AbstractIntroductionMethodResultsConclusionIntroductionMaterials and methodsMetaplastic breast carcinoma samplesImmunohistochemical and chromogenic in situ hybridization analysisCorrelation between EGFR overexpression and amplification and clinicopathological parameters and survivalResultsTable 2 DiscussionConclusionCompeting interestsAuthors' contributionsAcknowledgementsReferencesOpen AccessAvailable online http://breast-cancer-research.com/content/7/6/R1028R1028Vol 7 No 6Research articleMetaplastic breast carcinomas exhibit EGFR, but not HER2, gene amplification and overexpression: immunohistochemical and chromogenic in situ hybridization analysisJorge S Reis-Filho1,2,3*, Fernanda Milanezi2,3*, Silvia Carvalho2,3*, Pete T Simpson4, Dawn Steele1, Kay Savage1, Maryou BK Lambros1, Emilio M Pereira5, Jahn M Nesland6, Sunil R Lakhani4 and Fernando C Schmitt21The Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, London, UK2IPATIMUP – Institute of Molecular Pathology and Immunology, University of Porto, Porto, Portugal3Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal4Molecular & Cellular Pathology, Mayne Medical School, University of Queensland, Queensland Institute of Medical Research and Royal Brisbane and Women's Hospital, Brisbane, Australia5Laboratório Salomão & Zoppi, São Paulo, Brazil6The Norwegian Radium Hospital, University of Oslo, Montebello, Norway* Contributed equallyCorresponding author: Jorge S Reis-Filho, [email protected]: 17 Jul 2005 Revisions requested: 31 Aug 2005 Revisions received: 12 Sep 2005 Accepted: 29 Sep 2005 Published: 25 Oct 2005Breast Cancer Research 2005, 7:R1028-R1035 (DOI 10.1186/bcr1341)This article is online at: http://breast-cancer-research.com/content/7/6/R1028© 2005 Reis-Filho et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.AbstractIntroduction Metaplastic breast carcinomas constitute aheterogeneous group of neoplasms, accounting for less than1% of all invasive mammary carcinomas. Approximately 70–80% of metaplastic breast carcinomas overexpress theepidermal growth factor receptor (EGFR). Human epidermalgrowth factor receptor (HER)2 and EGFR have attracted muchattention in the medical literature over the past few years owingto the fact that humanized monoclonal antibodies against HER2and therapies directed against the extracellular ligand-bindingdomain or the intracellular tyrosine kinase domain of EGFR haveproven successful in treating certain types of human cancer. Weinvestigated whether HER2 and EGFR overexpression waspresent and evaluated gene amplification in a series ofmetaplastic breast carcinomas.Method Twenty-five metaplastic breast carcinomas wereimmunohistochemically analyzed using a monoclonal antibody(31G7) for EGFR and two antibodies for HER2 (Herceptest andCB11) and scored using the Herceptest scoring system. Geneamplification was evaluated by chromogenic in situ hybridizationusing Zymed Spot-Light EGFR and HER2 amplification probe.The results were evaluated by bright field microscopy under40× and 63× objective lenses.Results Nineteen (76%) metaplastic breast carcinomasexhibited EGFR ovexpression, and among these EGFRamplification (defined either by large gene clusters or >5signals/nucleus in >50% of neoplastic cells) was detected inseven cases (37%): three carcinomas with squamousdifferentiation and four spindle cell carcinomas. One caseexhibited HER2 overexpression of grade 2+ (>10% of cells withweak to moderate complete membrane staining), but HER2gene amplification was not detected.Conclusion Metaplastic breast carcinomas frequentlyoverexpressed EGFR, which was associated with EGFR geneamplification in one-third of cases. Our findings suggest thatsome patients with metaplastic breast carcinomas might benefitfrom novel therapies targeting EGFR. Because mostmetaplastic breast carcinomas overexpress EGFR without geneamplification, further studies to evaluate EGFR activatingmutations are warranted.CISH = chromogenic in situ hybridization; DFS = disease-free survival; EGFR = epidermal growth factor receptor; HER = human epidermal growth factor receptor; MBC = metaplastic breast carcinoma; OS = overall survival.Breast Cancer Research Vol 7 No 6 Reis-Filho et al.R1029IntroductionIn recent years, the family of epidermal growth factor receptors(EGFRs) or ERBB receptors has attracted great attention inthe literature [1]. This family includes four tyrosine kinasereceptors: EGFR (HER1/c-erbB1), human epidermal growthfactor receptor (HER)2/neu (c-erbB2), HER3 (c-erbB3) andHER4(c-erbB4). All members of this family are characterizedby an extracellular ligand-binding region, a single membranespanning region, and a cytoplasmic tyrosine kinase containingdomain [1]. Although expression of the four members of theERBB family has been studied in several types of humantumours [1], only EGFR and HER2 have been proven to playmajor roles in different histological types of breast cancer [1-12]. Thus far, only these two receptors have successfully beentargeted as therapy for lung [13-17] and breast cancer[18,19].EGFR was the first tyrosine kinase receptor to be directlylinked with human cancer. The EGFR gene maps to 7p11.2-p2 and encodes a 170 kDa transmembrane protein [1]. EGFRgene amplification has been described in oligodendrogliomas[20], glioblastomas [21], lung carcinomas [13,14,22], gastriccarcinomas [23] and, recently, breast carcinomas [8,24,25].The HER2 gene maps to chromosome 17q21 and encodes a185 kDa glycoprotein. It is reported to be amplified and over-expressed in several types of human tumours, including about30% of all breast carcinomas [1,18,19]. Most importantly, inrecent years EGFR tyrosine kinase inhibitors and humanizedmonoclonal antibodies against HER2 have received US Foodand Drug Administration approval and are currently beingtested in patients with lung and breast cancer.Data on the response of patients with lung cancer have dem-onstrated that approximately 10–15% of patients with EGFR-positive lung carcinomas have a dramatic response to EGFRtyrosine kinase inhibitors [15-17,22]. Interestingly, responsewas linked to the presence of an activating somatic