ErbB2-positive Tumors Research Articles

Lrig1 Is an Estrogen-Regulated Growth Suppressor and Correlates with Longer Relapse-Free Survival in ERα-Positive Breast Cancer

Lrig1 is the founding member of the Lrig family and has been implicated in the negative regulation of several oncogenic receptor tyrosine kinases including ErbB2. Lrig1 is expressed at low levels in several cancer types but is overexpressed in some prostate and colorectal tumors. Given this heterogeneity, whether Lrig1 functions to suppress or promote tumor growth remains a critical question. Previously, we found that Lrig1 was poorly expressed in ErbB2-positive breast cancer, suggesting that Lrig1 has a growth-inhibitory role in this tumor type. However, breast cancer is a complex disease, with ErbB2-positive tumors accounting for just 25% of all breast cancers. To gain a better understanding of the role of Lrig1 in breast cancer, we examined its expression in estrogen receptor α (ERα)-positive disease which accounts for the majority of breast cancers. We find that Lrig1 is expressed at significantly higher levels in ERα-positive disease than in ERα-negative disease. Our study provides a molecular rationale for Lrig1 enrichment in ERα-positive disease by showing that Lrig1 is a target of ERα. Estrogen stimulates Lrig1 accumulation and disruption of this induction enhances estrogen-dependent tumor cell growth, suggesting that Lrig1 functions as an estrogen-regulated growth suppressor. In addition, we find that Lrig1 expression correlates with prolonged relapse-free survival in ERα-positive breast cancer, identifying Lrig1 as a new prognostic marker in this setting. Finally, we show that ErbB2 activation antagonizes ERα-driven Lrig1 expression, providing a mechanistic explanation for Lrig1 loss in ErbB2-positive breast cancer. This work provides strong evidence for a growth-inhibitory role for Lrig1 in breast cancer.

Potent anti-proliferative effects of metformin on trastuzumab-resistant breast cancer cells via inhibition of erbB2/IGF-1 receptor interactions

We have shown that erbB2 altered breast cancer cells are less sensitive to the anti-proliferative effects of metformin than triple negative cells, and have described the differences of molecular mechanisms of metformin action by tumor subtypes. We hypothesized that metformin may be more effective against trastuzumab-resistant erbB2-overexpressing breast cancer cells because it targets the critical signaling pathways that are altered with resistance. BT474, SKBR3 and derived trastuzumab-resistant sublines BT474-HR20 (HR20) and SKBR3-pool2 (pool2) were used to test this hypothesis. Metformin treatment resulted in significantly more inhibition of proliferation and clonogenicity in resistant sublines. It decreased erbB2/insulin-like growth factor-1 receptor (IGF-1R) complexes (present only in the resistant sublines) without altering erbB2 expression, and reduced the expression and activity of erbB3 and IGF-1R in the trastuzumab-resistant but not parental cells. Trastuzumab-resistant sublines were resistant to rapamycin induced changes in mTOR activity and cell growth. In contrast, both BT474 and HR20 cells were highly sensitive to inhibitors of Src (Dasatinib) and PI-3K (LY294002). The pool2 cells showed higher sensitivity than SKBR3 cells to LY294002, but not Dasatinib. On the basis of these data, metformin appears to be significantly more effective against trastuzumab-resistant as compared to sensitive breast cancer cells. Metformin disrupts erbB2/IGF-1R complexes, erbB3 and IGF-1R expression and activity, as well as Src kinase and/or PI-3K/Akt signaling. This action appears to be independent of mTOR signaling. Our findings provide a rationale to study the effects of metformin on patients with erbB2 positive tumors treated with trastuzumab, with or without resistance.

