- Research article
- Open Access
Presence of HER4 associates with increased sensitivity to Herceptin™ in patients with metastatic breast cancer
Breast Cancer Research volume 11, Article number: R50 (2009)
HER2 overexpression, or rather HER2 gene amplification, is indicative for Herceptin therapy in both metastatic and pre-metastatic breast cancer patients. Patient's individual sensitivity to Herceptin treatment, however, varies enormously and spans from effectual responsiveness over acquired insensitivity to complete resistance from the outset. Thus no predictive information can be deduced from HER2 determination so that molecular biomarkers indicative for Herceptin sensitivity or resistance need to be identified. Both ErbB receptor-dependent signalling molecules as well as HER2-related ErbB receptor tyrosine kinases, known to mutually interact and to cross-regulate each other are prime candidates to be involved in cellular susceptibility to Herceptin.
Using immunohistochemistry and fluorescence in situ hybridisation, we retrospectively investigated primary breast cancer tissues from 48 patients who were under Herceptin treatment. We quantified the gene copy numbers of all HER receptors and evaluated their coexpression profile. Moreover the HER2 phosphorylation state, the ratio of native to truncated HER2, p27(kip1) and PTEN expression were objects of this study.
Above all markers investigated in this study Kaplan-Meier and Cox regression analysis revealed a significant positive impact of HER4 (co-)expression on overall survival from beginning of antibody therapy. Both HER4 expression and HER4 gene amplification emerged as independent prognostic markers in Herceptin-treated breast cancer patients and responsiveness to Herceptin turned out to be more efficient if tumour cells show HER4 expression.
Although HER4 is known to potentially exert a tumour cell killing activity and in turn to have a favourable impact in breast cancer patients we demonstrate here the first time that HER4 expression prolongs overall survival in Herceptin-treated patients. Elucidating HER4 receptor function in the context of Herceptin treatment will advance the design of highly efficient receptor targeting. By then we need to extend the analysis of breast cancer by allowing for HER2/HER4 coexpression by which valuable additional prognostic and predictive information might possibly be revealed.
The overexpressed human epidermal growth factor receptor (HER) 2 receptor tyrosine kinase (RTK) is a useful therapeutic target in breast cancer patients. In pathological diagnostics, the HER2 expression level is routinely evaluated and an elevated receptor expression (scored 3+) is usually based on HER2 gene amplification, which can reliably be identified by in situ hybridisation . Without contraindication HER2 protein overexpression (or HER2 gene amplification) represents the worldwide accepted rationale for antibody-targeted therapy using trastuzumab (Herceptin™). Herceptin™ was approved in Europe in 2000 for the treatment of breast cancer patients suffering from metastatic and aggressive tumour growth that is indicative for a poor disease outcome . In fall 2006, Herceptin™ treatment was extended by the approval for adjuvant patient treatment . Statistically considered, antigen-specific targeting of tumour cells has brought significant clinical benefit to these patients, both in terms of overall survival (OS) and recurrence free survival (RFS) documented in many clinical trials [4, 5]. Although HER2 overexpression or HER2 gene amplification is a prerequisite for therapeutic antibody treatment, the extent of sensitivity to Herceptin™ therapy varies enormously and ranges from excellent individual responsiveness over acquired insensitivity to complete resistance from the outset [6–9]. Consequently, HER2 overexpression does not represent a reliable predictive marker for responsiveness to Herceptin™.
Numerous molecular biomarkers implicated in individual sensitivity to Herceptin™ treatment have been suggested, but have not been precisely and reproducibly defined to date. The panel of suggested molecules comprises proteins involved in intracellular survival signalling (PTEN)  and cell proliferation (p21, p27) [11–15], as well as cell membrane located molecules featuring the capacity to directly interact with HER2 and thereby modulate lateral and transversal HER2 activity . Among them the HER2-related RTK family members HER1 (also known as epidermal growth factor receptor (EGFR)), HER3 and HER4 are in the focus of interest . Depending on their coexpression profile HER receptors act in concert on ligand binding and trigger transmembraneous signal transduction as a functional unit rather than individually and independently. The recruitment of intracellular signalling pathways is a result of intensively cross-talking receptors and well-defined three-dimensional intracellular activation domains . Hence a highly organised spatiotemporal coexpression of HER receptors plays a pivotal role in growth, development and differentiation both on the cellular and organ level in healthy organisms [19, 20]. However, HER1 and/or HER2 overexpression, as well as atypical HER receptor coexpression profiles, causing receptor hyperactivation and enhanced mitogenic signalling, could have been frequently attributed to malignant cell growth .
Moreover, HER3 has been found to preferentially interact with HER2  and, although kinase deficient, to significantly enhance mitogenic signalling . As a consequence even antiproliferative effects mediated by Herceptin™ can be extenuated by HER3 coexpression . Remarkably, receptor coexpression does not necessarily result in hyperstimulation and hypermitogenic cellular activity. Signal modulation, fine tuning and control can also be achieved by extensive receptor communication and even antiproliferative and induction of differentiation responses on growth factor binding (i.e. heregulin) to HER4 have been previously described [24, 25].
Conceivably the tissue-specific coexpression of HER receptors has immediate impact on the cellular response to Herceptin™, which specifically binds to HER2. In a previous study we have experimentally shown that the cellular sensitivity to Herceptin™ treatment is directly contingent on the EGFR coexpression density and can be enhanced by EGFR downregulation . In another study undertaken in our laboratory, we have shown that amplified HER3 and HER4 gene copy numbers have additional prognostic impact on the course of breast cancer disease in patients both with and without HER2 gene amplification .
