`overexpression
`
`Soonmyoung Paik, Elizabeth Burkhard, and Marc E. Lippman
`
`1. Introduction
`
`Originally described as a homologue of the EGF receptor gene amplified in
`breast cancer [1], erbB-Z protein is now thought to be a cell membrane
`receptor for a newly described ligand—gp30 [2]. There is some confusion in
`the literature about the terminology for erbB-Z. In brief, erbB-Z, c-erbB-Z,
`and HER-2 refer to the same human gene residing in the long arm of the
`chromosome 17, which has homology to human EGF receptor gene (c-erbB)
`[3]. erbB-2 protein, p18SerbB-2, or p185HER-2 refer to the transmembrane
`receptor protein translated from the erbB-2 gene, which has a molecular
`weight of 185 kDa. c-neu refers to the rat counterpart of the erbB—2 gene [4].
`Carcinomas from many organs have frequent overexpression of erbB-Z
`protein. These include carcinomas of the breast [5],
`lung [6], ovary [7],
`stomach [8], pancreas [9], and endometrium [10]. The overexpression
`of erbB-Z protein is usually accompanied by amplification of the gene.
`However, in about 10% of the tumors that overexpress erbB—Z protein, no
`amplification is found, as in the T-47—D breast cancer cell line [11].
`In this review, we will concentrate mainly on the clinical importance of
`erbB-2 protein overexpression in human cancers.
`
`2. Function of erbB-Z
`
`As shown in Figure 1, erbB-2 protein is thought to be composed of three
`domains —- the extracellular ligand binding domain,
`transmembrane do—
`main, and intracellular tyrosine kinase domain [3]. Since the ligand was
`identified only recently, signal transduction through erbB-Z was still poorly
`understood. However, studies utilizing hybrid receptors (the extracellular
`domain of EGF receptor/intracellular domain of erbB-Z, see Chapter 11)
`have demonstrated that stimulation of erbB-2 may result in activation of
`early-response genes, such as fun and fos [12].
`
`RB. Dickson and ME. Lippman (eds), Genes, Oncogenes, and Hormones: Advances in Cellular and
`Molecular Biology of Breast Cancer. © 1991 Kluwer Academic Publishers. Boston. All rights reserved.
`
`Genentech 2116
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`Celltrion v. Genentech
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`|PR2017-01122
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`Genentech 2116
`Celltrion v. Genentech
`IPR2017-01122
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`182
`
`Shed
`
`extracellular
`domain
`
`flgpw
`
`Shed
`extracellular
`domain 30
`
`
`
`Extracellular Ligand
`Binding Domain
`
`
`
`
`
`
`tyrosine kinase domain
`
`
`Intracellular signalling
`
`
`
`
`Activation of important
`genes (rat, jun, fos, ...)
`
`
`Figure 1. Schematic representation of the postulated domains of erbB-Z protein and its
`interaction with ligand and ligand-like molecules; shed extracellular domain may directly
`interact with the receptor or may block the binding of the gp30 ligand to the receptor.
`Heterodimerization with the other receptor may also be important in the regulation of signal
`transduction as well as the phosphatases.
`
`3. Ligand and ligand-like activities for erbB-2
`
`Recently a putative ligand for erbB-Z has been described by Lupu et al. [2].
`This gp30 ligand activates phosphorylation of both EGF receptor (EGF-R)
`and erbB-Z. Although it
`is known that EGF-R can crossphosphorylate
`erbB-Z [13,14], gp30 showed both biological and biochemical effects on cells
`with erbB-2 overexpression but without EGF-R [2]. Thus gp30 seems to act
`as a ligand for erbB-Z. Intriguingly, gp30 ligand did not show any demon-
`strable effect on cells with normal
`levels of erbB-Z [2]. The shed extra-
`cellular domain of erbB-2 represents another Class of ligand-like activity,
`which might play a role in vivo [15]. Theoretically such a shed domain can
`directly interact with membrane—bound erbB-Z protein, with or without
`
`
`
`183
`
`Normal Duct
`
`w Regresswn
`erbB-2
`overexpression Aligand
`
`
`
`Co-expression
`'
`Intraductal Carcmoma of Ligand
`
`\h»
`
`Over 50% of DCIS
`overexpress erbB-2
`
`Invasion
`
`Only 20 to 30% of
`infiltrating ductal carcinoma
`overexpress erbB-2
`
`Figure 2. Hypothesized role of the ligand in the diseasse progression in breast cancer; tumor
`cells that express both the receptor and the ligand would result in infiltrating ductal carcinoma.