Assessment of ErbB2 (Her2) in oesophageal adenocarcinomas: summary of a revised immunohistochemical evaluation system, bright field double in situ hybridisation and fluorescence in situ hybridisation

Amplification and overexpression of ErbB2 (Her2) is a frequent event in oesophageal adenocarcinomas. Assessment of ErbB2 status is crucial for identifying patients who are likely to benefit from treatment with trastuzumab. In this study, we performed a comprehensive analysis of ErbB2 amplification and expression in 142 oesophageal adenocarcinomas by comparing the most commonly used methods for ErbB2 assessment: ErbB2 expression was determined by immunohistochemistry and was scored (0, 1+, 2+ and 3+) according to a recently described modified scoring system for gastric cancer. ErbB2 amplification was evaluated by bright field double in situ hybridisation. The results were compared with pathologic features, patients' survival and previously published data from fluorescence in situ hybridisation analysis. On the basis of immunohistochemistry, which was applicable in 110 cores of the cases, 83 tumours (75%) had a score of 0 or 1+ (immunohistochemistry negative), 13 tumours (12%) were scored as 2+ and 14 tumours (13%) were scored as 3+. In situ hybridisation data were obtained from 142 cases. There was a highly significant correlation of immunohistochemistry, bright field in situ hybridisation and fluorescent in situ hybridisation (P<0.001 each). In total, 41 tumours (29%) were categorised as ErbB2 positive, which was defined as immunohistochemistry 3+ and/or an ErbB2/Chr17 quotient of ≥2 as assessed by either bright field double in situ hybridisation or fluorescence in situ hybridisation. ErbB2 positivity was observed more frequently in tumours with lower differentiation grades (P=0.029). Patients with ErbB2-positive tumours had a significantly worse prognosis, both in univariate analysis (P=0.004) and in multivariate analysis (P=0.03). In conclusion, we demonstrate that a significant number of oesophageal adenocarcinomas are positive for ErbB2. Assessment of ErbB2 amplification can be equivalently performed by conventional fluorescence in situ hybridisation or other light-microscopy-based methods, such as the novel bright field double in situ hybridisation technique.

A novel fully human antitumour immunoRNase targeting ErbB2-positive tumours.

Background:ErbB2 is an attractive target for immunotherapy, as it is a tyrosine kinase receptor overexpressed on tumour cells of different origin, with a key role in the development of malignancy. Trastuzumab, the only humanised anti-ErbB2 antibody currently used in breast cancer with success, can engender cardiotoxicity and a high fraction of patients is resistant to Trastuzumab treatment.Methods:A novel human immunoRNase, called anti-ErbB2 human compact antibody-RNase (Erb-hcAb-RNase), made up of the compact anti-ErbB2 antibody Erbicin-human-compact Antibody (Erb-hcAb) and human pancreatic RNase (HP-RNase), has been designed, expressed in mammalian cell cultures and purified. The immunoRNase was then characterised as an enzymatic protein, and tested for its biological actions in vitro and in vivo on ErbB2-positive tumour cells.Results:Erb-hcAb-RNase retains the enzymatic activity of HP-RNase and specifically binds to ErbB2-positive cells with an affinity comparable with that of the parental Erb-hcAb. Moreover, this novel immunoRNase is endowed with an effective and selective antiproliferative action for ErbB2-positive tumour cells both in vitro and in vivo. Its antitumour activity is more potent than that of the parental Erb-hcAb as the novel immunoconjugate has acquired RNase-based cytotoxicity in addition to the inhibitory growth effects, antibody-dependent and complement-dependent cytotoxicity of Erb-hcAb.Conclusion:Erb-hcAb-RNase could be a promising candidate for the immunotherapy of ErbB2-positive tumours.

Abstract 4042: Activation of ErbB2 results in decreased Par6-associated aPKC kinase activity

Abstract Loss of epithelial cell polarity is often associated with the development of carcinoma. Polarized MDCK cells and MCF-10A mammary epithelial cells grown in 3D are useful model systems to study the regulation of cell polarity in vitro and to investigate the interaction of oncogenes and cell polarity proteins. Using these experimental models, we have previously demonstrated that activation of the oncogene ErbB2 disrupts apical-basal polarity in mammary epithelial cells. Activated ErbB2 associates with the Par6-aPKC polarity complex and the interaction between Par6 and aPKC is indispensible for ErbB2-mediated disruption of cell polarity and inhibition of apoptosis. Following ErbB2 activation, we find a decrease in Par6-associated aPKC kinase activity. Furthermore, kinase dead aPKC cooperates with activated ErbB2 to transform mammary epithelial cells in vitro, without inducing any changes in proliferation of cells grown on plastic dishes. Taken together, our data implicate a target (or targets) of aPKC as a likely determinant of ErbB2-mediated disruption of cell polarity and transformation. Identification of these substrates could lead to the development of novel biomarkers to monitor the progression of ErbB2 positive tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4042. doi:10.1158/1538-7445.AM2011-4042

