ERBB2 Gene Amplification Research Articles

Sequencing of 279 cancer genes in ampullary carcinoma reveals trends relating to histologic subtypes and frequent amplification and overexpression of ERBB2 (HER2)

The biological relevance of histological subtyping of ampullary carcinoma into intestinal vs pancreaticobiliary types remains to be determined. In an effort to molecularly profile these subtypes of ampullary carcinomas, we conducted a two-phase study. In the discovery phase, we identified 18 pancreatobiliary-type ampullary carcinomas and 14 intestinal-type ampullary carcinomas using stringent pathologic criteria and performed next-generation sequencing targeting 279 cancer-associated genes on these tumors. Although the results showed overlapping of genomic alterations between the two subtypes, trends including more frequent KRAS alterations in pancreatobiliary-type ampullary carcinoma (61 vs 29%) and more frequent mutations in APC in intestinal-type ampullary carcinoma (43 vs 17%) were observed. Of the entire cohort of 32 tumors, the most frequently mutated gene was TP53 (n=17); the most frequently amplified gene was ERBB2 (n=5); and the most frequently deleted gene was CDKN2A (n=6). In the second phase of the study, we aimed at validating our observation on ERBB2 and assessed ERBB2 amplification and protein overexpression in a series of 100 ampullary carcinomas. We found that (1) gene amplification and immunohistochemical overexpression of ERBB2 occurred in 13% of all ampullary carcinomas, therefore providing a potential target for anti-HER2 therapy in these tumors; (2) amplification and immunohistochemical expression correlated in all cases, thus indicating that immunohistochemistry could be used to screen tumors; and (3) none of the 14 ERBB2-amplified tumors harbored any downstream driver mutations in KRAS/NRAS, whereas 56% of the cases negative for ERBB2 amplification did, an observation clinically pertinent as downstream mutations may cause primary resistance to inhibition of EGFR family members.

Abstract 1101: Functional genomics of HER2 and HER3 mutations and response to neratinib

Abstract Selective kinase inhibitors, such as erolotinib and lapatinib, and monoclonal antibodies, such as cetuximab, trastuzumab and pertuzumab, targeting EGFR and ERBB2 have elicited significant response rates in lung cancer patients harboring mutant EGFR alleles and breast cancer patients whose tumors harbor gene amplification of ERBB2. To date, only a small number of ERBB2 and ERBB3 mutations have been reported and biologically characterized. Moreover, the therapeutic impact of ERBB2 or ERBB3 mutant alleles and their value as an actionable target in patients has not been validated. While ERBB2 itself cannot bind ligand, it is a potent receptor that integrates growth signals via homo- or heterodimerization with other ERBB family members. Genomic alterations increase ERBB2 kinase activity by constitutively activating the kinase domain, enabling ligand-independent receptor activation, by promoting enhanced dimerization or other mechanisms that have yet to be elucidated. Although ERBB3 has limited inherent kinase activity and cannot homodimerize, ligand stimulation promotes dimerization with active kinases like ERBB2 and EGFR that can phosphorylate ERBB2 which then promotes transformation. Thus genomic alterations in ERBB3 may be a key means of promoting oncogenic signaling despite the protein lacking robust enzymatic activity. We performed an analysis of ∼100 large-scale next generation sequencing datasets newly generated by our group (bladder), our institution (MSKCC-IMPACT), or found in recently published repositories (The Cancer Genome Atlas (TCGA), Broad, Genentech, Sanger, etc) to characterize the distribution and spectrum of mutations in the ERBB2 and ERBB3 genes. Data generated suggests that ERBB2 and ERBB3 mutations are highly prevalent in bladder cancer (each ∼10%), and recurrent, but with less frequency (<5%), in stomach, breast, colon and lung cancers. Hotspots include mutation of S310F and a cluster of residues in the kinase domain in ERBB2 and the V104M mutation in ERBB3. Preliminary analysis of ∼300 tumors demonstrated that ERBB2 and ERBB3 mutations present in a typically in a mutually exclusive pattern suggesting that these may be driver events with overlapping biologic effects. Generation and expression of several of these ERBB2 and ERBB3 mutants induced receptor activation, cell transformation and tumor growth in isogenic cell line and xenograft models. Treatment with the dual EGFR/HER2 inhibitor, neratinib, in isogenic cell lines abrogated colony formation in soft agar and receptor activation and downstream signaling. We hope to correlate the in vitro potency of specific ERBB2 and ERBB3 mutations with drug response in patients on ongoing or future clinical trials of targeted ERBB therapies and determine the whether lineage, subclonality and co-altered genes, if present, predict for drug resistance. Citation Format: Aphrothiti J. Hanrahan, David M. Hyman, John Sfakianos, Alexis Jones, Ricardo Ramirez, Hannah Johnsen, Gopakumar Iyer, Hikmat A. Al-Ahmadie, Dean Bajorin, Bernard H. Bochner, Jonathan A. Coleman, Jonathan E. Rosenberg, Michael F. Berger, Sarat Chandarlapaty, Jose Baselga, David B. Solit. Functional genomics of HER2 and HER3 mutations and response to neratinib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1101. doi:10.1158/1538-7445.AM2015-1101