`Some tumors may begin to express both and bypass the intraductal stage,
`thus resulting in
`infiltrating ductal carcinoma without the intraductal component.
`
`biological effects, or may even block the interaction between gp30 ligand
`and the receptor (see Figure 1). The shed extracellular domain may even be
`useful for patient follow—up during and after treatment. Thus it is apparent
`that the erbB-2 signal transduction pathway presents an extremely complex
`interaction of receptors, ligands, and ligand-like molecules. We are still far
`from understanding the role of each component.
`As will be discussed later, the incidence of erbB-Z overexpression is much
`higher in intraductal breast cancer compared to infiltrating ductal carcinoma
`[16]. We have frequently seen cases in which only the intraductal component
`is positive, whereas the infiltrating component is negative. Based on these
`findings, we propose an ‘erbB-Z ligand invasion’ hypothesis, as shown in
`Figure 2. According to this hypothesis, overexpression of erbB-Z is a fairly
`early event in the progression and does not result in an invasive phenotype
`on its own. It is believed that about one third of the intraductal cancers
`
`actually progress to infiltrating ductal carcinoma. Only when the gp30 ligand
`is coexpressed could erbB—Z-mediated cell invasion be initiated and tumor
`become invasive. Generation of cDNA probe and antibodies for the ligand(s)
`will be required to test such a hypothesis. Of course it should be remem-
`bered that not all
`tumors with erbB—Z overexpression have intraductal
`components.
`In such tumors, erbB-Z and the ligand might have been
`expressed together at an early stage, according to this model.
`
`
`
`184
`
`4. Role of the transmembrane domain in transformation
`
`Although the transforming neu gene (the rat homologue of the human
`erbB-2) has a point mutation in the transmembrane domain [4], no such
`transforming mutations have been found yet for the human erbB-Z (17).
`While this may be partly due to the technical difficulties involved in de-
`tecting mutations in amplified gene, overexpression of the erbB-2 protein
`alone without any mutation have been shown to transform NIH—3T3 cells
`[18]. However, possibilities for mutations are not completely excluded.
`
`5. Immunostaining for erbB-2 protein
`
`Although Slamon and coworkers were the first to show the potential clinical
`importance of erbB—2 gene amplification [19], the development of immuno-
`staining assays that allowed the detection of erbB-Z overexpression in
`paraffin—embedded sections has been the major breakthrough in studying
`the clinical importance of erbB—Z [5]. Immunostaining assays detect only the
`tumor cells with overexpression of erbB-Z protein, not the cells with normal
`levels of erbB-Z. The breast-cancer cell lines MCF-7 or MDA-MB-468 are
`
`examples of tumor cells expressing only the normal (unamplified) amount of
`erbB-Z protein. Shown in Figure 3b is an example of a breast cancer with
`erbB-Z protein overexpression. Note the strong membrane staining of all
`tumor cells in the section. Usually all tumor cells stain positive when a given
`tumor is stained positive, including both in situ and invasive components, as
`well as the metastatic site. Many studies have shown a paradoxically higher
`incidence of erbB-Z overexpression in intraductal cancer of the breast [20].
`This disease is believed to be in situ and a preinvasive cancer. We have
`never observed a case in which the invasive component is positive when the
`in situ component is negative. However, we have seen cases in which the in
`situ component is positive but the invasive component is negative, or in
`which the primary tumor is positive but the metastatic site is negative. Thus
`overexpression of erbB-Z seems to be an early event in the progression of
`breast cancer.