The prevalence and assessment of ErbB2‐positive breast cancer in Asia

Overexpression of the epidermal growth factor receptor-related gene ErbB2 occurs in 18% to 25% of patients with breast cancer in Western countries and is associated with a poor prognosis. The prevalence of ErbB2-positive tumors in Asia is unclear, partly because data are limited. The objective of this review was to summarize the reported prevalence of ErbB2-positive tumors from a large sample of Asian patients and to examine ErbB2 assessment methods in Asia. From searches of MEDLINE, local language journals, and local and international conference proceedings as well as locoregional breast cancer experts' recommendations, the authors selected up to 5 studies each from India, Korea, Malaysia, the Philippines, Singapore, Taiwan, and Thailand that reported ErbB2 results based on assessment with immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH). The reported prevalence of ErbB2-positive tumors in 22 studies on 24,671 patients, of whom 14,398 patients were assessed for ErbB2 status, varied widely (range, 6%-65%) as did the assessment methods used. Most studies (n = 21) used IHC to assess ErbB2 status, but definitions for positivity varied. When robust assessment methods were used, the median prevalence was 19% based on strong IHC staining (IHC3+; n = 9812 patients) and 25% based on FISH (n = 681 patients). Data on the prevalence of ErbB2-positive breast cancer in Asia are limited. The current survey indicated that the prevalence in Asia may be similar to that in Western countries; thus, up to 1 in 4 Asian patients with breast cancer potentially could benefit from ErbB2-targeted treatment. A standard, reliable ErbB2 assessment method available to patients across Asia is urgently required. Cancer 2010;116:5348–57. © 2010 American Cancer Society.

Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition

BackgroundErbB2-positive breast cancer is characterized by highly aggressive phenotypes and reduced responsiveness to standard therapies. Although specific ErbB2-targeted therapies have been designed, only a small percentage of patients respond to these treatments and most of them eventually relapse. The existence of this population of particularly aggressive and non-responding or relapsing patients urges the search for novel therapies. The purpose of this study was to determine whether cannabinoids might constitute a new therapeutic tool for the treatment of ErbB2-positive breast tumors. We analyzed their antitumor potential in a well established and clinically relevant model of ErbB2-driven metastatic breast cancer: the MMTV-neu mouse. We also analyzed the expression of cannabinoid targets in a series of 87 human breast tumors.ResultsOur results show that both Δ9-tetrahydrocannabinol, the most abundant and potent cannabinoid in marijuana, and JWH-133, a non-psychotropic CB2 receptor-selective agonist, reduce tumor growth, tumor number, and the amount/severity of lung metastases in MMTV-neu mice. Histological analyses of the tumors revealed that cannabinoids inhibit cancer cell proliferation, induce cancer cell apoptosis, and impair tumor angiogenesis. Cannabinoid antitumoral action relies, at least partially, on the inhibition of the pro-tumorigenic Akt pathway. We also found that 91% of ErbB2-positive tumors express the non-psychotropic cannabinoid receptor CB2.ConclusionsTaken together, these results provide a strong preclinical evidence for the use of cannabinoid-based therapies for the management of ErbB2-positive breast cancer.

Deletion of thePER3Gene on Chromosome 1p36 in Recurrent ER-Positive Breast Cancer

To investigate the role of the PER3 circadian rhythm gene, located within the commonly deleted region of chromosome 1p36, in human breast cancer development. The frequency of genetic alterations at 1p36 and PER3 gene copy number status were analyzed in 180 lymph node-negative breast cancers from patients who had received treatment with chemotherapy and/or tamoxifen. The expression levels of PER3 were also analyzed using published microarray profiles from > 400 breast cancer samples. Finally, the effect of loss of Per3 on tumor susceptibility was tested using two mouse models of breast cancer. Deletion of PER3 is directly related to tumor recurrence in patients with estrogen receptor (ER) - positive breast cancers treated with tamoxifen. Low expression of PER3 mRNA is associated with poor prognosis, particularly in a subset of tumors that are ER positive, and either luminal A or ERBB2-positive tumors. Mice deficient in Per3 showed increased susceptibility to breast cancer induced by carcinogen treatment or by overexpression of Erbb2. Disruption of PER3 function may serve as an indicator of probability of tumor recurrence in patients with ER-positive tumors. Further investigations of this pathway may reveal links between deregulation of sleep homeostasis and breast tumorigenesis.