Abstract 3897: High-throughput profiling of actionable mutations in salivary duct carcinoma

Abstract Salivary duct carcinoma (SDC) is a highly aggressive subtype of salivary gland cancers and there is no established standard therapy for this disease. Thus, development of molecular markers for SDC will be important to guide the diagnosis and therapy of this aggressive tumor. We performed next-generation sequencing using the Ion Torrent AmpliSeq cancer panel, which explores the mutational status of hotspot regions in 50 cancer-associated genes, and we analyzed copy number variations (CNVs) of 21 genes by NanoString nCounter for 37 patients with SDC. Fluorescent in situ hybridization was also conducted to confirm ERBB2 gene amplification. Clinical records and tumor histopathology of the patients were retrospectively reviewed. Genetic alterations were detected in 29 of 37 (78.3%) tumors, including mutations in PIK3CA (N = 9, 24.3%), ERBB2 (N = 4, 10.8%), and EGFR (N = 4, 10.8%). To our knowledge, this is the first time that ERBB2 mutations have been reported in this tumor type. Both PIK3CA and ERBB2 mutation status were associated with poor overall survival, but without statistical significance. ERBB2 amplification was strong and common in SDC and almost all cases also exhibited EGFR and ERBB3 amplifications. This study reports the largest and most comprehensive analysis of DNA aberrations in SDC. Our results show that PIK3CA and/or ERBB2 alterations in the development of SDC might be a useful diagnostic tool and could serve as a potential therapeutic target. Key words: Salivary duct carcinoma, next-generation sequencing, molecular markers, PIK3CA, ERBB2, EGFR Citation Format: Bo Mi Ku, Yeon-Hee Bae, Hyun Ae Jung, Jong-Mu Sun, Young Hyeh Ko, Han-Sin Jeong, Young-Ik Son, Chung-Hwan Baek, Keunchil Park, Myung-Ju Ahn. High-throughput profiling of actionable mutations in salivary duct carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3897. doi:10.1158/1538-7445.AM2015-3897

ERBB2 gene amplification increases during the transition of proximal EGFR+ to distal HLA-G+ first trimester cell column trophoblasts

IntroductionAlthough, DNA copy-number alterations (CNAs) have been well documented in a number of adverse phenotypic conditions, accumulating data suggest that CNAs also occur during physiological processes. Interestingly, extravillous trophoblasts induce the expression of the transforming, proto-oncogene ERBB2, which is frequently amplified in human cancer. However, no data are available to address whether trophoblast-related ERBB2 expression might also be linked to genomic amplification. MethodsDual color silver as well as fluorescence in situ hybridization analyses were carried out to evaluate frequency and degree of ERBB2 gene and chromosome 17 copy numbers in first trimester placental cell columns and isolated trophoblasts. Proliferative EGFR+ and differentiated HLA-G+ trophoblasts were identified or separated by means of in situ immunofluorescence co-stainings and magnetic beads cell isolation, respectively. ResultsERBB2 gene amplification is detected in approximately 40% of isolated HLA-G+ trophoblasts. Although already detectable in EGFR+ cells, the percentage and extent of ERBB2 amplification was markedly increased in HLA-G+ trophoblasts in situ and after isolation. Accordingly, HLA-G+ trophoblasts highly express ERBB2 on protein level. Finally, ERBB2 copy number variations occur independently of aneuploidy as the majority of ERBB2 amplifying cells were cytogenetically diploid for chromosome 17. DiscussionERBB2 gene amplification is a frequent event during EVT differentiation. This finding challenges the long standing paradigm, which associates gene amplification with pathological conditions and further supports recent evidences suggesting that CNAs are a normal feature of developmental processes.

Measurement of Domain-Specific HER2 (ERBB2) Expression May Classify Benefit From Trastuzumab in Breast Cancer.

