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Clinical Cancer Research Vol. 12, 424-431, January 2006
© 2006 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

p95HER-2 Predicts Worse Outcome in Patients with HER-2-Positive Breast Cancer

Rosana Sáez1, Miguel A. Molina3, Elizabeth E. Ramsey4, Federico Rojo3, Edward J. Keenan4, Joan Albanell3, Ana Lluch2, Javier García-Conde2, José Baselga3 and Gail M. Clinton5

Authors' Affiliations:1 Cell Biology Department, Faculty of Biological Sciences, University of Valencia, Burjasot; 2 Hematology and Medical Oncology Service, Hospital Clínico Universitario, Valencia; 3 Laboratory of Oncology Research, Medical Oncology Service, Vall d'Hebron Hospital, Barcelona, Spain; and Departments of 4 Physiology and Pharmacology and 5 Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon

Requests for reprints: Gail M. Clinton, Department of Biochemistry and Molecular Biology, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239. Phone: 503-494-2543; Fax: 503-494-8393; E-mail: clinton{at}ohsu.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Background: The HER-2 receptor undergoes a proteolytic cleavage generating an NH2-terminally truncated fragment, p95HER-2, that is membrane-associated and tyrosine-phosphorylated. We have reported that p95HER-2, but not the full-length receptor, p185HER-2, correlated with the extent of lymph node involvement in patients with breast cancer and its expression was significantly enhanced in nodal metastatic tissue. These facts suggested an important role for p95HER-2 either as a marker or cause of metastasis and poor outcome in breast cancer. In this work, we have studied the prognostic value of p95HER-2 in breast cancer.

Methods: Primary breast tumor tissues (n = 483) were from surgical resections conducted in hospitals in two different countries: the U.S. (n = 334) and Spain (n = 149). HER-2 protein forms, including p185HER-2 and p95HER-2, were examined in extracts of primary breast tumors by Western blot analysis. The levels of the two forms (high or low) were tested for association with other clinicopathologic factors and for correlation with disease-free survival.

Results: The median follow-up was 46 months. A high level of p95HER-2 in primary tumor tissue correlated with reduced 5-year disease-free survival (hazard ratio, 2.55; 95% confidence interval, 2.13-8.01; P < 0.0001). The median time for disease-free survival was 32 versus 139 months in patients with low levels of p95HER-2. In comparison, high levels of the full-length p185HER-2 did not significantly correlate with poor outcome (P > 0.1). Multivariate analysis revealed that high p95HER-2 was an independent predictor of disease-free survival (hazard ratio, 1.59; 95% confidence interval, 1.246-1.990; P = 0.0004).

Conclusions: p95HER-2 expression is an independent prognostic factor in breast cancer and defines a group of patients with HER-2-positive breast cancer with significantly worse outcome.

The HER (or ErbB) family of transmembrane tyrosine kinase receptors is composed of four members, HER1 to HER4. The important biological role of ErbB receptors in the signaling network that drives epithelial cell proliferation and transformation has led to an extensive investigation about its potential as prognostic or diagnostic indicators for human cancer. In addition, its extracellular accessibility and its overexpression in some human tumors make them appropriate targets for tumor-specific therapies. In this regard, several inhibitors of the tyrosine-kinase domain of HER receptors and monoclonal antibodies directed against ectodomains have been developed, and some of them have shown clinical efficacy (1–6).

Overexpression of HER-2 (erbB2), a 185 kDa membrane receptor tyrosine kinase, is found in 25% to 30% of breast cancers and has been associated with an increased rate of relapse and death (7). However, the significance of HER-2, particularly in early stage cancer, prior to metastasis, is controversial, and thus far, most studies have reported only weak predictive value in this group (7, 8). Indeed, a large consensus study by the American Society of Clinical Oncology did not recommend HER-2 as a prognostic factor in breast cancer (7). Although HER-2 may have limited value as a prognostic indicator, it is nonetheless clinically very relevant. Several studies have suggested that HER-2 overexpression correlates with response to other adjuvant therapy regimes (8, 9). Moreover, the HER-2 receptor is routinely measured in patients to predict the likelihood of their response to the therapeutic monoclonal antibody trastuzumab (Herceptin), which binds to p185HER-2 at the tumor cell surface. Other inhibitors that function by binding to the extracellular domain of the HER-2 receptor are under investigation as potential anticancer agents (10, 11).