Suppression of the NF-κB co-factor, Bcl3, delays the metastatic progression of breast cancer

A large proportion of breast cancers overexpress the HER receptors, HER1 or HER2. Generally these patients have a poor prognosis, exhibit resistance to first-line anti-cancer drugs, and frequently develop metastatic disease - the most common cause of patient death. NF-κB transcription factors lie downstream of HER1/2 signalling pathways and are aberrantly activated in the majority of these breast tumours. We have found that a constitutive deficiency in Bcl3 (an NF-κB co-factor that modifies NF-κB signalling) delayed HER2 (ErbB2) tumour onset and inhibited metastasis of mammary tumours in mice while growth of primary tumours was unaffected. In those Bcl3-null animals that did acquire metastases, the size of secondary tumours was significantly reduced compared with controls. Critically, Bcl3 deficiency did not affect normal mammary function other than having a transitory effect on apoptosis of epithelial cells in the post-lactational mammary gland. Therefore, unlike other NF-κB regulators, Bcl3 exhibited tumour-specific effects in vivo. The pro-metastatic properties of Bcl3 were confirmed in several human breast cancer cell lines exhibiting elevated HER2 and/or HER1 levels, including aggressive basal-like tumour cell types. These observations are significant because they suggest it may be possible to target Bcl3 in established tumour cells to reduce metastatic behaviour, and furthermore that Bcl3's effects are not restricted to ERBB2-positive breast tumours, consistent with the observed increase in NF-κB activity seen in both ERBB1-positive and ERBB2-positive breast tumours.

Abstract 3256: Investigating the intrinsic signaling differences in MET- and ERBB2-driven breast cancers

Abstract Understanding the signaling pathways that drive aggressive breast cancers is critical to the development of effective therapeutics. Over 20% of breast cancers overexpress ERBB2 and are associated with an aggressive tumor phenotype and poor outcome. Patients with ERBB2-positive tumors are often treated with the humanized monoclonal antibody trastuzumab, yet the majority of patients develop resistance. Recent studies show that the receptor tyrosine kinase MET is expressed in ERBB2 positive tumors and contributes to trastuzumab resistance in ERBB2-expressing cell lines. Other studies have shown that MET correlates with poor clinical outcome independent of ERBB2. We are utilizing mouse models to examine the intrinsic signaling differences between MET and ERBB2-mediated breast cancers. We have developed a novel mouse model of mutationally activated MET (Metmut) that develops a high incidence of diverse mammary tumors with basal characteristics. Using this mouse model and human breast cancer tissue, we have examined the role of MET in aggressive breast cancers. By gene expression and tissue microarray analysis, we observe that high MET expression in human breast cancers significantly correlates with estrogen receptor negative/ERBB2 negative tumors and with basal breast cancers (Graveel et al., 2009). We performed gene expression analysis on mammary tumors from Metmut mice and activated ErbB2 (Neu-Ndl2-5) mice. Unsupervised hierarchical analysis revealed that solid Metmut tumors clustered separately from Metmut tumors with a mixed pathology and that solid Metmut tumors have expression patterns very similar to ERBB2 tumors. The similarity of solid Metmut tumors to ErbB2 tumors suggests that MET signaling may be influential in ERBB2-driven tumors. To investigate this hypothesis further, we performed a parametric gene set enrichment analysis (PGSEA) and observed that the Ras activation signature was significantly associated with the Metmut mixed tumors. Currently, we are also investigating expression patterns of MET and ERBB2 in human breast cancers by coimmunostaining. Collectively, our studies indicate that MET may play a critical role in the development of the most aggressive breast cancers and may be a rational therapeutic target for trastuzumab-resistant ERBB2 breast cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3256.