Studies have shown that antibodies targeting the intracellular (ICD) or extracellular domains (ECD) of human epidermal growth factor receptor 2 (HER2) are equivalent when traditional methods are used. We describe a new method to quantify ICD and ECD expression separately and assess the prognostic value of domain-specific HER2 results in patients who received adjuvant trastuzumab therapy. We measured HER2 protein expression with quantitative immunofluorescence (QIF) in tissue microarrays (TMA) using two different antibodies targeting the ICD (CB11 and A0485) and ECD (SP3 and D8F12). We assessed the prognostic value of ICD and ECD expression in 180 patients from a clinical trial of adjuvant chemotherapy followed by trastuzumab (HeCOG 10/05). We performed an exploratory univariate domain-specific, disease-free survival (DFS) analysis and compared DFS functions with Kaplan-Meier estimates. All statistical tests were two-sided. HER2 ICD expression by QIF showed slightly higher sensitivity to predict ERBB2 (HER2) gene amplification than ECD expression, which was more specific and had higher positive predictive value. In the HeCOG 10/05 trial specimens, 15% of cases showed discordant results for ICD and ECD expression. High ECD was statistically associated with longer DFS (log-rank P = .049, HR = 0.31, 95% CI = 0.144 to 0.997), while ICD status was not. Among patients with low ECD, there was no difference in DFS by ICD status. However, when ICD was high, high ECD was statistically associated with longer DFS (log-rank P = .027, HR = 0.23, 95% CI = 0.037 to 0.82) compared with low ECD. Quantitative measurements of HER2 ICD and ECD expression in breast cancer suggest a subclassification of HER2-positive tumors. Trastuzumab-treated patients with high ECD showed better DFS than patients with low ECD. This suggests differential benefit from trastuzumab therapy based on HER2 ECD expression.

Abstract P6-07-04: Targeting ErbB2 with human PEPD

Abstract ErbB2, also known as Her2 or Neu, belongs to the ErbB family of plasma membrane-bound receptor tyrosine kinases, which also include ErbB1, ErbB3 and ErbB4. ErbB2 is best known for its involvement in human breast cancer. ErbB2 gene amplification occurs in ∼20% of breast cancer, and ErbB2 amplification or overexpression is a strong predictor of poor disease prognosis. ErbB2-targeted therapies, particularly humanized monoclonal antibody trastuzumab (Ttzm) in combination with chemotherapy, have shown considerable clinical efficacy. However, primary and secondary resistance remains a clinical challenge, and Ttzm, produced in mammalian cells, is very expensive. We have found that human prolidase, also known as peptidase D (PEPD) among several other names, binds to ErbB2 with high affinity (Kd = ∼7 nM) and binds as a homodimer (493 amino acids per subunit) to subdomain 3 in the extracellular domain of ErbB2. Each monomer of PEPD binds to one copy of ErbB2. However, PEPD is a weak ErbB1 binder (Kd = ∼5 μM) and does not bind to ErbB3 or ErbB4. PEPD is the first-ever natural ligand of ErbB2, and unlike the other ligands of ErbB receptors, it is devoid of an EGF motif. PEPD has been long known to hydrolyze dipeptides with proline or hydroxylproline at the carboxy terminus, but the dipeptidase activity of PEPD is not involved in ErbB2/ErbB1 modulation. In cells overexpressing ErbB2, where both activated dimers and inactive monomers of ErbB2 exist, as ErbB2 overexpression causes spontaneous dimerization, auto-tyrosine phosphorylation and recruitment and activation of downstream signals, PEPD rapidly binds to ErbB2 homodimers (<∼10 min) and silences the existing ErbB2-SRC signaling, a key oncogenic pathway of ErbB2, by disrupting SRC association with ErbB2. In contrast, PEPD binds to ErbB2 monomers relatively slowly (>∼30 min), but this binding causes ErbB2 dimerization, ErbB2 phosphorylation and downstream signaling. PEPD binding to ErbB2 subsequently causes pronounced ErbB2 depletion, resulting from its internalization and degradation. PEPD also strongly inhibits the DNA synthesis, anchorage-independent growth and invasion of cells that overexpress ErbB2, but has no effect on cells without overexpression of ErbB2. In fact, cells become sensitized to inhibition by PEPD upon achieving stable ErbB2 overexpression. Thus, the overall impact of PEPD on ErbB2 is inhibitory, and PEPD targets cells addicted to ErbB2. In ErbB2-overexpressing cells, at equimolar concentrations, PEPD was more effective than Ttzm in driving ErbB2 depletion, but is weaker than Ttzm in stimulating ErbB2 phosphorylation. In mouse tumor models, PEPD administered by intraperitoneal injection (Monday, Wednesday, Friday) at 0.2-2 mg/kg body weight strongly inhibited the growth of ErbB2-overexpressing tumors, but had no impact on tumors without ErbB2 overexpression, and the PEPD-treated mice showed no adverse effects. Given that the findings described above were made using human PEPD generated in bacteria, there is a distinct possibility that recombinant human PEPD may be a low cost alternative to Ttzm. Further investigation of the antitumor activity of PEPD and its modulation of ErbB2 signaling is warranted. Citation Format: Lu Yang, Yun Li, Arup Bhattacharya, Yuesheng Zhang. Targeting ErbB2 with human PEPD [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-07-04.