Here, in a series of 48 breast cancer patients with HER2 gene amplification we retrospectively investigated numerous additional biomolecular parameters to have potential impact on the course of disease under Herceptin™ treatment (prognostic value) and to be implicated in responsiveness to Herceptin™ treatment (predictive value). Therefore, we quantified the gene copy numbers of all HER receptors via fluorescent in situ hybridisation (FISH) and immunohistochemically investigated the coexpression profile of all HER receptors. In addition, the HER2 phosphorylation state, the ratio of native to truncated HER2, the expression of p27kip1 and PTEN, and the presence of the proliferation associated antigen Ki-67 were studied. As inhibition of activated HER2 might be primarily responsible for Herceptin™ sensitivity rather than the total receptor targeting , we evaluated the pY1248 phosphorylation, which has been linked to the mitogenic ras/MAP kinase signalling pathway . Herceptin™ is targeted to the juxtamembrane domain of HER2 , so the loss of binding site by receptor truncation could be suggestive for antibody resistance . Furthermore, the identification of proteolytically shed HER2 (sHER2/p95) in relation to total HER2 expression could be informative in terms of receptor activation and consequently the efficacy of Herceptin™ binding. Moreover PTEN, a negative regulator of PKB/Akt, and p27kip1 both downstream targets of HER2, have been proposed to be involved in Herceptin™ resistance [10, 32].
The aim of this study was to cytogenetically and immunohistochemically identify molecular biomarkers within the context of HER receptor signalling, which affects the susceptibility of metastatic breast cancer to Herceptin™ treatment. Of all the markers investigated in this study, the presence of truncated and phosphorylated receptors appeared to have an impact by trend on the course of disease of Herceptin™-treated (HT) patients. All Kaplan-Meier and Cox regression analyses revealed a significant positive impact of HER4 (co-)expression on OS from the beginning of antibody therapy. Finally both HER4 expression and HER4 gene amplification emerged as independent prognostic markers in HT breast cancer patients. HER4 receptor expression is even supposed to provide predictive value because it apparently affects susceptibility to Herceptin™ treatment.
Materials and methods
This study was approved by the Institutional Review Board of the University of Regensburg, Germany.
Breast tumour samples and patient characteristics
Formalin-fixed paraffin-embedded tissue blocks from 48 female patients with invasive lobular or ductal unilateral primary breast cancer (median age, 56.0 years; range 31.4 to 80.9) were obtained from the archives of the Institute of Pathology, Regensburg, and the Institute of Pathology, Hospital Weiden, Germany. All patients were diagnosed between March 1997 and October 2005 and underwent Herceptin™ therapy between September 2000 and November 2005. The patients were not involved in any clinical trial. Clinical data were acquired by the Tumour Centre Inc., Regensburg. The median follow-up period was 55.8 months (range 28.4 to 116.6). The median OS time was 41.7 months (range 10.6 to 116.6), median RFS was 21.9 months (range 1.5 to 63.6). A total of 36 patients (75%) died and 12 patients survived until the end of monitoring. Of the 48 patients, 31 (65%) suffered from a recurrence of breast cancer including the subgroups of 4 of the 12 surviving patients (33.3%) plus 27 of the 36 (75%) patients who passed away.
Fluorescence in situhybridisation
Tissue microarrays (TMAs) were constructed and handled as described previously . Briefly, after dewaxing and rehydrating, slides were steamed in sodium citrate. Cell structures were digested in pepsin and hydrochloric acid, then washed and dehydrated.
For FISH analyses, 5 μm sections of the TMAs directly labelled dual-colour DNA probes for HER1 (7p11.2), HER2 (17q21-22), HER3 (12q13.2), HER4 (2q33.3-34), and oestrogen receptor α (ERα, 6q25) (ZytoVision Ltd., Bremerhaven, Germany) were used. The probes identified locus-specific sequences for both the genes and the corresponding centromeres 7, 17, 12, 2, and 6 to differentiate between gene amplification and polysomy of the respective chromosome. Respective DNA probe sets were applied to the TMA area and incubated overnight at 37°C. Subsequent to several washing steps, nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole) and analysed by epifluorescence microscopy. FISH scoring was performed by counting fluorescence signals in 25 malignant, non-overlapping cell nuclei for each case by two independent interpreters (AS, MB). The FISH ratio was assessed as the number of genes proportional to the number of centromeres. Patients with HER2 ratios, that is total gene/total centromere relations, of 1.5 or more were considered as amplified based on recent statistical approaches of our group  and included in this study.
TMA sections stained with H&E were used for reference histology.
Microscopy, fluorescence in situhybridisation scoring and digital imaging
Slides were imaged as described previously  with an Axio Imager Z.1 (Zeiss, Göttingen, Germany), equipped with specific filter sets (AHF, Tübingen, Germany) and the plug-in module ApoTome™ for taking pseudoconfocal images. Three-dimensional z-stacks were generated, colours were separately recorded and digitally processed. Corresponding images were superimposed.
Immunostaining with anti-HER-receptor antibodies, anti-phosphoHER2, anti-extracellular HER2, anti-ER, anti-progesterone receptor (PR), anti-p27kip1, and anti-PTEN was performed on 5 μm sections of the TMAs and applied in accordance with the manufacturer's instructions. Table 1 explicitly shows the antibody-specific staining and scoring characteristics. Interpretation was performed independently by two experienced pathologists (SS, FH). Stably transfected mouse fibroblasts proved specific immunostaining of HER1 to HER4 .