ErbB2 requires integrin α5 for anoikis resistance via Src regulation of receptor activity in human mammary epithelial cells

ErbB2, a receptor tyrosine kinase highly expressed in many tumors, is known to inhibit apoptotic signals. Overexpression of ErbB2 causes anoikis resistance that contributes to luminal filling in three-dimensional mammary epithelial acinar structures in vitro. Given that integrins and growth factor receptors are highly interdependent for function, we examined the role of integrin subunits in ErbB2-mediated survival signaling. Here, we show that MCF-10A cells overexpressing ErbB2 upregulate integrin alpha5 via the MAP-kinase pathway in three-dimensional acini and found elevated integrin alpha5 levels associated with ErbB2 status in human breast cancer. Integrin alpha5 is required for ErbB2-mediated anoikis resistance and for optimal ErbB2 signaling to the Mek-Erk-Bim axis as depletion of integrin alpha5 reverses anoikis resistance and Bim inhibition. Integrin alpha5 is required for full activation of ErbB2 tyrosine phosphorylation on Y877 and ErbB2 phosphorylation is associated with increased activity of Src in the absence of adhesion. Indeed, we show that blocking elevated Src activity during cell detachment reverses ErbB2-mediated survival and Bim repression. Thus, integrin alpha5 serves as a key mediator of Src and ErbB2-survival signaling in low adhesion states, which are necessary to block the pro-anoikis mediator Bim, and we suggest that this pathway represents a potential novel therapeutic target in ErbB2-positive tumors.

Gemini vitamin D analog suppresses ErbB2-positive mammary tumor growth via inhibition of ErbB2/AKT/ERK signaling

Numerous synthetic vitamin D analogs have been studied for their effects on the prevention and treatment of breast cancer. However, the inhibitory effects of naturally occurring 1α,25-dihydroxyvitamin D 3 or its synthetic analogs on ErbB2 overexpressing mammary tumorigenesis have not been reported. Gemini vitamin D analogs are novel synthetic vitamin D derivatives with a unique structure of two six-carbon chains at C-20. We have previously shown that Gemini vitamin D analogs significantly inhibited carcinogen-induced estrogen receptor (ER)-positive mammary tumorigenesis and reduced ER-negative MCF10DCIS.com xenograft tumor growth without hypercalcemic toxicity. In the present study, we have determined the inhibitory effect of a potent Gemini vitamin D analog BXL0124 (1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol) on the ErbB2/Her-2/neu overexpressing mammary tumorigenesis. The Gemini BXL0124 inhibits ErbB2-positive mammary tumor growth and down-regulates the phosphorylation of ErbB2, ERK and AKT in tumors of MMTV-ErbB2/neu transgenic mice. These effects of Gemini BXL0124 in vivo were confirmed by using the ErbB2 overexpressing tumor cells derived from the mammary tumors of MMTV-ErbB2/neu mice. In conclusion, the Gemini vitamin D analog BXL0124 inhibits the growth of ErbB2 overexpressing mammary tumors through regulating the ErbB2/AKT/ERK signaling pathways, suggesting that Gemini vitamin D analog may be considered for translational studies.

An RNA Interference Screen Identifies Metabolic Regulators NR1D1 and PBP as Novel Survival Factors for Breast Cancer Cells with the ERBB2 Signature

Overexpression of the adverse prognostic marker ERBB2 occurs in 30% of breast cancers; however, therapies targeting this gene have not proved to be as effective as was initially hoped. Transcriptional profiling meta-analyses have shown that there are approximately 150 genes co-overexpressed with ERBB2, suggesting that these genes may represent alternative factors influencing ERBB2-positive tumors. Here we describe an RNA interference-based analysis of these genes that identifies transcriptional regulators of fat synthesis and storage as being critical for the survival of these cells. These transcription factors, nuclear receptor subfamily 1, group D, member 1 (NR1D1) and peroxisome proliferator activated receptor gamma binding protein (PBP), both reside on ERBB2-containing 17q12-21 amplicons and are part of the ERBB2 expression signature. We show that NR1D1 and PBP act through a common pathway in upregulating several genes in the de novo fatty acid synthesis network, which is highly active in ERBB2-positive breast cancer cells. Malate dehydrogenase 1 and malic enzyme 1, enzymes that link glycolysis and fatty acid synthesis, are also regulated by NR1D1. The resulting high-level fat production from increased expression of these genes likely contributes to an abnormal cellular energy metabolism based on aerobic glycolysis. Together, these results show that the cells of this aggressive form of breast cancer are genetically preprogrammed to depend on NR1D1 and PBP for the energy production necessary for survival.