The HER-2 receptor, p185HER-2, undergoes a slow proteolytic shedding of its ectodomain in cultured cells (12–14). The HER-2 ectodomain has also been found in the serum of patients with advanced breast cancer, where it correlates with recurrence (15) and with decreased responsiveness to endocrine therapy and chemotherapy (15–23). In addition, proteolytic shedding of the HER-2 ectodomain from cultured tumor cells generates an NH2-terminally truncated fragment, p95HER-2, that is membrane-associated, has in vitro kinase activity, and is tyrosine-phosphorylated (24, 25). Membrane bound forms of ErbB receptors with NH2-terminal deletions, analogous to p95HER-2, have been shown to be constitutively active (26, 27), and a truncated form of epidermal growth factor receptor (EGFR) has been shown to be resistant to the effects of Herstatin, an autoinhibitor of the EGFR family (28), suggesting that the ectodomain exerts a negative regulatory constraint. A dimerization motif in the cytoplasmic domain of p185HER-2 may be responsible for constitutive activation following removal of the ectodomain (29). Further evidence that this shedding event may be biologically relevant comes from the finding that trastuzumab blocks proteolytic release of the HER-2 ectodomain (25) and has been suggested to be one of the mechanisms responsible for efficacy against HER-2 overexpressing breast cancer (30). Finally, it has recently been shown that p95HER-2 has distinct biological properties in breast cancer cells. The truncated receptor forms heterodimers with HER-3, but not EGFR, whereas p185HER-2 heterodimerizes with both EGFR and HER3; and heregulin—a HER-3 and HER-4 ligand—but not EGF, stimulates p95HER-2 phosphorylation. This effect could be abrogated by GW572016—an inhibitor of EGFR/HER2 kinase activity—but not by trastuzumab, thus suggesting that resistance to this therapeutic antibody could be mediated in part through selection of p95HER-2 expressing breast cancer cells (31).

The methods currently used to measure HER-2 status for clinical purposes are fluorescence in situ hybridization and immunohistochemistry. For this reason, most prognostic or predictive studies only analyze total levels of the receptor, without quantifying its activation status or discriminating the truncated forms generated by proteolysis. Using Western blotting, we have recently described p95HER-2 in a subset of primary and metastatic human breast cancer tissues and showed its tyrosine phosphorylation (24, 25). In primary breast cancer, p95HER-2, but not the full-length receptor, significantly correlated with the extent of lymph node involvement (32). Importantly, p95HER-2 was significantly enhanced in tissue from metastatic nodes, whereas p185HER-2 was at similar levels in primary and metastatic nodal tissue. This is in agreement with the results reported by Simon et al. (33), that p185HER-2 status of primary tumors is maintained in nodal metastases. Because lymph node involvement is the single strongest indicator of disease-free and overall survival in breast cancer (7), the association of p95HER-2 with lymph node involvement suggests an important role either as a marker or cause of metastasis and poor outcome in breast cancer (32).

In the present study, we aimed to determine the value of p95HER-2 as a possible prognostic indicator in breast cancer. We studied its association with outcome and investigated the significance of p95HER-2 as an independent prognostic marker. The results of this study indicate that high levels of p95HER-2 in primary breast tumor tissue correlated with reduced overall and disease-free survival and is a stronger prognostic factor than p185HER-2 overexpression. These results suggest that p95HER-2, which might have relevance to oncogenic signaling pathways, is an independent prognostic factor that marks a subset of HER-2-positive breast cancer for worse outcome. These findings raise the possibility that p95HER-2, distinct from the full-length receptor, may represent an important target for therapeutic intervention.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
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Cells. The BT-474 cell line was provided by the American Type Culture Collection (Rockville, MD) and grown in DMEM/F-12 cell culture medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, and 10 µg/mL insulin (Life Technologies, Inc., Ltd., Paisley, United Kingdom). NIH-3T3 cells transfected with HER-2/neu cDNA (17-3-1 cells) were obtained from Applied Biotechnology, Inc. (Cambridge, MA) and cultured in DMEM supplemented with 5% fetal bovine serum containing 0.4 mg/mL genecitin (G418, Life Technologies).

