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Targeted Therapy in Breast Cancer

The HER-2/neu Gene and Protein
Open AccessPublished:February 03, 2004DOI:https://doi.org/10.1074/mcp.R400001-MCP200
      The HER-2/neu oncogene, a member of the epidermal growth factor receptor or erb gene family, encodes a transmembrane tyrosine kinase receptor that has been linked to prognosis and response to therapy with the anti-HER-2-humanized monoclonal antibody, trastuzumab (Herceptin, Genentech, South San Francisco, CA) in patients with advanced metastatic breast cancer. HER-2/neu status has also been tested for its ability to predict the response of breast cancer to other therapies including hormonal therapies, topoisomerase inhibitors, and anthracyclines. This review includes an analysis of 80 published studies encompassing more than 25,000 patients designed to consider the relative advantages and disadvantages of the various methods of measuring HER-2/neu in clinical breast cancer specimens. Southern blotting, PCR amplification detection, and fluorescence in situ hybridization assays designed to detect HER-2/neu gene amplification are compared with HER-2/neu protein overexpression assays performed by immunohistochemical techniques applied to frozen and paraffin-embedded tissues and enzyme immunoassays performed on tumor cytosols. The significance of HER-2/neu overexpression in ductal carcinoma in situ and the HER-2/neu status in uncommon female breast conditions and male breast cancer are also considered. The role of HER-2/neu testing for the prediction of response to trastuzumab therapy in breast cancer is reviewed along with the current studies designed to test whether HER-2/neu status can predict the response to standard and newer hormonal therapies, cytotoxic chemotherapy, and radiation. The review will also evaluate the status of serum-based testing for circulating HER-2/neu receptor protein and its ability to predict disease outcome and therapy response.
      The HER-2/neu (C-erbB-2) gene is localized to chromosome 17q and encodes a transmembrane tyrosine kinase receptor protein (Fig. 1) that is a member of the epidermal growth factor receptor (EGFR)
      The abbreviations used are: EGFR, epidermal growth factor receptor; IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; RT-PCR, reverse transcription PCR; CISH, chromogenic in situ hybridization; ER, estrogen receptor; PR, progesterone receptor; ELISA, enzyme-linked immunosorbent assay; CTA, clinical trial assay.
      1The abbreviations used are: EGFR, epidermal growth factor receptor; IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; RT-PCR, reverse transcription PCR; CISH, chromogenic in situ hybridization; ER, estrogen receptor; PR, progesterone receptor; ELISA, enzyme-linked immunosorbent assay; CTA, clinical trial assay.
      or HER family (Fig. 2) (
      • Navolanic P.M.
      • Steelman L.S.
      • McCubrey J.A.
      EGFR family signaling and its association with breast cancer development and resistance to chemotherapy..
      ). This family of receptors is involved in cell-cell and cell-stromal communication primarily through a process known as signal transduction, in which external growth factors, or ligands, affect the transcription of various genes by phosphorylating or dephosphorylating a series of transmembrane proteins and intracellular signaling intermediates, many of which possess enzymatic activity. Signal propagation occurs as the enzymatic activity of one protein turns on the enzymatic activity of the next protein in the pathway (
      • Karunagaran D.
      • Tzahar E.
      • Beerli R.R.
      • Chen X.
      • Graus-Porta D.
      • Ratzkin B.J.
      • Seger R.
      • Hynes N.E.
      • Yarden Y.
      ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: Implications for breast cancer..
      ). Major pathways involved in signal transduction, including the Ras/mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, the Janus kinase(JAK)/signal transducers and activators of transcription (STAT) pathway, and the phospholipase Cγ (PLC-γ) pathway, ultimately affect cell proliferation, survival, motility, and adhesion (Fig. 3).
      Figure thumbnail gr1
      Fig. 1The HER-2/neu tyrosine kinase receptor. The extracellular domain is recognized by the antibody 4D5, which is the murine antigen binding component of the humanized anti-HER-2/neu antibody trastuzumab (Herceptin™).
      Figure thumbnail gr2
      Fig. 2The HER (erb) gene family. Note that HER-2/neu has no known ligands and that HER-3 has no intrinsic tyrosine kinase activity.
      Figure thumbnail gr3
      Fig. 3Dimerization and downstream signaling of the HER (EGFR) family. Major pathways involved in signal transduction, including the Ras, MAPK, PI3K/Akt, JAK/STAT, and the PLC-γ pathway ultimately affect cell proliferation, cell survival, motility and adhesion.
      Receptor activation requires three variables, a ligand, a receptor, and a dimerization partner (Fig. 2) (
      • Yarden Y.
      • Sliwkowski M.X.
      Untangling the ErbB signalling network..
      ). After a ligand binds to a receptor, that receptor must interact with another receptor of identical or related structure in a process known as dimerization, in order to trigger phosphorylation and activate signaling cascades. Therefore, after ligand binding to an EGFR family member, the receptor can dimerize with various members of the family (EGFR, HER-2, HER3, or HER-4). It may dimerize with a like member of the family (homodimerization), or it may dimerize with a different member of the family (heterodimerization) (Table I). The specific tyrosine residues on the intracellular portion of the HER-2/neu receptor protein that are phosphorylated, and hence the signaling pathways that are activated, depends on the ligand and dimerization partner. The wide variety of ligands and intracellular cross talk with other pathways allow for significant diversity in signaling. While no known ligand for the HER-2/neu receptor has been identified, it is the preferred dimerization partner of the other family members. HER-2/neu heterodimers are more stable (
      • Tzahar E.
      • Waterman H.
      • Chen X.
      • Levkowitz G.
      • Karunagaran D.
      • Lavi S.
      • Ratzkin B.J.
      • Yarden Y.
      A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor..
      ) and their signaling is more potent (
      • Craven R.J.
      • Lightfoot H.
      • Cance W.G.A.
      A decade of tyrosine kinases: From gene discovery to therapeutics..
      ) than receptor combinations without HER-2/neu. HER-2/neu gene amplification and/or protein overexpression has been identified in 10–34% of invasive breast cancers (
      • Navolanic P.M.
      • Steelman L.S.
      • McCubrey J.A.
      EGFR family signaling and its association with breast cancer development and resistance to chemotherapy..
      ).
      Table IDimerization of the HER-2/neu receptor and signaling pathway
      Dimerization partnerHER-1 (EGFR)HER-2HER-3HER-4
      LigandsTGF-?NoneNRG1NRG1
      EGFHeregulinNRG2
      HB-EGFHeregulin
      Epiregulin
      Amphiregulin
      ?-cellulin
      HER-1SRCP13K: CytoskeletonNoneNone
      PI3KAKT: Anti-apoptosis
      AKT
      HER-2NoneRAS: TranscriptionRAS: TranscriptionNone
      RAS: Cell cycle progressionRAS: Cell cycle progression
      P13KP13K
      HER-3NoneP13K: CytoskeletonNoneNone
      AKT: Anti-apoptosis
      None
      HER-4NoneNoneNone

      BACKGROUND AND CLINICAL RELEVANCE

      Serum HER-2/neu Antigen Levels as a Tumor Marker—

      Circulating HER-2/neu receptor protein levels have successfully predicted the presence and progression of HER-2/neu-positive breast cancer. In 20 published studies on 4,088 patients, 16 (73%) studies involving 3,458 (85%) of the patients reported a significant correlation of serum HER-2/neu protein levels with disease recurrence, metastasis, or shortened survival (
      • Isola J.J.
      • Holli K.
      • Oksa H.
      • Teramoto Y.
      • Kallioniemi O.P.
      Elevated erb B-2 oncoprotein levels in preoperative and follow-up serum samples defined in aggressive course in patients with breast cancer..
      ,
      • Andersen T.I.
      • Paus E.
      • Nesland J.M.
      • McKenzie S.J.
      • Borresen A.L.
      Detection of C-erb B2 related proteins in sera from breast cancer patients. Relationship to ERBB2 gene amplification and Cerb B2 protein overexpression in tumour..
      ,
      • Willsher P.C.
      • Beaver J.
      • Pinder S.
      • Bell J.A.
      • Ellis I.O.
      • Blamey R.W.
      • Robertson J.F.
      Prognostic significance of serum CerbB2 protein in breast cancer patients..
      ,
      • Fehm T.
      • Maimonis P.
      • Wetz S.
      • Teramoto Y.
      • Katalinic A.
      • Jager W.
      Influence of circulating C-erbB-2 serum protein on response to adjuvant chemotherapy in node-positive breast cancer patients..
      ,
      • Mansour O.A.
      • Zekri A.R.
      • Harvey J.
      • Teramoto Y.
      • el-Ahmady O.
      Tissue in serum CerbB2 and tissue EGFR in breast carcinoma: Three years follow-up..
      ,
      • Disis M.L.
      • Pupa S.M.
      • Gralow J.R.
      • Dittadi R.
      • Menard S.
      • Cheever M.A.
      High-titer HER-2/neu protein-specific antibody can be detected in patients with early-stage breast cancer..
      ,
      • Krainer M.
      • Brodowicz T.
      • Zeillinger R.
      • Wiltschke C.
      • Scholten C.
      • Seifert M.
      • Kubista E.
      • Zielinski C.C.
      Tissue expression and serum levels of HER-2/neu in patients with breast cancer..
      ,
      • Burke H.B.
      • Hoang A.
      • Iglehart J.D.
      • Marks J.R.
      Predicting response to adjuvant and radiation therapy in patients with early stage breast carcinoma..
      ,
      • Fehm T.
      • Maimonis P.
      • Katalinic A.
      • Marks J.R.
      The prognostic significance of c erbB-2 serum protein in metastatic breast cancer..
      ,
      • Mehta R.R.
      • McDermott J.H.
      • Hieken T.J.
      • Marler K.C.
      • Patel M.K.
      • Wild L.D.
      • Das Gupta T.K.
      Plasma c-erbB-2 levels in breast cancer patients: Prognostic significance in predicting response to chemotherapy..
      ,
      • Colomer R.
      • Montero S.
      • Lluch A.
      • Ojeda B.
      • Barnadas A.
      • Casado A.
      • Massuti B.
      • Cortes-Funes H.
      • Lloveras B.
      Circulating HER2 extracellular domain and resistance to chemotherapy in advanced breast cancer..
      ,
      • Bewick M.
      • Conlon M.
      • Gerard S.
      • Lee H.
      • Parissenti A.M.
      • Zhang L.
      • Gluck S.
      • Lafrenie R.M.
      HER-2 expression is a prognostic factor in patients with metastatic breast cancer treated with a combination of high-dose cyclophosphamide, mitoxantrone, paclitaxel and autologous blood stem cell support..
      ,
      • Harris L.N.
      • Liotcheva V.
      • Broadwater G.
      • Ramirez M.J.
      • Maimonis P.
      • Anderson S.
      • Everett T.
      • Harpole D.
      • Moore M.B.
      • Berry D.A.
      • Rizzeri D.
      • Vredenburgh J.J.
      • Bentley R.C.
      Comparison of methods of measuring HER-2 in metastatic breast cancer patients treated with high-dose chemotherapy..
      ,
      • Ali S.M.
      • Leitzel K.
      • Chinchilli V.M.
      • Engle L.
      • Demers L.
      • Harvey H.A.
      • Carney W.
      • Allard J.W.
      • Lipton A.
      Relationship of serum HER-2/neu and serum CA 15-3 in patients with metastatic breast cancer..
      ,
      • Classen S.
      • Kopp R.
      • Possinger K.
      • Weidenhagen R.
      • Eiermann W.
      • Wilmanns W.
      Clinical relevance of soluble c-erbB-2 for patients with metastatic breast cancer predicting the response to second-line hormone or chemotherapy..
      ,
      • Lipton A.
      • Ali S.M.
      • Leitzel K.
      • Demers L.
      • Chinchilli V.
      • Engle L.
      • Harvey H.A.
      • Brady C.
      • Nalin C.M.
      • Dugan M.
      • Carney W.
      • Allard J.
      Elevated serum Her-2/neu level predicts decreased response to hormone therapy in metastatic breast cancer..
      ,
      • Kandl H.
      • Seymour L.
      • Bezwoda W.R.
      Soluble c-erb B-2 fragments in serum correlates with disease stage and predicts more shortened survival in patients with early-stage and advanced breast cancer..
      ,
      • Volas G.H.
      • Leitzel K.
      • Teramoto Y.
      • Grossberg H.
      • Demers L.
      • Lipton A.
      Serial serum C-erbB-2 levels in patients with breast carcinoma..
      ,
      • Carney W.P.
      • Neumann R.
      • Lipton A.
      • Leitzel K.
      • Ali S.
      • Price C.P.
      Potential clinical utility of serum HER-2/neu oncoprotein concentrations in patients with breast cancer..
      ,
      • Revillion F.
      • Hebbar M.
      • Boneterre J.
      • Peyrat J.P.
      Plasma CerbB2 concentrations in relations to chemotherapy in breast cancer patients..
      ). Two studies involving 379 patients reported no significant association of serum levels with prognosis (
      • Kandl H.
      • Seymour L.
      • Bezwoda W.R.
      Soluble c-erb B-2 fragments in serum correlates with disease stage and predicts more shortened survival in patients with early-stage and advanced breast cancer..
      ,
      • Volas G.H.
      • Leitzel K.
      • Teramoto Y.
      • Grossberg H.
      • Demers L.
      • Lipton A.
      Serial serum C-erbB-2 levels in patients with breast carcinoma..
      ). Of the 11 studies in which serum HER-2/neu protein levels were tested for their ability to predict response to therapy, 8 (73%) of the studies found that elevated serum HER-2/neu protein levels predicted therapy resistance (
      • Willsher P.C.
      • Beaver J.
      • Pinder S.
      • Bell J.A.
      • Ellis I.O.
      • Blamey R.W.
      • Robertson J.F.
      Prognostic significance of serum CerbB2 protein in breast cancer patients..
      ,
      • Colomer R.
      • Montero S.
      • Lluch A.
      • Ojeda B.
      • Barnadas A.
      • Casado A.
      • Massuti B.
      • Cortes-Funes H.
      • Lloveras B.
      Circulating HER2 extracellular domain and resistance to chemotherapy in advanced breast cancer..
      ,
      • Bewick M.
      • Conlon M.
      • Gerard S.
      • Lee H.
      • Parissenti A.M.
      • Zhang L.
      • Gluck S.
      • Lafrenie R.M.
      HER-2 expression is a prognostic factor in patients with metastatic breast cancer treated with a combination of high-dose cyclophosphamide, mitoxantrone, paclitaxel and autologous blood stem cell support..
      ,
      • Harris L.N.
      • Liotcheva V.
      • Broadwater G.
      • Ramirez M.J.
      • Maimonis P.
      • Anderson S.
      • Everett T.
      • Harpole D.
      • Moore M.B.
      • Berry D.A.
      • Rizzeri D.
      • Vredenburgh J.J.
      • Bentley R.C.
      Comparison of methods of measuring HER-2 in metastatic breast cancer patients treated with high-dose chemotherapy..
      ,
      • Lipton A.
      • Ali S.M.
      • Leitzel K.
      • Demers L.
      • Chinchilli V.
      • Engle L.
      • Harvey H.A.
      • Brady C.
      • Nalin C.M.
      • Dugan M.
      • Carney W.
      • Allard J.
      Elevated serum Her-2/neu level predicts decreased response to hormone therapy in metastatic breast cancer..
      ), whereas three additional studies did not demonstrate this association (
      • Mehta R.R.
      • McDermott J.H.
      • Hieken T.J.
      • Marler K.C.
      • Patel M.K.
      • Wild L.D.
      • Das Gupta T.K.
      Plasma c-erbB-2 levels in breast cancer patients: Prognostic significance in predicting response to chemotherapy..
      ,
      • Volas G.H.
      • Leitzel K.
      • Teramoto Y.
      • Grossberg H.
      • Demers L.
      • Lipton A.
      Serial serum C-erbB-2 levels in patients with breast carcinoma..
      ,
      • Revillion F.
      • Hebbar M.
      • Boneterre J.
      • Peyrat J.P.
      Plasma CerbB2 concentrations in relations to chemotherapy in breast cancer patients..
      ). Serum HER-2/neu levels have correlated with decreased survival and absence of clinical response to hormonal therapy in estrogen receptor (ER)-positive tumors in some studies (
      • Burke H.B.
      • Hoang A.
      • Iglehart J.D.
      • Marks J.R.
      Predicting response to adjuvant and radiation therapy in patients with early stage breast carcinoma..
      ,
      • Lipton A.
      • Ali S.M.
      • Leitzel K.
      • Demers L.
      • Chinchilli V.
      • Engle L.
      • Harvey H.A.
      • Brady C.
      • Nalin C.M.
      • Dugan M.
      • Carney W.
      • Allard J.
      Elevated serum Her-2/neu level predicts decreased response to hormone therapy in metastatic breast cancer..
      ), but not in others (
      • Volas G.H.
      • Leitzel K.
      • Teramoto Y.
      • Grossberg H.
      • Demers L.
      • Lipton A.
      Serial serum C-erbB-2 levels in patients with breast carcinoma..
      ). Serum HER-2/neu protein measurements have successfully predicted resistance to high-dose chemotherapy (
      • Colomer R.
      • Montero S.
      • Lluch A.
      • Ojeda B.
      • Barnadas A.
      • Casado A.
      • Massuti B.
      • Cortes-Funes H.
      • Lloveras B.
      Circulating HER2 extracellular domain and resistance to chemotherapy in advanced breast cancer..
      ,
      • Bewick M.
      • Conlon M.
      • Gerard S.
      • Lee H.
      • Parissenti A.M.
      • Zhang L.
      • Gluck S.
      • Lafrenie R.M.
      HER-2 expression is a prognostic factor in patients with metastatic breast cancer treated with a combination of high-dose cyclophosphamide, mitoxantrone, paclitaxel and autologous blood stem cell support..
      ,
      • Harris L.N.
      • Liotcheva V.
      • Broadwater G.
      • Ramirez M.J.
      • Maimonis P.
      • Anderson S.
      • Everett T.
      • Harpole D.
      • Moore M.B.
      • Berry D.A.
      • Rizzeri D.
      • Vredenburgh J.J.
      • Bentley R.C.
      Comparison of methods of measuring HER-2 in metastatic breast cancer patients treated with high-dose chemotherapy..
      ), bone marrow transplantation (
      • Bewick M.
      • Conlon M.
      • Gerard S.
      • Lee H.
      • Parissenti A.M.
      • Zhang L.
      • Gluck S.
      • Lafrenie R.M.
      HER-2 expression is a prognostic factor in patients with metastatic breast cancer treated with a combination of high-dose cyclophosphamide, mitoxantrone, paclitaxel and autologous blood stem cell support..
      ), and response to trastuzumab single agent and combination treatment for metastatic HER-2/neu-positive disease (
      • Ali S.M.
      • Leitzel K.
      • Chinchilli V.M.
      • Engle L.
      • Demers L.
      • Harvey H.A.
      • Carney W.
      • Allard J.W.
      • Lipton A.
      Relationship of serum HER-2/neu and serum CA 15-3 in patients with metastatic breast cancer..
      ,
      • Lipton A.
      • Ali S.M.
      • Leitzel K.
      • Demers L.
      • Harvey H.A.
      • Chaudri-Ross H.A.
      • Brady C.
      • Wyld P.
      • Carney W.
      Serum HER-2/neu and response to the aromatase inhibitor letrozole versus tamoxifen..
      ,
      • Nunes R.A.
      • Harris L.N.
      The HER2 extracellular domain as a prognostic and predictive factor in breast cancer..
      ,
      • Slamon D.
      • Pegram M.
      Rationale for trastuzumab (Herceptin) in adjuvant breast cancer trials..
      ).