SA02 Trial: A Genomics-Based Prospective Study of Adjuvant Chemotherapy in Node-Positive Early Breast Cancer with «Good-Prognosis Signature».

Abstract Background: Adjuvant chemotherapy (CT) for node-positive (N+) early breast cancer (EBC) is based upon an anthracycline-taxane combination. However, the benefit of taxane is limited to a small population, but associated to morbid and financial costs, making crucial the identification of patients likely to benefit or not from anthracycline-based regimen without taxane. Using DNA microarrays to profile a retrospective series of 498 patients (pts) treated with adjuvant anthracycline-based CT without taxane, we identified and validated a gene expression signature (GES) associated with metastatic relapse. The corresponding Relapse Score (RS) sorted the pts in two groups: the “good-prognosis” group (75% of pts) with a 5-year metastasis-free survival (MFS) of 82%, and the “poor-prognosis” group (25% of pts) with a 56% 5-year MFS. A prospective multicentric cohort of 220 pts, SA02, was initiated to analyze anthracycline-based adjuvant CT without taxane in N+ EBC patients with a “good-prognosis” RS, with the aim of confirming their good prognosis.Methods: Women with surgical EBC were screened for inclusion in 4 French hospitals. After diagnosis of node positivity, frozen samples were used for hybridizationon Affymetrix U133 Plus 2.0 microarrays. “Good-prognosis” pts were included in the SA02 cohort and received 6 FEC100 cycles (Fluoro-uracile 500 mg/m2, Epirubicin 100 mg/m2, Cyclophosphamide 500 mg/m2, every 21 days), followed by adjuvant radiotherapy, hormone therapy and/or trastuzumab according to standard guidelines. Pts with a “poor-prognosis” RS were not included in the cohort.Results: Between April 2007 and May 2009, samples from 106 eligible N+ EBC pts were collected for gene profiling. The profiling failed for 34 samples (32%), due to insufficient RNA amount (6%), poor RNA quality (17%), or technical problems (9%). Sixty-six pts (92%) were defined as “good-prognosis” and enrolled in the SA02 cohort, while only 2 pts (3%) were defined as “poor-prognosis”; 4 pts could not be assigned a prognostic group. The percentage of “good-prognosis” RS was higher than observed in the identification and validation retrospective series. Comparison of histo-clinical data between these retrospective series and the 72 samples successfully profiled in the present study showed more favourable features in the current series, with fewer positive lymph nodes, lower grade, more HR-positive and less ERBB2-positive tumors. After a median follow-up of 1 year, no relapse or death was observed in the SA02 cohort.Conclusion: This interim analysis confirms that genomic analyses are feasible in clinical practice, even if more challenging than in a retrospective academic environment. A shift in histo-clinical features of N+ EBC was observed between the retrospective series and the current prospective series; this could be due to the evolution of EBC diagnostic methods and treatment strategies. Study is ongoing, and final follow-up data will be available in 2014. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4041.

Prognostic value of gene expression of p63 by microarray analysis in estrogen receptor positive and negative breast cancer