The prognostic impact of EGFR, ErbB2 and MET gene amplification in human gastric carcinomas as measured by quantitative Real-Time PCR.

Identification of critical genes which play pivotal roles in controlling tumor growth and survival will establish the basis for developing therapeutic targets. In this study, we focused on frequencies of EGFR, ErbB2 and MET gene amplification in gastric cancer patients to develop personalized medicine to improve the treatment. EGFR, ErbB2 and MET gene amplification, and mRNA expression were analyzed by the quantitative Real-Time PCR in paraffin-embedded samples from 115 patients with gastric cancer. EGFR, ErbB2 and MET genes were amplified in 11.3% (13/115), 6.1% (7/115) and 19.1% (22/115) of cancerous specimens, respectively. The correlation coefficient test clearly indicated that gene amplification in these three genes was positively correlated with mRNA transcription (EGFR: R=0.631, p=0.009; ErbB2: R=0.652, p=0.023; MET: R=0.715, p<0.001). EGFR and MET gene amplification was significantly associated with Ki-67 MI (p=0.022 and p=0.015). MET amplification was also significantly associated with age of≥60years (p=0.021) and tumor size of≥5cm (p=0.032). MET amplification, but not EGFR and ErbB2, was a significant prognostic factor in poor survival among patients with gastric cancer. EGFR, ErbB2 and MET genes are frequently amplified in gastric carcinoma. EGFR, ErbB2 and MET gene amplification is positively correlated with mRNA transcription. MET gene amplification correlates with a poor prognosis and poor survival in gastric carcinomas.

Multiomics profiling for gastric cancer using high-tech omics-based patient evaluation (HOPE).

95 Background: The Project HOPE (High-tech Omics-based Patient Evaluation) is an ongoing comprehensive platform that analyzes genome and transcriptome of fresh surgical specimens and blood samples obtained from cancer patients using whole-exome sequencing (WES, AmpliSeq Exome kit, Life Technology) and gene expression profiling (GEP, DNA microarray, Agilent Technologies). The research plan was approved by institutional review board (IRB) in Shizuoka Cancer Center, and total number of patients evaluated in this plan was about 3000 during the 3-year period of the research. Here, we focused on gastric cancer to investigate its characteristic profile. Methods: Tumor sample and peripheral blood was obtained from a total of 19 gastric cancer patients. At the initial stage of the study, the known mutation sites were analyzed by the WES, and expression level of cancer-related genes was evaluated by GEP. Results: Fourteen patients out of 19 patients (74 %) were categorized as advanced gastric cancer. The patients with single nucleotide variations (SNVs) in tumor protein P53 (TP53) accounted for 37% (7/19), and 21% (4/19) of patients had multiple SNVs in the known mutation sites. Furthermore, in two patients with gene amplification of ERBB2 or EGFR, mRNA expression corresponding these genes was markedly up-regulated. Conclusions: WES and GEP analyses may play an important role in interpretation of genetic changes of cancer tissues and personalized medicine.

Genomic markers of panitumumab resistance including ERBB2/HER2 in a phase II study of KRAS wild-type (wt) metastatic colorectal cancer (mCRC).

