9. Clinical significance of erbBZ protein
`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.
`
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`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
`
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`

`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
`
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`

`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
`
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`

`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,
`
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