`
`the
`Although some investigators have questioned the sensitivity of
`immunostaining assay for paraffin-embedded sections, we have found that a
`cocktail preparation of antibodies directed against
`the extracellular and
`intracellular domains (pAb-l plus mAb-l, Triton Biosciences) can be used
`to increase the sensitivity of the assay almost threefold compared to poly-
`clonal antibody against the cytoplasmic domain (pAb-l) used alone. Figure
`4 shows an example of cases that were strongly stained with the cocktail
`preparation (Figure 4a) but did not show staining at ail with pAb-l alone
`(Figure 4b). Furthermore, even in cases that showed heterogenous staining
`by one antibody (an example is shown in Figure 3a), the cocktail antibody
`stained all tumor cells in the section (Figure 3b), thus improving the objec-
`
`
`
`185
`
`
`
`B F
`
`immunostaining of monoclonal antibody by the addition of
`igure 3. Enhancement of
`polyclonal antiserum. A: Staining with monoclonal antibody (mAb-l) alone resulted in
`heterogenous staining due to the failure to recognize the epitope in the center of the formalin-
`fixed and paraffin-embedded tissue section (20X magnification) B: Addition of the polyclonal
`antiserum (pAb-l) resulted in homogenous staining of all tumor cells in the section.
`
`
`
`186
`
`B
`
`
`
`Figure 4. Comparison of immunostaining by the cocktail antibody (mAb—l plus pAb—l) vs. the
`polyclonal antiserum (pAb-l), which has been commonly used in the published studies. A: The
`cocktail antibody stains
`all
`tumor cells
`in this case of gastric adenocarcinoma (20X
`magnification). B: Polyclonal antiserum fails to stain any tumor cells in the same section.
`
`
`
`187
`
`tivity of the interpretation of the assay result. The immunostaining assay is
`now generally accepted as the method of choice for the detection of erbB-2
`overexpression in the clinical tumor samples [21]. Obviously, this method
`has many practical advantages over DNA, RNA, or protein blotting tech—
`niques. Recent progress in automatic immunohistochemistry promises a
`bright future for the routine clinical application of this assay.
`
`6. Clinical importance of erbB-Z protein overexpression
`
`Among the tumors that overexpress erbB—2, breast cancer has been most
`extensively examined. Slamon is responsible for many of the initial studies.
`By screening a variety of human tumor samples for the existence of possible
`gene amplification or overexpression of almost all known oncogenes,
`Slamon has successfully shown that erbB—2 gene amplification is frequent in
`breast cancer and is a potentially important prognostic indicator in stage 2
`breast cancer. However, much of the clinical interest in erbB—2 was mainly
`due to the hope that one might be able to use the assay to identify the
`patients with node-negative breast cancer who are at high risk and should be
`treated with adjuvant systemic treatments. Indeed many studies, including
`our own, have shown the prognostic value of erbB-2 overexpression/gene
`amplification in breast cancer [reviewed in 21]. Patients with erbB-2-positive
`tumors are probably at higher risk and should be treated with adjuvant
`therapy. This may be especially true in the case of those with node-negative
`breast cancer with good nuclear grade or ER—positive status, who otherwise
`would not be candidates for chemotherapy [5,22]. However, what has been
`emerging from recent studies is the realization that, in addition to its role as
`an indicator for poor prognosis after surgical treatment without adjuvant
`therapy [23], erbB—2 is an indicator for poor prognosis even for patients
`treated with adjuvant regimens — PF (L-PAM, S-FU) [5], CMF (cytoxan,
`methotrexate, S-FU), CMFP [24], or tamoxifen [25]. There is preliminary
`evidence that suggests erbB-2 overexpression correlates with a lack of
`response to at
`least some of these regimens [26,27]. If erbB-2-positive
`tumors still have a poor prognosis when treated, other approaches will have
`to be employed. For the latter goal, in vitro studies have been carried out