      HER-2/neu Expression and Breast Pathology—

      HER-2/neu overexpression has been consistently associated with higher grade and extensive forms of ductal carcinoma in situ (
      • Bose S.
      • Lesser M.L.
      • Norton L.
      • Rosen P.P.
      Immunophenotype of intraductal carcinoma..
      ,
      • Moreno A.
      • Lloveras B.
      • Figueras A.
      • Escobedo A.
      • Ramon J.M.
      • Sierra A.
      • Fabra A.
      Ductal carcinoma in-situ of the breast: Correlation between histologic classification and biologic markers..
      ,
      • Mack L.
      • Kerkzelit N.
      • Doig G.
      • O’Malley F.P.
      Relationship of a new histological categorization of ductal carcinoma in-situ of the breast with size and the immunohistochemical expression of p53, C-erb B2, bcl2 and ki-67..
      ). HER-2/neu gene amplification occurs at a lower rate (less than 10%) and has been linked to an adverse outcome in invasive lobular carcinoma (
      • Rosenthal S.I.
      • Depowski P.L.
      • Sheehan C.E.
      • Ross J.S.
      Comparison of HER-2/neu oncogene amplification detected by fluorescence in situ hybridization in lobular and ductal breast cancer..
      ). The frequency of HER-2/neu gene amplification appears to be strongly correlated with tumor grade and ductal versus lobular status. Only 1 of 73 grade I invasive ductal carcinomas and 1 of 67 classic lobular carcinomas showed amplification of the HER-2 gene. HER-2/neu overexpression has been a consistent feature of both mammary and extramammary Paget’s disease (
      • Wolber R.A.
      • DuPuis B.A.
      • Wick M.R.
      Expression of cerb B2 oncoprotein in mammary and extramammary Paget’s disease..
      ,
      • Fu W.
      • Lobocki C.A.
      • Silberberg B.K.
      Molecular markers in Paget disease of the breast..
      ,
      • Hanna W.
      • Alowami S.
      • Malik A.
      The role of HER-2/neu oncogene and vimentin filaments in the production of the Paget’s phenotype..
      ). The majority of studies that have compared the HER-2/neu status in paired primary and metastatic tumor tissues have found an overwhelming consistency of the patient’s status regardless of the method of testing (immunohistochemistry (IHC) versus fluorescence in situ hybridization (FISH)) (
      • Masood S.
      • Bui M.M.
      Assessment of Her-2/neu overexpression in primary breast cancers and their metastatic lesions: An immunohistochemical study..
      ,
      • Dittadi R.
      • Zancan M.
      • Perasole A.
      • Gion M.
      Evaluation of HER-2/neu in serum and tissue of primary and metastatic breast cancer patients using an automated enzyme immunoassay..
      ,
      • Simon R.
      • Nocito A.
      • Hubscher T.
      • Bucher C.
      • Torhorst J.
      • Schraml P.
      • Bubendorf L.
      • Mihatsch M.M.
      • Moch H.
      • Wilber K.
      • Schotzau A.
      • Kononen J.
      • Sauter G.
      Patterns of her-2/neu amplification and overexpression in primary and metastatic breast cancer..
      ,
      • Vincent-Salomon A.
      • Jouve M.
      • Genin P.
      • Freneaux P.
      • Sigal-Zafrani B.
      • Caly M.
      • Beuzeboc P.
      • Pouillart P.
      • Sastre-Garau X.
      HER2 status in patients with breast carcinoma is not modified selectively by preoperative chemotherapy and is stable during the metastatic process..
      ,
      • Xu R.
      • Perle M.A.
      • Inghirami G.
      • Chan W.
      • Delgado Y.
      • Feiner H.
      Amplification of Her-2/neu gene in Her-2/neu-overexpressing and -nonexpressing breast carcinomas and their synchronous benign, premalignant, and metastatic lesions detected by FISH in archival material..
      ,
      • Symmans W.F.
      • Liu J.
      • Knowles D.M.
      • Inghirami G.
      Breast cancer heterogeneity: Evaluation of clonality in primary and metastatic lesions..
      ). In one study of node-positive tumors that were defined as biclonal by DNA ploidy profile, HER-2/neu status was determined by IHC in 17 primary tumors and their 82 axillary lymph node metastases (
      • Symmans W.F.
      • Liu J.
      • Knowles D.M.
      • Inghirami G.
      Breast cancer heterogeneity: Evaluation of clonality in primary and metastatic lesions..
      ). Despite this apparent heterogeneity of the predominant clone measured by ploidy status, in each metastatic site the HER-2/neu status was consistent between primary tumors and their corresponding metastases (
      • Symmans W.F.
      • Liu J.
      • Knowles D.M.
      • Inghirami G.
      Breast cancer heterogeneity: Evaluation of clonality in primary and metastatic lesions..
      ). HER-2/neu amplification and overexpression has been associated with adverse outcome in some studies of male breast carcinoma (
      • Gattuso P.
      • Reddy V.B.
      • Green L.K.
      • Bloom K.J.
      Prognostic factors for carcinoma of the male breast..
      ,
      • Joshi M.G.
      • Lee A.K.
      • Loda M.
      • Camus M.G.
      • Pedersen C.
      • Heatley G.J.
      • Hughes K.S.
      Male breast carcinoma: An evaluation of prognostic factors contributing to a poorer outcome..
      ,
      • Pich A.
      • Margaria E.
      • Chiusa L.
      Oncogenes and male breast carcinoma: c-erbB-2 and p53 coexpression predicts a poor survival..
      ,
      • Wang-Rodriguez J.
      • Cross K.
      • Gallagher S.
      • Djahanban M.
      • Armstrong J.M.
      • Wiedner N.
      • Shapiro D.H.
      Male breast carcinoma: Correlation of ER, PR, Ki-67, Her2-Neu, and p53 with treatment and survival, a study of 65 cases..
      ), but not in others (
      • Rayson D.
      • Erlichman C.
      • Suman V.J.
      • Roche P.C.
      • Wold L.E.
      • Ingle J.N.
      • Donohue J.H.
      Molecular markers in male breast carcinoma..
      ,
      • Shpitz B.
      • Bomstein Y.
      • Sternberg A.
      • Klein E.
      • Liverant S.
      • Groisman G.
      • Bernheim J.
      Angiogenesis, p53, and c-erbB-2 immunoreactivity and clinicopathological features in male breast cancer..
      ,
      • Bloom K.J.
      • Govil H.
      • Gattuso P.
      • Reddy V.
      • Francescatti D.
      Status of HER-2 in male and female breast carcinoma..
      ). Finally, low-level HER-2/neu overexpression has been identified in benign breast disease biopsies and associated with an increased risk of subsequent invasive breast cancer (
      • Stark A.
      • Hulka B.S.
      • Joens S.
      • Novotny D.
      • Thor A.D.
      • Wold L.E.
      • Schell M.J.
      • Melton 3rd, L.J.
      • Liu E.T.
      • Conway K.
      HER-2/neu amplification in benign breast disease and the risk of subsequent breast cancer..
      ).

      Interaction of HER-2/neu Expression with Other Prognosis Variables—

      HER-2/neu gene amplification and protein overexpression have been associated consistently with high tumor grade, DNA aneuploidy, high cell proliferation rate, negative assays for nuclear protein receptors for estrogen and progesterone, p53 mutation, topoisomerase IIa amplification, and alterations in a variety of other molecular biomarkers of breast cancer invasiveness and metastasis (
      • Navolanic P.M.
      • Steelman L.S.
      • McCubrey J.A.
      EGFR family signaling and its association with breast cancer development and resistance to chemotherapy..
      ,
      • Stark A.
      • Hulka B.S.
      • Joens S.
      • Novotny D.
      • Thor A.D.
      • Wold L.E.
      • Schell M.J.
      • Melton 3rd, L.J.
      • Liu E.T.
      • Conway K.
      HER-2/neu amplification in benign breast disease and the risk of subsequent breast cancer..
      ,
      • Hayes D.F.
      • Thor A.D.
      c-erbB-2 in breast cancer: Development of a clinically useful marker..
      ,
      • Masood S.
      • Bui M.M.
      Prognostic and predictive value of HER2/neu oncogene in breast cancer..
      ,
      • Eccles S.A.
      The role of c-erbB-2/HER2/neu in breast cancer progression and metastasis..
      ,
      • Piccart M.
      • Lohrisch C.
      • Di Leo A.
      • Larsimont D.
      The predictive value of HER2 in breast cancer..
      ,
      • Yarden Y.
      Biology of HER2 and its importance in breast cancer..
      ,
      • Zemzoum I.
      • Kates R.E.
      • Ross J.S.
      • Dettmar P.
      • Dutta M.
      • Henrichs C.
      • Yurdseven S.
      • Hofler H.
      • Kiechle M.
      • Schmitt M.
      • Harbeck N.
      Invasion factors uPA/PAI-1 and HER2 status provide independent and complementary information on patient outcome in node-negative breast cancer..
      ).

      Phosphorylated HER2 as a Potential Prognostic and Predictive Marker—

      It has been argued that one potential confounding aspect of the existing HER-2 tests is that they only detect gene amplification or protein overexpression that does not necessarily reflect the functional activity of the receptor. If HER-2 is truly important in the pathobiology of breast cancer, the receptor must be activated to exert its effects. A common feature of signal transduction through membrane-bound receptor tyrosine kinases is autophosphorylation of the receptor. Autophosphorylation of HER-2, therefore, may be used as a surrogate for active signaling. Monoclonal antibodies have been developed to detect autophosphorylated HER-2 by IHC (
      • DiGiovanna M.P.
      • Stern D.F.
      Activation state-specific monoclonal antibody detects tyrosine phosphorylated p185neu/erbB-2 in a subset of human breast tumors overexpressing this receptor..
      ). In invasive breast cancer with HER-2 overexpression, the receptor appears to be activated only in a small subset (12%) of patients (
      • DiGiovanna M.P.
      • Stern D.F.
      Activation state-specific monoclonal antibody detects tyrosine phosphorylated p185neu/erbB-2 in a subset of human breast tumors overexpressing this receptor..
      ,
      • Thor A.D.
      • Liu S.
      • Edgerton S.
      • Moore 2nd, D.
      • Kasowitz K.M.
      • Benz C.C.
      • Stern D.F.
      • DiGiovanna M.P.
      Activation (tyrosine phosphorylation) of ErbB-2 (HER-2/neu): A study of incidence and correlation with outcome in breast cancer..
      ). Interestingly, the proportion of cases with phosphorylated HER-2 appears to be greater (58%) in ductal carcinoma in situ (
      • DiGiovanna M.P.
      • Chu P.
      • Davison T.L.
      • Howe C.L.
      • Carter D.
      • Claus E.B.
      • Stern D.F.
      Active signaling by HER-2/neu in a subpopulation of HER-2/neu-overexpressing ductal carcinoma in situ: Clinicopathological correlates..
      ). In one large study of 800 cases of invasive breast cancer with HER-2 overexpression, only cases with phosphorylated HER-2 displayed adverse prognosis (
      • Thor A.D.
      • Liu S.
      • Edgerton S.
      • Moore 2nd, D.
      • Kasowitz K.M.
      • Benz C.C.
      • Stern D.F.
      • DiGiovanna M.P.
      Activation (tyrosine phosphorylation) of ErbB-2 (HER-2/neu): A study of incidence and correlation with outcome in breast cancer..
      ). Cases with overexpressed but unphosphorylated receptor had a prognosis as favorable as non-HER-2-overexpressing cases, which supports the concept that phopsho-HER-2 may be a more powerful prognostic marker than overall HER-2 protein overexpression. The role of phosphorylated HER-2 as a predictor to trastuzumab therapy is currently unknown. Tissue specimens from the pivotal Herceptin studies are being re-analyzed to answer this very important question.

