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© 2000 American Society for Clinical Oncology Activation (Tyrosine Phosphorylation) of ErbB-2 (HER-2/neu): A Study of Incidence and Correlation With Outcome in Breast CancerFrom the Evanston Hospital/Northwestern University, Evanston, IL; California Pacific Medical Center, University of California at San Francisco, and University of California at San Francisco Medical Center, San Francisco, CA; and Yale University School of Medicine, New Haven, CT. Address reprint requests to A.D. Thor, MD, Department of Pathology, Evanston Hospital, 2650 Ridge Ave, Evanston, IL 60201; email a-thor{at}nwu.edu
PURPOSE: We hypothesize that phosphorylated ErbB-2 (P-ErbB-2, identified by a novel antibody PN2A) may provide either more significant or additional prognostic marker data for breast cancer patients. This study was designed to compare the incidence and prognostic value of ErbB-2 (HER-2/neu) and P-ErbB-2 immunoexpression in archival breast cancer samples. MATERIALS AND METHODS: Eight hundred sixteen invasive breast cancers with a median of 16.3 years of follow-up were immunostained for ErbB-2 (using antibody CB11) and P-ErbB-2 (using antibody PN2A). ErbB-2 and P-ErbB-2 data were compared with clinical, histologic, immunohistochemical, and outcome variables.
RESULTS: Of 816 primary breast cancers, 307 (38%) were positive for ErbB-2 and 37 (12% of ErbB-2 positive and 5% of the study population) expressed P-ErbB-2. P-ErbB-2 was not detected in ErbB-2–negative cases (n = 509). ErbB-2 immunohistochemical data were bimodal; patients with
CONCLUSION: PN2A immunostaining identified a subset (approximately 12% of ErbB-2–positive breast cancers) with activation (phosphorylation) of the receptor ErbB-2. P-ErbB-2 expression was strongly associated with higher levels of ErbB-2 expression (
ErbB-2 (HER-2/neu) IS A member of the epidermal growth factor receptor (EGFR) family of growth factor receptor tyrosine kinases, which includes EGFR, HER-3/ErbB-3, and HER-4/ErbB-4.1 ErbB-2 is overexpressed in approximately one third of breast cancers, usually as a result of gene amplification.1,2 ErbB-2 overexpression/amplification has been associated with increased rates of relapse and death, particularly for patients with node-positive disease.1,2 It has been more difficult to demonstrate the influence of ErbB-2 on outcomes among node-negative patients, although some larger, recent studies have demonstrated prognostic value in this subset of patients as well.1,2 The predictive value of ErbB-2 to identify patients more likely to respond to trastuzumab (Herceptin; Genentech, Inc, South San Francisco, CA), doxorubicin, or other agents may be more clinically relevant than prognostic value to justify ErbB-2 testing.1,2 Laboratory methods for detection of ErbB-2 alterations include tests to detect the ErbB-2 protein or quantitate copies of the erbB-2 gene. There is currently great debate regarding the best method (immunohistochemistry or fluorescence in situ hybridization) or reagents to measure ErbB-2 alteration for clinical purposes on fixed embedded breast tissues. A commonly used cut point for determining ErbB-2 positivity by immunohistochemistry is 10% (for the HercepTest; Dako Corp, Copenhagen, Denmark). Consensus panels have recommended the reporting of the percentage of positive invasive breast cancer cells as well as the reagents and methodology used. ErbB-2 phosphorylation, which is indicative of activation, can be detected by immunohistochemistry or immunoblotting methods using the monoclonal antibody PN2A, which specifically recognizes Tyr1248-phosphorylated ErbB-2.3 Members of the receptor tyrosine kinases superfamily are highly regulated in their activities. Prognostic or predictive studies of ErbB-2 have not used methods to specifically quantitate the activity of ErbB-2. ErbB-2 is as of yet an orphan receptor, in that a ligand, which binds directly to ErbB-2, has not yet been identified. However, ErbB-2 can be activated via heterodimerization with EGFR, HER-3/ErbB-3, or HER-4/ErbB-4 when any of the latter receptors are coexpressed and activated by their cognate ligands.4-12 In cell culture, high-level overexpression of ErbB-2, even without the addition of activating ligands, can result in basal receptor phosphorylation and activation.13-16 High levels can effect cellular transformation.13-15 Activating mutations of ErbB-2 have been described experimentally,16-21 but none have been described in human breast tumors. Natural variations in splicing or proteolytic cleavage can generate activated or inhibitory variant receptors,22-25 although the biologic effect of these in vivo is unclear. Activation via any of these mechanisms results in autophosphorylation