`exploring monoclonal antibodies against erbB-Z protein [28 and Chapter 10]
`and antisense oligodeoxynucleotides directed against erbB-2 mRNA [29]. At
`the same time, alternate conventional chemotherapeutic regimens that
`might be effective for erbB-Z—positive tumors should be explored. Insight
`into this possibility can be accomplished by retrospective screening of the
`completed clinical trials comparing different adjuvant regimens. We have
`observed that breast cancer cell lines with erbB-2 overexpression tend to be
`resistant
`in vitro to suprapharmacological doses of S-FU, L-PAM, and
`mitomycin C, but not to anthracyclines (doxorubicin and mitoxantrone) in a
`drug resistance assay [30]. Thus we hypothesize that erbB—2-positive tumors
`
`
`
`188
`
`should respond as well to doxorubicin-containing regimen as erbB-Z-negative
`tumors. If the latter hypothesis is true, erbB-Z determination would not be
`a prognostic indicator
`for
`tumors treated with doxorubicin-containing
`regimens, while being a prognostic indicator for tumors treated with surgery
`only or nondoxorubicin adjuvant chemotherapeutic regimens. Such studies
`are in progress for breast cancer in collaboration with the NSABP (National
`Surgical Adjuvant Breast Project) — NSABP protocols B—11 and B-12 —— in
`which more than 1400 node-positive patients were randomized to receive
`either PF or PAF. While such studies are in progress we have also examined
`this hypothesis in gastric cancer. Although included in the earliest screening
`studies [31,32], erbB-2 has not been studied extensively in gastric cancer.
`Most of the studies examining gene amplification observed amplification
`only in well-differentiated (or tubular) adenocarcinoma but not in poorly
`differentiated adenocarcinomas
`[33,34]. More
`recently,
`studies using
`immunohistochemistry have shown that overexpression is not confined to
`tubular cancer, and the incidence is between 10 and 50%, depending on the
`study [8,35,36]. There are only two studies in the literature that have
`examined the effect of erbB-2 overexpression on patient outcome. Of these
`the study by Yonemura and coworkers deserves particular attention [8].
`They reported that erbB—Z was found to be an independent indicator for
`poor prognosis for patients treated with adjuvant regimes — MF (mitomycin
`C plus oral S-FU) or oral S-FU alone. A gastric cancer cell
`line that
`overexpresses the erbB-2 protein (NCI-N87) is also resistant to S-FU [37]. In
`a retrospective study with small number of cases, we found that erbB—Z is a
`prognostic indicator for patients treated with surgery alone, but not for
`patients treated with a doxorubicin-containing regimen [38]. Together with
`the data by Yonemura et al.,
`the data suggest that tumors with erbB-2
`overexpression may be resistant to an MF regimen but not to doxorubicin-
`containing regimens and that further studies in this direction are required.
`
`7. Therapeutic importance of erbB-Z
`
`Whether erbB-2 overexpression is a causative variable or an associated
`variable for a poor drug response is a clinically important question. This is
`true because if overexpression of erbB-2 is causative, inhibition of erbB-Z
`may result
`in reversion of the drug-resistant phenotype. Hancock and
`coworkers have shown that an anitbody against erbB-Z can enhance the
`cytocidal effect of cisplatin on ovarian cancer cell line with erbB-Z over-
`expression [39]. Benz and coworkers have also shown that transfection of
`erbB—Z into MCF-7 cells results in a decrease in sensitivity to tamoxifen and
`cisplatin [27]. Thus it seems possible that erbB-2 is at least associated with, if
`not responsible for, decreased sensitivity to various chemotherapeutic drugs,
`which can be overcome by interfering with the erbB-2 signal transduction
`pathway. This suggests the possibility that therapeutic approaches can be
`
`
`
`189
`
`developed by combining conventional drugs plus reagents that will block
`erbB-2, such as antibodies or antisense oligodeoxynucleotides.