      TESTING FOR HER-2/neu STATUS IN BREAST CANCER

      Both morphology-based and molecular-based techniques have been used to measure HER-2/neu status in breast cancer clinical samples (Table II) (
      • Rosenthal S.I.
      • Depowski P.L.
      • Sheehan C.E.
      • Ross J.S.
      Comparison of HER-2/neu oncogene amplification detected by fluorescence in situ hybridization in lobular and ductal breast cancer..
      ,
      • Slamon D.J.
      • Clark G.M.
      • Wong S.G.
      • Levin W.J.
      • Ullrich A.
      • McGuire W.L.
      Human breast cancer: Correlation of relapse and survival with amplification of the Her-2/neu oncogene..
      ,
      • Berger M.S.
      • Locher G.W.
      • Saurer S.
      • Gullick W.J.
      • Waterfield M.D.
      • Groner B.
      • Hynes N.E.
      Correlation of c-erb B2 gene amplification and protein expression in human breast carcinoma with nodal status and nuclear grading..
      ,
      • van de Vivjer M.J.
      • Peterse J.L.
      • Mooi W.J.
      • Wisman P.
      • Lomans J.
      • Dalesio O.
      • Nusse R.
      Neu-protein overexpression in breast cancer..
      ,
      • Heintz N.H.
      • Leslie K.O.
      • Rogers L.A.
      • Howard P.L.
      Amplification of the c-erb B 2 oncogene in prognosis of breast adenocarcinoma..
      ,
      • Wright C.
      • Angus B.
      • Nicholson S.
      • Sainsbury J.R.
      • Cairns J.
      • Gullick W.J.
      • Kelly P.
      • Harris A.L.
      • Horne C.H.
      Expression of c-erbB-2 oncoprotein: A prognostic indicator in human breast cancer..
      ,
      • Tsuda H.
      • Hirohashi S.
      • Shimosato Y.
      • Hirota T.
      • Tsugane S.
      • Watanabe S.
      • Terada M.
      • Yamamoto H.
      Correlation between histologic grade of malignancy and copy number of c-erbB-2 gene in breast carcinoma. A retrospective analysis of 176 cases..
      ,
      • Borg A.
      • Tandon A.K.
      • Sigurdsson H.
      • Clark G.M.
      • Ferno M.
      • Fuqua S.A.
      • Killander D.
      • McGuire W.L.
      HER-2/neu amplification predicts poor survival in node-positive breast cancer..
      ,
      • Paik S.
      • Hazan R.
      • Fisher E.R.
      • Sass R.E.
      • Fisher B.
      • Redmond C.
      • Schlessinger J.
      • Lippman M.E.
      • King C.R.
      Pathologic findings from the nations surgical adjuvant breast and bowel project: Prognostic significance of erb B2 protein overexpression in primary breast cancer..
      ,
      • Battifora H.
      • Gaffey M.
      • Esteban J.
      • Mehta P.
      • Bailey A.
      • Faucett C.
      • Niland J.
      Immunohistochemical assay of neu/c-erb B-2 oncogene product in paraffin-embedded tissues in early breast cancer: Retrospective follow-up study of 245 stage I and II cases..
      ,
      • Kallioniemi O.P.
      • Holli K.
      • Visakorpi T.
      • Koivula T.
      • Helin H.H.
      • Isola J.J.
      Association of C-erb B2 protein over-expression with high rate of cell proliferation, increased risk of visceral metastasis and poor long-term survival in breast cancer..
      ,
      • Gullick W.J.
      • Love S.B.
      • Wright C.
      • Barnes D.M.
      • Gusterson B.
      • Harris A.L.
      • Altman D.G.
      c-erbB-2 protein overexpression in breast cancer is a risk factor in patients with involved and uninvolved lymph nodes..
      ,
      • Clark G.M.
      • McGuire W.L.
      Follow-up study of HER-2/neu amplification in primary breast cancer..
      ,
      • Lovekin C.
      • Ellis I.O.
      • Locker A.
      • Robertson J.F.
      • Bell J.
      • Nicholson R.
      • Gullick W.J.
      • Elston C.W.
      • Blamey R.W.
      C-erb B2 oncoprotein expression in primary and advanced breast cancer..
      ,
      • McCann A.H.
      • DeDervan T.A.
      • O’Regan M.
      • Codd M.B.
      • Gullick W.J.
      • Tobin B.M.
      • Carney D.N.
      Prognostic significance of C-erb B2 and estrogen receptor status in human breast cancer..
      ,
      • Dykens R.
      • Corbett I.P.
      • Henry J.
      • Wright C.
      • Yuan J.
      • Hennessy C.
      • Lennard T.J.
      • Angus B.
      • Horne C.H.
      Long term survival in breast cancer related to overexpression of the C-erb B2 oncoprotein: An immunohistochemical study using monoclonal antibody NCL-CB11..
      ,
      • Rilke F.
      • Colnaghi M.I.
      • Cascinelli N.
      • Andreola S.
      • Baldini M.T.
      • Bufalino R.
      • Della Porta G.
      • Menard S.
      • Pierotti M.A.
      • Testori A.
      Prognostic significance of HER-2/neu expression in breast cancer and its relationship to other prognostic factors..
      ,
      • Winstanley J.
      • Cooke T.
      • Murray G.D.
      • Platt-Higgins A.
      • George W.D.
      • Holt S.
      • Myskov M.
      • Spedding A.
      • Barraclough B.R.
      • Rudland P.S.
      The long term prognostic significance of C-erb B2 in primary breast cancer..
      ,
      • O’Reilly S.M.
      • Barnes D.M.
      • Camplejohn R.S.
      • Bartkova J.
      • Gregory W.M.
      • Richards M.A.
      The relationship between C-erb B2 expression, and s-phase fraction in prognosis in breast cancer..
      ,
      • Paterson M.C.
      • Dietrich K.D.
      • Danyluk J.
      • Paterson A.H.
      • Lees A.W.
      • Jamil N.
      • Hanson J.
      • Jenkins H.
      • Krause B.E.
      • McBlain W.A.
      • et al.
      Correlation between C erb B2 amplification and risk of recurrent disease in node-negative breast cancer..
      ,
      • Toikkanen S.
      • Helin H.
      • Isola J.
      • Joensuu H.
      Prognostic significance of Her-2 oncoprotein expression in breast cancer: A 30-year follow up..
      ,
      • Molina R.
      • Ciocca D.R.
      • Candon A.K.
      • Allred D.C.
      • Clark G.M.
      • Chamness G.C.
      • Gullick W.J.
      • McGuire W.L.
      Expression of HER-2/neu oncoprotein in breast cancer: A comparison of immunohistochemical and western blot techniques..
      ,
      • Noguchi M.
      • Koyasaki M.
      • Ohta N.
      • Kitagawa H.
      • Earashi M.
      • Thomas M.
      • Miyazaki I.
      • Mizukami Y.
      c-erb B-2 oncoprotein expression versus internal mammary lymph node metastases as additional prognostic factors in patients with axillary lymph node-positive breast cancer..
      ,
      • Allred D.C.
      • Clark G.M.
      • Tandon A.K.
      • Molina R.
      • Tormey D.C.
      • Osborne C.K.
      • Gilchrist K.W.
      • Mansour E.G.
      • Abeloff M.
      • Eudey L.
      • et al.
      HER-2/neu node-negative breast cancer: Prognostic significance of overexpression influenced by the presence of in-situ carcinoma..
      ,
      • Babiak J.
      • Hugh J.
      • Poppeme S.
      Significance of c-erb B-2 amplification in DNA aneuploidy. Analysis in 78 patients with node-negative breast cancer..
      ,
      • Tiwari R.K.
      • Borgen P.I.
      • Wong G.Y.
      • Cordon-Cardo C.
      • Osborne M.P.
      HER-2/neu amplification and overexpression in primary human breast cancer is associated with early metastasis..
      ,
      • Gusterson B.A.
      • Gelber R.D.
      • Goldhirsch A.
      • Price K.N.
      • Save-Soderborgh J.
      • Anbazhagan R.
      • Styles J.
      • Rudenstam C.M.
      • Golouh R.
      • Reed R.
      • et al.
      Prognostic importance of C-erb B2 expression in breast cancer..
      ,
      • Bianchi S.
      • Paglierani M.
      • Zampi G.
      • Cardona G.
      • Cataliotti L.
      • Bonardi R.
      • Ciatto S.
      Prognostic significance of C-erb B2 expression in node negative breast cancer..
      ,
      • Press M.F.
      • Pike M.C.
      • Chazin V.R.
      • Hung G.
      • Udove J.A.
      • Markowicz M.
      • Danyluk J.
      • Godolphin W.
      • Sliwkowski M.
      • Akita R.
      • et al.
      Her-2/neu expression in node-negative breast cancer: Direct tissue quantification by computerized image analysis and association of overexpression with increased risk of recurrent disease..
      ,
      • Seshadri R.
      • Firgaira F.A.
      • Horsfall D.J.
      • McCaul K.
      • Setlur V.
      • Kitchen P.
      Clinical significance of Her-2/neu oncogene amplification in primary breast cancer..
      ,
      • Descotes F.
      • Pavy J.-J.
      • Adessi G.L.
      Human breast cancer: Correlation study between Her-2/neu amplification and prognostic factors in an unselected population..
      ,
      • Giai M.
      • Roagna R.
      • Ponzone R.
      • De Bortoli M.
      • Dati C.
      • Sismondi P.
      Prognostic and predictive relevance of C-erb B2 and ras expression in node positive and negative breast cancer..
      ,
      • Muss H.B.
      • Thor A.D.
      • Berry D.A.
      • Kute T.
      • Liu E.T.
      • Koerner F.
      • Cirrincione C.T.
      • Budman D.R.
      • Wood W.C.
      • Barcos M.
      • et al.
      Cerb-B2 expression and response to adjuvant therapy in women with node-positive early breast cancer..
      ,
      • Tetu B.
      • Brisson J.
      Prognostic significance of Her-2/neu oncogene expression in node-positive breast cancer. The influence of the pattern of immunostaining and adjuvant therapy..
      ,
      • Hartmann L.C.
      • Ingle J.N.
      • Wold L.E.
      • Farr Jr., G.H.
      • Grill J.P.
      • Su J.Q.
      • Maihle N.J.
      • Krook J.E.
      • Witzig T.E.
      • Roche P.C.
      Prognostic value of CerbB2 overexpression in axillary lymph node positive breast cancer. Results from a randomized adjuvant treatment protocol..
      ,
      • Jacquemeier J.
      • Penault-Llorca P.
      • Viens P.
      • Houvenaeghel G.
      • Hassoun J.
      • Torrente M.
      • Adelaide J.
      • Birnbaum D.
      Breast cancer response to adjuvant chemotherapy in correlation with erb B2 and p53 expression..
      ,
      • Marks J.R.
      • Humphrey P.A.
      • Wu K.
      • Berry D.
      • Bandarenko N.
      • Kerns B.J.
      • Iglehart J.D.
      Overexpression of p53 and Her 2/neu proteins as prognostic markers in early stage breast cancer..
      ,
      • Rosen P.P.
      • Lesser M.L.
      • Arroyo C.D.
      • Cranor M.
      • Borgen P.
      • Norton L.
      • et al.
      Immunohistochemical detection of Her-2/neu expression in patients with axillary lymph node-negative breast carcinoma. A study of epidemiologic risk factors, histologic features and prognosis..
      ,
      • Quenel N.
      • Wafflart J.
      • Bonichon F.
      • de Mascarel I.
      • Trojani M.
      • Durand M.
      • Avril A.
      • Coindre J.M.
      The prognostic value of c-erbB2 in primary breast carcinomas: A study on 942 cases..
      ,
      • Sundblad A.S.
      • Pellicer E.M.
      • Ricci L.
      Carcinoembryonic expression in stages I and II breast cancer; its relationship with clinicopathologic factors..
      ,
      • O’Malley F.P.
      • Saad Z.
      • Kerkvliet N.
      • Doig G.
      • Stitt L.
      • Ainsworth P.
      • Hundal H.
      • Chambers A.F.
      • Turnbull D.I.
      • Bramwell V.
      The predictive power of semiquantitative immunohistochemical assessment of p53 and C-erbB2 in lymph node-negative breast cancer..
      ,
      • Hieken T.J.
      • Mehta R.R.
      • Shilkaitis A.
      Her-2/neu and p53 expression in breast cancer: Valid prognostic markers when assessed by direct immunoassay, but not by immunochemistry..
      ,
      • Xing W.-R.
      • Gilchrist K.W.
      • Harris C.P.
      • Samson W.
      • Meisner L.F.
      FISH detection of HER-2/neu oncogene amplification in early onset breast cancer..
      ,
      • Dittadi R.
      • Brazzale A.
      • Pappagallo G.
      • Salbe C.
      • Nascimben O.
      • Rosabian A.
      • Gion M.
      ErbB2 assay in breast cancer: Possibly improved clinical information using a quantitative method..
      ,
      • Fernandez Acenero M.J.
      • Farina Gonzalez J.
      • Arangoncillo Ballesteros P.
      Immunohistochemical expression of p53 and c-erbB-2 in breast carcinoma: Relation with epidemiologic factors, histologic features and prognosis..
      ,
      • Eissa S.
      • Khalifa A.
      • el-Gharib A.
      • Salah N.
      • Mohamed M.K.
      Multivariate analysis of DNA ploidy, p53, c-erbB-2 proteins, EGFR, and steroid hormone receptors for short-term prognosis in breast cancer..
      ,
      • Charpin C.
      • Garcia S.
      • Bouvier C.
      • Martini F.
      • Lavaut M.N.
      • Allasia C.
      • Bonnier P.
      • Andrac L.
      c-erbB-2 oncoprotein detected by automated quantitative immunocytochemistry in breast carcinomas correlates with patients’ overall and disease-free survival..
      ,
      • Press M.J.
      • Bernstein L.
      • Thomas P.A.
      • Meisner L.F.
      • Zhou J.Y.
      • Ma Y.
      • Hung G.
      • Robinson R.A.
      • Harris C.
      • El-Naggar A.
      • Slamon D.J.
      • Phillips R.N.
      • Ross J.S.
      • Wolman S.R.
      • Flom K.J.
      Her-2/neu gene amplification characterized by fluorescence in situ hybridization: Poor prognosis in node negative breast carcinomas..
      ,
      • Ross J.S.
      • Muraca P.J.
      • Jaffe D.
      • Morgan T.
      • Fletcher J.A.
      Multivariate analysis of prognostic factors in lymph node negative breast cancer..
      ,
      • Depowski P.L.
      • Brien T.P.
      • Sheehan C.E.
      • Stylos S.
      • Johnson R.L.
      • Ross J.S.
      Prognostic significance of p34cdc2 cyclin-dependent kinase and MIB1 overexpression, and HER-2/neu gene amplification detected by fluorescence in situ hybridization in breast cancer..
      ,
      • Querzoli P.
      • Albonico G.
      • Ferretti S.
      • Rinaldi R.
      • Beccati D.
      • Corcione S.
      • Indelli M.
      • Nenci I.
      Modulation of biomarkers in minimal breast carcinoma..
      ,
      • Andrulis I.L.
      • Bull S.B.
      • Blasckstein M.E.
      • Sutherland D.
      • Mak C.
      • Sidlofsky S.
      • Pritzker K.P.
      • Hartwick R.W.
      • Hanna W.
      • Lickley L.
      • Wilkinson R.
      • Qizilbash A.
      • Ambus U.
      • Lipa M.
      • Weizel H.
      • Katz A.
      • Baida M.
      • Mariz S.
      • Stoik G.
      • Dacamara P.
      • Strongitharm D.
      • Geddie W.
      • McCready D.
      Neu/erbB-2 amplification identifies a poor-prognosis group of women with node-negative breast cancer..
      ,
      • Sjogren S.
      • Inganas M.
      • Lindgren A.
      • Holmberg L.
      • Bergh J.
      Prognostic and predictive value of c-erbB-2 overexpression in primary breast cancer, alone and in combination with other prognostic markers..
      ,
      • Harbeck N.
      • Ross J.S.
      • Yurdseven S.
      • Dettmar P.
      • Polcher M.
      • Kuhn W.
      • Ulm K.
      • Graeff H.
      • Schmitt M.
      HER-2/neu gene amplification by fluorescence in situ hybridization allows risk-group assessment in node-negative breast cancer..
      ,
      • Scorilas A.
      • Yotis J.
      • Pateras C.
      • Trangas T.
      • Talieri M.
      Predictive value of c-erbB-2 and cathepsin-D for Greek breast cancer patients using univariate and multivariate analysis..
      ,
      • Rudolph P.
      • Olsson H.
      • Bonatz G.
      • Ratjen V.
      • Bolte H.
      • Baldetorp B.
      • Ferno M.
      • Parwaresch R.
      • Alm P.
      Correlation between p53, c-erbB-2, and topoisomerase II alpha expression, DNA ploidy, hormonal receptor status and proliferation in 356 node-negative breast carcinomas: Prognostic implications..
      ,
      • Reed W.
      • Hannisdal E.
      • Boehler P.J.
      • Gundersen S.
      • Host H.
      • Marthin J.
      The prognostic value of p53 and c-erb B-2 immunostaining is overrated for patients with lymph node negative breast carcinoma: A multivariate analysis of prognostic factors in 613 patients with a follow-up of 14–30 years..
      ,
      • Pauletti G.
      • Dandekar S.
      • Rong H.
      • Ramos L.
      • Peng H.
      • Seshadri R.
      • Slamon D.J.
      Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: A direct comparison of fluorescence in situ hybridization and immunohistochemistry..
      ,
      • Kakar S.
      • Puangsuvan N.
      • Stevens J.M.
      • Serenas R.
      • Mangan G.
      • Sahai S.
      • Mihalov M.L.
      HER-2/neu assessment in breast cancer by immunohistochemistry and fluorescence in situ hybridization: Comparison of results and correlation with survival..
      ,
      • Agrup M.
      • Stal O.
      • Olsen K.
      • Wingren S.
      C-erbB-2 overexpression and survival in early onset breast cancer..
      ,
      • Umekita Y.
      • Ohi Y.
      • Sagara Y.
      • Yoshida H.
      Co-expression of epidermal growth factor receptor and transforming growth factor-alpha predicts worse prognosis in breast-cancer patients..
      ,
      • Pawlowski V.
      • Revillion F.
      • Hebbar M.
      • Hornez L.
      • Peyrat J.P.
      Prognostic value of the type I growth factor receptors in a large series of human primary breast cancers quantified with a real-time reverse transcription-polymerase chain reaction assay..
      ,
      • Volpi A.
      • De Paola F.
      • Nanni O.
      • Granato A.M.
      • Bajorko P.
      • Becciolini A.
      • Scarpi E.
      • Riccobon A.
      • Balzi M.
      • Amadori D.
      Prognostic significance of biologic markers in node-negative breast cancer patients: A prospective study..
      ,
      • Carr J.A.
      • Havstad S.
      • Zarbo R.J.
      • Divine G.
      • Mackowiak P.
      • Velanovich V.
      The association of HER-2/neu amplification with breast cancer recurrence..
      ,
      • Ferrero-Pous M.
      • Hacene K.
      • Bouchet C.
      • Le Doussal V.
      • Tubiana-Hulin M.
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      Relationship between c-erbB-2 and other tumor characteristics in breast cancer prognosis..
      ,
      • Platt-Higgins A.M.
      • Renshaw C.A.
      • West C.R.
      • Winstanley J.H.
      • De Silva Rudland S.
      • Barraclough R.
      • Rudland P.S.