of tyrosine residues. Autophosphorylation allows docking of other proteins involved in complex signal transduction pathways.26-30 Given these considerations, the number of receptors in the activated (phosphorylated) state may be more important biologically than the total number of receptors present. We therefore postulated that receptor phosphorylation (indicative of signaling activity), either in addition to or rather than receptor overexpression data, might improve the value of ErbB-2 immunostaining as a prognostic test.31 To study ErbB-2 activation in clinical tumor specimens, a monoclonal antibody PN2A was developed which specifically detects activated ErbB-2 in fixed, archival tissues.3,32 The specificity of PN2A for phosphorylated ErbB-2 on fixed embedded tissues has been demonstrated using Western blots of fresh cell lysates, as well as immunohistochemistry with and without appropriate peptide blocks. The antibody recognizes only one of five reported phosphorylation sites. Of the five reported autophosphorylation sites, the specific phosphopeptide recognized by PN2A is known to be significant for transformation.33-35 Some data suggest that this site is the sole autophosphorylation site responsible for oncogenicity as well as coupling to the ras/MAP kinase signaling pathway.36 Using this antibody, we have previously shown that receptor phosphorylation is highly variable among different human breast cancers that express high levels of the receptor.3,32 In preliminary studies, we found the receptor to be in the phosphorylated state in only seven of 20 invasive breast carcinomas in which ErbB-2 was overexpressed.32 Therefore, PN2A can be used with immunohistochemistry to specifically separate ErbB-2 overexpression from site-specific phosphorylation. Reagents that detect activated proteins allow greater assessment of receptor function and serve as a surrogate for downstream activation. In the present study, we compared a commonly used anti–ErbB-2 antibody CB11 with PN2A (anti–P-ErbB-2) using immunohistochemical methods on a large archival bank of human breast cancers with long-term follow-up. These cases included ErbB-2–positive (low, moderate, and high) and ErbB-2–negative cases to determine the added or replacement value of P-ErbB-2 as a prognostic factor.
Patient Population Eight hundred sixteen retrospectively identified, available archival formalin-fixed paraffin-embedded primary breast cancer cases diagnosed between 1976 and 1983 at the Massachusetts General Hospital, Boston, MA, were used for this study. Tumors have been classified according to the World Health Organization schema37; 784 (96%) of 816 were classified as invasive ductal (including specialized subtypes) and 32 (4%) of 816 were invasive lobular carcinomas. Tumors were graded using the Nottingham combined histologic grading scheme38: 74 tumors (10%) were classified as grade 1, 363 tumors (47%) were grade 2, and 333 tumors (43%) were grade 3. Estrogen receptor (ER) status was determined by either charcoal dextran assay or immunohistochemical assay.39,40 ER was positive in 448 (55%), negative in 359 (44%), and unknown in nine cases (1%). Tumors ranged in size from 0.2 to 16 cm in maximum dimension, with a mean of 2.91 cm. Patient age ranged from 24 to 95 years, with a mean of 60 years. Mitotic index (MI) ranged from 0 to 213 mitoses per 10 high-powered fields (hpf), with a median of 6 mitoses/hpf. MIB-1 immunostaining was performed on 795 patients, and scores ranged from 0 to 95.7 positive cells per 100 cells counted. Lymph node status was determined by node dissection and examination in 694 patients (85%); 351 (51%) were node-negative, and 343 (49%) were lymph node–positive. The numbers of positive nodes and total lymph nodes examined were also recorded. Tumors were surgically removed with modified radical or total mastectomy in 70% (573 patients), a simple mastectomy in 2% (15), or a lumpectomy with or without axillary dissection in 28% (228). Adjuvant radiation therapy was given to 25% (201) and chemotherapy was administered to 38% (311). The type of chemotherapy was very diverse, ranging from single agent to multiple agents, both on and off clinical trials. Common chemotherapy drug combinations included cyclophosphamide, doxorubicin, and fluorouracil (5-FU) (CAF); cyclophosphamide, methotrexate, and 5-FU; cyclophosphamide, methotrexate, 5-FU, vincristine, and prednisone; vinblastine, doxorubicin, thiotepa, and fluoxymesterone; and cyclophosphamide, doxorubicin, 5-FU, vincristine, and prednisone. There were a number of other drugs and combinations given to individual patients. Doxorubicin-containing therapies were given to 21% (64) of the patients treated with chemotherapy. Most of the doxorubicin dosages