646 Background: Epidermal Growth Factor Receptor Inhibitors (EGFRi) are effective agents in the treatment of mCRC but up to one third of KRAS wt tumors do not respond. The primary objective was to identify a gene expression signature associated with primary resistance to panitumumab (Pmab) monotherapy and identify genomic markers associated with resistance. Methods: Patients with previously treated codon 12/13 KRAS wt mCRC were administered Pmab 6mg/kg IV q2weeks in a prospective phase II study. The primary endpoint was tumor response by RECIST and patients with progressive disease 8 weeks after initial Pmab were classified as resistant. Gene expression analysis was performed using a Nanostring-based assay with candidate genes implied in EGFRi resistance expression patterns. Differentially expressed genes were identified by Significance Analysis of Microarrays. Further interrogation of the HER2 signaling pathway was performed by immunohistochemistry and in situ hybridization. Illumina-based targeted resequencing was conducted on 20 cancer-implicated genes. Results: Of 34 patients treated with Pmab, 11 (32%) had progressive disease at 8 weeks and were classified Pmab resistant. A 5-gene expression signature was significantly associated with Pmab resistance (p = 0.001), including ERBB2, MLPH, IRX3, c11orf9, and KLK6. HER2 protein was overexpressed in 4/11 resistant and 0/21 responding cases (p = 0.035). Two (2) resistant tumors had ERBB2 gene amplification only, and one demonstrated both ERBB2 amplification and mutation. A non-codon 12/13 RAS mutation occurred in one treatment-resistant patient and was mutually exclusive with ERBB2 mutation, amplification, or HER2 overexpression. Four BRAF mutations were detected, two occurring in resistant patients. Conclusions: In this prospective study, a 5-gene signature including ERBB2 was associated with primary resistance to single agent Pmab. A subgroup of patients had evidence of aberrant HER2/ERBB2 signaling by protein over-expression and gene amplification. Results suggest a significant portion of KRAS wt tumors unresponsive to EGFRi may be identified by gene signature analysis. Clinical trial information: NCT00853931.

Could Pharmacogenomics Improve Efficacy and Safety of Trastuzumab?

Copyright: © 2015 Yiannakopoulou EC. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Trastuzumab (HerceptinTM) is a humanized anti-ErbB2 monoclonal antibody that has been approved for the treatment in the adjuvant and the metastatic setting of breast cancer patients that either overexpress ErbB2, or demonstrate ErbB2 gene amplification [1]. An important serious adverse event associated with trastuzumab is the reduction of left heart ejection fraction resulting to congestive heart failure. The role of pharmacogenomics in breast cancer targeted treatment has been well recognized [2]. However, data on the role of pharmacogenomics on the efficacy and safety of trastuzumab are still scarce.

High-throughput profiling identifies clinically actionable mutations in salivary duct carcinoma

BackgroundSalivary duct carcinoma (SDC) is a highly aggressive subtype of salivary gland cancers and there is no established standard therapy for this disease. Thus, development of molecular markers for SDC will be important to guide the diagnosis and therapy of this aggressive tumor.MethodsWe performed next-generation sequencing using the Ion Torrent AmpliSeq cancer panel, which explores the mutational status of hotspot regions in 50 cancer-associated genes, and we analyzed copy number variations (CNVs) of 21 genes by NanoString nCounter for 37 patients with SDC. Fluorescent in situ hybridization was also conducted to confirm ERBB2 gene amplification. Clinical records and tumor histopathology of the patients were retrospectively reviewed.ResultsGenetic alterations were detected in 29 of 37 (78.3%) tumors, including mutations in PIK3CA (N = 9, 24.3%), ERBB2 (N = 4, 10.8%), and EGFR (N = 4, 10.8%). To our knowledge, this is the first time that ERBB2 mutations have been reported in this tumor type. Both PIK3CA and ERBB2 mutation status were associated with poor overall survival, but without statistical significance. ERBB2 amplification was strong and common in SDC and almost all cases also exhibited EGFR and ERBB3 amplifications.ConclusionsThis study reports the largest and most comprehensive analysis of DNA aberrations in SDC. Our results show that PIK3CA and/or ERBB2 alterations in the development of SDC might be a useful diagnostic tool and could serve as a potential therapeutic target.