      Comparison of the metastasis-inducing protein S100A4 (p9ka) with other prognostic markers in human breast cancer..
      ,
      • Eppenberger-Castori S.
      • Kueng W.
      • Benz C.
      • Caduff R.
      • Varga Z.
      • Bannwart F.
      • Fink D.
      • Dieterich H.
      • Hohl M.
      • Muller H.
      • Paris K.
      • Schoumacher F.
      • Eppenberger U.
      Prognostic and predictive significance of ErbB-2 breast tumor levels measured by enzyme immunoassay..
      ,
      • Jukkola A.
      • Bloigu R.
      • Soini Y.
      • Savolainen E.R.
      • Holli K.
      • Blanco G.
      c-erbB-2 positivity is a factor for poor prognosis in breast cancer and poor response to hormonal or chemotherapy treatment in advanced disease..
      ,
      • Gaci Z.
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      • Gaci M.
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      • Ingrand P.
      • Metaye T.
      Prognostic impact of cathepsin D and c-erbB-2 oncoprotein in a subgroup of node-negative breast cancer patients with low histological grade tumors..
      ,
      • Rudolph P.
      • Alm P.
      • Olsson H.
      • Heidebrecht H.J.
      • Ferno M.
      • Baldetorp B.
      • Parwaresch R.
      Concurrent overexpression of p53 and c-erbB-2 correlates with accelerated cycling and concomitant poor prognosis in node-negative breast cancer..
      ,
      • Beenken S.W.
      • Grizzle W.E.
      • Crowe D.R.
      • Conner M.G.
      • Weiss H.L.
      • Sellers M.T.
      • Krontiras H.
      • Urist M.M.
      • Bland K.I.
      Molecular biomarkers for breast cancer prognosis: Coexpression of c-erbB-2 and p53..
      ,
      • Pinto A.E.
      • Andre S.
      • Pereira T.
      • Nobrega S.
      • Soares J.
      C-erbB-2 oncoprotein overexpression identifies a subgroup of estrogen receptor positive (ER+) breast cancer patients with poor prognosis..
      ,
      • Riou G.
      • Mathieu M.C.
      • Barrois M.
      • Le Bihan M.L.
      • Ahomadegbe J.C.
      • Benard J.
      • Le M.G.
      c-erbB-2 (HER-2/neu) gene amplification is a better indicator of poor prognosis than protein over-expression in operable breast-cancer patients..
      ,
      • Horita K.
      • Yamaguchi A.
      • Hirose K.
      • Ishida M.
      • Noriki S.
      • Imamura Y.
      • Fukuda M.
      Prognostic factors affecting disease-free survival rate following surgical resection of primary breast cancer..
      ,
      • Suo Z.
      • Risberg B.
      • Kalsson M.G.
      • Willman K.
      • Tierens A.
      • Skovlund E.
      • Nesland J.M.
      EGFR family expression in breast carcinomas. c-erbB-2 and c-erbB-4 receptors have different effects on survival..
      ,
      • Ristimaki A.
      • Sivula A.
      • Lundin J.
      • Lundin M.
      • Salminen T.
      • Haglund C.
      • Joensuu H.
      • Isola J.
      Prognostic significance of elevated cyclooxygenase-2 expression in breast cancer..
      ,
      • Tsutsui S.
      • Ohno S.
      • Murakami S.
      • Hachitanda Y.
      • Oda S.
      Prognostic value of c-erbB2 expression in breast cancer..
      ,
      • Spizzo G.
      • Obrist P.
      • Ensinger C.
      • Theurl I.
      • Dunser M.
      • Ramoni A.
      • Gunsilius E.
      • Eibl G.
      • Mikuz G.
      • Gastl G.
      Prognostic significance of Ep-CAM AND Her-2/neu overexpression in invasive breast cancer..
      ,
      • Kato T.
      • Kameoka S.
      • Kimura T.
      • Nishikawa T.
      • Kobayashi M.
      C-erbB-2 and PCNA as prognostic indicators of long-term survival in breast cancer..
      ,
      • el-Ahmady O.
      • el-Salahy E.
      • Mahmoud M.
      • Wahab M.A.
      • Eissa S.
      • Khalifa A.
      Multivariate analysis of bcl-2, apoptosis, P53 and HER-2/neu in breast cancer: A short-term follow-up..
      ). Of the 80 studies considering 26,309 patients listed in Table III, 72 (90%) of the studies and 24,314 (92%) of the cases found that either HER-2/neu gene amplification or HER-2 (p185 neu) protein overexpression predicted breast cancer outcome on either univariate or multivariate analysis. In 51 (71%) of the 72 studies that featured multivariate analysis of outcome data, the adverse prognostic significance of HER-2/neu gene, message, or protein overexpression was independent of all other prognostic variables. Thirteen (16%) of the studies reported prognostic significance on univariate analysis only (in eight studies multivariate analysis was not performed). Only eight (10%) of the studies, encompassing 1,995 (8%) of the patients, showed no correlation between HER-2/neu status and outcome. Of these eight studies, five (63%) used IHC on paraffin-embedded tissues as the HER-2/protein detection technique, two (25%) used Southern analysis, and one (13%) used a reverse transcription PCR (RT-PCR) technique. All eight studies that used the FISH technique showed univariate prognostic significance of gene amplification, and seven of these (83%) showed prognostic significance on multivariate analysis as well.
      Table IIMethods of detection of HER-2/neu status in breast cancer
      MethodTargetFDA-approvedSlide-based
      IHCProteinYes
      For prognosis and prediction of response and eligibility to receive trastuzumab therapy.
      Yes
      FISHGeneYes
      For prognosis and prediction of response and eligibility to receive trastuzumab therapy.
      Yes
      CISHGeneNoYes
      Southern blotGeneNoNo
      RT-PCRmRNANoNo
      Microarray TP
      TP, transcriptional profiling.
      mRNANoNo
      Tumor ELISAProteinNoNo
      Serum ELISAProteinYes
      For monitoring response of breast cancer to treatment.
      No
      a For prognosis and prediction of response and eligibility to receive trastuzumab therapy.
      b TP, transcriptional profiling.
      c For monitoring response of breast cancer to treatment.
      Table IIIHER-2/neu status and prognosis in breast cancer
      Study/yearFirst author/ref.No. of casesSpecimen typeMethod(s) of analysisUnivariate significanceMultivariate significanceComment
      1 1987Slamon
      • Slamon D.J.
      • Clark G.M.
      • Wong S.G.
      • Levin W.J.
      • Ullrich A.
      • McGuire W.L.
      Human breast cancer: Correlation of relapse and survival with amplification of the Her-2/neu oncogene..
      189FrozenSouthern blotYesYesHER-2 amplification predicted overall survival and time to relapse
      2 1988Berger
      • Berger M.S.
      • Locher G.W.
      • Saurer S.
      • Gullick W.J.
      • Waterfield M.D.
      • Groner B.
      • Hynes N.E.
      Correlation of c-erb B2 gene amplification and protein expression in human breast carcinoma with nodal status and nuclear grading..
      51Frozen paraffinSouthern blot IHCYesHER-2 protein by IHC correlated with node status and tumor grade
      3 1988van de Vijver
      • van de Vivjer M.J.
      • Peterse J.L.
      • Mooi W.J.
      • Wisman P.
      • Lomans J.
      • Dalesio O.
      • Nusse R.
      Neu-protein overexpression in breast cancer..
      189ParaffinIHCNoNoCorrelated with size, comedocarcinoma
      4 1989Wright
      • Heintz N.H.
      • Leslie K.O.
      • Rogers L.A.
      • Howard P.L.
      Amplification of the c-erb B 2 oncogene in prognosis of breast adenocarcinoma..
      185ParaffinIHCYesYesOverexpression correlated with high grade, negative ER, not size or node status
      5 1990Heintz
      • Wright C.
      • Angus B.
      • Nicholson S.
      • Sainsbury J.R.
      • Cairns J.
      • Gullick W.J.
      • Kelly P.
      • Harris A.L.
      • Horne C.H.
      Expression of c-erbB-2 oncoprotein: A prognostic indicator in human breast cancer..
      50FrozenSouthern analysisNoNoMitoses, ER/PR-negative
      6 1990Tsuda
      • Tsuda H.
      • Hirohashi S.
      • Shimosato Y.
      • Hirota T.
      • Tsugane S.
      • Watanabe S.
      • Terada M.
      • Yamamoto H.
      Correlation between histologic grade of malignancy and copy number of c-erbB-2 gene in breast carcinoma. A retrospective analysis of 176 cases..
      176ParaffinSouthern analysisYesNoImpact on prognosis absorbed by grade
      7 1990Borg
      • Borg A.
      • Tandon A.K.
      • Sigurdsson H.
      • Clark G.M.
      • Ferno M.
      • Fuqua S.A.
      • Killander D.
      • McGuire W.L.
      HER-2/neu amplification predicts poor survival in node-positive breast cancer..
      300FreshWesternYesNoGene amplification predicted relapse and death in node-negative on univariate only
      8 1990Paik
      • Paik S.
      • Hazan R.
      • Fisher E.R.
      • Sass R.E.
      • Fisher B.
      • Redmond C.
      • Schlessinger J.
      • Lippman M.E.
      • King C.R.
      Pathologic findings from the nations surgical adjuvant breast and bowel project: Prognostic significance of erb B2 protein overexpression in primary breast cancer..
      292ParaffinIHCYesYesHER-2 protein overexpression second only to nodal status in predicting outcome
      9 1991Battifora
      • Battifora H.
      • Gaffey M.
      • Esteban J.
      • Mehta P.
      • Bailey A.
      • Faucett C.
      • Niland J.
      Immunohistochemical assay of neu/c-erb B-2 oncogene product in paraffin-embedded tissues in early breast cancer: Retrospective follow-up study of 245 stage I and II cases..
      245ParaffinIHCYesYesSignificant only for low-grade, low-stage cases
      10 1991Kallionemi
      • Kallioniemi O.P.
      • Holli K.
      • Visakorpi T.
      • Koivula T.
      • Helin H.H.
      • Isola J.J.
      Association of C-erb B2 protein over-expression with high rate of cell proliferation, increased risk of visceral metastasis and poor long-term survival in breast cancer..
      319ParaffinIHCYesYesIndependent predictor in node-negative and -positive
      11 1991Gullick
      • Gullick W.J.
      • Love S.B.
      • Wright C.
      • Barnes D.M.
      • Gusterson B.
      • Harris A.L.
      • Altman D.G.
      c-erbB-2 protein overexpression in breast cancer is a risk factor in patients with involved and uninvolved lymph nodes..
      483ParaffinIHCYesYesOverexpression correlated with grade, but not size, ER, or nodal status
      12 1991Clark
      • Clark G.M.
      • McGuire W.L.
      Follow-up study of HER-2/neu amplification in primary breast cancer..
      362FreshSlot blotNoNoAmplification marginally predictive in node-positive only
      13 1991Lovekin
      • Lovekin C.
      • Ellis I.O.
      • Locker A.
      • Robertson J.F.
      • Bell J.
      • Nicholson R.
      • Gullick W.J.
      • Elston C.W.
      • Blamey R.W.
      C-erb B2 oncoprotein expression in primary and advanced breast cancer..
      782ParaffinIHCYesYesHER-2 overexpression predicted outcome in early and advanced cases; grade more significant predictor
      14 1991McCann
      • McCann A.H.
      • DeDervan T.A.
      • O’Regan M.
      • Codd M.B.
      • Gullick W.J.
      • Tobin B.M.
      • Carney D.N.
      Prognostic significance of C-erb B2 and estrogen receptor status in human breast cancer..
      314ParaffinIHCYesYesPredicted outcome in node-negative and node-positive
      15 1991Dykens
      • Dykens R.
      • Corbett I.P.
      • Henry J.
      • Wright C.
      • Yuan J.
      • Hennessy C.
      • Lennard T.J.
      • Angus B.
      • Horne C.H.
      Long term survival in breast cancer related to overexpression of the C-erb B2 oncoprotein: An immunohistochemical study using monoclonal antibody NCL-CB11..
      178ParaffinIHCYesYesOverexpression predicted shortened survival in node-negative, but not in node-positive patients
      16 1991Rilke
      • Rilke F.
      • Colnaghi M.I.
      • Cascinelli N.
      • Andreola S.
      • Baldini M.T.
      • Bufalino R.
      • Della Porta G.
      • Menard S.
      • Pierotti M.A.
      • Testori A.
      Prognostic significance of HER-2/neu expression in breast cancer and its relationship to other prognostic factors..
      1,210ParaffinIHCYesNoProtein overexpression predicted outcome in node-positive only
      17 1991Winstanley
      • Winstanley J.
      • Cooke T.
      • Murray G.D.
      • Platt-Higgins A.
      • George W.D.
      • Holt S.
      • Myskov M.
      • Spedding A.
      • Barraclough B.R.
      • Rudland P.S.
      The long term prognostic significance of C-erb B2 in primary breast cancer..
      465ParaffinIHCYesYesHER-2 protein staining independently predicted survival
      18 1991O’Reily
      • O’Reilly S.M.
      • Barnes D.M.
      • Camplejohn R.S.
      • Bartkova J.
      • Gregory W.M.
      • Richards M.A.
      The relationship between C-erb B2 expression, and s-phase fraction in prognosis in breast cancer..
      172ParaffinIHCYesYesHER-2 protein overexpression predicted outcome in node-positive, but not in node-negative disease
      19 1991Patterson
      • Paterson M.C.
      • Dietrich K.D.
      • Danyluk J.
      • Paterson A.H.
      • Lees A.W.
      • Jamil N.
      • Hanson J.
      • Jenkins H.
      • Krause B.E.
      • McBlain W.A.
      • et al.
      Correlation between C erb B2 amplification and risk of recurrent disease in node-negative breast cancer..
      115ParaffinSlot blotYesYesGene amplification predicted disease-free interval in node-negative patients
      20 1992Toikkanen
      • Toikkanen S.
      • Helin H.
      • Isola J.
      • Joensuu H.
      Prognostic significance of Her-2 oncoprotein expression in breast cancer: A 30-year follow up..
      209ParaffinIHCYesYesProtein overexpression predicted shortened survival in node-positive patients
      21 1992Molina
      • Molina R.
      • Ciocca D.R.
      • Candon A.K.
      • Allred D.C.
      • Clark G.M.
      • Chamness G.C.
      • Gullick W.J.
      • McGuire W.L.
      Expression of HER-2/neu oncoprotein in breast cancer: A comparison of immunohistochemical and western blot techniques..
      301ParaffinIHCYesYesBoth methods of protein levels predicted worse overall survival
      22 1992Noguchi
      • Noguchi M.
      • Koyasaki M.
      • Ohta N.
      • Kitagawa H.
      • Earashi M.
      • Thomas M.
      • Miyazaki I.
      • Mizukami Y.
      c-erb B-2 oncoprotein expression versus internal mammary lymph node metastases as additional prognostic factors in patients with axillary lymph node-positive breast cancer..
      234ParaffinIHCYesNoOnly nodal status correlated independently
      23 1992Allred
      • Allred D.C.
      • Clark G.M.
      • Tandon A.K.
      • Molina R.
      • Tormey D.C.
      • Osborne C.K.
      • Gilchrist K.W.
      • Mansour E.G.
      • Abeloff M.
      • Eudey L.
      • et al.
      HER-2/neu node-negative breast cancer: Prognostic significance of overexpression influenced by the presence of in-situ carcinoma..
      613ParaffinIHCYesSignificant for node-negative low-risk only
      24 1992Babiak
      • Babiak J.
      • Hugh J.
      • Poppeme S.
      Significance of c-erb B-2 amplification in DNA aneuploidy. Analysis in 78 patients with node-negative breast cancer..
      78ParaffinSlot blotYesOnly when combined with aneuploid status
      25 1992Tiwari
      • Tiwari R.K.
      • Borgen P.I.
      • Wong G.Y.
      • Cordon-Cardo C.
      • Osborne M.P.
      HER-2/neu amplification and overexpression in primary human breast cancer is associated with early metastasis..
      61FrozenSouthernYesHER-2 amplification associated with nodal metastasis
      26 1992Gusterson
      • Gusterson B.A.
      • Gelber R.D.
      • Goldhirsch A.
      • Price K.N.
      • Save-Soderborgh J.
      • Anbazhagan R.
      • Styles J.
      • Rudenstam C.M.
      • Golouh R.
      • Reed R.
      • et al.
      Prognostic importance of C-erb B2 expression in breast cancer..
      1,506ParaffinIHCYesYesPredicted outcome in node-positive only
      27 1993Bianchi
      • Bianchi S.
      • Paglierani M.
      • Zampi G.
      • Cardona G.
      • Cataliotti L.
      • Bonardi R.
      • Ciatto S.
      Prognostic significance of C-erb B2 expression in node negative breast cancer..
      230ParaffinIHCYesNoOnly diffuse intense staining correlated
      28 1993Press
      • Press M.F.
      • Pike M.C.
      • Chazin V.R.
      • Hung G.
      • Udove J.A.
      • Markowicz M.
      • Danyluk J.
      • Godolphin W.
      • Sliwkowski M.
      • Akita R.
      • et al.
      Her-2/neu expression in node-negative breast cancer: Direct tissue quantification by computerized image analysis and association of overexpression with increased risk of recurrent disease..
      210ParaffinIHCYesYesPredicts relapse in node-negative
      29 1993Seshadri
      • Seshadri R.
      • Firgaira F.A.
      • Horsfall D.J.
      • McCaul K.
      • Setlur V.
      • Kitchen P.
      Clinical significance of Her-2/neu oncogene amplification in primary breast cancer..
      1,056FreshSlot blotYesYesIndependent for both node-negative and -positive
      30 1993Descotes
      • Descotes F.
      • Pavy J.-J.
      • Adessi G.L.
      Human breast cancer: Correlation study between Her-2/neu amplification and prognostic factors in an unselected population..
      298FrozenSlot blotYesYesWith PR-negative, node status
      31 1994Giai
      • Giai M.
      • Roagna R.
      • Ponzone R.
      • De Bortoli M.
      • Dati C.
      • Sismondi P.
      Prognostic and predictive relevance of C-erb B2 and ras expression in node positive and negative breast cancer..
      159ParaffinWestern blotYesYesIndependent predictor in node-negative
      32 1994Muss
      • Muss H.B.
      • Thor A.D.
      • Berry D.A.
      • Kute T.
      • Liu E.T.
      • Koerner F.
      • Cirrincione C.T.
      • Budman D.R.
      • Wood W.C.
      • Barcos M.
      • et al.
      Cerb-B2 expression and response to adjuvant therapy in women with node-positive early breast cancer..
      442ParaffinIHCYesPredicts response to chemotherapy
      33 1994Tetu
      • Tetu B.
      • Brisson J.
      Prognostic significance of Her-2/neu oncogene expression in node-positive breast cancer. The influence of the pattern of immunostaining and adjuvant therapy..
      888ParaffinIHCYesYesSurvival in node-positive, only membranous pattern correlates
      34 1994Hartmann
      • Hartmann L.C.
      • Ingle J.N.
      • Wold L.E.
