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Originally published In Press as doi:10.1074/mcp.R400001-MCP200 on February 3, 2004.
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Molecular & Cellular Proteomics 3:379-398, 2004.
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Review

Targeted Therapy in Breast Cancer

The HER-2/neu Gene and Protein

Jeffrey S. Ross{ddagger},§,, Jonathan A. Fletcher||, Kenneth J. Bloom**, Gerald P. Linette§,{ddagger}{ddagger}, James Stec§, W. Fraser Symmans§§, Lajos Pusztai§§ and Gabriel N. Hortobagyi§§

From the {ddagger} Department of Pathology and Laboratory Medicine, Albany Medical College, Albany, NY 12208; § Division of Molecular Medicine, Millennium Pharmaceuticals, Inc., Cambridge, MA 01238; || Department of Pathology, Brigham Women’s Hospital and Harvard Medical School, 75 Francis Street, Boston, MA 02115; ** US Labs, Inc., 2601 Campus Drive, Irvine, CA 92612; {ddagger}{ddagger} Department of Medicine, Washington University, St. Louis, MO 63110; §§ Departments of Medicine and Pathology, MD Anderson Cancer Center, Houston, TX 77030


   ABSTRACT
TOP
 ABSTRACT
BACKGROUND AND CLINICAL...
 TESTING FOR HER-2/neu STATUS...
 HER-2/neu TESTING TECHNIQUES
 TRASTUZUMAB (HERCEPTINTM)
 PREDICTING RESPONSE TO THERAPY
 SUMMARY AND FUTURE CLINICAL...
 REFERENCES
 
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)1 or HER family (Fig. 2) (1). 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 (2). 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 C{gamma} (PLC-{gamma}) pathway, ultimately affect cell proliferation, survival, motility, and adhesion (Fig. 3).



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  		FIG. 1. The 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 (HerceptinTM).

 


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  		FIG. 2. The HER (erb) gene family. Note that HER-2/neu has no known ligands and that HER-3 has no intrinsic tyrosine kinase activity.

 


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  		FIG. 3. Dimerization 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-{gamma} 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) (3). 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 (4) and their signaling is more potent (5) 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 (1).


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  		TABLE I Dimerization of the HER-2/neu receptor and signaling pathway

 

   BACKGROUND AND CLINICAL RELEVANCE
TOP
 ABSTRACT
 BACKGROUND AND CLINICAL...
TESTING FOR HER-2/neu STATUS...
 HER-2/neu TESTING TECHNIQUES
 TRASTUZUMAB (HERCEPTINTM)
 PREDICTING RESPONSE TO THERAPY
 SUMMARY AND FUTURE CLINICAL...
 REFERENCES
 
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 (6–25). Two studies involving 379 patients reported no significant association of serum levels with prognosis (22, 23). 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 (8, 16–18, 21), whereas three additional studies did not demonstrate this association (15, 23, 25). 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 (13, 21), but not in others (23). Serum HER-2/neu protein measurements have successfully predicted resistance to high-dose chemotherapy (16–18), bone marrow transplantation (17), and response to trastuzumab single agent and combination treatment for metastatic HER-2/neu-positive disease (26–29).

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 (30–32). 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 (33). 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 (34–36). 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)) (37–42). 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 (42). 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 (42). HER-2/neu amplification and overexpression has been associated with adverse outcome in some studies of male breast carcinoma (43–46), but not in others (47–49). 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 (50).

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 (1, 50–56).

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 (57). In invasive breast cancer with HER-2 overexpression, the receptor appears to be activated only in a small subset (12%) of patients (57, 58). Interestingly, the proportion of cases with phosphorylated HER-2 appears to be greater (58%) in ductal carcinoma in situ (59). In one large study of 800 cases of invasive breast cancer with HER-2 overexpression, only cases with phosphorylated HER-2 displayed adverse prognosis (58). 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
TOP
 ABSTRACT
 BACKGROUND AND CLINICAL...
 TESTING FOR HER-2/neu STATUS...
HER-2/neu TESTING TECHNIQUES
 TRASTUZUMAB (HERCEPTINTM)
 PREDICTING RESPONSE TO THERAPY
 SUMMARY AND FUTURE CLINICAL...
 REFERENCES
 
Both morphology-based and molecular-based techniques have been used to measure HER-2/neu status in breast cancer clinical samples (Table II) (33, 60–138). 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.


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  		TABLE II Methods of detection of HER-2/neu status in breast cancer

 

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  		TABLE III HER-2/neu status and prognosis in breast cancer

 

   HER-2/neu TESTING TECHNIQUES
TOP
 ABSTRACT
 BACKGROUND AND CLINICAL...
 TESTING FOR HER-2/neu STATUS...
 HER-2/neu TESTING TECHNIQUES
TRASTUZUMAB (HERCEPTINTM)
 PREDICTING RESPONSE TO THERAPY
 SUMMARY AND FUTURE CLINICAL...
 REFERENCES
 
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 (1, 51, 52, 139). 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 (140). 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 (141). 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 (142). 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 (143). 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 (144). The use of a quantitative image analysis system can reduce slide-scoring variability among pathologists, especially in 2+ cases (145). 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) (146). Two commercially available HER-2/neu IHC kits, the Dako HerceptestTM and the Ventana PathwayTM, are approved for sale for determining eligibility for patients to receive HerceptinTM by the Food and Drug Administration (FDA).



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  		FIG. 4. HER-2/neu testing in breast cancer. A, HER-2/neu protein expression in infiltrating ductal breast cancer measured by immunohistochemistry using the HerceptestTM 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 HerceptestTM antibody x200). (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 PathvysionTM 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 InformTM 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 (SpotLightTM HER-2/neu probe, Zymed Laboratories Inc., South San Francisco, CA).