`The data by Bacus and coworkers on erbB-2 and ploidy deserves atten-
`tion from the therapeutic point of View [41]. They showed in small number
`of breast cancer cases that erbB—Z overexpression is highly associated with
`hypertetraploid DNA content and a low proliferation rate. If these data
`hold true,
`then cell-cycle-specific drugs, as suggested by Clark and co-
`workers, may not be an ideal treatment strategy [42]. However, there are
`many larger studies that contradict these results, although at least one study
`showed that erbB-Z overexpression is associated with hypertetraploidy [25].
`This question should be more carefully addressed in the future studies in
`light of the therapeutic viewpoint.
`Regardless of its relationship with the chemotherapeutic responses,
`erbB-Z seem to be an extremely attractive target for experimental thera—
`peutics. Since (1) only the cells with erbB—2 overexpression respond to the
`growth-inhibitory effects of monoclonal antibodies or gp30 ligand, and (2)
`when a given tumor is positive for erbB-2 overexpression all tumor cells
`overexpress erbB—2, one can easily visualize the possibility of the clinical
`application of such molecules.
`
`8. Future directions
`
`During the past several years we have learned much about the clinical
`importance of erbB-2 protein overexpression. However, we do not know the
`cellular and biochemical mechanisms responsible for such clinical behavior.
`As such mechanisms are elucidated, we will be able to take adventage 0f the
`situation and use overexpressed erbB-Z as a therapeutic target for novel
`agents, as well as a guide for custom-tailored therapies for individual
`tumors,
`
`References
`
`1. King, C.R., Kraus, M.H., Aaronson, SA. (1985) Amplification of a novel v-erbB related
`gene in a human mammary carcinoma. Science 229:974—976.
`2. Lupu, R., Colomer, R., Zugmaier, (3., et al. (1990) Direct interaction of a ligand for
`erbB-2 oncogene product with the EGF receptor and p185erbB-2. Science 249:1552—1555.
`3. Coussens, L., Yang-Feng, T.L., Liao, Y.C., et al. (1985) Tyrosine kinase receptor with
`extensive homology to EGF receptor shares chromosomal location with neu oncogene.
`Science 230:1130—1139.
`
`(1986) The neu oncogene encodes an
`4. Bargman, C..I., Hung, M.C., Weinberg, RA.
`epidermal growth factor receptor related protein. Nature 319:230—234.
`5. Paik, S.M., Hazan, R., Fisher, E.R., et al. (1990) Pathologic findings from the NSABP:
`Prognostic significance of erbB-2 protein overexpression in primary breast cancer. J. Clin.
`Oncol. 81103—112.
`
`
`
`190
`
`. Kern, J.A., Schwartz, D.A., Nordberg, JR, et al. (1990) p185neu expression in human
`lung adenocarcinomas predicts shortened survival. Cancer Res. 50:5184—5187.
`. Slamon, D.J., Godolphin, W., Jones, L.A., et al.
`(1989) Studies of the HER-Zlneu
`prom-oncogene in human breast and ovarian cancer. Science 224:707-712.
`. Yonemura, Y., Ninomiya, 1., Yamaguchi, A., et a1. (1991) Evaluation of immunoreactivity
`for erbB-2 protein as a marker of poor short term prognosis in gastric cancer. Cancer Res.
`51:1034—1038.
`
`10.
`
`. Hall, P.A., Hughes, C.M., Staddon, S.L., et al. (1990) The c-erbB-2 protooncogene in
`human pancreatic cancer. J. Pathol. 161:195—200.
`Berchuck, A., Rodriguez, 6., Kinney, R.B., et a1. (1991) Overexpression of HER-Z/neu in
`endometrial cancer is associated with advanced stage disease. Am. J. Obstet. Gynecol.
`164215—21.
`
`11.
`
`12.
`
`13.
`
`14.
`
`15.
`
`16.
`
`17.
`
`18.
`
`19.
`
`20.
`
`21.
`
`22.
`
`23.
`
`24.
`
`25.
`
`26.
`
`Kraus, M.H., Popescu, N.C., Amsbaugh, C., et a1. (1987) Overexpression of the EGF
`receptor-related proto-oncogene erbB-2 in human mammary carcinoma cell
`lines by
`different molecular mechanisms. EMBO J. 62605—610.