given were between 15 and 45 mg/m2, which is considered low by current protocols. In addition, a number of patients given doxorubicin were on a clinical protocol in which the doxorubicin was given 6 to 9 months postsurgery. The follow-up intervals were calculated from date of biopsy/lumpectomy/mastectomy to the last recorded follow-up (mean 15.6, years; median, 16.3 years). Local recurrence and distant metastasis intervals were calculated from biopsy/lumpectomy/mastectomy date to first documented failure date (surgical, clinical, radiologic). Follow-up information was obtained from annual review of patient charts or tumor registry data for patients alive at the previous annual review. For the total patient population, 61% were disease-free at 5 years, 53% were disease-free at 10 years, and 48% were disease-free at last follow-up (median, 16.3 years). Disease-specific survival (DSS) for all patients at 5 years was 76%, at 10 years was 63%, and at last follow-up was 59%.
Immunohistochemical Staining Activated ErbB-2 by PN2A. Monoclonal antibody PN2A was raised against a synthetic, tyrosine-phosphorylated peptide corresponding to the COOH-terminal 14 amino acids (plus the NH2-terminal lysine for coupling) of the ErbB-2 protein.3 Four-µm formalin-fixed paraffin-embedded sections of breast cancer were deparaffinized, rehydrated, and endogenous peroxidase blocked with 2% hydrogen peroxide. Antigen retrieval was performed by placing sections in 10 mmol/L citrate buffer (pH 6.0) and microwaving for 15 minutes. Slides were allowed to cool to room temperature, washed with distilled water and phosphate-buffered saline in 0.05% Tween-20 (Fisher Scientific, Pittsburgh, PA), and blocked with normal horse serum (10%). PN2A (5 µg/mL) was applied and sections were incubated at 4°C overnight. After additional phosphate-buffered saline/Tween-20 washes, sections were sequentially incubated at room temperature for 30 minutes with biotinylated horse-anti-mouse (dilution 1:200; Vector Labs, Burlingame, CA) and streptavidin-HRP (dilution 1:400; Zymed, South San Francisco, CA). Slides were incubated with diaminobenzidine, counterstained with hematoxylin, dehydrated, cleared, and cover-slipped. Microscopic examination of the entire slide was performed and slides were scored using the same system as for ErbB-2. Faint cytoplasmic staining (present in a few cases) was not considered positive. For each ErbB-2 and P-ErbB-2 assay, pelleted, formalin-fixed, and paraffin-embedded control cell lines (American Type Culture Collection, Manassas, MD) were used for controls. These included MDA-MB-231 and MDA-MB-453 (negative and positive controls for CB11 assays) and MCF10A and epidermal growth factor–stimulated SKBR3 (100 ng/mL x 7 minutes at room temperature3) for negative and positive P-ErbB-2 controls, respectively. Blocking experiments to demonstrate specificity of P-ErbB-2. The antibody PN2A has previously been demonstrated to be specific for site-specific (Tyr1248) phosphorylation (an activated form) of ErbB-2 using Western blots (of fresh-cell lysates) and immunohistochemistry (formalin-fixed paraffin-embedded human breast tumors) using peptide blocking experiments.3 Similar blocking experiments were performed on cell lines and MGH tumor tissues and specificity was confirmed in the laboratory of A.D.T. (data not shown). p53 Immunohistochemistry. p53 staining was performed as previously reported for some of these cases using monoclonal anti-p53 antibody PAb 1801 (Genesis Bio-Pharmaceuticals, Inc, Tenefly, NJ; dilution 1:4,000).42 The entire slide was evaluated for p53 scoring. Invasive carcinoma only was considered, although benign breast epithelium was also incidentally evaluated and was consistently negative. Any nuclear staining visible at x10 magnification was considered positive. The percentage of invasive tumor cells with brown nuclear staining was estimated by visual review of the entire slide, similar to the scoring systems used for ErbB-2 and P-ErbB-2. Proliferation markers. MI and MIB-1 staining was performed as previously published.43 In brief, 4-µm sections were stained with anti–MIB-1 antibody (AMAC, Inc., Westbrook, ME) used at a concentration of 1:200 at 4°C overnight after antigen retrieval.43 Nuclear staining visible at x10 magnification was considered positive for MIB-1. A minimum of 1,000 tumor cells were counted for MIB-1, and 10 separate hpf were examined for MI using an eyepiece grid after examining the entire slide for fields with the highest percentage of MIB-1 staining. MI was scored according to the guidelines for the Nottingham combined histologic grading system, but was separately considered (in addition to its inclusion in the grading system) as an estimate of cellular proliferation.