 

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  		TABLE V Mechanism of action of trastuzumaba

 


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  		FIG. 5. Image 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 InformTM test that measures only HER-2/neu gene copies and the Abbott-Vysis PathvysionTM test that includes a chromosome 17 probe in a dual color format. Published studies indicate that the two assays are highly correlative (147). The advantages and disadvantages of IHC- and FISH-based HER-2/neu testing are summarized in Table IV.


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  		TABLE IV Advantages and disadvantages of IHC and FISH-based testing for HER-2/neu in breast cancer

 
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 (148, 149). 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 (150). 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.



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  		FIG. 6. Simultaneous 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 (151, 152) 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 (153), 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 (121). 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 (154–156). 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 (154). 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 (157–163).



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  		FIG. 7. HER-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 GeneChipTM 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. 114). 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. 114.)

 
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 (102, 104, 123, 125, 127, 138). 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 (164). 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 (HERCEPTINTM)
TOP
 ABSTRACT
 BACKGROUND AND CLINICAL...
 TESTING FOR HER-2/neu STATUS...
 HER-2/neu TESTING TECHNIQUES
 TRASTUZUMAB (HERCEPTINTM)
PREDICTING RESPONSE TO THERAPY
 SUMMARY AND FUTURE CLINICAL...
 REFERENCES
 
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 (165). 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 (166–169). 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 (170). Although not completely understood at this time, mechanisms believed to be associated with the anti-tumor effects of trastuzumab are listed in Table V.



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  		FIG. 8. Trastuzumab (HerceptinTM). 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 (170). Cardiac toxicity has remained a significant limiting factor for the use of trastuzumab since its FDA approval in late 1998 (171). 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 (172). This has led to the development of nonanthracycline-containing regimens combining trastuzumab with taxanes and platinins (173). Preliminary data from phase II studies of these combinations have generated promising results (174), 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 (175).


   PREDICTING RESPONSE TO THERAPY
TOP
 ABSTRACT
 BACKGROUND AND CLINICAL...
 TESTING FOR HER-2/neu STATUS...
 HER-2/neu TESTING TECHNIQUES
 TRASTUZUMAB (HERCEPTINTM)
 PREDICTING RESPONSE TO THERAPY
SUMMARY AND FUTURE CLINICAL...
 REFERENCES
 
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 HerceptinTM 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 (PathwayTM, 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® (176). After its FDA-approval and launch, the HercepTest® assay was initially criticized for yielding false-positive results (177), 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 (141) 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) (178) and well-documented lower response rates of 2+ IHC staining versus 3+ staining tumors (128) 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 (179). 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 (180). 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 (181). The CB11 and TAB250 antibodies for IHC and FISH featured the strongest significance (181). 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 (182). 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 (183). In summary, while the superiority of one method versus the other remains controversial (139, 184–186), 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.



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  		FIG. 9. FISH 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 HerceptinTM alone or HerceptinTM plus chemotherapy in patients whose primary tumors were classified by FISH (PathvysionTM 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-HerceptinTM therapy response is the reported resistance of HER-2/neu-positive patients to hormonal therapy alone (28, 187–189). 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 (190). In some studies, HER-2/neu-positive tumors were specifically resistant to tamoxifen therapy (13, 111, 123, 126, 130, 191). However, in other studies, HER-2/neu status failed to predict tamoxifen resistance in ER-positive cases (192). 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 (193). However, this finding has not, to date, been confirmed by large intergroup studies in the United States (194). 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 (195). HER-2/neu protein overexpression has also been linked to resistance of tumors in patients treated with cytoxan, methotrexate, 5-fluorouracil (CMF) adjuvant chemotherapy (90, 193) as well as to taxane-based regimens (196–198). However, in another study, HER-2/neu-positive breast cancers were three times more sensitive to taxol (199). HER-2/neu overexpression has also been associated with enhanced response rates to anthracycline-containing chemotherapy regimens in some, but not all, studies (91, 169, 200–204). 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 (205).

Because anthracyclines are topoisomerase inhibitors and topoisomerase II{alpha} (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 (206). 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 (207). 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 (208). However, other studies have consistently linked co-expression and co-amplification of the topoisomerase II{alpha} and HER-2/neu genes with adverse prognosis and sensitivity to anthracycline drugs (209–214). Finally, HER-2/neu immunostaining has successfully predicted local recurrence in patients receiving surgery and radiation (215–216). 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
TOP
 ABSTRACT
 BACKGROUND AND CLINICAL...
 TESTING FOR HER-2/neu STATUS...
 HER-2/neu TESTING TECHNIQUES
 TRASTUZUMAB (HERCEPTINTM)
 PREDICTING RESPONSE TO THERAPY
 SUMMARY AND FUTURE CLINICAL...
REFERENCES
 
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.


   FOOTNOTES
 
Received, January 20, 2004, and in revised form, February 2, 2004.

Published, MCP Papers in Press, February 3, 2004, DOI 10.1074/mcp.R400001-MCP200

1 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. Back

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Albany Medical College, Department of Pathology, Mail Code 81, 47 New Scotland Avenue, Albany, NY 12208. Tel.: 518-262-5461; Fax: 518-262-8092; E-mail: rossj{at}mail.amc.edu


   REFERENCES
TOP
 ABSTRACT
 BACKGROUND AND CLINICAL...
 TESTING FOR HER-2/neu STATUS...
 HER-2/neu TESTING TECHNIQUES
 TRASTUZUMAB (HERCEPTINTM)
 PREDICTING RESPONSE TO THERAPY
 SUMMARY AND FUTURE CLINICAL...
 REFERENCES
 

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