      • Farr Jr., G.H.
      • Grill J.P.
      • Su J.Q.
      • Maihle N.J.
      • Krook J.E.
      • Witzig T.E.
      • Roche P.C.
      Prognostic value of CerbB2 overexpression in axillary lymph node positive breast cancer. Results from a randomized adjuvant treatment protocol..
      340ParaffinIHCYesNoPredicts lymph node status
      35 1994Jaquemeier
      • Jacquemeier J.
      • Penault-Llorca P.
      • Viens P.
      • Houvenaeghel G.
      • Hassoun J.
      • Torrente M.
      • Adelaide J.
      • Birnbaum D.
      Breast cancer response to adjuvant chemotherapy in correlation with erb B2 and p53 expression..
      81ParaffinIHCNoNoNo correlation with therapy response
      36 1994Marks
      • Marks J.R.
      • Humphrey P.A.
      • Wu K.
      • Berry D.
      • Bandarenko N.
      • Kerns B.J.
      • Iglehart J.D.
      Overexpression of p53 and Her 2/neu proteins as prognostic markers in early stage breast cancer..
      230ParaffinIHCYesYesp53 and node status also independent
      37 1994Rosen
      • Rosen P.P.
      • Lesser M.L.
      • Arroyo C.D.
      • Cranor M.
      • Borgen P.
      • Norton L.
      • et al.
      Immunohistochemical detection of Her-2/neu expression in patients with axillary lymph node-negative breast carcinoma. A study of epidemiologic risk factors, histologic features and prognosis..
      440ParaffinIHCNoNoMedullary carcinoma is negative
      38 1995Quenel
      • Quenel N.
      • Wafflart J.
      • Bonichon F.
      • de Mascarel I.
      • Trojani M.
      • Durand M.
      • Avril A.
      • Coindre J.M.
      The prognostic value of c-erbB2 in primary breast carcinomas: A study on 942 cases..
      942ParaffinIHCYesYesHER-2 predicted relapse-free and metastasis-free survival
      39 1996Sundblad
      • Sundblad A.S.
      • Pellicer E.M.
      • Ricci L.
      Carcinoembryonic expression in stages I and II breast cancer; its relationship with clinicopathologic factors..
      271ParaffinIHCYesYesNode-positive, HER-2/neu-negative, CEA-positive and bcl-2-positive had the best prognosis
      40 1996O’Malley
      • O’Malley F.P.
      • Saad Z.
      • Kerkvliet N.
      • Doig G.
      • Stitt L.
      • Ainsworth P.
      • Hundal H.
      • Chambers A.F.
      • Turnbull D.I.
      • Bramwell V.
      The predictive power of semiquantitative immunohistochemical assessment of p53 and C-erbB2 in lymph node-negative breast cancer..
      107ParaffinIHCYesYesBoth p53 and HER-2/neu staining independently predicted outcome
      41 1996Hieken
      • Hieken T.J.
      • Mehta R.R.
      • Shilkaitis A.
      Her-2/neu and p53 expression in breast cancer: Valid prognostic markers when assessed by direct immunoassay, but not by immunochemistry..
      100Paraffin; cytosolIHCNoNoHER-2 protein by ELISA, not IHC predictive
      42 1996Xing
      • Xing W.-R.
      • Gilchrist K.W.
      • Harris C.P.
      • Samson W.
      • Meisner L.F.
      FISH detection of HER-2/neu oncogene amplification in early onset breast cancer..
      37ParaffinFISHYesHER-2 amplification more predictive than nodal status in young patients
      43 1997Dittadi
      • Dittadi R.
      • Brazzale A.
      • Pappagallo G.
      • Salbe C.
      • Nascimben O.
      • Rosabian A.
      • Gion M.
      ErbB2 assay in breast cancer: Possibly improved clinical information using a quantitative method..
      115CytosolELISAYesYesHER-2 protein levels predicted disease-free survival
      44 1997Fernandez-Acenero
      • Fernandez Acenero M.J.
      • Farina Gonzalez J.
      • Arangoncillo Ballesteros P.
      Immunohistochemical expression of p53 and c-erbB-2 in breast carcinoma: Relation with epidemiologic factors, histologic features and prognosis..
      112ParaffinIHCYesNoOnly TNM stage independent predictor
      45 1997Eissa
      • Eissa S.
      • Khalifa A.
      • el-Gharib A.
      • Salah N.
      • Mohamed M.K.
      Multivariate analysis of DNA ploidy, p53, c-erbB-2 proteins, EGFR, and steroid hormone receptors for short-term prognosis in breast cancer..
      100CytosolELISAYesYesHER-2 strongest predictor of recurrence in node-negative
      46 1997Charpin
      • Charpin C.
      • Garcia S.
      • Bouvier C.
      • Martini F.
      • Lavaut M.N.
      • Allasia C.
      • Bonnier P.
      • Andrac L.
      c-erbB-2 oncoprotein detected by automated quantitative immunocytochemistry in breast carcinomas correlates with patients’ overall and disease-free survival..
      148FrozenIHCYesYesOverall and disease-free survival independent of nodal status
      47 1997Press
      • Press M.J.
      • Bernstein L.
      • Thomas P.A.
      • Meisner L.F.
      • Zhou J.Y.
      • Ma Y.
      • Hung G.
      • Robinson R.A.
      • Harris C.
      • El-Naggar A.
      • Slamon D.J.
      • Phillips R.N.
      • Ross J.S.
      • Wolman S.R.
      • Flom K.J.
      Her-2/neu gene amplification characterized by fluorescence in situ hybridization: Poor prognosis in node negative breast carcinomas..
      324ParaffinFISHYesYesHER-2/neu gene amplification predicts recurrence and death
      48 1998Ross
      • Ross J.S.
      • Muraca P.J.
      • Jaffe D.
      • Morgan T.
      • Fletcher J.A.
      Multivariate analysis of prognostic factors in lymph node negative breast cancer..
      224ParaffinFISHYesYesThree-tiered amplification system
      49 1998Depowski
      • Depowski P.L.
      • Brien T.P.
      • Sheehan C.E.
      • Stylos S.
      • Johnson R.L.
      • Ross J.S.
      Prognostic significance of p34cdc2 cyclin-dependent kinase and MIB1 overexpression, and HER-2/neu gene amplification detected by fluorescence in situ hybridization in breast cancer..
      145ParaffinFISHYesYesHER-2 amplification predicted death independent of nodal status
      50 1998Querzoli
      • Querzoli P.
      • Albonico G.
      • Ferretti S.
      • Rinaldi R.
      • Beccati D.
      • Corcione S.
      • Indelli M.
      • Nenci I.
      Modulation of biomarkers in minimal breast carcinoma..
      164ParaffinIHCYesYesHER-2 overexpression independently predicted recurrence and survival
      51 1998Andrulis
      • Andrulis I.L.
      • Bull S.B.
      • Blasckstein M.E.
      • Sutherland D.
      • Mak C.
      • Sidlofsky S.
      • Pritzker K.P.
      • Hartwick R.W.
      • Hanna W.
      • Lickley L.
      • Wilkinson R.
      • Qizilbash A.
      • Ambus U.
      • Lipa M.
      • Weizel H.
      • Katz A.
      • Baida M.
      • Mariz S.
      • Stoik G.
      • Dacamara P.
      • Strongitharm D.
      • Geddie W.
      • McCready D.
      Neu/erbB-2 amplification identifies a poor-prognosis group of women with node-negative breast cancer..
      580FreshSouthernYesYesHER-2 amplification independently predicted recurrence in node-negative; impact more in adjuvant treated cases
      52 1998Sjogren
      • Sjogren S.
      • Inganas M.
      • Lindgren A.
      • Holmberg L.
      • Bergh J.
      Prognostic and predictive value of c-erbB-2 overexpression in primary breast cancer, alone and in combination with other prognostic markers..
      315ParaffinIHCYesYesCB-11 antibody IHC predicted overall survival independent of age, node status, size, grade, hormone receptor status, S phase, p53 status, and adjuvant therapy
      53 1999Harbeck
      • Harbeck N.
      • Ross J.S.
      • Yurdseven S.
      • Dettmar P.
      • Polcher M.
      • Kuhn W.
      • Ulm K.
      • Graeff H.
      • Schmitt M.
      HER-2/neu gene amplification by fluorescence in situ hybridization allows risk-group assessment in node-negative breast cancer..
      112ParaffinFISHYesYesHER-2 amplification by FISH and [plasminogen protease (uPA, PAI-1) levels were both independent predictors in node-negative patients
      54 1999Scorilas
      • Scorilas A.
      • Yotis J.
      • Pateras C.
      • Trangas T.
      • Talieri M.
      Predictive value of c-erbB-2 and cathepsin-D for Greek breast cancer patients using univariate and multivariate analysis..
      136FreshSouthernYesNoCathepsin D was an independent predictor, but HER-2 was not
      55 1999Rudolph
      • Rudolph P.
      • Olsson H.
      • Bonatz G.
      • Ratjen V.
      • Bolte H.
      • Baldetorp B.
      • Ferno M.
      • Parwaresch R.
      • Alm P.
      Correlation between p53, c-erbB-2, and topoisomerase II alpha expression, DNA ploidy, hormonal receptor status and proliferation in 356 node-negative breast carcinomas: Prognostic implications..
      356ParaffinIHCYesNoHER-2 overexpression not an independent predictor of prognosis
      56 2000Reed
      • Reed W.
      • Hannisdal E.
      • Boehler P.J.
      • Gundersen S.
      • Host H.
      • Marthin J.
      The prognostic value of p53 and c-erb B-2 immunostaining is overrated for patients with lymph node negative breast carcinoma: A multivariate analysis of prognostic factors in 613 patients with a follow-up of 14–30 years..
      385ParaffinIHCNoNoThe HER-2-positive rate was only 10% in the non-correlating IHC study
      57 2000Pauletti
      • Pauletti G.
      • Dandekar S.
      • Rong H.
      • Ramos L.
      • Peng H.
      • Seshadri R.
      • Slamon D.J.
      Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: A direct comparison of fluorescence in situ hybridization and immunohistochemistry..
      189ParaffinIHCYesYesAmplification detected by FISH was consistently predictive of outcome, but IHC testing was not
      58 2000Kakar
      • Kakar S.
      • Puangsuvan N.
      • Stevens J.M.
      • Serenas R.
      • Mangan G.
      • Sahai S.
      • Mihalov M.L.
      HER-2/neu assessment in breast cancer by immunohistochemistry and fluorescence in situ hybridization: Comparison of results and correlation with survival..
      117ParaffinIHCYesIHC 3+-positive and FISH-positive cases correlated with outcome; IHC 2+-positive cases did not
      59 2000Agrup
      • Agrup M.
      • Stal O.
      • Olsen K.
      • Wingren S.
      C-erbB-2 overexpression and survival in early onset breast cancer..
      110ParaffinIHCYesYesHER-2 overexpression predicted survival in node-positive disease in young women independent of adjuvant therapy
      60 2000Umekita
      • Umekita Y.
      • Ohi Y.
      • Sagara Y.
      • Yoshida H.
      Co-expression of epidermal growth factor receptor and transforming growth factor-alpha predicts worse prognosis in breast-cancer patients..
      159ParaffinIHCYesNoCo-expression of TGF-α and EGFR had worst prognosis
      61 2000Pawlowski
      • Pawlowski V.
      • Revillion F.
      • Hebbar M.
      • Hornez L.
      • Peyrat J.P.
      Prognostic value of the type I growth factor receptors in a large series of human primary breast cancers quantified with a real-time reverse transcription-polymerase chain reaction assay..
      365FreshRT-PCRNoNoHER-2 mRNA levels correlated with grade and ER/PR status but not survival; increased HER-4 levels were a favorable prognostic finding
      62 2000Volpi
      • Volpi A.
      • De Paola F.
      • Nanni O.
      • Granato A.M.
      • Bajorko P.
      • Becciolini A.
      • Scarpi E.
      • Riccobon A.
      • Balzi M.
      • Amadori D.
      Prognostic significance of biologic markers in node-negative breast cancer patients: A prospective study..
      286ParaffinIHCYesNoHER-2 status predictive of short-term prognosis only and not an independent predictor
      63 2000Carr
      • Carr J.A.
      • Havstad S.
      • Zarbo R.J.
      • Divine G.
      • Mackowiak P.
      • Velanovich V.
      The association of HER-2/neu amplification with breast cancer recurrence..
      190ParaffinFISHYesYesHER-2 amplification predicted recurrence independent of ER and LN status
      64 2000Ferrero-Pous
      • Ferrero-Pous M.
      • Hacene K.
      • Bouchet C.
      • Le Doussal V.
      • Tubiana-Hulin M.
      • Spyratos F.
      Relationship between c-erbB-2 and other tumor characteristics in breast cancer prognosis..
      488FreshELISAYesYesHER-2 overexpression also predicted reduced response to Tamoxifen
      65 2000Platt-Higgins
      • Platt-Higgins A.M.
      • Renshaw C.A.
      • West C.R.
      • Winstanley J.H.
      • De Silva Rudland S.
      • Barraclough R.
      • Rudland P.S.
      Comparison of the metastasis-inducing protein S100A4 (p9ka) with other prognostic markers in human breast cancer..
      349ParaffinIHCYesNoS1004A protein marker was an independent predictor; HDER-2 expression was significant on univariate analysis only
      66 2001Eppenberger- Castori
      • Eppenberger-Castori S.
      • Kueng W.
      • Benz C.
      • Caduff R.
      • Varga Z.
      • Bannwart F.
      • Fink D.
      • Dieterich H.
      • Hohl M.
      • Muller H.
      • Paris K.
      • Schoumacher F.
      • Eppenberger U.
      Prognostic and predictive significance of ErbB-2 breast tumor levels measured by enzyme immunoassay..
      1,123FreshELISAYesYesHER-2 levels predicted survival independent of hormonal or cytotoxic therapy
      67 2001Jukkola
      • Jukkola A.
      • Bloigu R.
      • Soini Y.
      • Savolainen E.R.
      • Holli K.
      • Blanco G.
      c-erbB-2 positivity is a factor for poor prognosis in breast cancer and poor response to hormonal or chemotherapy treatment in advanced disease..
      650FreshSouthernYesYesHER-2-positive patients also had reduced responses to both hormonal and cytotoxic therapies
      68 2001Gaci
      • Gaci Z.
      • Bouin-Pineau M.H.
      • Gaci M.
      • Daban A.
      • Ingrand P.
      • Metaye T.
      Prognostic impact of cathepsin D and c-erbB-2 oncoprotein in a subgroup of node-negative breast cancer patients with low histological grade tumors..
      100FreshELISAYesYesHER-2 and cathepsin D expression were both independent predictors of OS
      69 2001Rudolph
      • Rudolph P.
      • Alm P.
      • Olsson H.
      • Heidebrecht H.J.
      • Ferno M.
      • Baldetorp B.
      • Parwaresch R.
      Concurrent overexpression of p53 and c-erbB-2 correlates with accelerated cycling and concomitant poor prognosis in node-negative breast cancer..
      261ParaffinIHCYesYesCell cycling ratio was most predictive factor in multivariate analysis
      70 2001Beenken
      • Beenken S.W.
      • Grizzle W.E.
      • Crowe D.R.
      • Conner M.G.
      • Weiss H.L.
      • Sellers M.T.
      • Krontiras H.
      • Urist M.M.
      • Bland K.I.
      Molecular biomarkers for breast cancer prognosis: Coexpression of c-erbB-2 and p53..
      90ParaffinIHCYesYesHER-2 and p53 co-expression had the worst prognosis
      71 2001Pinto
      • Pinto A.E.
      • Andre S.
      • Pereira T.
      • Nobrega S.
      • Soares J.
      C-erbB-2 oncoprotein overexpression identifies a subgroup of estrogen receptor positive (ER+) breast cancer patients with poor prognosis..
      306ParaffinIHCYesYesHER-2-positive ER-positive patients had reduced response rate to Tamoxifen
      72 2001Riou
      • Riou G.
      • Mathieu M.C.
      • Barrois M.
      • Le Bihan M.L.
      • Ahomadegbe J.C.
      • Benard J.
      • Le M.G.
      c-erbB-2 (HER-2/neu) gene amplification is a better indicator of poor prognosis than protein over-expression in operable breast-cancer patients..
      172FreshSouthernYesYesHE-2 amplification independently predicted survival; no correlation with survival for IHC positive cases with no gene amplification
      73 2001Horita
      • Horita K.
      • Yamaguchi A.
      • Hirose K.
      • Ishida M.
      • Noriki S.
      • Imamura Y.
      • Fukuda M.
      Prognostic factors affecting disease-free survival rate following surgical resection of primary breast cancer..
      76ParaffinIHCYesYesHER-2 expression correlated with PCNA expression
      74 2002Suo
      • Suo Z.
      • Risberg B.
      • Kalsson M.G.
      • Willman K.
      • Tierens A.
      • Skovlund E.
      • Nesland J.M.
      EGFR family expression in breast carcinomas. c-erbB-2 and c-erbB-4 receptors have different effects on survival..
      100ParaffinIHCYesYesCo-expression of HER-2 with EGFR (HER-1) had worse DFS
      75 2002Ristimaki
      • Ristimaki A.
      • Sivula A.
      • Lundin J.
      • Lundin M.
      • Salminen T.
      • Haglund C.
      • Joensuu H.
      • Isola J.
      Prognostic significance of elevated cyclooxygenase-2 expression in breast cancer..
      1,576ParaffinFISHYesYesHER-2 amplification was an independent predictor of survival and correlated with Cox-2 expression
      76 2002Rosenthal
      • Rosenthal S.I.
      • Depowski P.L.
      • Sheehan C.E.
      • Ross J.S.
      Comparison of HER-2/neu oncogene amplification detected by fluorescence in situ hybridization in lobular and ductal breast cancer..
      71ParaffinFISHYesNoInvasive lobular cancer had lower HER-2 amplification rate (<10%) than invasive ductal cancer and reached near significance as an independent predictor of OS
      77 2002Tsutsui
      • Tsutsui S.
      • Ohno S.
      • Murakami S.
      • Hachitanda Y.
      • Oda S.
      Prognostic value of c-erbB2 expression in breast cancer..
      698ParaffinIHCYesYesCombined HER-2-positive and ER-negative had the worst outcome
      78 2002Spizzo
      • Spizzo G.
      • Obrist P.
      • Ensinger C.
      • Theurl I.
      • Dunser M.
      • Ramoni A.
      • Gunsilius E.
      • Eibl G.
      • Mikuz G.
      • Gastl G.
      Prognostic significance of Ep-CAM AND Her-2/neu overexpression in invasive breast cancer..
      205ParaffinIHCYesYesBoth HER-2 and Ep-CAM expression were independent predictors of OS
      79 2002Kato
      • Kato T.
      • Kameoka S.
      • Kimura T.
      • Nishikawa T.
      • Kobayashi M.
      C-erbB-2 and PCNA as prognostic indicators of long-term survival in breast cancer..
      408ParaffinIHCYesNoHER-2 status was an independent predictor in some but not all patient cohorts
      80 2002El-Amady
      • el-Ahmady O.
      • el-Salahy E.
      • Mahmoud M.
      • Wahab M.A.
      • Eissa S.
      • Khalifa A.
      Multivariate analysis of bcl-2, apoptosis, P53 and HER-2/neu in breast cancer: A short-term follow-up..
      94FreshELISAYesYesHER-2 protein level by ELISA most predictive factor for short-term prognosis
      TOTAL26,309

