then washed, de(cid:173)
`hydrated, and denatured in a 90° C oven for 12
`minutes. Hybridization was performed overnight
`(16 hr) in a 37° C incubator after slides were placed
`in a humid box. Post-hybridization washes with
`agitation were performed, after removal of the cov(cid:173)
`erslip, in 50% formamide/2X SSC (pH 7) at 42° C for
`15 minutes. This was followed by a second wash in
`0.1 x SSC at 37° C for 30 to 35 minutes. The bioti(cid:173)
`nylated probe was visualized with streptavidin-la(cid:173)
`beled fluorescein after amplification with anti-avi(cid:173)
`din-labeled fluorescein. Fluorescent slides were
`viewed with a Zeiss (Thornwood, NY) Axiophot flu(cid:173)
`orescent microscope using a triple dichroic (4' ,6-
`diamidine-2-phenylindole, fluorescein isothiocya(cid:173)
`nate, Texas Red)
`filter
`(Chroma Technology,
`Brattleboro, VT).
`
`12 48
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`Modern Patho log y
`
`Double Staining
`ISH was performed according to the program
`established for the BioTek 1000 using BioTek solu(cid:173)
`tions (water and phosphate buffers). Slides were
`placed in Buffer 2 (BioTek) three times, for 10 min(cid:173)
`utes each. Protein digestion was carried out with a
`prediluted Proteinase K enzyme solution (DAKO,
`Carpinteria, CA) at room temperature for 10 min(cid:173)
`utes Slides were then washed in Buffer 2 (BioTek)
`three times, for 10 minutes each. This was followed
`by three washes in 100% alcohol, 10 seconds each.
`Slides were then removed from the machine for
`addition of the probe.
`Slides were air dried until the tissue was opaci(cid:173)
`fied. Her-2/ neu probe, prediluted 1 :5 in Hybrisol
`VII (Oncor) , was applied according to the manufac(cid:173)
`turer's directions, approximately 10 µL per 25 x
`25-mm surface area. Slides were coverslipped and
`sealed with rubber cement. After applying the rub(cid:173)
`ber cement, the slides were incubated at 37° C until
`the rubber cement was clear. The slides were then
`denatured at 98° C on a hot plate for 13 minutes,
`and transferred to a 42° C hot plate for 2 hours.
`Slides were removed from the hot plate and
`placed in a Coplin jar filled with low-wash solution,
`2 x SSC plus 0.2% Tween 20, for removal of the
`coverslip. Slides were then reloaded in the Tech(cid:173)
`mate oven slide holder, again with care to avoid air
`bubbles, and placed back on the BioTek 1000. Four
`low-stringency washes, 2X SSC plus 0.2% Tween
`20, at 10 seconds each were followed by three high (cid:173)
`stringency washes, 0.1 x SSC and 0.2% Tween 20, at
`5 minutes each. The high-stringency washes were
`followed by two Buffer 2 (BioTek) washes, 10 sec(cid:173)
`onds each. Slides were then placed in streptavidin/
`alkaline phosphatase for 25 minutes. Slides were
`washed in two Buffer 2 (BioTek) washes, 10 seconds
`each, and then four Buffer 3 (BioTek) washes, also
`10 seconds each. Slides were then stained with
`three changes of BT Red chromogen (BioTek) for 7
`minutes each. Slides were washed in distilled water.
`
`lmmun ohi stoch e mical Exa minati o n
`Slides were removed after staining with chromo (cid:173)
`gen and placed in a plastic Tissue Tek Coplin jar
`filled with citrate buffer (citrate acid monohydrate
`10 mM adjusted to pH 6.0 with 2 N sodium hydrox(cid:173)
`ide). Slides were microwaved (Model NN-5602; Pa(cid:173)
`nasonic, Danville, KY) on the high setting for 10
`minutes, with care used to avoid evaporation dur(cid:173)
`ing microwaving. After microwave heating, slides
`were allowed to cool for 20 minutes. They were
`then rinsed in several changes ofdistilled water to
`remove citrate. After rinsing, slides were reloaded
`on the BioTek 1000.