Abstract 3419: Frequency and function of ERBB3 mutations in bladder cancer

Abstract Deregulation of the HER family of receptor tyrosine kinases has been widely implicated in cancer initiation and progression. Activating mutations or gene amplification of EGFR and ERBB2 (HER2) are frequently found in non-small cell lung, breast, colorectal, glioblastoma and head and neck cancers. However, little is known regarding the prevalence or functionality of genomic alterations in the ERBB3 (HER3) isoform. Although ERBB3 has limited inherent kinase activity, ligand stimulation promotes its dimerization with active kinases like ERBB2 and EGFR that can phosphorylate ERBB3 which then promotes transformation. Thus genomic alterations in ERBB3 may be a key means of promoting oncogenic signaling despite the protein lacking robust enzymatic activity. We performed a meta-analysis of 40 cross-cancer next generation sequencing and copy number genomics data sets newly generated by our group (bladder) or found in recently published repositories (The Cancer Genome Atlas (TCGA), Broad, Genentech, Sanger, etc). Analogous to the singular 2013 publication on ERBB3 mutations to date, we found frequent mutations in ERBB3 in gastric tumors (∼11%). Preliminary analysis of our novel and expanding cohort of 109 high-grade bladder cancer tumors suggests that ERBB3 mutations are equally frequent in bladder cancer (∼10-12%). Collectively, EGFR, ERBB2 and ERBB3 alterations were found in 23-30% of all high-grade bladder cancers, typically in a mutually exclusive pattern suggesting that these may be driver events. ERBB3 mutations were distributed across all structural domains of the protein. We identified more than 15 novel ERBB3 mutations in our bladder cohort, including hotspot mutations in the extracellular domain (V104L/M, D297Y, others) that were recurrent across multiple cancers types. Stable retroviral overexpression of ERBB3 mutants into NIH-3T3 cells induced phosphorylation of ERBB3, activation of downstream signaling cascades and soft agar colony formation, all which were enhanced in the presence of neuregulin and inhibited by the dual EGFR/ERBB2 kinase inhibitor, neratinib, and a monoclonal antibody to ERBB3, AMG-888/U3-1287. These data highlight a large cohort of bladder cancer patients with novel, activating mutations in the tractable kinase ERBB3, that could potentially benefit from approved EGFR/ERBB2 targeted therapeutics in the clinic and novel ERBB3 therapeutics in early clinical trials. Citation Format: Aphrothiti J. Hanrahan, John P. Sfakianos, Ricardo Ramirez, Phillip H. Kim, Gopa Iyer, Hikmat A. Al-Ahmadie, Sasinya N. Scott, Dean F. Bajorin, Bernard H. Bochner, Jonathan A. Coleman, Jonathan E. Rosenberg, Michael F. Berger, Sarat Chandarlapaty, David B. Solit. Frequency and function of ERBB3 mutations in bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3419. doi:10.1158/1538-7445.AM2014-3419

Anti-metastasis activity of black rice anthocyanins against breast cancer: analyses using an ErbB2 positive breast cancer cell line and tumoral xenograft model.

Increasing evidence from animal, epidemiological and clinical investigations suggest that dietary anthocyanins have potential to prevent chronic diseases, including cancers. It is also noteworthy that human epidermal growth factor receptor 2 (ErbB2) protein overexpression or ErbB2 gene amplification has been included as an indicator for metastasis and higher risk of recurrence for breast cancer. The present experiments investigated the anti-metastasis effects of black rice anthocyanins (BRACs) on ErbB2 positive breast cancer cells in vivo and in vitro. Oral administration of BRACs (150 mg/kg/day) reduced transplanted tumor growth, inhibited pulmonary metastasis, and decreased lung tumor nodules in BALB/c nude mice bearing ErbB2 positive breast cancer cell MDA-MB-453 xenografts. The capacity for migration, adhesion, motility and invasion was also inhibited by BRACs in MDA-MB-453 cells in a concentration dependent manner, accompanied by decreased activity of a transfer promoting factor, urokinase-type plasminogen activator (u-PA). Together, our results indicated that BRACs possess anti-metastasis potential against ErbB2 positive human breast cancer cells in vivo and in vitro through inhibition of metastasis promoting molecules.

Therapeutic targeting of ERBB2 in breast cancer: understanding resistance in the laboratory and combating it in the clinic.

ERBB2 gene amplification occurs in about one quarter of breast carcinomas (BCs) and identifies a distinct clinical subset of BC. The introduction in the clinic of Trastuzumab, a humanized monoclonal antibody (mAb) directed to the ERBB2 extracellular domain, has had a great impact on the therapeutic management of ERBB2+ BC. Yet, not all patients respond to Trastuzumab and resistance develops also among patients that initially benefit from Trastuzumab-based regimens. Pre-clinical studies have discovered several mechanisms through which tumor cells may escape from Trastuzumab-mediated ERBB2 inhibition. These include rewiring of the ErbB signaling network, loss of ERBB2 expression, expression of ERBB2 isoforms refractory to Trastuzumab inhibition, vicarious signaling by non-ErbB tyrosine kinases and constitutive activation of downstream signaling routes, such as the PI3K pathway. While the relative contribution of each of these mechanisms to establishing Trastuzumab resistance in the clinical setting is not fully understood, much attention has been focused on abating resistance by achieving complete blockade of ERBB2-containing dimers. This approach, propelled by the development of novel anti-ERBB2 therapeutics, has led to the recent approval of Lapatinib, Pertuzumab and T-DM1 as additional anti-ERBB2 therapeutics in BC. However, full success is far from being achieved and resistance to ERBB2 targeting remains a relevant problem in the clinical management of BC. Herein, we provide an overview of biological and molecular bases underpinning resistance to ERBB2 therapeutics in BC, discuss outstanding issues in the field of ERBB2 therapeutic targeting and elaborate on future directions of translational research on ERBB2+ breast cancer.