566 Background: The protein 63 (p63) represents a member of the p53 family (p53/p63/p73) located on chromosome 3q27. This gene family seems to play an important role in carcinogenesis and its members may act as oncogenes or tumor suppressor genes. P63 is overexpressed in many different tumors like head and neck cancer, lung cancers, uterine tumors and breast cancer, and has been associated with poor prognosis in some studies. P63 was found to be overexpressed in a subset of highly aggressive breast cancers that represent a basal and myoepithelial phenotype and have a poor clinical outcome. This protein seems to be a specific myoepithelial cell marker in normal breast tissue and might represent a prognostic factor in breast cancer. Methods: Large scale analysis was performed using Affymetrix microarray data from n=1581 breast cancer patients to evaluate p63 expression. Results: P63 expression showed a strong correlation with patient's age (χ2-test, p &lt; 0.001), tumor size (p &lt; 0.003), proliferation rate (p &lt; 0.001), Topo2α expression (p = 0.001) and estrogen receptor expression (p = 0.017). Survival analysis of all patients with available follow up data (n = 1263) showed a significant difference due to high and low p63 expression (log rank p &lt; 0.001). Patients with a low p63 expression had the worst prognosis. In univariate Cox regression analysis p63 showed a hazard ratio (HR) of 1.61 (95% CI 1.31–2.00, p &lt; 0.001) for disease free survival. This prognostic impact remained significant when samples were stratified by estrogen receptor status. High expression of p63 was significantly associated with longer OS in both ER negative (n = 334, log rank p = 0.022) and ER positive (n = 929, log rank p &lt; 0,001) breast cancer. The prognostic impact of p63 expression was independent of Ki67 expression (p = 0.011 and p = 0.001 for high and low Ki67, respectively). Moreover a worse prognosis of low p63 expressing tumors was found in both subgroups of ErbB2 positive tumors (p &lt; 0.001) and ErbB2 negative tumors (p &lt; 0.001). Conclusions: P63 expression is a prognostic factor in both ER positive and negative breast cancer and could be helpful for risk assessment in breast cancer patients. No significant financial relationships to disclose.

Effect of Lapatinib on the Development of Estrogen Receptor–Negative Mammary Tumors in Mice

Lapatinib, a selective orally available inhibitor of epidermal growth factor receptor (EGFR) and ErbB2 receptor tyrosine kinases, is a promising agent for the treatment of breast cancer. We examined the effect of lapatinib on the development of mammary tumors in MMTV-erbB2 transgenic mice, which express wild-type ErbB2 under the control of the mouse mammary tumor virus promoter and spontaneously develop estrogen receptor (ER)-negative and ErbB2-positive mammary tumors by 14 months of age. Mice were treated from age 3 months to age 15 months with vehicle (n = 17) or lapatinib (30 or 75 mg/kg body weight; n = 16 mice per group) by oral gavage twice daily (6 d/wk). All statistical tests were two-sided. By 328 days after the start of treatment, all 17 (100%) of the vehicle-treated mice vs five (31%) of the 16 mice treated with high-dose lapatinib developed mammary tumors (P < .001). Among MMTV-erbB2 mice treated for 5 months (n = 20 mice per group), those treated with lapatinib had fewer premalignant lesions and noninvasive cancers in their mammary glands than those treated with vehicle (P = .02). Lapatinib also effectively blocked epidermal growth factor-induced signaling through the EGFR and ErbB2 receptors, suppressed cyclin D1 and epiregulin mRNA expression, and stimulated p27 mRNA expression in human mammary epithelial cells and in mammary epithelial cells from mice treated for 5 months with high-dose lapatinib. Thus, cyclin D1, epiregulin, and p27 may represent useful biomarkers of lapatinib response in patients. These data suggest that lapatinib is a promising agent for the prevention of ER-negative breast cancer.

Regulation of ERBB2 by oestrogen receptor–PAX2 determines response to tamoxifen

Crosstalk between the oestrogen receptor (ER) and ERBB2/HER-2 pathways has long been implicated in breast cancer aetiology and drug response, yet no direct connection at a transcriptional level has been shown. Here we show that oestrogen-ER and tamoxifen-ER complexes directly repress ERBB2 transcription by means of a cis-regulatory element within the ERBB2 gene in human cell lines. We implicate the paired box 2 gene product (PAX2), in a previously unrecognized role, as a crucial mediator of ER repression of ERBB2 by the anti-cancer drug tamoxifen. We show that PAX2 and the ER co-activator AIB-1/SRC-3 compete for binding and regulation of ERBB2 transcription, the outcome of which determines tamoxifen response in breast cancer cells. The repression of ERBB2 by ER-PAX2 links these two breast cancer subtypes and suggests that aggressive ERBB2-positive tumours can originate from ER-positive luminal tumours by circumventing this repressive mechanism. These data provide mechanistic insight into the molecular basis of endocrine resistance in breast cancer.