`Immunostaining was performed according to the
`protocol adapted at Dartmouth-Hitchcock Medical
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`Center for the BioTek 1000. Slides were placed in
`Buffer 2 (BioTek) for 10 seconds, followed by three
`changes of Buffer 1 (BioTek), 10 seconds each. Pri(cid:173)
`mary antibody was then applied (c-erbB-2 clone
`CBll (BioGenex) diluted 1:50) for 2 hours at 40° C.
`Primary antibody application was followed by five
`Buffer 1 (BioTek) washes, 10 seconds each, then
`three washes in Buffer 2 (BioTek), 10 seconds each.
`Secondary antibody was applied for 25 minutes at
`40° C. After the application of th e secondary anti(cid:173)
`body, the slides were washed two tim es in Buffer 2
`(BioTek) for 10 seconds each. The avidin-biotin
`complex (ABC) was applied for 25 minutes at 40° C.
`After application of the ABC, the slides were washed
`in Buffer 2 (BioTek) two times for 10 seconds each,
`followed by three Buffer 3 (BioTek) washes, 10 sec(cid:173)
`onds each. 3,3' -diaminobenzidine was then applied
`for 5 minutes, followed by two washes in distilled
`water for 10 seconds each. The slides were then
`counterstained with hematoxylin (BioTek) for 1
`minute. Slides were removed from the BioTek 1000,
`dehydrated in a series of 95% ethanol followed by
`three changes of absolute ethanol, and then cleared
`in four xylene changes. Slides were then cover(cid:173)
`slipped with Richard Allen mounting medium
`(Richard Allen Medical, Richland, Ml).
`
`RESULTS
`
`Of the 15 breast carcinomas studied, 10 were
`infiltrating ductal carcinoma, 4 were pure intraduc(cid:173)
`tal carcinomas, and 1 was a multifocal papillary
`carcinoma. Of the infiltrating ductal carcinoma
`cases, six also had an intraductal component. Over(cid:173)
`all, 4 (27%) of the 15 tumors were scored as positive
`for c-erbB-2 overexpression/amplification: 2 infil(cid:173)
`trating ductal carcinomas with an associated come(cid:173)
`do-pattern intraductal component, 1 multifocal in(cid:173)
`filtrating ductal carcinoma without an intraductal
`component, and 1 pure intraductal carcinoma with
`apocrine features.
`Nuclear staining of the Her2/ neu oncogene was
`concordant in tumor populations between FISH
`and the alkaline phosphatase detection used in the
`double-staining method (Fig. 1). Double staining
`demonstrated specific nuclear signal only in cells
`that also showed positive immunostaining for the
`c-erbB-2 protein (Fig. lC). Immunohistochemical
`techniques alone revealed membranous staining in
`populations similar to those viewed in double(cid:173)
`stained sections.
`Tumor populations were heterogeneous. Ampli(cid:173)
`fication of the gene, although detected in the ma(cid:173)
`jority of cells in tumors positive for overexpression
`of the c-erbB -2 protein, was conspicuously absent
`in scattered tumor cells with detectable membrane
`staining (Fig. 2B). In some tumors with very weak
`
`FIGURE 1. Infil trati ng ductal carcinoma of breast, highly amplified
`fo r the Her2/ neu. on cogen e. A, FISH for the Her2/ neu oncogene
`(propictium ioctide cow1terstain; original magnification, 200X). B
`demonstrates typ ical strong mem brane staining by
`immw10histochemical analysis alone (original magnification, 200X). C,
`doub le staining for th e Her2/ neu oncogene (pink nucleus) and its
`protein product c-erbB-2 (brown membrane) (ISH with alkaline
`phosphatase detection combined with avidi n-biotin inlmunopero:xidase
`and hematoxylin counterstain; original magnification, 300XJ.
`
`membrane immunostaining for the c-erbB-2 pro(cid:173)
`tein (Fig. 3A), double staining exhibited nuclear
`staining in only a few scattered cells (Fig. 3B) . This
`pattern of weak staining was also observed in adja(cid:173)
`cent histologically normal-appearing breast epithe(cid:173)
`lium (Fig. 4). The converse, gene amplification
`without protein expression, was not identified in
`this study.
`As predicted, the major factor in determining
`quality of staining was tissue fixation. In tissues less
`
`Double Staining f or Her2/neu (N. Ratcliffe et al.)