Tissues. Breast tissues for this study were surgical resection specimens from primary tumors obtained from hospitals in two different countries: the U.S. (n = 334) and Spain (n = 149). U.S. samples were randomly selected from a collection of previously frozen (–80°C) tumors of all clinical stages that had been submitted to the Oregon Health and Sciences University (Portland, OR) from 20 different local or regional hospitals in Oregon and Southern Washington State for routine estrogen receptor (ER) and progesterone receptor (PR) analysis during the period 1986 to 1996. Samples from the Hospital Clínico of Valencia (Spain) and Hospital Vall d'Hebron of Barcelona (Spain), were collected for similar purposes during the period 1983 to 1999, kept frozen at –80°C until the analysis and selected by following the same criteria as above. The Institutional Review Board Committee on Human Research of the respective hospitals approved collection of all tissues and procedures conducted on these tissues. The study of tissues collected in Spain was approved by U.S. assurance number FWA00003437.

Antibodies. The antibody against the COOH terminus of p185HER-2 and p95HER-2 [anti-neu (C)] has been previously described (34). This antibody has been found to be much more effective in detecting p95HER-2 than most commercially available antipeptide antibodies directed against the COOH terminus of p185HER-2.6 Secondary antibody, conjugated to horseradish peroxidase, was from Amersham Pharmacia Biotech (Little Chalfont, United Kingdom) or from Bio-Rad (Hercules, CA).

Immunohistochemistry. Slides were deparaffinized, and endogenous peroxidase activity was blocked by incubation in 3% H2O2 in methanol for 10 minutes at room temperature. Sections were then microwaved in PBS for 4 minutes for antigen retrieval and incubated with avidin and then biotin for 15 minutes each to block nonspecific binding. An immunoperoxidase technique was then done using a commercial kit (Vectastain ABC Elite, Vector Laboratories, Burlingame, CA). Primary antibody was anti-neu (C) against HER2 COOH terminus (34) and secondary antibody was from Amersham. Hematoxylin was used as a counterstain. As a negative control, primary antibody was omitted. Neoplasms (n = 40) were classified from 0 to +3 according to the extension and intensity of membrane staining. Study specimens were evaluated independently by two examiners (R. Sáez and M.A. Molina) and reviewed by a pathologist (F. Rojo). Samples showing moderate or intense complete membrane immunohistochemical staining in >10% of tumor cells (+2 and +3) were considered as HER2 overexpressers.

No labeling was ever observed in control experiments when primary antibodies were omitted or, alternatively, when normal nonimmune serum was used. Therefore, there was no evidence of cross-reactivity with the antibody used in this study.

Western blot analysis of p95HER-2 and p185HER-2. Western blot analysis was done exactly as previously described (24, 25, 32). Several controls and extraction procedures were conducted previously to characterize p95HER-2 and to ensure that it was not generated as a degradation artifact. Approximately 0.1 g of fresh-frozen tumor tissue was thawed and minced on dry ice and suspended in homogenization buffer containing protease inhibitors. Following homogenization and fractionation, equal amounts of protein (20 µg) were resolved under denaturing and reducing conditions by SDS-PAGE in 7.5% gels. Each gel also contained 3 µg of protein extracted from 17-3-1 (NIH3T3 cells transfected with HER-2 cDNA provided by Applied Biotechnology, Inc., Paisley, United Kingdom) or 4-amino-phenylmercuric acetate–treated BT474 cells (25) as a marker for p95HER-2 generated by ectodomain proteolytic shedding and to provide a standard for the entire study. Electrotransfer to nitrocellulose membranes (Bio-Rad) was followed by immunoblotting with the anti-neu (C) antibody. Finally, membranes were incubated with antirabbit secondary antibody conjugated to horseradish peroxidase, which was detected by chemiluminescence methods (Pierce, Rockford, IL) and exposure to X-ray film.