Statistical Methods Survival analysis was focused on comparing the prognostic power of P-ErbB-2 and ErbB-2 data in the presence of other markers. Outcomes selected for study included disease-free survival (DFS), defined as diagnosis date to recurrence date (local or regional recurrence or distant metastasis), and DSS, defined as diagnosis date to date of death due to disease. Patients who died from causes unrelated to breast cancer were considered as censored at the time of death and deaths from unknown causes (n = 20) were excluded from DSS analysis. For univariate survival analysis, the log-rank test was used to test for statistical significance.
We constructed multivariate Cox proportional hazards models by first building a baseline model from factors excluding P-ErbB-2 and ErbB-2.44 Initially, all univariately significant factors were included in the model. Then factors were removed one at a time, based on the Wald statistic for each variable, until only statistically significant factors remained (P < .05). Finally, factors of focused interest, eg, P-ErbB-2 and ErbB-2, were added to the baseline model to assess statistical significance in the presence of other markers. This assessment was based on the change in the log likelihood between the baseline model and the model including the factor(s) of focused interest. A The number of positive lymph nodes was entered into the Cox models as a logarithm because this transformation gave a better linear relationship between risk and nodes. Tumor size, grade and age were entered as continuous variables, and biochemical ER was dichotomized with a cut point of 10 fmol/mg protein. p53 was entered into the statistical models as a binary variable for which any staining was considered as positive and no staining was considered as negative. To compare P-ErbB-2 with ErbB-2 data, a scatterplot of P-ErbB-2 by ErbB-2 staining was generated. Distribution data were used to select a cut point for ErbB-2 that would maximize the correlation with P-ErbB-2.
Clinicohistologic Correlates With ErbB-2 and P-ErbB-2 The 816 cases demonstrated heterogeneous ErbB-2 staining of the invasive component (median, 0% ErbB-2; range, 0% to 100%). Of the 307 ErbB-2–positive cases, the median percentage positive was 40%. The distribution of ErbB-2 positivity was bimodal, with peak frequencies at less than 10% and  90% (data not shown). Twelve percent (37 of 307) of ErbB-2–positive cases were positive for P-ErbB-2 by immunohistochemistry (positivity illustrated; Fig 1). None of the 509 ErbB-2–negative cases expressed detectable P-ErbB-2. P-ErbB-2 positivity was observed in seven (5%) of node-negative and ErbB-2–positive and 25 (18%) of node-positive and ErbB-2–positive tumors. P-ErbB-2 staining was absent in 779 (95%) of tumors (ErbB-2–positive and –negative) and positive in 37 (5%) of cases.
For survival analysis, the 816 patients could be divided into three general groups based on ErbB-2 and P-ErbB-2 data. A cut point of 0% was used for both ErbB-2 and P-ErbB-2. The groups were as follows: group A, ErbB-2 = 0% and P-ErbB-2 = 0%; group B, ErbB-2 more than 0% and P-ErbB-2 = 0%; and group C, ErbB-2 more than 0% and P-ErbB-2 more than 0%. As observed in Table 1, there was a gradient in covariate values in going from group A to group C. Patients in group A tended to be older, with smaller, low-grade tumors that were ER-positive. Patients in group B also tended to be older, with smaller, moderate-grade, ER-positive tumors. Patients in group C were younger, with high-grade, larger, ER-negative tumors. Of interest, differences in the number of positive lymph nodes and proliferation rates for these groups were not significant (Table 1). The log of the number of positive nodes increased across the patient groups from A to B to C.