Concomitant mutation and amplification of the ERBB2 (HER2) gene in human tumors.

11071 Background: Amplification of the ERBB2 (HER2) gene is a well-known genetic aberration in many tumor entities. Up to 20% of breast and gastric cancer show ERBB2 gene amplification and anti-HER...

Neuregulin-1/erbB activities with focus on the susceptibility of the heart to anthracyclines.

Neuregulin-1 (NRG1) signaling through the tyrosine kinase receptors erbB2 and erbB4 is required for cardiac morphogenesis, and it plays an essential role in maintaining the myocardial architecture during adulthood. The tyrosine kinase receptor erbB2 was first linked to the amplification and overexpression of erbb2 gene in a subtype of breast tumor cells, which is indicative of highly proliferative cells and likely a poor prognosis following conventional chemotherapy. The development of targeted therapies to block the survival of erbB2-positive cancer cells revealed that impaired NRG1 signaling through erbB2/erbB4 heterodimers combined with anthracycline chemotherapy may lead to dilated cardiomyopathy in a subpopulation of treated patients. The ventricular-specific deletion of either erbb2 or erbb4 manifested dilated cardiomyopathy, which is aggravated by the administration of doxorubicin. Based on the exacerbated toxicity displayed by the combined treatment, it is expected that the relevant pathways would be affected in a synergistic manner. This review examines the NRG1 activities that were monitored in different model systems, focusing on the emerging pathways and molecular targets, which may aid in understanding the acquired dilated cardiomyopathy that occurs under the conditions of NRG1-deficient signaling.

Chromatin Modifications Sequentially Enhance ErbB2 Expression in ErbB2-Positive Breast Cancers

ErbB2 gene amplification occurs in 20%-25% of breast cancers, and its therapeutic targeting has markedly improved survival of patients with breast cancer in the adjuvant setting. However, resistance to these therapies can develop. Because epigenetic mechanisms can importantly influence oncogene expression and be druggable as well, we investigated histone modifications that influence ErbB2 overexpression, independent of gene amplification. We demonstrate here that ErbB2-overexpressing breast carcinomas acquire the H3K4me3 mark on the erbB2 promoter and that receptor-amplified tumors further acquire the H3K9ac mark, which is dependent on H3K4me3 mark acquisition. Targeting WD repeat domain 5 (Wdr5), which is absolutely required for H3K4me3 enrichment, decreased ErbB2 overexpression, associated with a decrease in the H3K4me3 mark on the erbB2 promoter. Of note, Wdr5 silencing cooperated with trastuzumab or chemotherapy in specifically inhibiting the growth of ErbB2-positive breast tumor cells. Thus, our studies illuminate epigenetic steps in the selection for ErbB2 activation.

Quantitative proteomic analysis of HER2 normal and overexpressing MCF-7 breast cancer cells revealed proteomic changes accompanied with HER2 gene amplification

Molecular classification of breast cancer is based, in part, on the presence or absence of amplification of the human epidermal growth factor receptor 2 (ERBB2) gene, which leads to HER2 protein overproduction. While the presence of the overexpressed HER2 protein is a necessary precondition for sensitivity to anti-HER2 therapies, many patients develop resistance. Thus, identification of the downstream effectors of this pathway will help in understanding mechanism(s) of chemoresistance and further, the identified molecules themselves may have the potential to be used as therapeutic targets. In this work, we studied the proteomic changes that accompany the HER2 gene amplification to identify potential new therapeutic targets and biomarkers. We analyzed bio-triplicate proteome samples extracted from wild-type MCF-7 human breast cancer cells and their isogenic stably overexpressing HER2 (amplified) transfectants. In total, 2455 unique proteins were quantified with 1278 of them differentially expressed in HER2 normal and HER2 overexpressing MCF-7 cells. Select biomarker candidates of particular interest were validated by western blotting, and evaluated for clinical relevance by the immunohistochemical assessment of protein abundance in breast tumor biopsies. HER2 transfection produced marked changes in proteins related to multiple aspects of cancer biology, and the identified expression patterns were recapitulated in the clinical samples. Breast cancer is a major cause of death in women. Molecular classification of breast cancer is based, in part, on the presence or absence of amplification of the human epidermal growth factor receptor 2 (ERBB2) gene, which leads to HER2 protein overproduction that triggers intracellular signaling events that drive proliferation, invasion, metastases, and resistance to apoptosis. While the presence of the overexpressed HER2 gene product, HER2 protein, is a necessary precondition for sensitivity to the therapeutic monoclonal antibody trastuzumab, the downstream effects of HER2 protein overexpression are incompletely understood. In this work, we applied quantitative proteomics to identify proteomic changes accompanying ERBB2 gene amplification. The significance of this work includes 1) identification of new biomarkers associated with the HER2 phenotype, 2) measurement of the magnitude of the proteomic changes triggered by the amplification of this single gene, and 3) better understanding of the downstream biological changes triggered by HER2 overexpression.