`
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`FIGURE 2. lnfiltrating ductal carcinoma of breast with less
`amplification of the Her2/ neu oncogene. A, FISH for the Her2 / neu
`oncogene (propiclium iodide counterstain; original magnification,
`200 X) . B, double staining for the Her2/ neu oncogene (pink nucleus)
`and its protein product c-erbB-2 (brown membrane). Note several
`tumor cells with membrane staining and no nuclear signal
`(arrowheads) (ISH with alkaline phosphatase detection combined with
`avidin-biotin immunoperox.idase and hematoxylin counterstain; original
`magnification, 250 X).
`
`well fixed, greater than one-half of the protein sig(cid:173)
`nal was lost after increasing the time of Proteinase
`K digestion. Conversely, decreasing protein diges(cid:173)
`tion by decreasing the time of digestion resulted in
`loss of ISH. Additional dilution of the prediluted
`Proteinase K, 1:3 and 1:5, resulted in preservation of
`the antigen but loss of hybridization signal. Al(cid:173)
`though time and concentration must be deter(cid:173)
`mined empirically, our best results to date far have
`been obtained with the DAKO prediluted Protein(cid:173)
`ase K used with no additional dilution at room
`temperature, followed by antigen retrieval. Gener(cid:173)
`ally, the double-stained slides contained more cy(cid:173)
`toplasmic signal than the same tissue stained by
`immunohistochernical techniques alone. This pat(cid:173)
`tern was most likely related to the Proteinase K
`digestion step required for ISH. The CBll antibody
`recognizes an intracytoplasmic component of the
`membrane-bound c-erbB -2 protein, and cleavage
`during digestion could easily result in a less crisp
`membrane stain.
`
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`
`FIGURE 3. [nfiltrating ductal carcinoma, male breast. A demonstrates
`weak membrane staining by inmrnnohistochemical analysis alone
`(original magnification, 150X). B, double staining showing nuclear
`signal (arrow) in scattered cells (!SH with alkaline phosphatase
`detection combined with avidin-biotin immunoperox.idase and
`hematoxylin counterstain; original magnification of all panels, 200 X) .
`
`FIGURE 4 . Normal-appearing breast lobule with weak membrane
`and cytoplasmic staining for c-erbB-2 (brown) but only scattered nuclei
`positive for the Her2/ neu oncogene (pink) (!SH with alkaline
`phosphatase detection combined with avidin-biotin immunoperox:idase
`and hematoxylin counterstain; original magnification, 200X).
`
`CONCLUSIONS
`
`Detection of Her2/neul c-erbB-2 by alkaline
`phosphatase ISH coupled with ABC 3,3' -diarnino-
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`benzidine immunohistochemical analysis in tumor
`populations correlated with single staining by ei(cid:173)
`ther immunohistochemical techniques alone or
`FISH alone. Heterogeneity for gene amplification
`within the tumor population was demonstrated by
`double staining as a lack of nuclear signal in indi(cid:173)
`vidual tumor cells that demonstrated clear mem(cid:173)
`brane immunostaining. This phenomenon was
`found within tumors that had strong membrane
`staining revealed by immunohistochemical meth(cid:173)
`ods as well as in tumors with less staining. In pop(cid:173)
`ulations with faint membrane staining, however,
`there were fewer cells with nuclear signal than there
`were in the populations with strong membrane
`staining. Heterogeneity for gene amplification
`within tumor populations has been described using
`FISH, but intratumoral heterogeneity is often not
`recognized by
`immunohistochemical analysis
`alone (14, 15). In this study, immunohistochemical
`techniques by themselves did not equate to the
`presence of gene amplification as detected by ISH
`for an individual tumor cell.
`Tumor populations that overexpress the c-erbB-2
`protein without gene amplification have been de(cid:173)
`scribed in approximately 3 to 10% of breast carci(cid:173)
`nomas studied by a combination of methods, i.e.,
`FISH,
`immunohistochemical
`techniques,
`and
`Southern blot analysis (1, 8, 14, 16). Hollywood and
`Hurst (17) demonstrated a novel DNA binding pro (cid:173)
`tein, OB2-l, in cell lines overexpressing the c(cid:173)
`erbB-2 protein and its corresponding mRNA but
`containing a single gene copy. This DNA-binding
`protein was increased in amplified tumors as well.