`
`Pandiella, A., Lehvaslaiho, H., Magni, M., Alitalo, K., Meldolesi, J. (1989) Activation of
`an EGFP/neu chimeric receptor: Early intracellular signals and cell proliferation responses.
`Oncogene 4:1299— 1305.
`King, C.R., Borello, 1., Bellot, F., et a1. (1988) EGF binding to its receptor triggers a rapid
`tyrosine phosphorylation of the erbB-2 protein in the mammary tumor cell line SKmBR-3.
`EMBO J. 721647-1651.
`
`(1988) Tumor
`Akiyama T., Saito, T., Ogawara, H., Toyoshima, K., Yamamoto, T.
`promoter and epidermal growth factor stimulate phosphorylation of the c—erbB-Z gene
`product in MKN-7 human adenocarcinoma cells. Mol. Cell. Biol. 8:1019— 1026.
`Langton, B.C., Crenshaw, M.C., Chao, L.A., et al. (1991) An antigen immunologically
`related to the external domain of gp185 is shed from nude mouse tumors overexpressing
`the c-erbB-Z (HERvZ/neu) oncogene. Cancer Res. 51:2593u2598.
`(1990)
`Ramachandra, S., Machin, L., Ashley, 8., Monaghan, P., Gusterson, B.A.
`Immunohistochemical distribution of c-erbB—2 in in situ breast carcinoma — a detailed
`
`morphological analysis. J. Pathol. 161:7— 14.
`Saya, H., Ara, S., Lee, P.S.Y., R0, J.S., Hung, MC. (1990) Direct sequencing analysis of
`transmembrane region of human Neu gene by polymerase chain reaction. M01. Carcinog.
`3:198—201.
`
`DiFiore, P.P., Pierce, J.H.G., Kraus, M.H., et al. (1986) erbB-2 is a potent oncogene
`when overexpressed in NIH/3T3 cells. Science 237:178— 182.
`Slamon, D.J., Clark, G.M., Wong, S.G., et a1. (1987) Human breast cancer: Correlation of
`relapse and survival with amplification of the HER—Z/neu oncogene. Science 235:177—182.
`van de Vijver, M.J., Peterse, J.L., Mooi, W.J., Wisman, P., Lomans, J., Delesio, 0.,
`Nusse, R. (1988) Neu-protein overexpression in breast cancer. Association with comedo-
`type ductal carcinoma is situ and limited prognostic value in stage 11 breast cancer. N. Engl.
`J. Med. 319:1239— 1245.
`
`Perren, T.J. (1991) c-erbB-2 oncogene as a prognostic marker in breast cancer (editorial).
`Br. J. Cancer 63:328—332.
`
`Battifora, H., Gaffey, M., Esteban, J., et al. (1991) Immunohistochemical assay of neu/c-
`erbB-2 oncogene product in paraffin-embedded tissues in early breast cancer: Retrospective
`follow-up study of 245 stage I and 11 cases. Modern Pathol. 42466-474.
`Winstanley, J ., Cooke, T., Murray, G.D., et al. (1991) The long term progostic significance
`of c-erbB-2 in primary breast cancer. Br. J. Cancer 63:447—450.
`Anbazhagan, R., Gelber, R.D., Bettelheim, R., et al.
`(1991) Association of c-erbB-2
`expression and S-phase fraction in the prognosis of node positive breast cancer. Ann.
`Oncol. 2:47—53.
`
`Borg, A., Baldetorp, B., Ferno, M., et al. (1991) erbB-2 amplification in breast cancer with
`a higher rate of proliferation. Oncogene 62137—143.
`Wright, C., Nicholson, S., Angus, B., et al. (1991) Association Of c-erbB-2 oncoprotein
`
`
`
`191
`
`27.
`
`28.
`
`29.
`
`30.
`
`31.
`
`32.
`
`33.
`
`34.
`
`35.
`
`36.
`
`37.
`
`38.
`
`39.
`
`40.
`
`41.