Anti HER1 to 4 and anti-Ki67 antibodies were assessed as described previously . HER2 phosphorylation status was scored as suggested in the Herceptest™ guidelines. The classification into p27Kip1 positive (n = 24) and negative cases (n = 24) was performed using an immunoreactive score (IRS), defined by the following formula: nuclear staining (0 to 100%) × intensity of staining (0 = no staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining) plus percentage of cells with cytoplasmic staining × medial staining intensity. An IRS of at least 200 was regarded as positive. Anti-PTEN staining (Rabbit monoclonal antibody, clone 138G6, Cell Signaling, Boston, MA, USA) detected endogeneous levels of total protein analogously to the above mentioned procedure considering nuclear and cytoplasmic staining, respectively. ER and PR scoring was performed according to Remmele and Stegner . Anything but IRS 0 was considered positive.
The primary outcome measure, OS, was calculated as the time from the beginning of Herceptin™ treatment to death from any cause or to the date on which the patient was last known to be alive. Patients lost to follow-up were treated as censored cases on the basis of the last contact date. A secondary outcome measure, RFS, was omitted because in our series Herceptin™ treatment was primarily indicated after diagnosis of relapse and/or in patients with metastatic breast cancer (M1, R2) so that complete remission did not occur.
Survival curves were generated using the Kaplan-Meier method, and log-rank tests compared the distributions between groups. In addition, hazard ratios (HR) with 95% confidence intervals (CI) were estimated for covariates (treated as continuous, and where appropriate as a dichotomous variable) using the Cox proportional-hazards model. Non-parametric correlations were analysed by Spearman tests. In all analyses, P ≤ 0.05 (two-tailed) was considered significant. Statistical analyses were performed with SPSS version 16.0 (SPSS Inc., Chicago, IL, USA).
TMAs of paraffin-embedded samples from 48 HT breast cancer patients were used to analyse gene amplification and protein expression of each member of the HER family. Metastasis as a prerequisite for patients receiving Herceptin™ therapy is referred to as stage IV (defined as Tx, Nx, M1). We found one grade 1 tumour (not included in the statistics), but 11 graded 2 (according to ) and 35 graded 3 with a trend for longer survival with tumours graded 2 (P = 0.116, Figure 1). For OS, patients age at various dichotomisations (cut-off points at 45, 50, 55 and 60 years) played no significant role (< 50 years, n = 20 vs. ≥ 50 years, n = 28; P = 0.399).
Univariate Cox analysis (Table 2) revealed HER4 (FISH and immunohistochemistry analysed), phosphoHER2 and p27Kip1 (immunohistochemistry) all to have a positive prognostic impact on OS. A multivariate Cox analysis (backstep: LR) was accomplished enclosing all parameters including HER1, 3, 4, and ERα, FISH, HER1, 3, 4 immunohistochemistry, Ki67 immunohistochemistry, ER and PR immunohistochemistry, grading, and age. The final step is shown in Table 3.
Fluorescence in situhybridisation
HER1, HER3, HER4, and ERα were processed as continuous variables instead of dichotomising the data due to indefinable cut-off points . HER1, HER3, and ERα FISH provided no further information whereas HER4 gene amplification was a significant positive marker for OS under Herceptin™ treatment (HER4 continuous: HR = 0.003 (95% CI < 0.01 to 0.17), P = 0.005, Table 3) in multivariate cox analysis.
Immunohistochemistry of HER1, HER3, and HER4
All immunohistochemical results are shown in Table 4. Immunostaining of all four HER receptors was performed. The specificity of applied antibodies was proved by staining stably transfected mouse fibroblasts (NIH 3T3, kindly provided by Roche Diagnostics, Penzberg, Germany, data shown elsewhere ).
By immunohistochemistry, 22.9% (11 of 48) were identified as HER1 positive (score 1+, 2+ and 3+) and 77.1% (37 of 48) as negative. In 22.4% (48 of 214) HER2 was overexpressed (score 2+ and 3+) and 77.6% (166 of 214) were unaltered. Immunohistochemistry of HER3 resulted in 93% (39 of 47) positive (score 1+, 2+ and 3+) and 17% (8 of 47) negative patients. Of the total number of cases, 54.2% (26 of 48) expressed HER4 (score 1+, 2+ and 3+), whereas 45.8% (22 of 48) did not.
As in the FISH results, HER1 and HER3 immunohistochemistry did not give further information (HER1 P = 0.278, HER3 P = 0.982; Kaplan-Meier curves not shown). Remarkably, HER4 immunohistochemistry corroborates HER4 FISH findings. HER4 expression resulted in significant longer survival times of HT patients (P = 0.013; Figure 2).
Within the patient collective investigated in this study, besides HER4 gene amplification and grading, HER4 immunohistochemistry was found to have significant positive impact on OS under Herceptin™ treatment (HR = 0.383 (95% CI 0.180 to 0.815), P = 0.013).
To evaluate Herceptin™ therapy, we matched the HT series (n = 48) with a cluster of patients (n = 20) originating from a recent study with no antibody-treated patients  and performed Kaplan-Meier survival analyses. The criterion for patient inclusion was their metastatic status (stage IV, M1 tumours).
Conventionally treated (CT) HER2-positive patients have the shortest survival time. Herceptin™ therapy improves their OS, but CT HER2-negative patients show the longest survival times (Figure 3). Irrespective of CT HER2-positive patients, a significant difference remains between HT HER2-positive patients and CT HER2-negative patients (P = 0.037). Thus, our statistical analysis is in agreement with the frequently found observation that HER2 positivity is a negative prognostic factor independent of therapy.
Considering therapy options (CT and HT), Herceptin™ clearly ameliorated OS (Figure 4).