`OB2-l was not increased in populations of cells
`with weak membrane staining. These observations
`led some authors to conclude that it is the overex(cid:173)
`pression of the membrane protein detected by im(cid:173)
`munohistochemical means that best predicts tu(cid:173)
`mor behavior (4). In support of this opinion, tumor
`cells that had Her2 / neu gene amplification but no
`protein expression were not identified in this study.
`The appearance of nuclear signal in scattered
`epithelial cells in the adjacent normal-appearing
`breast epithelium that showed weaker membrane
`staining by the CBll antibody was even more in(cid:173)
`teresting. Wealc membrane staining for c-erbB -2
`protein in normal breast epithelium has been pre(cid:173)
`viously described, and this level of staining is
`thought to correspond to the level identified in
`nonamplified breast cancers that contain a single
`copy of the gene by Southern blot analysis (18, 19).
`Identification of a few morphologically benign cells
`with nuclear signal might indicate an early increase
`in gene copy number, which could herald neoplas(cid:173)
`tic transformation. This observation is concordant
`with that of Kallioniemi et al. (15), who found no
`extrachromosomal erbB -2 genes in amplified pop(cid:173)
`ulations. They suggested that if extrachromosomal
`
`amplification occurred, it would have happened in
`a preclinical phase of tumor growth. Accumulation
`of genetic abnormalities as loss of heterozygosity in
`morphologically normal-appearing breast tissue
`was recently described (20), and our results provide
`additional evidence of genetic changes in histolog(cid:173)
`ically normal-appearing breast epithelium adjacent
`to tumor.
`Methodologic problems encountered were not
`unique to double staining. Fundamentally, the
`quality of the tissue fixation was the key to good
`results. The biggest drawback to performing this
`procedure using an automated stainer was the
`amount of probe required for coverage with the
`capillary gap action. The cost of the commercially
`available probe made it prohibitive to use the
`stainer during hybridization. Slides were removed
`from the stainer just before denaturation, to con(cid:173)
`serve probe, and the procedure continued manu(cid:173)
`ally. The slides were returned to the machine after
`hybridization for the rest of the procedure.
`After double staining, more cytoplasmic signal
`was present in some of the tissues than was found
`in the same tissue stained with immunohistochem(cid:173)
`ical methods alone, which gave only membrane
`staining. A likely explanation for this additional cy(cid:173)
`toplasmic staining is that the Proteinase K digestion
`is cleaving some of the membrane-bound protein,
`which is then detected in the cytoplasm, because
`the CBll antibody is targeted to an intracytoplas(cid:173)
`mic portion of this molecule. The level of mem(cid:173)
`brane staining correlated with the intensity of nu(cid:173)
`clear staining in the majority of the tumor cells,
`thus reconfirming a relationship between gene am(cid:173)
`plification and membrane protein expression.
`In conclusion, the ability to study both the phe(cid:173)
`notype and genotypic expression of c-erbB-2 in
`single cells within various tumor and nontumorous
`populations offers a unique view of its expression in
`the breast. The majority, approximately 70%, of the
`carcinomas that we studied were considered nega(cid:173)
`tive for overexpression of the oncogene, which is
`consistent with data reported by other investiga(cid:173)
`tors. Nevertheless, as demonstrated in Figure 3,
`most of the tumors we studied did show some
`staining with the CBll antibody and scattered pos(cid:173)
`itive nuclei by ISH. Our data suggest that amplifi(cid:173)
`cation of the gene is an early event that might in
`some cases precede overexpression of the protein,
`because we did not see hybridization in scattered
`individual cells in both nontumor and tumor pop(cid:173)
`ulations that showed weak expression of the pro(cid:173)
`tein. These findings are an additional confirmation
`of the themy that breast carcinoma, much like car(cid:173)
`cinoma in the colon, results from a multistage pro(cid:173)
`cess, with the progressive accumulation of genetic
`alterations along the way.
`
`Double Sta i ning for Her2/ neu (N. Ratc liffe et al. )
`
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`Acknowledgments: The authors thank Ken Orn(cid:173)
`dorff, Englert Cell Analysis Laboratory, Norris Cotton
`Cancer Center, Lebanon, New Hampshire for his
`assistance with the fluorescent images.
`
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