HER-2 protein scoring. Scoring of p95HER-2 and p185HER-2 levels was done as previously described (32). Samples were scored as p185HER-2 overexpressers if the p185HER-2 signal was greater than or equal to the p185HER-2 level in 3 µg of 17-3-1 or BT-474 cells. Specimens were scored as highly positive for p95HER-2 if this band was greater than or equal to the p95HER-2 level in 3 µg of 17-3-1 or 4-amino-phenylmercuric acetate–treated BT-474 cell extracts. The levels of p95HER-2 in tumor samples were usually lower than those of p185HER-2. There were rarely any background bands under our experimental conditions.

Estrogen and progesterone receptor analysis. Cytosolic steroid hormone receptors were quantitated by conventional steroid binding assay (titration analysis) and Scatchard plot analysis or by ELISA. For immunoassays, human ER and PR kits were obtained from Abbott Laboratories and assays were conducted according to the manufacturer's specifications. Results of 10 fmol/mg protein or more were considered positive for both receptors.

Clinical data. Clinical data including patient age, nodal status, histologic type, tumor size, stage at diagnosis, surgical procedure, and systemic treatment therapies were compiled from hospital tumor registries, coded to protect individual identity, and entered into a computer database. Complete clinical data were not available for all patients because of differences in data collection methods used by community hospital tumor registries. U.S. tumor samples (n = 334) were collected from multiple hospitals in Oregon, and patients from Spain (n = 149) were from two major urban hospitals. Because U.S. tumor samples were collected from a number of community hospital sites, systemic therapies varied according to patient age, hormone receptor status, clinical stage, and hospital of origin. The treatment distribution of patients from the U.S. (Oregon) included no systemic therapy (n = 116 patients), chemotherapy (n = 91 patients), hormone therapy, mainly tamoxifen (n = 85 patients), and chemotherapy plus tamoxifen (n = 43 patients). The treatment distribution for patients from Spain included no therapy (n = 4 patients), adjuvant chemotherapy (n = 53 patients), or chemotherapy plus tamoxifen (n = 83 patients).

Statistical analysis. Associations between variables were determined by {chi}2 analysis using JMP software. Strength of association for study comparisons was calculated as odds ratios and 95% confidence interval (CI) using standard formulas. All P values reported are two-sided.

The effect of baseline characteristics on the risk of recurrence or death was calculated using Kaplan-Meier actuarial survival analysis and the log-rank test of significance. Disease-free survival is defined as the time elapsed in months from the date of surgery to first event, either recurrence (includes local recurrence, distant metastasis, or collateral breast cancer) or death from any cause or from breast cancer–related causes.

A principal objective of this analysis was to determine if p95HER-2 is a significant predictor of outcome after adjustment for confounders. Multivariate analysis was carried out using a Cox proportional-hazard model (JMP software) to evaluate the relation of p95HER-2 expression (highly positive) to disease-free survival. For this model, we adjusted for the clinical site (Oregon or Spain), age at diagnosis, clinical stage at diagnosis, ER and PR status (positive or negative), number of positive lymph nodes, tumor size, and p185HER-2 level (highly positive), and systemic treatment group. These factors were chosen because of biological importance in breast cancer and significance in the univariate analysis. To consider treatment in the multivariate analysis, patients were divided into the four groups mentioned above.


   Results
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 Materials and Methods
 Results
Discussion
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Characteristics of patients. The median age of the study population was 60 years with a range of 22 to 93 years. Histologic types included 78% infiltrating ductal, 7% infiltrating lobular, and 15% other. The majority of patients (76%) had stage I or II breast cancer and 66% of the tumors were ≥2 cm. Most patients, 74%, underwent a modified radical mastectomy whereas others were given partial mastectomy with axillary lymph node dissection. Finally, 120 patients (25%) received no systemic therapy; 30% received chemotherapy; 19% received hormone therapy, mainly tamoxifen; and the rest (26%) received chemotherapy plus tamoxifen.

p185HER-2 and p95HER-2 levels in breast tumor tissue. Primary tumor tissues were examined for HER-2/neu proteins by Western analysis using an antibody that recognizes the receptor COOH terminus. As previously reported in numerous studies, there were distinct expression patterns of p185HER-2 in the breast cancer samples and tumors were also heterogeneous for p95HER-2 expression (Fig. 1.).