Cancer Immunotherapy UsingIn vitroGenetically Modified Targeted Dendritic Cells

Modest clinical outcomes of dendritic cell (DC) vaccine trials call for novel strategies. In this study, we have created a chimeric CD40 molecule that incorporates a single chain Fv (scFv) molecule specific for human ErbB2 antigen and fusing to the membrane spanning and cytosolic domains of murine CD40. After adenoviral transfer to bone marrow-derived DC, this chimeric receptor (CR) induced nuclear factor-kappaB (NF-kappaB)-dependent DC activation and effector function when cultured with immobilized ErbB2 protein or ErbB2-positive tumor cells in vitro. In vivo migration assays showed that approximately 40% injected CR-modified DC (scFv-CD40-DC) effectively migrated to ErbB2-positive tumors, where they were activated after ErbB2 antigen stimulation, and sequentially homed into the draining lymph nodes. In murine ErbB2-positive D2F2/E2 breast tumor (BALB/c) and EL4/E2 thymoma (C57BL/6) models, i.v. injection of 1 x 10(6) scFv-CD40-DC significantly inhibited tumor growth and cured established tumors. Importantly, the cured mice treated by injection of scFv-CD40-DC were effective in preventing both ErbB2-positive and parental ErbB2-negative tumor rechallenge. Analysis of the underlying mechanism revealed that i.v. infusion of scFv-CD40-DC elicited tumor-specific CTL responses, and the transfer of CTLs from scFv-CD40-DC-treated mice protected naive mice against a subsequent tumor challenge. These results support the concept that genetic modification of DC with tumor-associated antigen-specific CD40 chimeric receptor might be a useful strategy for treatment of human cancers.

Role of Erbb2 in Selection for Adjuvant Tamoxifen or Aromatase Inhibitors

Evaluation of: Rasmussen BB, Regan MM, Lykkesfeldt AE et al.: Adjuvant letrozole versus tamoxifen according to centrally-assessed ERBB2 status for postmenopausal women with endocrine-responsive early breast cancer: supplementary results from the BIG 1-98 randomized trial. Lancet Oncol. 9, 23-28 (2008). This study analysed data after a median of 51 months follow-up from 4922 postmenopausal women with early hormone-sensitive breast cancer randomly assigned to 5 years of treatment with letrozole or tamoxifen. Tumors from 3650 (74%) patients were centrally assessed for estrogen receptor, progesterone receptor and ErbB2 status. By central assessment, 7% (257 of 3650) of tumors were classified as ErbB2-positive. Disease-free survival was significantly worse in patients with ErbB2-positive tumors (p < 0.0001). Letrozole improved disease-free survival compared with tamoxifen regardless of ErbB2 status.

Gene expression of topoisomerase II alpha (TOP2A) by microarray analysis is highly prognostic in estrogen receptor (ER) positive breast cancer

Overexpression of Topoisomerase II alpha (TOP2A) has been implicated with gene amplification of the 17q21 amplicon and consecutively with ErbB2 overexpression and amplification. However, gene amplification does not necessarily correlate with RNA and protein expression. There is growing evidence that TOP2A protein expression is a strong prognostic and TOP2A gene amplification might be a predictive marker (particularly for the use of anthracyclines). Large scale analysis was performed using Affymetrix microarray data from n = 1,681 breast cancer patients to evaluate TOP2A expression. TOP2A expression showed a strong correlation with tumor size (chi(2)-test, P < 0.001), grading (P < 0.001), ErbB2 (P < 0.001) and Ki67 expression (P < 0.001) as well as nodal status (P = 0.042). Survival analysis revealed a significant prognostic value in ER positive (n = 994; log rank P < 0.001), but not in ER negative breast cancer patients (n = 369, P = 0.35). The prognostic impact of TOP2A expression was independent of Ki67 expression in ER positive tumors (P = 0.002 and P = 0.007 for high and low Ki67, respectively). Moreover a worse prognosis of high TOP2A expressing tumors was found in the subgroup of ErbB2 negative tumors (P < 0.001) and a trend among ErbB2 positive tumors (P = 0.11). The prognostic value of TOP2A was independent of whether the patients were untreated or had received adjuvant therapy. In multivariate Cox regression analysis including standard parameters TOP2A emerged to be the top prognostic marker (HR 2.40, 95% CI 1.68-3.43, P < 0.001). TOP2A expression is an independent prognostic factor in ER positive breast cancer and could be helpful for risk assessment in ER positive breast cancer patients.