      HER-2/neu TESTING TECHNIQUES

      IHC staining (Fig. 4A) has been the predominant method utilized. Unlike most immunohistochemical assays, the assessment of HER-2/neu status is quantitative rather than qualitative, because HER-2/neu is expressed in all breast epithelial cells. In order to provide a meaningful interpretation of a HER-2/neu immunostain, it was necessary to establish a relationship between the number of HER-2/neu receptors on a cell’s surface and the distribution and intensity of the immunostain. Utilizing cell lines, it was possible to establish a standardized immunohistochemical procedure and scoring system in which cells containing less than 20,000 receptors would show no staining (0), cells containing ∼100,000 receptors would show partial membrane staining with less than 10% of the cells showing complete membrane staining (1+), cells containing ∼500,000 receptors would show light to moderate complete membrane staining in more than 10% of the cells (2+), and cells containing ∼2,300,000 receptors would show strong, complete membrane staining in more than 10% of the cells (3+). Studies have shown that when a standardized IHC assay is performed on specimens that are carefully fixed, processed, and embedded, there is excellent correlation between gene copy status and protein expression levels (
      • Navolanic P.M.
      • Steelman L.S.
      • McCubrey J.A.
      EGFR family signaling and its association with breast cancer development and resistance to chemotherapy..
      ,
      • Hayes D.F.
      • Thor A.D.
      c-erbB-2 in breast cancer: Development of a clinically useful marker..
      ,
      • Masood S.
      • Bui M.M.
      Prognostic and predictive value of HER2/neu oncogene in breast cancer..
      ,
      • Schnitt S.J.
      • Jacobs T.W.
      Current status of HER2 testing: Caught between a rock and a hard place..
      ). However, alterations can be significantly impacted by technical issues, especially in archival fixed paraffin-embedded tissues. Advantages of IHC testing (see Table V) include its wide availability, relatively low cost, easy preservation of stained slides, and use of a familiar routine microscope. Disadvantages of IHC include the impact of preanalytic issues including storage, duration and type of fixation, intensity of antigen retrieval, type of antibody (polyclonal versus monoclonal), nature of system control samples, and, most importantly, the difficulties in applying a subjective slide scoring system. In a study of a large panel of antibodies, Press et al. reported a significant variation in detection rates for HER-2/neu protein by IHC using a large tissue block containing multiple breast tumors (
      • Press M.F.
      • Hung G.
      • Godolphin W.
      • Slamon D.J.
      Sensitivity of HER-2/neu antibodies in archival tissue samples: Potential source of error in immunohistochemical studies of oncogene expression..
      ). Problems with standardization in slide scoring have been recently highlighted in reference to the best method for using HER-2/neu status to predict response to the anti-HER-2/neu antibody therapeutic, trastuzumab (
      • Paik S.
      • Bryant J.
      • Tan-Chiu E.
      • Romond E.
      • Hiller W.
      • Park K.
      • Brown A.
      • Yothers G.
      • Anderson S.
      • Smith R.
      • Wickerham D.L.
      • Wolmark N.
      Real-world performance of HER2 testing—National Surgical Adjuvant Breast and Bowel Project experience..
      ). Slide scoring can be improved by avoiding specimen edges, retraction artifacts, under- or overfixation, cases with significant staining of benign elements, and tumor cells lacking a complete membranous staining pattern (the so-called “chicken wire” appearance). Recent data presented by the National Surgical Adjuvant Breast Program (NSABP) has shown that certified laboratories, defined as those laboratories performing high-volume HER-2/neu testing and demonstrating high concordance between IHC and FISH results, approach 98% interlaboratory concordance when tumors assessed as 3+ were reanalyzed by both IHC and FISH testing at the NSABP laboratory (
      • Paik S.
      • Tan-chui E.
      • Bryant J.
      • Hiller W.
      • Park K.
      • Wolmark N.
      Successful quality assurance program for HER-2 testing in the NSAPB trial for Herceptin..
      ). Because most of the submitting laboratories were reference laboratories that cannot control tissue fixation or storage, it has been suggested that preanalytical issues may not be the major cause of interlaboratory variability. Results from the United Kingdom National External Quality Assessment Scheme for Immunohistochemistry (UK NEQAS-ICC) also suggested that the lack of reproducibility of HER-2/neu scoring between laboratories was not the result of tumor heterogeneity or differences in fixation or processing but rather the result of how the scoring system was applied (
      • Rhodes A.
      • Jasani B.
      • Anderson E.
      • Dodson A.R.
      • Balaton A.J.
      Evaluation of HER-2/neu immunohistochemical assay sensitivity and scoring on formalin-fixed and paraffin-processed cell lines and breast tumors: A comparative study involving results from laboratories in 21 countries..
      ). However, in a recent report, prolonged formalin fixation did not impact on the staining for cell cycle markers such as Ki-67 and p27, but did cause significant degradation in hormone receptor proteins and HER-2/neu (
      • Arber D.A.
      Effect of prolonged formalin fixation on the immunohistochemical reactivity of breast markers..
      ). The use of a quantitative image analysis system can reduce slide-scoring variability among pathologists, especially in 2+ cases (
      • Wang S.
      • Saboorian M.H.
      • Frenkel E.P.
      • Haley B.B.
      • Siddiqui M.T.
      • Gokaslan S.
      • Wians Jr., F.H.
      • Hynan L.
      • Ashfaq R.
      Assessment of HER-2/neu status in breast cancer. Automated Cellular Imaging System (ACIS)-assisted quantitation of immunohistochemical assay achieves high accuracy in comparison with fluorescence in situ hybridization assay as the standard..
      ). When 130 HER-2/neu-immunostained slides were reviewed by 10 pathologists and then were later reviewed with the aid of image analysis, the use of image analysis eliminated most of the interobserver variability, which was significant by routine microscopy (Fig. 5) (
      • Bloom K.J.
      • Torre-Bueno J.
      • Press M.
      • Gown A.
      • Bauer K.
      • Harrington D.
      Comparison of HER-2/neu analysis using FISH and IHC when HercepTest is scored using conventional microscopy and image analysis..
      ). Two commercially available HER-2/neu IHC kits, the Dako Herceptest™ and the Ventana Pathway™, are approved for sale for determining eligibility for patients to receive Herceptin™ by the Food and Drug Administration (FDA).
      Figure thumbnail gr4
      Fig. 4HER-2/neu testing in breast cancer.A, HER-2/neu protein expression in infiltrating ductal breast cancer measured by immunohistochemistry using the Herceptest™ slide scoring system. Upper left, 0+ (negative) staining for HER-2/neu protein. This level of staining is typically associated with 15,000–25,000 surface receptor molecules per cell and HER-2/neu gene copy to chromosome 17 copy ratios measured by FISH of 1.0–1.2. Upper right, 1+ staining associated with 80,000–110,000 receptors and gene ratio of 1.2–1.4. Lower left, 2+ staining with membranous distribution, but no total cell encirclement associated with 370,000–630,000 receptors and gene ratio of 1.4–2.4. Lower right, 3+ staining with diffuse positive membranous distribution, total cell encirclement and “chicken wire” appearance associated with 2,000,000–10,000,000 receptors and gene ratio of 3.4–5.6 (peroxidase-anti-peroxidase with Herceptest™ antibody ×200). (Receptor count and FISH gene ratio data provided by Dr. Kenneth Bloom, US Labs, Inc., Irvine, CA.) B, HER-2/neu gene amplification in infiltrating ductal breast cancer detected by FISH. Left, HER-2/neu gene amplification demonstrated by the Abbott-Vysis Pathvysion™ method showing significant increase in HER-2/neu gene signals (red) compared with chromosome 17 signals (green) with a HER-2/neu gene ratio of 3.9. Right, HER-2/neu gene amplification using the Ventana Inform™ method showing another breast cancer specimen with an absolute (raw) HER-2/neu gene copy number of 24. C, HER-2/neu gene amplification in infiltrating breast cancer detected by CISH using anti-HER-2/neu probe and IHC with diaminobenzidine chromagen (SpotLight™ HER-2/neu probe, Zymed Laboratories Inc., South San Francisco, CA).
      Table VMechanism of action of trastuzumab
      Modified from Nahata, R., and Esteva, F. J. (2003) HER-2 targeted therapy: Lessons learned and future directions. Clin. Cancer Res. 9, 5078–5084.
      Antibody-dependent cellular cytotoxicity
      Stimulation of natural killer cells
      Immune system mediated
      Prevention of HER-2/neu receptor dimerization
      Downstream signaling is reduced or completely interrupted
      Direct cell cycle inhibitor
      Induces p27kip1 cyclin-dependent kinase inhibitor to slow G1 phase
      Anti-angiogenesis
      Reduces angiogenic factors: VEGF, TGF-α and PAI-1
      Pro-apoptotic
      Inhibits Akt signaling
      a Modified from Nahata, R., and Esteva, F. J. (2003) HER-2 targeted therapy: Lessons learned and future directions. Clin. Cancer Res. 9, 5078–5084.
      Figure thumbnail gr5
      Fig. 5Image analysis and HER-2/neu slide scoring. Interobserver variability among 10 pathologists reviewing the same 130 HER-2/neu immunostained slides. Concordance with gene amplification status, assessed by FISH, is plotted on the y-axis. Slides assessed as 2+ or 3+ were called positive. Manual assessment showed significant interobserver variability, while assessment with the aid of image analysis showed little variability. Additionally all pathologists improved their concordance with the aid of image analysis.
      Southern and slot blotting were the first gene-based HER-2/neu detection methods used in breast cancer specimens. These methods can be significantly hampered when tumor cell DNA extracted from the primary carcinoma sample is diluted by DNA from benign breast tissue and inflammatory cells. The FISH technique (Fig. 4B), which is morphology-driven and like IHC can be automated, has the advantages of a more objective scoring system and the presence of a built-in internal control consisting of the two HER-2/neu gene signals present in all non-neoplastic cells in the specimen. Disadvantages of FISH testing include the higher cost of each test, longer time required for slide scoring, requirement of a fluorescent microscope, the inability to preserve the slides for storage and review, and occasionally in identifying the invasive tumor cells. Two versions of the FISH assay are FDA-approved; the Ventana Inform™ test that measures only HER-2/neu gene copies and the Abbott-Vysis Pathvysion™ test that includes a chromosome 17 probe in a dual color format. Published studies indicate that the two assays are highly correlative (
      • Wang S.
      • Saboorian M.H.
      • Frenkel E.
      • Hynan L.
      • Gokaslan S.T.
      • Ashfaq R.
      Laboratory assessment of the status of Her-2/neu protein and oncogene in breast cancer specimens: Comparison of immunohistochemistry assay with fluorescence in situ hybridization assays..
      ). The advantages and disadvantages of IHC- and FISH-based HER-2/neu testing are summarized in Table IV.
      Table IVAdvantages and disadvantages of IHC and FISH-based testing for HER-2/neu in breast cancer
      IHCFISH
      Advantages• Widely available• DNA is a stable target
      • Rapid procedure• Standardized threshold for positivity
      • Light microscope based• Built-in internal control
      • Hercep Test™ and Pathway™ FDA-approved assays for Herceptin eligibility selection• Low interlaboratory variability
      • High accuracy (sensitivity and specificity)
      Disadvantages• Variable antibody sensitivity and specificity• Fluorescence microscope equipped with correct filter sets is required
      • Highly impacted by tissue processing variables• Certain fixatives interfere with assay (noninformative result)
      • Affected by antigen retrieval and reagent variability• Limited community experience with tissue-based FISH
      • Non-FDA-approved assays in routine use
      • Subjective scoring criteria
      • Low pathologist concordance and high interlaboratory variability
      The chromogenic in situ hybridization method or CISH technique (Fig. 4C) features the advantages of both IHC (routine microscope, lower cost, familiarity) and FISH (built-in internal control, subjective scoring, the more robust DNA target), but is not, to date, FDA-approved for selecting patient eligibility for trastuzumab treatment (
      • Tanner M.
      • Gancberg D.
      • Di Leo A.
      • Larsimont D.
      • Rouas G.
      • Piccart M.J.
      • Isola J.
      Chromogenic in situ hybridization: a practical alternative for fluorescence in situ hybridization to detect HER-2/neu oncogene amplification in archival breast cancer samples..
      ,
      • Zhao J.
      • Wu R.
      • Au A.
      • Marquez A.
      • Yu Y.
      • Shi Z.
      Determination of HER2 gene amplification by chromogenic in situ hybridization (CISH) in archival breast carcinoma..
      ). In a recent study using both FISH and CISH to assay 31 cases of infiltrating breast carcinoma with testing performed in laboratories at two institutions, identical results for both methods were found in 26 (84%) of the cases (
      • Gupta D.
      • Middleton L.P.
      • Whitaker M.J.
      • Abrams J.
      Comparison of fluorescence and chromogenic in situ hybridization for detection of HER-2/neu oncogene in breast cancer..
      ). The CISH and IHC detection methods can be combined, so as to provide simultaneous evaluation of gene copy number and protein expression (Fig. 6), but such methods are experimental and have not yet been adopted in clinical practice.
      Figure thumbnail gr6
      Fig. 6Simultaneous HER-2/neu gene amplification and protein overexpression determination. HER-2/neu combined gene and protein evaluation in pleural effusion by CISH (DAB, brown) and immunohistochemistry (vector, blue), respectively. A single breast carcinoma cell with HER-2/neu gene amplification and protein overexpression is seen among nonneoplastic cells.
      The RT-PCR technique (
      • Pawlowski V.
      • Revillion F.
      • Hornez L.
      • Peyrat J.P.
      A real-time one-step reverse transcriptase-polymerase chain reaction method to quantify c-erbB-2 expression in human breast cancer..
      ,
      • Bieche I.
      • Onody P.
      • Laurendeau I.
      • Olivi M.
      • Vidaud D.
      • Lidereau R.
      • Vidaud M.
      Real-time reverse transcription-PCR assay for future management of ERBB2-based clinical applications..
      ) has predominantly been used to detect HER-2/neu mRNA in peripheral blood and bone marrow samples, has correlated more with gene amplification status than IHC levels (
      • Tubbs R.R.
      • Pettay J.D.
      • Roche P.C.
      • Stoler M.H.
      • Jenkins R.B.
      • Grogan T.M.
      Discrepancies in clinical laboratory testing of eligibility for trastuzumab therapy: Apparent immunohistochemical false-positives do not get the message..
      ), and has failed to predict survival but did correlate with ER/progesterone receptor (PR) and tumor grade status in one breast cancer outcome study of 365 patients (
      • Volpi A.
      • De Paola F.
      • Nanni O.
      • Granato A.M.
      • Bajorko P.
      • Becciolini A.
      • Scarpi E.
      • Riccobon A.
      • Balzi M.
      • Amadori D.
      Prognostic significance of biologic markers in node-negative breast cancer patients: A prospective study..
      ). With the advent of laser capture microscopy and the acceptance of RT-PCR as a routine and reproducible laboratory technique, the use RT-PCR to assess HER-2/neu status may increase in the future.
      The cDNA microarray-based method (Fig. 7) of detecting HER-2/neu mRNA expression levels has recently achieved interest as an alternative method for measuring HER-2/neu status in breast cancer (
      • Pusztai L.
      • Ayers M.
      • Stec J.
      • Clark E.
      • Hess K.
      • Stivers D.
      • Damokosh A.
      • Sneige N.
      • Buchholz T.A.
      • Esteva F.J.
      • Arun B.
      • Cristofanilli M.
      • Booser D.
      • Rosales M.
      • Valero V.
      • Adams C.
      • Hortobagyi G.N.
      • Symmans W.F.
      Gene expression profiles obtained from fine-needle aspirations of breast cancer reliably identify routine prognostic markers and reveal large-scale molecular differences between estrogen-negative and estrogen-positive tumors..
      ,
      • Mackay A.
      • Jones C.
      • Dexter T.
      • Silva R.L.
      • Bulmer K.
      • Jones A.
      • Simpson P.
      • Harris R.A.
      • Jat P.S.
      • Neville A.M.
      • Reis L.F.
      • Lakhani S.R.
      • O’Hare M.J.
      cDNA microarray analysis of genes associated with ERBB2 (HER2/neu) overexpression in human mammary luminal epithelial cells..
      ,
      • Kauraniemi P.
      • Barlund M.
      • Monni O.
      • Kallioniemi A.
      New amplified and highly expressed genes discovered in the ERBB2 amplicon in breast cancer by cDNA microarrays..
      ). This method has the advantage of being able to assess downstream signaling of the HER-2 pathway at the same time the level of HER-2 mRNA is measured. Other pathways relevant to HER-2 such as the ER pathway can also be assessed simultaneously with this technique. In a recent study, the HER-2/neu gene amplification status detected by FISH on 20 paraffin-embedded breast cancer core biopsy samples was correctly predicted in all cases by the quantification of the HER-2 mRNA levels obtained by expression profiling of mRNA extracted from paired fine needle aspiration biopsies from the same patients (
      • Pusztai L.
      • Ayers M.
      • Stec J.
      • Clark E.
      • Hess K.
      • Stivers D.
      • Damokosh A.
      • Sneige N.
      • Buchholz T.A.
      • Esteva F.J.
      • Arun B.
      • Cristofanilli M.
      • Booser D.
      • Rosales M.
      • Valero V.
      • Adams C.
      • Hortobagyi G.N.
      • Symmans W.F.
      Gene expression profiles obtained from fine-needle aspirations of breast cancer reliably identify routine prognostic markers and reveal large-scale molecular differences between estrogen-negative and estrogen-positive tumors..
      ). Tissue microarrays have also been recently introduced into breast cancer research and have shown excellent correlation with FISH and IHC HER-2/neu results obtained from the donor tissue blocks used to produce the array (
      • Kononen J.
      • Bubendorf L.
      • Kallioniemi A.
      • Barlund M.
      • Schraml P.
      • Leighton S.
      • Torhorst J.
      • Mihatsch M.J.
      • Sauter G.
      • Kallioniemi O.P.
      Tissue microarrays for high-throughput molecular profiling of tumor specimens..
      ,
      • Camp R.L.
      • Charette L.A.
      • Rimm D.L.
      Validation of tissue microarray technology in breast carcinoma..
      ,
      • Andersen C.L.
      • Hostetter G.
      • Grigoryan A.
      • Sauter G.
      • Kallioniemi A.
      Improved procedure for fluorescence in situ hybridization on tissue microarrays..
      ,
      • Gancberg D.
      • Di Leo A.
      • Rouas G.
      • Jarvinen T.
      • Verhest A.
      • Isola J.
      • Piccart M.J.
      • Larsimont D.
      Reliability of the tissue microarray based FISH for evaluation of the HER-2 oncogene in breast carcinoma..
      ,
      • O’Grady A.
      • Flahavan C.M.
      • Kay E.W.
      • Barrett H.L.
      • Leader M.B.
      HER-2 analysis in tissue microarrays of archival human breast cancer: Comparison of immunohistochemistry and fluorescence in situ hybridization..
      ,
      • Zhang D.
      • Salto-Tellez M.
      • Do E.
      • Putti T.C.
      • Koay E.S.
      Evaluation of HER-2/neu oncogene status in breast tumors on tissue microarrays..
      ,
      • Camp R.L.
      • Dolled-Filhart M.
      • King B.L.
      • Rimm D.L.
      Quantitative analysis of breast cancer tissue microarrays shows that both high and normal levels of HER2 expression are associated with poor outcome..
      ).
      Figure thumbnail gr7
      Fig. 7HER-2/neu mRNA detection by gene expression profiling.A, cluster analysis of transcriptional profile of a series of primary stages I-III invasive breast cancers that were sampled by fine needle aspiration (FNA) biopsy prior to the commencement of neoadjuvant chemotherapy. After mRNA extraction, mRNA expression profiling was performed using the Affymetrix u133 GeneChip™ oligonucleotide microarray system (Affymetrix, Santa Clara, CA). Heat map demonstrates a series of six genes at chromosomal locus 17q12 that were co-amplified with HER-2/neu in a significant number of cases. B, comparison of HER-2/neu mRNA expression levels measured by transcriptional profiling on 32 FNA samples and HER-2/neu gene amplification status determined by FISH on the corresponding core biopsy specimen. Note the close correlation of gene expression with amplification status. (Original data from Pusztai et al., Ref.
      • Rudolph P.
      • Olsson H.
      • Bonatz G.
      • Ratjen V.
      • Bolte H.
      • Baldetorp B.
      • Ferno M.
      • Parwaresch R.
      • Alm P.
      Correlation between p53, c-erbB-2, and topoisomerase II alpha expression, DNA ploidy, hormonal receptor status and proliferation in 356 node-negative breast carcinomas: Prognostic implications..
      ). C, hierarchical clustering after transcriptional profiling of 26 infiltrating breast cancer specimens sampled by FNA using a 62-gene model (25 genes for determining ER status and 37 genes for determining HER-2/neu status). Note the clustering of the HER-2/neu-positive cases and their separation from the HER-2/neu-negative cases. The HER-2/neu-negative ER-positive and HER-2/neu-negative ER-negative tumors can also be separated by this technique. (Original data from Pusztai et al., Ref.
      • Rudolph P.
      • Olsson H.
      • Bonatz G.
      • Ratjen V.
      • Bolte H.
      • Baldetorp B.
      • Ferno M.
      • Parwaresch R.
      • Alm P.
      Correlation between p53, c-erbB-2, and topoisomerase II alpha expression, DNA ploidy, hormonal receptor status and proliferation in 356 node-negative breast carcinomas: Prognostic implications..
      .)
      The enzyme-linked immunosorbent assay (ELISA) technique when performed on tumor cytosols made from fresh tissue samples avoids the potential antigen damage associated with fixation, embedding, and uncontrolled storage. In the six published studies listed in Table III, ELISA-based measurements of HER-2/neu protein in tumor cytosols, mostly performed in Europe, have uniformly correlated with disease outcome (
      • Dittadi R.
      • Brazzale A.
      • Pappagallo G.
      • Salbe C.
      • Nascimben O.
      • Rosabian A.
      • Gion M.
      ErbB2 assay in breast cancer: Possibly improved clinical information using a quantitative method..
      ,
      • Eissa S.
      • Khalifa A.
      • el-Gharib A.
      • Salah N.
      • Mohamed M.K.
      Multivariate analysis of DNA ploidy, p53, c-erbB-2 proteins, EGFR, and steroid hormone receptors for short-term prognosis in breast cancer..
      ,
      • Ferrero-Pous M.
      • Hacene K.
      • Bouchet C.
      • Le Doussal V.
      • Tubiana-Hulin M.
      • Spyratos F.
      Relationship between c-erbB-2 and other tumor characteristics in breast cancer prognosis..
      ,
      • Eppenberger-Castori S.
      • Kueng W.
      • Benz C.
      • Caduff R.
      • Varga Z.
      • Bannwart F.
      • Fink D.
      • Dieterich H.
      • Hohl M.
      • Muller H.
      • Paris K.
      • Schoumacher F.
      • Eppenberger U.
      Prognostic and predictive significance of ErbB-2 breast tumor levels measured by enzyme immunoassay..
      ,
      • Gaci Z.
      • Bouin-Pineau M.H.
      • Gaci M.
      • Daban A.
      • Ingrand P.
      • Metaye T.
      Prognostic impact of cathepsin D and c-erbB-2 oncoprotein in a subgroup of node-negative breast cancer patients with low histological grade tumors..
      ,
      • el-Ahmady O.
      • el-Salahy E.
      • Mahmoud M.
      • Wahab M.A.
      • Eissa S.
      • Khalifa A.
      Multivariate analysis of bcl-2, apoptosis, P53 and HER-2/neu in breast cancer: A short-term follow-up..
      ). In a recent study from Europe, the results of ELISA measurements on tumor extracts correlated closely with both HER-2/neu gene amplification results detected by FISH and protein expression results measured by IHC (
      • Muller V.
      • Thomssen C.
      • Karakas C.
      • Eustermann I.
      • Ramirez Porras J.
      • Coith C.
      • Berger J.
      • Loning T.
      • Janicke F.
      • Pantel K.
      Quantitative assessment of HER-2/neu protein concentration in breast cancer by enzyme-linked immunosorbent assay..
      ). However, the small size of breast cancers associated with expanded screening programs in the United States generally precludes tumor tissue ELISA methods because insufficient tumor tissue is available to produce a cytosol.