Gene amplification and immunohistochemical expression of ERBB2 and EGFR in cervical carcinogenesis. Correlation with cell-cycle markers and HPV presence

Although the members of the epidermal growth factor receptor family ERBB2 and EGFR are important therapeutic targets in the treatment of malignant neoplasias, little is known about their role in cervical carcinogenesis. Our objective was to evaluate the dysfunction of ERBB2 and EGFR at the gene copy number and protein expression level in neoplastic lesions of the uterine cervix with the aim of obtaining information about its role in cervical carcinogenesis and their possible use as therapeutic targets in these diseases. We studied gene amplification and protein expression of ERBB2 and EGFR and their relationship with Ki67, p16 and p53 and HPV presence in 22 normal/benign (N/B) cervices, 20 low-grade squamous intraepithelial lesions (LSILs), 70 high-grade SILs (HSILs) and 32 invasive squamous cervical carcinomas (ISCCs). No cases showed selective amplification of ERBB2 or EGFR but corresponding chromosome-specific probes displayed chromosome 17 and 7 polyploidy associated with the grade of the lesion (p<0.0001 and p=0.004, respectively) and with the positive expression of Ki67 and p16 (p<0.01). Concurrent polyploidy for both chromosomes was statistically related (p<0.0001). ERBB2 immunohistochemical expression was not observed in any of the study cases except for one ISCC but EGFR was associated with higher-grade lesions (N/B plus LSIL 21.4% vs. HSIL plus ISCC 45.5%; p=0.007). No association was observed between EGFR expression and that of cell-cycle markers or HPV presence. Increased copy number of EGFR and ERBB2 is due to polyploidy of 7 and 17 chromosomes, this being a phenomenon associated with lesion severity and with an increase in the expression of cell-cycle markers. EGFR, but not ERBB2, is expressed in precursor lesions of squamous cervical neoplasia and is related to the neoplastic progression but not to proliferation marker expression and therefore ERBB2 and this calls into question the usefulness of ERBB2 as a therapeutic target.

Heterogeneity of ERBB2 in gastric carcinomas: a study of tissue microarray and matched primary and metastatic carcinomas

Trastuzumab in association with systemic cytotoxic chemotherapy is a therapeutic option for patients with advanced or metastatic ERBB2+ gastric carcinoma. The status of the ERBB2 overexpression or gene amplification is an important predictive marker in gastric cancer. However, it is controversial whether the primary tumor is representative of distant metastases in terms of ERBB2 status. Quadruplicated tissue microarrays from formalin-fixed paraffin-embedded tissues from 498 advanced primary gastric carcinomas and 97 matched metastatic lymph nodes were investigated by immunohistochemistry with HercepTest and silver in situ hybridization. For further comparison, another set of 41 paired primary and distant metastatic gastric carcinomas were also tested. Intratumoral heterogeneity was defined as different results between tissue microarray cores. ERBB2-positivity was observed in 52 gastric carcinomas (10%) and was not associated with recurrence of disease or survival of patients. In ERBB2-positive primary gastric carcinomas, heterogeneous ERBB2 overexpression was observed in 21/63 (33%) gastric carcinomas and heterogeneous ERBB2 gene amplification in 14/62 (23%) cases. Repeated immunohistochemistry and silver in situ hybridization in representative paraffin tumor blocks confirmed focal ERBB2 overexpression and ERBB2 gene amplification and did not change the final results. Discrepancies in ERBB2 results between primary and paired metastatic lymph nodes were observed in 11% of cases by immunohistochemistry and 7% by silver in situ hybridization. Out of the 41 paired primary and distant metastases, 5 (12%) cases were ERBB2-positive, and discrepancy was observed in one case. Intratumoral heterogeneity and discrepant ERBB2 results in primary and metastatic tumor are not uncommon in gastric carcinoma. Results of silver in situ hybridization showed less frequent heterogeneity compared with immunohistochemistry. Wherever possible, ERBB2 immunohistochemistry testing should be performed in both primary and distant metastatic sites.