With given HER2 overexpression but independent of treatment, HER4 positivity lengthened survival times. HER4 positivity showed a positive impact on OS both in CT and in HT patients (curve 3 to 4, log rank, P = 0.013; and curve 1 to 2, log rank, P = 0.083).
Comparing patients with HT and HER4 negativity and patients with CT but HER4 positivity, no significant difference could be found (log rank, P = 0.351).
Other immunohistochemical investigations
Regarding the HT patient series, p27Kip1 immunohistochemistry positive patients (n = 24) had a significantly better outcome concerning OS compared with p27Kip1 negative (n = 24) patients (Figure 5a, log rank, P = 0.012).
Phosphorylated HER2 receptor (Figure 5b) was associated with increased OS (P = 0.016). In 16% of cases (7 of 43) we documented a positive staining with a significant positive effect on OS, whereas 84% of cases (36 of 43) showed less or no staining.
To examine the proliferation status in our patient cohort we assessed Ki-67 immunohistochemistry. The proliferative index had no significant impact on the OS of patients (data not shown).
All tumours were stained with two anti-HER2 antibodies. The routinely used antibody binds an intracellular target and showed a positive staining as expected in a HT patients series. We compared the staining intensity and pattern with staining obtained by using the SP3 antibody which targets a protruding extracellular HER2 domain (Table 4). In 22 cases we found differences; in 20 cases the extracellular staining was weaker than the intracellular, pointing out a shedding of the extracellular receptor domain. Comparing survival times of these 20 patients with those with homogeneous staining (n = 24), the latter demonstrated a trend for decreased RFS times (P = 0.096).
Immunohistochemical staining of ER and PR did not yield any supplemental information (ER P = 0.631, PR P = 0.593). The anti-PTEN antibody did not reveal any interpretable results and was eliminated from evaluation.
HER2 and ER immunohistochemistry were negatively correlated (P = 0.011, correlation coefficient = -0.374).
Screening of HER2 aberration (both on a protein or DNA level) as a prerequisite for Herceptin™ therapy does not allow the courses of disease or individual therapy responses to be predicted, so numerous biomarkers with potential impact on Herceptin™ responsiveness have moved into the focus of interest. Utilising immunohistochemistry and FISH, we retrospectively investigated the most likely parameters with immediate impact on HER2 activity and Herceptin™ responsiveness in a cohort of 48 HT breast cancer patients with HER2 amplification. Here, we present first-hand data comprising expression of all HER receptors as well as p27Kip1 receptor phosphorylation and native vs. truncated HER2 staining.
Our study demonstrates for the first time that HER4 expression improves the outcome in a sub-collective of HER2-positive, HT breast cancer patients. First evidence indicating that, irrespective of therapy, HER4 coexpression has a positive impact on the outcome of breast cancer patients was published in 2003 , indicating a potential anti-tumourigenic effect mediated by the fourth member of the HER receptor family discovered in 1993 . From there on a significant reduction of the proliferation indices in HER4-positive invasive breast carcinomas was reported, suggesting HER4 itself has a functional anti-proliferative or even protective capacity . An association of HER4 expression and a favourable outcome in breast cancer patients has independently been described by Suo and colleagues  who found that HER4 coexpression obviously antagonises the effect of HER2 (over-)expression on the course of breast cancer disease.
Our observation that HER4 coexpression apparently favours the outcome of HER2-positive, HT breast cancer patients indicates that HER4 coexpression favours the HER2 targeting with Herceptin™. This suggests a non-autonomous role of HER4 but rather some degree of HER4/HER2 interplay what is in agreement with data provided by Sartor and colleagues . They transfected HER2-positive cancer cells to overexpress HER4, achieving a reduction in proliferation and an increase in apoptosis which is suggestive of HER4 slowing down HER2 signalling activity. Moreover, a differentiation inducing effect by heregulin treatment in HER4 expressing SUM44 and SUM102 breast cancer cells has been experimentally demonstrated. This observation appeared to be independent of HER2 expression, because HER2 elimination could not eradicate the HER4 dependent decrease in cell growth. However, we previously found the coexpressed EGFR to contribute to cell susceptibility to Herceptin™ (and pertuzumab) and thereby provided evidence for EGFR/HER2 interaction playing a pivotal role in anti-HER2 targeting  in which HER4 is also most likely to be involved in.
Although HER4 expression has been frequently attributed to low-grade tumours with low proliferation activity , the discussion on the impact of HER4 on breast cancer progression and outcome of disease appeared contradictory over the past couple of years. This debate can be attributed to the ambivalent function of HER4 representing either oncogenic or tumour-suppressing activity . Four differentially spliced HER4 isoforms have been identified but never been distinguished in descriptive studies addressing HER4 expression in primary cancer . The JM-a/CYT-1 and JM-a/CYT-2 isoforms can be proteolytically cleaved by tumour necrosis factor-α converting enzyme [40, 42] and subsequently by γ-secretase , whereas JM-b/CYT-1 and JM-b/CYT-2 variants represent non-cleavable counterparts  characterised by ligand-independent activity that promotes cancer cell growth . Commonly, only JM-a/CYT-1 and JM-a/CYT-2 variants are expressed in tumour tissues at the same time . The intracellular CYT isoforms differ by having (CYT-1) or not having (CYT-2) a cytoplasmic binding site for phosphoinositide 3-kinase. Upon cleavage of JM-a isoforms these intracellular released HER4 domains (4ICD) can be differentially translocated into intracellular compartments and, if cytoplasmically located, subsequently trigger anti-proliferative and pro-apoptotic signals [46, 47]. In contrast, if translocated into the nucleus, they have the capacity to trigger pro-proliferative and survival promoting signalling [48–51] and accordingly a nuclear localisation of 4ICD has been associated to shortened survival of breast cancer patients . In contrast, a mitochondrial accumulation of 4ICD, which evidently shares structural and functional homology with BH3-only pro-apoptotic BCL-2 family members, has also been reported . This in turn results in a disturbance of mitochondrial membrane integrity. This process typically entails an apoptotic cell death characterised by mitochondrial permeabilisation, cytochrome-c efflux and caspase activation, and can explain the protective role of HER4.