      TRASTUZUMAB (HERCEPTIN™)

      Using recombinant technologies, trastuzumab, a monoclonal IgG1 class-humanized murine antibody (Fig. 8), was developed by Genentech (South San Francisco, CA) to specifically bind the extracellular portion of HER-2/neu. This antibody therapy was initially targeted specifically for patients with advanced relapsed breast cancer that overexpressed the HER-2/neu protein (
      • Huston J.S.
      • George A.J.
      Engineered antibodies take center stage..
      ). Since its launch in 1998, trastuzumab has become an important therapeutic option for patients with HER-2/neu-positive breast cancer. Trastuzumab is widely used for its approved indication as a second line of treatment for advanced metastatic disease, and is also being studied in adjuvant treatment for earlier-stage disease and in neo-adjuvant treatment protocols (
      • Hortobagyi G.N.
      Overview of treatment results with trastuzumab (Herceptin) in metastatic breast cancer..
      ,
      • McKeage K.
      • Perry C.M.
      Trastuzumab: A review of its use in the treatment of metastatic breast cancer overexpressing HER2..
      ,
      • Shawver L.K.
      • Slamon D.
      • Ullrich A.
      Smart drugs: Tyrosine kinase inhibitors in cancer therapy..
      ,
      • Ligibel J.A.
      • Winer E.P.
      Trastuzumab/chemotherapy combinations in metastatic breast cancer..
      ). Using a clinical trial IHC assay to select patients for the phase III pivot trial, the addition of trastuzumab to chemotherapy (either anthracycline plus cyclophosphamide or taxane) was associated with a longer time to disease progression (median 7.4 versus 4.6 months; p < 0.001), a higher rate of objective response (50% versus 32%; p < 0.001), a longer duration of response (median 9.1 versus 6.1 months; p < 0.001), a lower rate of death at 1 year (22% versus 33%; p = 0.008), longer survival (median survival 25.1 versus 20.3 months; p = 0.01), and a 20% reduction in the risk of death (
      • Slamon D.J.
      • 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..
      ). Although not completely understood at this time, mechanisms believed to be associated with the anti-tumor effects of trastuzumab are listed in Table V.
      Figure thumbnail gr8
      Fig. 8Trastuzumab (Herceptin™). The trastuzumab molecule includes the murine 4D5 antigen recognition antibody and the human IgG1 class antibody structure.
      In the original pivot trial, class III or IV cardiac dysfunction occurred in 27% of the anthracycline and cyclophosphamide plus trastuzumab-treated group compared with 8% of the group given an anthracycline and cyclophosphamide alone (
      • Slamon D.J.
      • 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..
      ). Cardiac toxicity has remained a significant limiting factor for the use of trastuzumab since its FDA approval in late 1998 (
      • Schneider J.W.
      • Chang A.Y.
      • Garratt A.
      Trastuzumab cardiotoxicity: Speculations regarding pathophysiology and targets for further study..
      ). Studies have demonstrated that HER-2 and HER-4 with its ligand heregulin are necessary for normal development of the heart, and knockout mice that lack the HER-2/neu gene expression in their cardiac myocytes develop progressive dilated cardiomyopathy (
      • Ozcelik C.
      • Erdmann B.
      • Pilz B.
      • Wettschureck N.
      • Britsch S.
      • Hubner N.
      • Chien K.R.
      • Birchmeier C.
      • Garratt A.N.
      Conditional mutation of the ErbB2 (HER2) receptor in cardiomyocytes leads to dilated cardiomyopathy..
      ). This has led to the development of nonanthracycline-containing regimens combining trastuzumab with taxanes and platinins (
      • Nahta R.
      • Esteva F.J.
      HER-2-targeted therapy: Lessons learned and future directions..
      ). Preliminary data from phase II studies of these combinations have generated promising results (
      • Nabholtz J.M.
      • Crown J.
      Phase III studies of single-agent docetaxel in patients with metastatic breast cancer who have progressed despite previous chemotherapy regimens: preliminary results..
      ), and a recent report listed a time to progression of 17 months for patients with HER-2/neu-amplified metastatic breast cancer treated with the combination of docetaxel, carboplatin, and trastuzumab (
      • Slamon D.J.
      • Patel R.
      • Northfelt R.
      • Pegram M.
      • Rubin J.
      • Sebastian G.
      • Tannenbaum S.
      • Sanchez J.
      • Quan E.
      • Toppmeyer D.
      • Overmoyer B.
      • Nabholtz J.M.
      Phase II pilot study of Herceptin combined with taxotere and carboplatin in metastatic breast cancer patients overexpressing the HER-2/neu proto-oncogene: A pilot study of the UCLA network..
      ).

      PREDICTING RESPONSE TO THERAPY

      HER-2/neu Status and the Prediction of Response to Trastuzumab Therapy—

      The best method to identify patients who may respond to trastuzumab therapy has been a source of controversy. The original IHC technique used in the trastuzumab pivot trial was the clinical trial assay (CTA), which consisted of two antibodies: 1) 4D5, the monoclonal antibody that is the actual antigen-binding murine component of Herceptin™ and is not commercially available, and 2) CB-11, a monoclonal antibody directed toward the internal domain of the p185neu receptor that is commercially available both as a research reagent and as an FDA-approved diagnostic (Pathway™, Ventana Medical Systems, Tucson, AZ). The original CTA was succeeded by the FDA-approved polyclonal HercepTest® (Dako, Glostrup, Denmark). There is good concordance between the CTA and HercepTest®, although 58 of 274 tumors that scored as positive with the CTA were scored as negative with HercepTest® and 59 of 274 tumors that scored as negative with the CTA were scored as positive with HercepTest® (

      HercepTest package insert. Dako Corporation, Glostrup, Denmark.

      ). After its FDA-approval and launch, the HercepTest® assay was initially criticized for yielding false-positive results (
      • Roche P.C.
      • Ingle J.N.
      Increased HER2 with U.S. Food and Drug Administration-approved antibody..
      ), although better performance was ultimately achieved when the test was performed exactly according to the manufacturer’s instructions. Concern over IHC accuracy using standard formalin-fixed paraffin-embedded tissue sections (
      • Paik S.
      • Bryant J.
      • Tan-Chiu E.
      • Romond E.
      • Hiller W.
      • Park K.
      • Brown A.
      • Yothers G.
      • Anderson S.
      • Smith R.
      • Wickerham D.L.
      • Wolmark N.
      Real-world performance of HER2 testing—National Surgical Adjuvant Breast and Bowel Project experience..
      ) has encouraged the use of the FISH assay for its ability to predict trastuzumab response rates. Reports that FISH could out-perform IHC in predicting trastuzumab response (Fig. 9) (
      • Mass R.D.
      • Press M.F.
      • Anderson S.
      • Murphy M.
      • Slamon D.
      Improved survival benefit from Herceptin (Trastuzumab) in patients selected by fluorescence in situ hybridization (FISH).
      ) and well-documented lower response rates of 2+ IHC staining versus 3+ staining tumors (
      • Rudolph P.
      • Alm P.
      • Olsson H.
      • Heidebrecht H.J.
      • Ferno M.
      • Baldetorp B.
      • Parwaresch R.
      Concurrent overexpression of p53 and c-erbB-2 correlates with accelerated cycling and concomitant poor prognosis in node-negative breast cancer..
      ) has resulted in an approach that either uses IHC as a primary screen with FISH testing of all 2+ cases or primary FISH-based testing (
      • Ogura H.
      • Akiyama F.
      • Kasumi F.
      • Kazui T.
      • Sakamoto G.
      Evaluation of HER-2 status in breast carcinoma by fluorescence in situ hybridization and immunohistochemistry..
      ). In a recently published study where trastuzumab was used as a single agent, the response rates in 111 assessable patients with 3+ IHC staining was 35% and the response rates for 2+ cases was 0%; the response rates in patients with and without HER-2/neu gene amplification detected by FISH were 34 and 7%, respectively (
      • Vogel C.L.
      • Cobleigh M.A.
      • Tripathy D.
      • Gutheil J.C.
      • Harris L.N.
      • Fehrenbacher L.
      • Slamon D.J.
      • Murphy M.
      • Novotny W.F.
      • Burchmore M.
      • Shak S.
      • Stewart S.J.
      • Press M.
      Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer..
      ). In another study of breast cancer treated with trastuzumab plus paclitaxel, in patients with HER2/neu-overexpressing tumors, overall response rates ranged from 67% to 81% compared with 41% to 46% in patients with normal expression of HER2/neu (
      • Seidman A.D.
      • Fornier M.N.
      • Esteva F.J.
      • Tan L.
      • Kaptain S.
      • Bach A.
      • Panageas K.S.
      • Arroyo C.
      • Valero V.
      • Currie V.
      • Gilewski T.
      • Theodoulou M.
      • Moynahan M.E.
      • Moasser M.
      • Sklarin N.
      • Dickler M.
      • D’Andrea G.
      • Cristofanilli M.
      • Rivera E.
      • Hortobagyi G.N.
      • Norton L.
      • Hudis C.A.
      Weekly trastuzumab and paclitaxel therapy for metastatic breast cancer with analysis of efficacy by HER2 immunophenotype and gene amplification..
      ). The CB11 and TAB250 antibodies for IHC and FISH featured the strongest significance (
      • Seidman A.D.
      • Fornier M.N.
      • Esteva F.J.
      • Tan L.
      • Kaptain S.
      • Bach A.
      • Panageas K.S.
      • Arroyo C.
      • Valero V.
      • Currie V.
      • Gilewski T.
      • Theodoulou M.
      • Moynahan M.E.
      • Moasser M.
      • Sklarin N.
      • Dickler M.
      • D’Andrea G.
      • Cristofanilli M.
      • Rivera E.
      • Hortobagyi G.N.
      • Norton L.
      • Hudis C.A.
      Weekly trastuzumab and paclitaxel therapy for metastatic breast cancer with analysis of efficacy by HER2 immunophenotype and gene amplification..
      ). Interestingly, in a recent published review from New York and Italy, it was noted that although FISH-based testing is more expensive and not as widely available as IHC, the data suggested that FISH was actually the most cost-effective option (
      • Fornier M.
      • Risio M.
      • Van Poznak C.
      • Seidman A.
      HER-2 testing and correlation with efficacy in Trastuzumab therapy..
      ). Other recent studies have favored the FISH approach not only to confirm 2+ IHC cases but to also confirm 3+ and prevent the use of potentially toxic trastuzumab to patients with false-positive IHC results who are unlikely to benefit from this therapy (
      • Hammock L.
      • Lewis M.
      • Phillips C.
      • Cohen C.
      Strong HER-2/neu protein overexpression by immunohistochemistry often does not predict oncogene amplification by fluorescence in situ hybridization..
      ). In summary, while the superiority of one method versus the other remains controversial (
      • Schnitt S.J.
      • Jacobs T.W.
      Current status of HER2 testing: Caught between a rock and a hard place..
      ,
      • Nichols D.W.
      • Wolff D.J.
      • Self S.
      • Metcalf J.S.
      • Jacobs D.
      • Kneuper-Hall R.
      • Cate 4th., J.C.
      A testing algorithm for determination of HER2 status in patients with breast cancer..
      ,
      • Yaziji H.
      • Gown A.M.
      Testing for HER-2/neu in breast cancer: Is fluorescence in situ hybridization superior in predicting outcome?.
      ,
      • Press M.F.
      • Slamon D.J.
      • Flom K.J.
      Evaluation of HER-2/neu gene amplification and overexpression: Comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens..
      ), most laboratories are either screening all cases with IHC and triaging selected cases for FISH testing or using FISH as the only method for HER-2/neu testing. For the laboratories that use IHC as the primary screen, the decision as to when to triage to FISH testing is also controversial. Some laboratories refer only their 2+ IHC cases, some triage 1+ and 2+, and others refer 1+, 2+, and any other cases where the HER-2/neu IHC results are not consistent with other disease parameters such as grade, stage, ploidy, S phase, and hormone receptor status.
      Figure thumbnail gr9
      Fig. 9FISH versus IHC and survival in trastuzumab single-agent and combination chemotherapy for advanced metastatic breast cancer. Survival curves demonstrate a significant (p < 0.05) increase in median duration of survival for patients treated with either Herceptin™ alone or Herceptin™ plus chemotherapy in patients whose primary tumors were classified by FISH (Pathvysion™ test) for HER-2/neu gene amplification status versus patients who were classified by IHC (Genentech CTA with antibodies 4D5 and CB-11). (Data from Genentech Clinical Trial H-0648 cited by Mass R et al. in Ref. 127.)