Figure 1
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  		Fig. 1. HER-2 expression in breast tumor tissue. A, representative results of HER-2 immunostaining in breast tumor tissue. B, Western blotting analyses of breast tumor samples.

 
Scoring of p95HER-2 and p185HER-2 levels was done by Western blot analysis of tumor tissue extracts and assigned high or low values by comparison with protein standards as previously described (32). The number of patients overexpressing p185HER-2 was 145 of 483 (30%), and 44 of 483 (9%) were highly positive for p95HER-2. Of those overexpressing p95HER-2, 93% (n = 41 patients) were also highly positive for p185HER-2 (P = 0.0001).

Forty tumor samples were also analyzed by immunohistochemistry, and scored 0 to +3. There was a strong correlation between the results obtained by both techniques. Tumors classified as p185HER-2 overexpressers by Western blot analysis, scored +2 or +3 by immunohistochemistry, showing moderate or intense complete membrane immunohistochemical staining in >10% of tumor cells. Neoplasms with no detectable p185HER-2 levels scored 0 by immunohistochemistry (Fig. 1).

Association of p95HER-2 and p185HER-2 with risk factors. Table 1 describes the association of p95HER-2 and p185HER-2 with breast cancer risk factors. Overexpression of p95HER-2 was inversely associated with ER (P = 0.013) and PR-positive (P = 0.001) status, and with extent of nodal involvement (P = 0.035). p95HER-2 was not differentially expressed in tumors of <2 cm versus larger tumors, with age of the patient (<50 versus ≥50 years) or clinical stage (I and II versus III and IV). p185HER-2 did not correlate with any of the risk factors analyzed in this study: size, stage, ER or PR expression.


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  		Table 1. Relationship between p95HER-2, p185HER-2, and other risk factors

 
Association of p95HER-2, p185HER-2, and other risk factors with clinical outcome. The results of the analysis of disease-free survival are summarized in Table 2. Univariate analysis revealed that high levels of p95HER-2 were significantly associated with reduced disease-free survival (P < 0.0001). Seventy-one percent of patients with low p95HER-2 versus only 42% of those that were highly positive for p95HER-2 survived disease-free for 5 years. Disease-free survival (recurrence of breast cancer or death from any cause) was also worse for patients with high p95HER-2 (P < 0.0001). Figure 2 illustrates the Kaplan Meier plot of the proportion of patients who had breast cancer recurrence or death from breast cancer–related causes with time following surgery, according to the level of p95HER-2 (high or low; hazard ratio, 2.55; 95% CI, 2.13-8.01). In contrast, p185HER-2 overexpression was not associated with reduced disease-free survival (P = 0.261). Several other well-established risk factors were also associated with disease-free survival in this study population. Breast cancer patients with one or more positive lymph nodes (data not shown) had significantly reduced disease-free survival compared with patients with node-negative disease (hazard ratio, 2.7; 95% CI, 1.67-3.67; P < 0.0001).


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  		Table 2. Univariate analysis of prognostic factors for disease-free survival

 

Figure 2
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  		Fig. 2. Kaplan-Meier analysis of disease-free survival. Patients were grouped according to high or low p95HER-2 expression levels on the top row or p185HER-2 levels on the bottom, as determined by Western blot analysis. For recurrence or any death in the high versus low p95HER-2 (hazard ratio, 2.46; 95% CI, 2.14-6.94; P < 0.0001). For recurrence or death due to breast cancer in the high versus low p95HER-2 (hazard ratio, 2.55; 95% CI, 2.13-8.01; P < 0.0001). For high versus low p185HER-2, in the recurrence or death from any cause (hazard ratio, 1.29; 95% CI, 0.94-1.82; P = 0.108). In the low versus high p185HER-2 group that was analyzed by recurrence or death due to breast cancer (hazard ratio, 1.23; 95% CI, 0.85-1.81; P = 0.261).