The positive impact of HER4 on a patient's course of disease as shown in this study is not only suggestive of its differentiation promoting and potentially pro-apoptotic function but rather for a Herceptin™ augmenting effect. As HER2 is primarily phosphorylated on Herceptin™ treatment, as we have previously shown [52, 16], it might be conceivable that HER4 can be crossactivated and a subsequent release of 4ICD might possibly enhance Herceptin™/HER2 triggered signalling. HER4, if coexpressed to HER2, could synergistically extenuate proliferative and survival signalling, for example via downregulation of ERK1/2 and PKB/Akt activity, respectively . Numerous observations suggest that irrespective of any therapeutic treatment HER4 expression is associated with reduced tumour breast cancer aggressiveness, which is indicative of its prognostic impact [27, 35–39, 51, 54]. Moreover, based on Kaplan-Meier analysis as illustrated in Figure 4, we here provide evidence that the outcome of HER2/HER4 double-positive patients is significantly better both under CT and HT compared with those patients who were HER2-positive but HER4 negative. The latter observation does not necessarily designate a predictive value of HER4 but is indicative of the positive effect of HER4 among patients characterised by a well defined therapeutic setting (either CT or HT treatment). Nevertheless, the molecular signalling background responsible for the higher efficiency of Herceptin™ treatment in HER2-positive/HER4-positive patients versus HER2-positive/HER4-negative patients needs to be experimentally elucidated in detail and the experimental design should allow for investigating the predictive relevance of HER4 in consideration of its oncogenic and tumour suppressing potential, respectively. Deciphering the molecular effect of HER4, which enhances therapy efficiency of Herceptin™ treatment, would affect the decision to undergo Herceptin™-related therapy but the presence of HER4 could be potentially therapeutically addressed in the future as well. It would not be advisable, for instance, to use novel anti-HER2 drugs, which would eradicate the therapy-enhancing effect of HER4. Alternatively one could consider therapeutically eliciting or intensifying the Herceptin™ enhancing effect of HER4.
The induction of the cyclin-dependent kinase inhibitor p27Kip1 protein is considered as a key mechanism on anti-HER2 targeting using Herceptin™ by which at least six signalling targets and pathways might be modulated . Also, in this study HER2 overexpressing patients with upregulated p27Kip1, benefit significantly from antibody therapy indicating a prognostic value of this protein in HT patients, which is in agreement with other reports [55, 56].
Patients respond more efficiently to Herceptin™ treatment if HER2-Tyr1248  is phosphorylated suggesting that Herceptin™ affects an activated rather than an inactive receptor. However, other sites may influence the response to Herceptin™ .
Even if no correlation between HER4 and ER status could be found in our study, the signalling crosstalk of HER receptors with the ERα needs to be functionally explored in more detail. 4ICD has been shown to potentially activate ERα and in turn to stimulate tumour cell proliferation . Accordingly, Tovey and colleagues showed a significantly poorer survival in ER-positive breast cancer patients by detecting nuclear HER4 localisation , highlighting the importance of 4ICD/ERα interplay.
Current approaches in developing novel anti-HER receptor targeted drugs that block the entire type 1 RTK family via 'pan-HER' blockade  may be counterproductive due to the ambivalent function of HER4. HER4 overexpression or amplification dependent on the prevalent isoform confers a reduced risk [27, 54]. Hence development of selective type 1 RTK inhibitors leaving HER4 signalling unaffected may prove more advantageous. On the contrary, antibodies selectively targeting HER4 JM-a, such as the recently introduced monoclonal antibody 1479 , could have therapeutic potential, assuming that JM-a-specific antibodies are expected not to cause adverse effects in tissues, such as the heart, that exclusively express ErbB4 JM-b.
Further studies addressed to elucidate HER4 receptor function in the context of Herceptin™ treatment are necessary to guide the design of highly efficient therapeutic strategies based on HER receptor targeting. Extending the data presented here by stratifying larger patient cohorts characterised by their HER2/HER4 coexpression profile will disclose the impact of HER4 on anti-HER2 targeted Herceptin™ treatment compared with conventional therapy of HER2-positive breast cancer patients. Potentially the evaluation of HER4 expression, by which valuable additional prognostic or even predictive information might possibly be revealed either from immunohistochemistry or FISH even without differentiating between HER4 splice variants, could contribute to therapy decisions and/or optimisation in the future.
epidermal growth factor receptor
fluorescence in situ hybridisation
haematoxylin and eosin
human epidermal growth factor receptor
recurrence free survival
receptor tyrosine kinase
Lottner C, Schwarz S, Diermeier S, Hartmann A, Knuechel R, Hofstaedter F, Brockhoff G: Simultaneous detection of HER2/neu gene amplification and protein overexpression in paraffin-embedded breast cancer. J Pathol. 2005, 205: 577-584. 10.1002/path.1742.
Ross JS: Breast cancer biomarkers and HER2 testing after 10 years of anti-HER2 therapy. Drug News Perspect. 2009, 22: 93-106. 10.1358/dnp.2009.22.2.1334452.