      Prediction of Response of Breast Cancer to Other Therapies—

      The best established correlate between HER-2/neu status and non-Herceptin™ therapy response is the reported resistance of HER-2/neu-positive patients to hormonal therapy alone (
      • Nunes R.A.
      • Harris L.N.
      The HER2 extracellular domain as a prognostic and predictive factor in breast cancer..
      ,
      • Dowsett M.
      Overexpression of HER-2 as a resistance mechanism to hormonal therapy for breast cancer..
      ,
      • Muss H.B.
      Role of adjuvant endocrine therapy in early-stage breast cancer..
      ,
      • Schmid P.
      • Wischnewsky M.B.
      • Sezer O.
      • Bohm R.
      • Possinger K.
      Prediction of response to hormonal treatment in metastatic breast cancer..
      ). Tumors that overexpress HER-2/neu are more likely to be ER-negative and PR-negative than tumors that do not show overexpression. In fact, when measured as continuous variables, the expression of HER-2/neu appears to be inversely related to the expression of ER and PR even in hormone receptor-positive tumors (
      • Konecny G.
      • Pauletti G.
      • Pegram M.
      • Untch M.
      • Dandekar S.
      • Aguilar Z.
      • Wilson C.
      • Rong H.M.
      • Bauerfeind I.
      • Felber M.
      • Wang H.J.
      • Beryt M.
      • Seshadri R.
      • Hepp H.
      • Slamon D.J.
      Quantitative association between HER-2/neu and steroid hormone receptors in hormone receptor-positive primary breast cancer..
      ). In some studies, HER-2/neu-positive tumors were specifically resistant to tamoxifen therapy (
      • Burke H.B.
      • Hoang A.
      • Iglehart J.D.
      • Marks J.R.
      Predicting response to adjuvant and radiation therapy in patients with early stage breast carcinoma..
      ,
      • Sjogren S.
      • Inganas M.
      • Lindgren A.
      • Holmberg L.
      • Bergh J.
      Prognostic and predictive value of c-erbB-2 overexpression in primary breast cancer, alone and in combination with other prognostic markers..
      ,
      • Ferrero-Pous M.
      • Hacene K.
      • Bouchet C.
      • Le Doussal V.
      • Tubiana-Hulin M.
      • Spyratos F.
      Relationship between c-erbB-2 and other tumor characteristics in breast cancer prognosis..
      ,
      • Jukkola A.
      • Bloigu R.
      • Soini Y.
      • Savolainen E.R.
      • Holli K.
      • Blanco G.
      c-erbB-2 positivity is a factor for poor prognosis in breast cancer and poor response to hormonal or chemotherapy treatment in advanced disease..
      ,
      • Pinto A.E.
      • Andre S.
      • Pereira T.
      • Nobrega S.
      • Soares J.
      C-erbB-2 oncoprotein overexpression identifies a subgroup of estrogen receptor positive (ER+) breast cancer patients with poor prognosis..
      ,
      • Carlomagno C.
      • Perrone F.
      • Gallo C.
      • De Laurentiis M.
      • Lauria R.
      • Morabito A.
      • Pettinato G.
      • Panico L.
      • D’Antonio A.
      • Bianco A.R.
      • De Placido S.
      CerbB2 overexpression decreases the benefit of adjuvant Tamoxifen in early-stage breast cancer without ancillary lymph node metastases..
      ). However, in other studies, HER-2/neu status failed to predict tamoxifen resistance in ER-positive cases (
      • Elledge R.M.
      • Green S.
      • Ciocca D.
      • Pugh R.
      • Allred D.C.
      • Clark G.M.
      • Hill J.
      • Ravdin P.
      • O’Sullivan J.
      • Martino S.
      • Osborne C.K.
      HER-2 expression and response to tamoxifen in estrogen receptor-positive breast cancer: A southwest oncology group study..
      ). In another study, ER-positive HER-2/neu-positive tumors were not only resistant to tamoxifen, single-agent tamoxifen treatment actually had an adverse impact compared with untreated patients (
      • De Placido S.
      • De Laurentiis M.
      • Carlomagno C.
      • Gallo C.
      • Perrone F.
      • Pepe S.
      • Ruggiero A.
      • Marinelli A.
      • Pagliarulo C.
      • Panico L.
      • Pettinato G.
      • Petrella G.
      • Bianco A.R.
      Twenty-year results of the Naples GUN randomized trial: predictive factors of adjuvant tamoxifen efficacy in early breast cancer..
      ). However, this finding has not, to date, been confirmed by large intergroup studies in the United States (
      • Ravdin P.M.
      • Green S.
      • Albain V.
      • Boucher V.
      • Ingle J.
      • Pritchard K.
      • Shepard L.
      • Davidson N.
      • Hayes D.F.
      • Clark G.M.
      • Martino S.
      • Osborne C.K.
      • Allred D.C.
      Initial report of the SWOG biologicalcorrelative study of c-ERBB2 expression as a predictor of outcome in a trial comparing adjuvant CAF with Tamoxifen alone..
      ). Most recently, ER-negative HER-2/neu-positive tumors have been associated with an improved response to alternative hormonal therapies such as the aromatase inhibitors (
      • Ellis M.J.
      • Coop A.
      • Singh B.
      • Mauriac L.
      • Llombert-Cussac A.
      • Janicke F.
      • Miller W.R.
      • Evans D.B.
      • Dugan M.
      • Brady C.
      • Quebe-Fehling E.
      • Borgs M.
      Letrozole is more effective neoadjuvant endocrine therapy than tamoxifen for ErbB-1- and/or ErbB-2-positive, estrogen receptor-positive primary breast cancer: Evidence from a phase III randomized trial..
      ). HER-2/neu protein overexpression has also been linked to resistance of tumors in patients treated with cytoxan, methotrexate, 5-fluorouracil (CMF) adjuvant chemotherapy (
      • Giai M.
      • Roagna R.
      • Ponzone R.
      • De Bortoli M.
      • Dati C.
      • Sismondi P.
      Prognostic and predictive relevance of C-erb B2 and ras expression in node positive and negative breast cancer..
      ,
      • De Placido S.
      • De Laurentiis M.
      • Carlomagno C.
      • Gallo C.
      • Perrone F.
      • Pepe S.
      • Ruggiero A.
      • Marinelli A.
      • Pagliarulo C.
      • Panico L.
      • Pettinato G.
      • Petrella G.
      • Bianco A.R.
      Twenty-year results of the Naples GUN randomized trial: predictive factors of adjuvant tamoxifen efficacy in early breast cancer..
      ) as well as to taxane-based regimens (
      • Berns E.M.
      • Foekens J.A.
      • van Staveren I.L.
      • van Putten W.L.
      • de Koning H.Y.
      • Portengen H.
      • Klijn J.G.
      Oncogene amplification and prognosis in breast cancer: Relationship with systemic treatment..
      ,
      • Sparano J.A.
      Taxanes for breast cancer: An evidence-based review of randomized phase II and phase III trials..
      ,
      • Yu D.
      Mechanisms of ErbB2-mediated paclitaxel resistance and trastuzumab-mediated paclitaxel sensitization in ErbB2-overexpressing breast cancers..
      ). However, in another study, HER-2/neu-positive breast cancers were three times more sensitive to taxol (
      • Baselga J.
      • Seidman A.D.
      • Rosen P.P.
      • Norton L.
      HER-2 overexpression and paclitazel sensitivity in breast cancer: therapeutic implications..
      ). HER-2/neu overexpression has also been associated with enhanced response rates to anthracycline-containing chemotherapy regimens in some, but not all, studies (
      • Muss H.B.
      • Thor A.D.
      • Berry D.A.
      • Kute T.
      • Liu E.T.
      • Koerner F.
      • Cirrincione C.T.
      • Budman D.R.
      • Wood W.C.
      • Barcos M.
      • et al.
      Cerb-B2 expression and response to adjuvant therapy in women with node-positive early breast cancer..
      ,
      • Ligibel J.A.
      • Winer E.P.
      Trastuzumab/chemotherapy combinations in metastatic breast cancer..
      ,
      • Kim R.
      • Tanabe K.
      • Uchida Y.
      The role of HER-2 oncoprotein in drug-sensitivity in breast cancer..
      ,
      • Hamilton A.
      • Larsimont D.
      • Paridaens R.
      • Drijkoningen M.
      • van de Vijver M.
      • Bruning P.
      • Hanby A.
      • Houston S.
      • Treilleux I.
      • Guastalla J.P.
      • Van Vreckem A.
      • Sylvester R.
      • Piccart M.
      A study of the value of p53, HER2, and Bcl-2 in the prediction of response to doxorubicin and paclitaxel as single agents in metastatic breast cancer: A companion study to EORTC 10923..
      ,
      • Di Leo A.
      • Larsimont D.
      • Gancberg D.
      • Jarvinen T.
      • Beauduin M.
      • Vindevoghel A.
      • Michel J.
      • Focan C.H.
      • Ries F.
      • Gobert P.H.
      • Closon-Dejardin M.T.
      • Dolci S.
      • Rouas G.
      • Paesmans M.
      • Lobelle J.P.
      • Isola J.
      • Piccart M.J.
      HER-2 and topo-isomerase II alpha as predictive markers in a population of node-positive breast cancer patients randomly treated with adjuvant CMF or epirubicin plus cyclophosphamide..
      ,
      • Petit T.
      • Borel C.
      • Ghnassia J.P.
      • Rodier J.F.
      • Escande A.
      • Mors R.
      • Haegele P.
      Chemotherapy response of breast cancer depends on HER-2 status and anthracycline dose intensity in the neoadjuvant setting..
      ,
      • Harris L.N.
      • Yang L.
      • Liotcheva V.
      • Pauli S.
      • Iglehart J.D.
      • Colvin O.M.
      • Hsieh T.S.
      Induction of topoisomerase II activity after ErbB2 activation is associated with a differential response to breast cancer chemotherapy..
      ). In a recent study of the response of locally advanced breast cancer to a neoadjuvant chemotherapy regimen consisting of 5-fluoruracil, doxorubicin, and cyclophosphamide, the pretreatment HER-2/neu status detected by IHC was not predictive of treatment response (
      • Zhang F.
      • Yang Y.
      • Smith T.
      • Kau S.W.
      • McConathy J.M.
      • Esteva F.J.
      • Kuerer H.M.
      • Symmans W.F.
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      • Hortobagyi G.N.
      • Pusztai L.
      Correlation between HER-2 expression and response to neoadjuvant chemotherapy with 5-fluorouracil, doxorubicin, and cyclophosphamide in patients with breast carcinoma..
      ).
      Because anthracyclines are topoisomerase inhibitors and topoisomerase IIα (TOP2A) is frequently co-amplified with HER-2/neu, it has been suggested that HER-2/neu may be serving as a surrogate marker. Cell lines transfected with HER-2/neu and then exposed to doxorubicin in vitro did not show enhanced sensitivity to the chemotherapy relative to the parent cell lines (
      • Pegram M.D.
      • Finn R.S.
      • Arzoo K.
      • Beryt M.
      • Pietras R.J.
      • Slamon D.J.
      The effect of HER-2/neu overexpression on chemotherapeutic drug sensitivity in human breast and ovarian cancer cells..
      ). Recently, it was shown that HER-2/neu protein expression, but not TOP2A expression, predicted the response of breast cancer to the anti-topoisomerase anthracycline, epirubicin (
      • Jarvinen T.A.
      • Holli K.
      • Kuukasjarvi T.
      • Isola J.J.
      Predictive value of topoisomerase II alpha and other prognostic factors for epirubicin chemo-therapy in advanced breast cancer..
      ). This is in contrast to another study that claimed that TOP2A gene amplification and deletion seem to account for both relative chemosensitivity and resistance to topoII inhibitors and that TOP2A status occurs in breast cancer independent from the HER-2/neu status (
      • Jarvinen T.A.
      • Liu E.T.
      Topoisomerase IIalpha gene (TOP2A) amplification and deletion in cancer—More common than anticipated..
      ). However, other studies have consistently linked co-expression and co-amplification of the topoisomerase IIα and HER-2/neu genes with adverse prognosis and sensitivity to anthracycline drugs (
      • Depowski P.L.
      • Rosenthal S.I.
      • Brien T.P.
      • Stylos S.
      • Johnson R.L.
      • Ross J.S.
      Topoisomerase II alpha expression in breast cancer: correlation with outcome variables..
      ,
      • Jarvinen T.A.
      • Tanner M.
      • Rantanen V.
      • Barlund M.
      • Borg A.
      • Grenman S.
      • Isola J.
      Amplification and deletion of topoisomerase II alpha associate with ErbB-2 amplification and affect sensitivity to topoisomerase II inhibitor doxorubicin in breast cancer..
      ,
      • Tanner M.
      • Jarvinen P.
      • Isola J.
      Amplification of HER-2/neu and topoisomerase II alpha in primary and metastatic breast cancer..
      ,
      • Park K.
      • Kim J.
      • Lim S.
      • Han S.
      Topoisomerase II-alpha (topoll) and HER2 amplification in breast cancers and response to preoperative doxorubicin chemotherapy..
      ,
      • Di Leo A.
      • Gancberg D.
      • Larsimont D.
      • Tanner M.
      • Jarvinen T.
      • Rouas G.
      • Dolci S.
      • Leroy J.Y.
      • Paesmans M.
      • Isola J.
      • Piccart M.J.
      HER-2 amplification and topoisomerase II alpha gene aberrations as predictive markers in node-positive breast cancer patients randomly treated either with an anthracycline-based therapy or with cyclophosphamide, methotrexate, and 5-fluorouracil..
      ,
      • Coon J.S.
      • Marcus E.
      • Gupta-Burt S.
      • Seelig S.
      • Jacobson K.
      • Chen S.
      • Renta V.
      • Fronda G.
      • Preisler H.D.
      Amplification and overexpression of topoisomerase II alpha predict response to anthracycline-based therapy in locally advanced breast cancer..
      ). Finally, HER-2/neu immunostaining has successfully predicted local recurrence in patients receiving surgery and radiation (
      • Haffty B.G.
      • Brown F.
      • Carter D.
      • Flynn S.
      Evaluation of HER-2/neu oncoprotein expression as a prognostic indicator of local recurrence in conservatively treated breast cancer: A case-control study..
      ,
      • Yamauchi H.
      • O’Neill A.
      • Gelman R.
      • Carney W.
      • Tenney D.Y.
      • Hosch S.
      • Hayes D.F.
      Prediction of response to antiestrogen therapy in advanced breast cancer patients by pretreatment circulating levels of extracellular domain of the HER-2/c-neu protein..
      ). In summary, although strong trends have been presented in the published studies, including the resistance to tamoxifen and sensitivity to anthracycline regimens for HER-2/neu-positive tumors, more studies are needed using appropriate control arms to confirm these important associations. Should this be accomplished, it would seem likely that HER-2/neu testing, which achieved standard of care status in the American Society of Clinical Oncology breast cancer clinical practice guidelines in 2001, would be of even greater value in the management of breast cancer patients.

      SUMMARY AND FUTURE CLINICAL POTENTIAL

      The testing of newly diagnosed breast cancer specimens for HER-2/neu status has now achieved “standard of practice” status for the management of breast cancer in the United States. The discussion as to the best method to determine HER-2/neu status in these samples continues with the FISH method gaining popularity due to the recent evidence that it, in comparison to IHC, may more accurately predict clinical responses to trastuzumab-based therapies. With trastuzumab achieving excellent results in the treatment of HER-2/neu-positive advanced disease and under extensive evaluation in major clinical trials for its potential efficacy when used earlier, the potential role(s) for HER-2/neu testing as a predictor(s) of responses to other therapies being resolved by large prospective clinical outcome studies and the more convenient gene-based CISH technique “waiting in the wings,” the “story” of HER-2/neu testing in breast cancer will continue to unfold over the next several years.

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