 
Multivariate analysis. Breast cancer risk factors listed in Table 2 as well as systemic treatment modalities described in Materials and Methods were analyzed using a Cox stratified regression model to identify factors with an independent effect on risk of recurrence or death from breast cancer (Table 3). After adjustment for significant confounders in a stratified Cox regression analysis of survival, p95HER-2 level (high) was a significant independent predictor of recurrence or death from breast cancer (P = 0.026). Moreover, p95HER-2 overexpression was also an independent predictor of recurrence or death due to any cause (P = 0.0009; Table 4). The results indicate that established risk factors for breast cancer including age (P = 0.005), number of positive lymph nodes (P = 0.009), stage (III or IV; P > 0.0001), and ER negativity (P = 0.0006) were all independent predictors. Unexpectedly, location was a significant independent risk factor (for the patients from Oregon; P = 0.027). Although we are uncertain why the cohort from Oregon fared differently than that from Spain, one possibility is that a greater proportion of patients from Spain received intervening systemic therapy as described in Materials and Methods. In contrast, p185HER-2 overexpression, tumor size, PR, and treatment regimen were not significant independent predictors of outcome in this model (P > 0.1).


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  		Table 3. Independent factors predictive of disease-free survival: recurrence or death from breast cancer

 

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  		Table 4. Independent factors predictive of disease-free survival: recurrence or death from any cause

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have analyzed the prognostic value of two different forms—full-length and truncated—of the HER-2 transmembrane receptor, in the entire cohort. The method of analysis of HER-2 status in our study—Western blotting—differs from most previous studies that have used either fluorescence in situ hybridization analysis to assess c-erbB-2 gene amplification or immunohistochemistry (35, 36). Western blotting is rarely employed to detect HER-2 because it is relatively cumbersome and impractical for routine specimens, but is the only currently available method able to discriminate between the full-length receptor molecule, p185HER-2, and the NH2-terminal truncated, p95HER-2.

Our previous results indicated that the significance of p95HER-2 and p185HER-2, with regard to breast cancer metastasis to the lymph nodes, was different. p95HER-2 expression was significantly more frequent in tissue from metastatic nodes, and its presence in primary tumor correlated with the extent of lymph node metastasis (32). In contrast, p185HER-2 overexpression was equally probable in primary versus metastatic tissue and in node-negative versus node-positive patients. Because lymph node involvement is the single strongest indicator of disease-free and overall survival in breast cancer (7), the association of p95HER-2 with lymph node involvement suggested an important role either as a marker or cause of metastasis and poor outcome in breast cancer (32).

Here, we have analyzed the association of p95HER-2 with outcome. In addition, we considered other risk factors in breast cancer including the expression levels of full-length p185HER-2, age of the patients, number of nodes affected, stage of the disease, hormonal status, and tumor size. We found that increased expression of p95HER-2, as measured by Western blotting of tumor lysates, is a powerful and independent predictor of poor outcome in patients with breast cancer. In a retrospective analysis of 483 patients with breast cancer with a median follow-up of 46 months, our results show that p95HER-2 overexpression predicted significantly reduced 5-year survival risk (42% high p95HER-2 versus 71% low p95HER-2). In contrast, p185HER-2 did not have significant prognostic value in the overall patient population.

To identify the independent factors predictive of disease-free survival, we conducted a multivariate analysis using a stratified Cox regression analysis of the samples included in our study. High expression of p95HER-2 was an independent predictor with a hazard ratio of 1.4 for disease-free survival. The age of the patient, stage of the disease, nodal status, and levels of ER were also independent predictive factors for disease-specific survival as previously described (37). Investigations over the past years have revealed that receptor tyrosine kinases are not only key regulators of normal cellular processes but are also critically involved in the development and progression of human tumors. Their activity is normally tightly controlled, and overactivation results in dysregulated cell growth and cancer (38–40). Constitutive receptor tyrosine kinase activation in tumor cells can occur by several mechanisms; one of them being the generation of truncated forms by either alternative splicing, missense mutations, or proteolytic shedding (38, 39). In this regard, a truncated form of vascular EGFR-3 has been proposed as a target for prevention of lymph node metastasis in prostate cancer (41) or inhibition of insulin-like growth factor receptor-1 proteolytic shedding has been shown to have antitumor effects (42). The association of the overexpression of p95HER-2 with reduced disease-free survival could also be related to its biological properties which are distinct from p185HER-2, such as increased signaling activity and enhanced oncogenic potential (32).