Bedard PL, Piccart-Gebhart MJ: Current paradigms for the use of HER2-targeted therapy in early-stage breast cancer. Clin Breast Cancer. 2008, 8 (Suppl 4): S157-S165. 10.3816/CBC.2008.s.012.
Pritchard KI, Shepherd LE, O'Malley FP, Andrulis IL, Tu D, Bramwell VH, Levine MN: HER2 and responsiveness of breast cancer to adjuvant chemotherapy. N Engl J Med. 2006, 354: 2103-2111. 10.1056/NEJMoa054504.
Nielsen DL, Andersson M, Kamby C: HER2-targeted therapy in breast cancer. monoclonal antibodies and tyrosine kinase inhibitors. Cancer Treat Rev. 2009, 35: 121-136. 10.1016/j.ctrv.2008.09.003.
Slamon D: Herceptin: increasing survival in metastatic breast cancer. Eur J Oncol Nurs. 2000, 4: 24-29. 10.1054/ejon.2000.0070.
Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, Baselga J, Norton L: Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med. 2001, 344: 783-792. 10.1056/NEJM200103153441101.
Plosker GL, Keam SJ: Trastuzumab: a review of its use in the management of HER2-positive metastatic and early-stage breast cancer. Drugs. 2006, 66: 449-475. 10.2165/00003495-200666040-00005.
Nahta R, Esteva FJ: HER2 therapy: molecular mechanisms of trastuzumab resistance. Breast Cancer Res. 2006, 8: 215-10.1186/bcr1612.
Nagata Y, Lan KH, Zhou X, Tan M, Esteva FJ, Sahin AA, Klos KS, Li P, Monia BP, Nguyen NT, Hortobagyi GN, Hung MC, Yu D: PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell. 2004, 6: 117-127. 10.1016/j.ccr.2004.06.022.
Nahta R, Takahashi T, Ueno NT, Hung MC, Esteva FJ: P27(kip1) down-regulation is associated with trastuzumab resistance in breast cancer cells. Cancer Res. 2004, 64: 3981-3986. 10.1158/0008-5472.CAN-03-3900.
Le XF, Pruefer F, Bast RC: HER2-targeting antibodies modulate the cyclin-dependent kinase inhibitor p27Kip1 via multiple signaling pathways. Cell Cycle. 2005, 4: 87-95.
Kute T, Lack CM, Willingham M, Bishwokama B, Williams H, Barrett K, Mitchell T, Vaughn JP: Development of Herceptin resistance in breast cancer cells. Cytometry A. 2004, 57: 86-93. 10.1002/cyto.a.10095.
Baselga J, Albanell J, Molina MA, Arribas J: Mechanism of action of trastuzumab and scientific update. Semin Oncol. 2001, 28: 4-11. 10.1016/S0093-7754(01)90276-3.
Lane HA, Motoyama AB, Beuvink I, Hynes NE: Modulation of p27/Cdk2 complex formation through 4D5-mediated inhibition of HER2 receptor signaling. Ann Oncol. 2001, 12 (Suppl 1): S21-S22. 10.1023/A:1011155606333.
Diermeier S, Horvath G, Knuechel-Clarke R, Hofstaedter F, Szollosi J, Brockhoff G: Epidermal growth factor receptor coexpression modulates susceptibility to Herceptin in HER2/neu overexpressing breast cancer cells via specific erbB-receptor interaction and activation. Exp Cell Res. 2005, 304: 604-619. 10.1016/j.yexcr.2004.12.008.
Yarden Y: Biology of HER2 and its importance in breast cancer. Oncology. 2001, 61 (Suppl 2): 1-13. 10.1159/000055396.
Stern DF: Tyrosine kinase signalling in breast cancer: ErbB family receptor tyrosine kinases. Breast Cancer Res. 2000, 2: 176-183. 10.1186/bcr51.
Muraoka-Cook RS, Feng SM, Strunk KE, Earp HS: ErbB4/HER4: role in mammary gland development, differentiation and growth inhibition. J Mammary Gland Biol Neoplasia. 2008, 13: 235-246. 10.1007/s10911-008-9080-x.
Casalini P, Iorio MV, Galmozzi E, Menard S: Role of HER receptors family in development and differentiation. J Cell Physiol. 2004, 200: 343-350. 10.1002/jcp.20007.
Graus-Porta D, Beerli RR, Daly JM, Hynes NE: ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J. 1997, 16: 1647-1655. 10.1093/emboj/16.7.1647.
Holbro T, Beerli RR, Maurer F, Koziczak M, Barbas CF, Hynes NE: The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. Proc Natl Acad Sci USA. 2003, 100: 8933-8938. 10.1073/pnas.1537685100.
Lee-Hoeflich ST, Crocker L, Yao E, Pham T, Munroe X, Hoeflich KP, Sliwkowski MX, Stern HM: A central role for HER3 in HER2-amplified breast cancer: implications for targeted therapy. Cancer Res. 2008, 68: 5878-5887. 10.1158/0008-5472.CAN-08-0380.
Plowman GD, Green JM, Culouscou JM, Carlton GW, Rothwell VM, Buckley S: Heregulin induces tyrosine phosphorylation of HER4/p180erbB4. Nature. 1993, 366: 473-475. 10.1038/366473a0.
Plowman GD, Culouscou JM, Whitney GS, Green JM, Carlton GW, Foy L, Neubauer MG, Shoyab M: Ligand-specific activation of HER4/p180erbB4, a fourth member of the epidermal growth factor receptor family. Proc Natl Acad Sci USA. 1993, 90: 1746-1750. 10.1073/pnas.90.5.1746.