`
`expression with lack of response to endocrine therapy in recurrent breast cancer (abstract).
`J. Pathol. 158:350A.
`
`Benz, C.C., Scott, G.K., Sarup, J.C., Shepard, H.M., Osborne, CK. (1991) Tamoxifen
`resistance associated with p185HER-2 overexpression in human breast cancer cells
`transfected with HER—Z/neu. Proc. Am. Assoc. Cancer Res. 32:1260A.
`
`Hudziak, R.M., Lewis, G.D., Winger, M., Fendly, BM. (1989) p185HER2 monoclonal
`antibody has antiproliferative effects in Vitro and sensitizes human breast tumor cells to
`tumor necrosis factor. Mol. Cell. Biol. 911165—1172.
`
`(1991) Inhibiting c-erbB-2
`Brysch, W., Magal, 13., Louis, J.C., Schilingensiepen, K.H.
`overexpression
`in
`human mammary
`carcinoma
`cells
`with
`phosphothioate
`oligodeoxynucleotides (abstract). Proc. Am. Assoc. Cancer Res. 32:2574A.
`Kern, D.H., Weisenthal, L.M.
`(1990) Highly specific prediction of antineoplastic drug
`resistance with an in vitro assay using suprapharmacologic drug exposures. J. Natl. Cancer
`Inst. 82:582—588.
`
`(1987) Light and electron microscopical
`Mori, S., Akiyama, T., Morishita, Y., et al.
`demonstration of c-erbB-2 gene product-like immunoreactivity in human malignant tumors.
`Virchows Archiv. B. 54:8—15.
`
`(1986) Amplification of c-erbB-2
`Yokota, J., Yamamoto, T., ToyOShima, K., et al.
`oncogene in human adenocarcinomas in vivo. Lancet 5:765—767.
`Park, J.B., Rhim, J.S., Park, SC, et al.
`(1989) Amplification, overexpression, and
`rearrangement of the erbB-2 protooncogene in primary human stomach carcinomas.
`Cancer Res. 49:6605h6609.
`
`Yokota, J., Yamamoto, T., Miyajirna, N., et al. (1988) Genetic alterations of c-erbB—2
`oncogene occur frequently in tubular adenocarcinoma of the stomach and are often
`accompanied by amplification of the v-erbA homologue. Oncogene 2:283—287.
`Falck, V.G., Gullick, W.J.
`(1989) c-erbB-2 oncogene product
`staining in gastric
`adenocarcinoma. An immunohistochemical study. J. Pathol. 159:107—111.
`Kameda, T., Yasui, W., Yoshida, K., et al. (1990) ExpreSSion of erbB-2 in human gastric
`carcinomas: Relationship between p185erbB-2 expression and the gene amplification.
`Cancer Res. 50:8002— 8009.
`
`Park, J.G., Kramer, BS, Lai, S.L., et al. (1990) Chemosensitivity patterns and expression
`of human multidrug resistance-associated MDR1 gene by human gastric and colorectal
`carcinoma cell lines. J. Natl. Cancer Inst. 82:193—198.
`
`Paik, S.M., Chung, H.C., Yang, W.I., et al. (1991) Overexpression of erbB-Z protein in
`gastric cancer (abstract). Proc. Annu. Meet. Am. Assoc. Cancer Res. 32:A1730.
`Hancock, M.C., Chan, A.W., Mischak, R.P., et al.
`(1990) Monoclonal antibodies to
`c-erbB-2 enhance the cytotoxicity of cisplatin against human breast and ovarian tumor cells.
`J. Cell. Biochem. Suppl 14B:CF203.
`(1990) HER-Z/neu oncogene
`Bacus, S.S., Bacus, J.W., Slamon, D.J., Press, M,F.
`expression and DNA ploidy analysis in breast cancer. Arch. Pathol. Lab. Med. 114:164—
`169.
`
`(1991) Follow-up study of HER-Z/neu amplification in
`Clark, G.M., McGuire, W.L.
`primary breast cancer. Cancer Res. 51:944—948.
`
`