These studies raise the question of the potential clinical utility of p95HER-2. One limitation is that p95HER-2 is overexpressed in only a small proportion, ~9%, of patients with breast cancer. On the other hand, ~30% of the HER-2-positive tumors express p95HER-2, which not only distinguishes a subgroup with worse outcome but may also be useful in predicting response to HER-2-targeted therapeutics. The association of p95HER-2 with reduced disease-free survival shown in this study, and prior evidence demonstrating its tyrosine phosphorylation and activity as a tyrosine kinase (24, 25) also suggest the potential importance of p95HER-2 itself as a therapeutic target. Because p95HER-2 is missing the HER-2 ectodomain but possesses an active kinase, tumors that express this truncated form of the HER-2 receptor may resist anticancer agents that target the ectodomain and respond to HER-2-specific small molecule kinase inhibitors (31). Identification of the specific sheddase, likely a metalloprotease (24, 25, 43), responsible for production of p95HER-2 may also offer a new target for treatment of HER-2-positive breast cancer. Inhibitors of the HER-2 sheddase may improve the efficacy of trastuzumab treatment because resistance may be related, in part, to the presence of p95HER-2, or to the selection of tumor cells that express this truncated HER-2 receptor. Perhaps a more serious limitation in the clinical utility is that p95HER-2 is currently detected only by Western blot analysis of tumor extract, a procedure that is easily done on <3 µg of tumor tissue in the research laboratory, but that may be impractical for routine assays in the clinical setting. Future development of monoclonal antibodies that specifically detect p95HER-2 by immunohistochemical analysis of tumor sections may be required to achieve clinical utility.

Although our study is the first to evaluate the prognostic value of p95HER-2, the association of p185HER-2 overexpression with prognosis in breast cancer has been extensively investigated (7–9). Whereas a correlation between p185HER-2 status and disease outcome has been reported in some studies for the overall population, the association has been observed more consistently in node-positive patients. Studies that have used either fluorescence in situ hybridization analysis to assess c-erbB-2 gene amplification or protein detection by ELISA have more frequently found association with outcome, whereas immunohistochemical analyses of formalin-fixed specimens have yielded contradictory results (9). For these reasons, HER-2 is generally considered to have limited value as a prognostic indicator in breast cancer, and a large consensus study by the American Society of Clinical Oncology did not recommend its use (7). In our case, we did not observe a significant correlation of p185HER-2 overexpression with prognosis in the overall patient population. Several factors might help to explain this finding. First, in our study group, there was a large proportion (50%) of node-negative patients; a group in which p185HER-2 seems to have less prognostic significance. Second, the median follow-up of the patients was only 3.8 years, a relatively short period for a disease prone to late recurrences. p185HER-2 overexpression may be more important in breast cancer that recurs more slowly (8).

In summary, we have shown that p95HER-2 expression defines a group of breast cancers with enhanced metastatic potential and significantly worse outcome. Moreover, these studies point to sharply distinct prognostic values for the truncated p95HER-2 versus the full-length HER-2 receptor. Our results suggest that truncated HER-2 may be useful as a prognostic indicator, helping physicians to identify the subgroup of patients who would benefit from adjuvant therapy.


   Footnotes
 
Grant support: National Cancer Institute grant no. 9346 from the Department of the Defense (G.M. Clinton) and by Spanish Health Ministry grant "Fondo de Investigaciones Sanitarias" 99/0020-01 (J. Albanell and A. Lluch), and 01/0040-01 (R. Sáez and J. Baselga).

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.

6 G.M. Clinton, unpublished observations. Back

Received 8/17/05; revised 11/ 3/05; accepted 11/10/05.


   References
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 Results
 Discussion
 References
 

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