Brockhoff G, Heckel B, Schmidt-Bruecken E, Plander M, Hofstaedter F, Vollmann A, Diermeier S: Differential impact of Cetuximab, Pertuzumab and Trastuzumab on BT474 and SK-BR-3 breast cancer cell proliferation. Cell Prolif. 2007, 40: 488-507. 10.1111/j.1365-2184.2007.00449.x.
Sassen A, Rochon J, Wild PJ, Hartmann A, Hofstaedter F, Schwarz S, Brockhoff G: Cytogenetic analysis of HER1/EGFR, HER2, HER3, AND HER4 in 278 breast cancer patients. Breast Cancer Res. 2008, 10: R2-10.1186/bcr1843.
DiGiovanna MP, Chu P, Davison TL, Howe CL, Carter D, Claus EB, Stern DF: Active signaling by HER-2/neu in a subpopulation of HER-2/neu-overexpressing ductal carcinoma in situ: clinicopathological correlates. Cancer Res. 2002, 62: 6667-6673.
DiGiovanna MP, Lerman MA, Coffey RJ, Muller WJ, Cardiff RD, Stern DF: Active signaling by Neu in transgenic mice. Oncogene. 1998, 17: 1877-1884. 10.1038/sj.onc.1202091.
Cho HS, Mason K, Ramyar KX, Stanley AM, Gabelli SB, Denney DW, Leahy DJ: Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab. Nature. 2003, 421: 756-760. 10.1038/nature01392.
Scaltriti M, Rojo F, Ocana A, Anido J, Guzman M, Cortes J, Di Cosimo S, Matias-Guiu X, Cajal S, Arribas J, Baselga J: Expression of p95HER2, a truncated form of the HER2 receptor, and response to anti-HER2 therapies in breast cancer. J Natl Cancer Inst. 2007, 99: 628-638. 10.1093/jnci/djk134.
Lu CH, Wyszomierski SL, Tseng LM, Sun MH, Lan KH, Neal CL, Mills GB, Hortobagyi GN, Esteva FJ, Yu D: Preclinical testing of clinically applicable strategies for overcoming trastuzumab resistance caused by PTEN deficiency. Clin Cancer Res. 2007, 13: 5883-5888. 10.1158/1078-0432.CCR-06-2837.
Remmele W, Stegner HE: [Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue]. Pathologe. 1987, 8: 138-140.
Elston CW, Ellis IO: Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology. 1991, 19: 403-410. 10.1111/j.1365-2559.1991.tb00229.x.
Witton CJ, Reeves JR, Going JJ, Cooke TG, Bartlett JM: Expression of the HER1-4 family of receptor tyrosine kinases in breast cancer. J Pathol. 2003, 200: 290-297. 10.1002/path.1370.
Tovey SM, Witton CJ, Bartlett JM, Stanton PD, Reeves JR, Cooke TG: Outcome and human epidermal growth factor receptor (HER) 1-4 status in invasive breast carcinomas with proliferation indices evaluated by bromodeoxyuridine labelling. Breast Cancer Res. 2004, 6: R246-R251. 10.1186/bcr783.
Suo Z, Risberg B, Kalsson MG, Willman K, Tierens A, Skovlund E, Nesland JM: EGFR family expression in breast carcinomas. c-erbB-2 and c-erbB-4 receptors have different effects on survival. J Pathol. 2002, 196: 17-25. 10.1002/path.1003.
Sartor CI, Zhou H, Kozlowska E, Guttridge K, Kawata E, Caskey L, Harrelson J, Hynes N, Ethier S, Calvo B, Earp HS: Her4 mediates ligand-dependent antiproliferative and differentiation responses in human breast cancer cells. Mol Cell Biol. 2001, 21: 4265-4275. 10.1128/MCB.21.13.4265-4275.2001.
Sundvall M, Iljin K, Kilpinen S, Sara H, Kallioniemi OP, Elenius K: Role of ErbB4 in breast cancer. J Mammary Gland Biol Neoplasia. 2008, 13: 259-268. 10.1007/s10911-008-9079-3.
Carpenter G: ErbB-4: mechanism of action and biology. Exp Cell Res. 2003, 284: 66-77. 10.1016/S0014-4827(02)00100-3.
Junttila TT, Sundvall M, Maatta JA, Elenius K: Erbb4 and its isoforms: selective regulation of growth factor responses by naturally occurring receptor variants. Trends Cardiovasc Med. 2000, 10: 304-310. 10.1016/S1050-1738(01)00065-2.
Rio C, Buxbaum JD, Peschon JJ, Corfas G: Tumor necrosis factor-alpha-converting enzyme is required for cleavage of erbB4/HER4. J Biol Chem. 2000, 275: 10379-10387. 10.1074/jbc.275.14.10379.
Maatta JA, Sundvall M, Junttila TT, Peri L, Laine VJ, Isola J, Egeblad M, Elenius K: Proteolytic cleavage and phosphorylation of a tumor-associated ErbB4 isoform promote ligand-independent survival and cancer cell growth. Mol Biol Cell. 2006, 17: 67-79. 10.1091/mbc.E05-05-0402.
Elenius K, Corfas G, Paul S, Choi CJ, Rio C, Plowman GD, Klagsbrun M: A novel juxtamembrane domain isoform of HER4/ErbB4. Isoform-specific tissue distribution and differential processing in response to phorbol ester. J Biol Chem. 1997, 272: 26761-26768. 10.1074/jbc.272.42.26761.
Junttila TT, Sundvall M, Lundin M, Lundin J, Tanner M, Harkonen P, Joensuu H, Isola J, Elenius K: Cleavable ErbB4 isoform in estrogen receptor-regulated growth of breast cancer cells. Cancer Res. 2005, 65: 1384-1393. 10.1158/0008-5472.CAN-04-3150.
Naresh A, Long W, Vidal GA, Wimley WC, Marrero L, Sartor CI, Tovey S, Cooke TG, Bartlett JM, Jones FE: The ERBB4/HER4 intracellular domain 4ICD is a BH3-only protein promoting apoptosis of breast cancer cells. Cancer Res. 2006, 66: 6412-6420. 10.1158/0008-5472.CAN-05-2368.
Vidal GA, Naresh A, Marrero L, Jones FE: Presenilin-dependent gamma-secretase processing regulates multiple ERBB4/HER4 activities. J Biol Chem. 2005, 280: 19777-19783. 10.1074/jbc.M412457200.
Zhu Y, Sullivan LL, Nair SS, Williams CC, Pandey AK, Marrero L, Vadlamudi RK, Jones FE: Coregulation of estrogen receptor by ERBB4/HER4 establishes a growth-promoting autocrine signal in breast tumor cells. Cancer Res. 2006, 66: 7991-7998. 10.1158/0008-5472.CAN-05-4397.
Williams CC, Allison JG, Vidal GA, Burow ME, Beckman BS, Marrero L, Jones FE: The ERBB4/HER4 receptor tyrosine kinase regulates gene expression by functioning as a STAT5A nuclear chaperone. J Cell Biol. 2004, 167: 469-478. 10.1083/jcb.200403155.
Elenius K, Choi CJ, Paul S, Santiestevan E, Nishi E, Klagsbrun M: Characterization of a naturally occurring ErbB4 isoform that does not bind or activate phosphatidyl inositol 3-kinase. Oncogene. 1999, 18: 2607-2615. 10.1038/sj.onc.1202612.
Jones FE: HER4 Intracellular Domain (4ICD) Activity in the Developing Mammary Gland and Breast Cancer. J Mammary Gland Biol Neoplasia. 2008, 13: 247-258. 10.1007/s10911-008-9076-6.
Diermeier-Daucher S, Hasmann M, Brockhoff G: Flow cytometric FRET analysis of erbB receptor interaction on a cell-by-cell basis. Ann N Y Acad Sci. 2008, 1130: 280-286. 10.1196/annals.1430.003.
Pickl M, Ries CH: Comparison of 3D and 2D tumor models reveals enhanced HER2 activation in 3D associated with an increased response to trastuzumab. Oncogene. 2009, 28: 461-468. 10.1038/onc.2008.394.
Barnes NL, Khavari S, Boland GP, Cramer A, Knox WF, Bundred NJ: Absence of HER4 expression predicts recurrence of ductal carcinoma in situ of the breast. Clin Cancer Res. 2005, 11: 2163-2168. 10.1158/1078-0432.CCR-04-1633.
Alkarain A, Slingerland J: Deregulation of p27 by oncogenic signaling and its prognostic significance in breast cancer. Breast Cancer Res. 2004, 6: 13-21. 10.1186/bcr722.
Chiarle R, Pagano M, Inghirami G: The cyclin dependent kinase inhibitor p27 and its prognostic role in breast cancer. Breast Cancer Res. 2001, 3: 91-94. 10.1186/bcr277.
DiGiovanna MP, Stern DF: Activation state-specific monoclonal antibody detects tyrosine phosphorylated p185neu/erbB-2 in a subset of human breast tumors overexpressing this receptor. Cancer Res. 1995, 55: 1946-1955.
Tovey SM, Dunne B, Witton CJ, Cooke TG, Bartlett JM: HER4 in breast cancer: comparison of antibodies against intra- and extra-cellular domains of HER4. Breast Cancer Res. 2006, 8: R19-10.1186/bcr1394.
Britten CD: Targeting ErbB receptor signaling: a pan-ErbB approach to cancer. Mol Cancer Ther. 2004, 3: 1335-1342.
Hollmen M, Maatta JA, Bald L, Sliwkowski MX, Elenius K: Suppression of breast cancer cell growth by a monoclonal antibody targeting cleavable ErbB4 isoforms. Oncogene. 2009
The authors thank Dr Monika Klinkhammer-Schalke, Tumour Centre Inc., Regensburg, for providing the clinical data. We also sincerely thank Marietta Bock for her excellent technical assistance. Furthermore, we appreciate Professor Dr Josef Giedl (Institute of Pathology, Hospital Weiden, Germany) for supporting the study by providing paraffin embedded material. This study was supported by the Faculty of Medicine, University of Regensburg (ReForM A, funded in 2008), Regensburg, Germany, and ZytoVision Ltd., Bremerhaven, Germany.
The authors declare that they have no competing interests.
AS wrote the manuscript, performed the image analysis and FISH evaluation and analysed the statistical data. MS helped to analyse the statistical data and performed FISH evaluation. SD-D helped to write the manuscript. OO performed the histological analysis and interpreted the data. FH provided material to be analysed and performed primary tissue-based diagnostics. SS performed the histological analysis and interpreted the data. GB is the research group leader, evaluated the data, and finally approved the manuscript to be published. All authors read and approved the final manuscript.
About this article
Cite this article
Sassen, A., Diermeier-Daucher, S., Sieben, M. et al. Presence of HER4 associates with increased sensitivity to Herceptin™ in patients with metastatic breast cancer. Breast Cancer Res 11, R50 (2009). https://doi.org/10.1186/bcr2339
- Overall Survival
- Epidermal Growth Factor Receptor
- Breast Cancer Patient
- Human Epidermal Growth Factor Receptor