Associations between ErbB-2 and P-ErbB-2 data using correlation coefficients are listed in Table 2. Both P-ErbB-2 and ErbB-2 overexpression were associated with a younger patient age, higher tumor grade, larger tumor size, ER negativity, p53 immunopositivity, and each other. Neither ErbB-2 nor P-ErbB-2 was associated with the number of positive lymph nodes.
Figure 2 shows a bivariate graph (scatterplot) comparing ErbB-2 and P-ErbB-2 data. This graph was used to determine the cut point to maximize the correlation between these two variables and to construct a binary factor for ErbB-2 used in the survival analyses. As shown in Fig 2, the majority of P-ErbB-2–positive tumors had 80% ErbB-2–positive tumor cells. Although many of the strongly ErbB-2 positive ( 80% cells stained for ErbB-2) cases were P-ErbB-2–positive (35 of 96), the majority of these were P-ErbB-2–negative. In addition, two cases with low ErbB-2 expression stained with PN2A for P-ErbB-2. Hence, this heterogeneity demonstrates that high ErbB-2 expression could not be used reliably as a surrogate for PN2A positivity.
Factors Associated With Outcome: Univariate Analyses Univariate analyses of factors associated with outcome for all patients are listed in Table 3. Factors associated with shortened DFS and DSS included higher tumor grade, larger tumor size, nodal positivity, higher numbers of positive lymph nodes, ER negativity, tumor proliferation (using either MIB-1 labeling or MI), ErbB-2 overexpression (using several different cut points), and P-ErbB-2 and p53 positivity (Table 3 lists odds ratios and P values for both DFS and DSS). Although the odds ratio is greater for P-ErbB-2 than it is for ErbB-2 ( 80%), it would be incorrect to state that P-ErbB-2 is a better marker of adverse prognosis, because the confidence intervals are overlapping (data not shown).
Univariate analyses of outcomes for node negative patients (n = 342) showed that for DFS, patient age, tumor grade, tumor size, p53 positivity, and cellular proliferation (MIB-1 and MI) were significantly associated with outcomes. Analyses of node-negative patients for DSS showed only tumor grade and cellular proliferation were significantly associated with outcome. ErbB-2 was not significantly associated with DFS or DSS in this subgroup. P-ErbB-2 could not be compared with outcomes, because only seven node-negative tumors expressed P-ErbB-2, which limited the power of statistical calculations (data not shown). Univariate analyses of outcomes of node-positive patients (n = 337) showed that for DFS and DSS, tumor grade, number of positive lymph nodes, ER status, cellular proliferation using MIB-1, and ErbB-2 overexpression (using multiple cut points or continuous data) were significantly associated with outcome (data not shown). P-ErbB-2 was associated with DFS (risk ratio, 2.851; P < .0001) and DSS (risk ratio, 3.767; P < .0001). In addition, p53 positivity and cellular proliferation using MI were also significantly associated with DSS but not DFS (data not shown in tabular form).
Multivariate Survival Analysis
The best-fit baseline model for DSS included the log of the number of positive lymph nodes, tumor size, histologic tumor grade, ER status, and patient age. The addition of ErbB-2 as a continuous variable, or dichotomized (< 80 v 80), again showed independent significance (Table 4). A trichotomized ErbB-2 variable was also independently significant (P = .0003). Using a cut point of either 0 versus more than 0 or less than 40 versus 40 failed to achieve independent significance (data not shown). P-ErbB-2 as either a continuous or dichotomized variable was associated with a statistically significant improvement in survival prediction. The addition of both dichotomized P-ErbB-2 and dichotomized ErbB-2 also provided a statistically significant improvement in predicting DSS. However, neither the addition of continuous P-ErbB-2 to continuous ErbB-2 nor the addition of either continuous variable to the dichotomized variables showed any significant improvement in survival prediction over the dichotomous variables alone (data not shown). Lymph node–negative patients. Among the node-negative cases, only tumor size and histologic grade were independently associated with DFS. Grade was the only factor that was independently associated with DSS. The addition of ErbB-2 or P-ErbB-2 (as either continuous or dichotomized variables) did not statistically improve survival prediction for DFS or DSS, although again, the small number of P-ErbB-2–positive cases precluded any sound statistical conclusions regarding P-ErbB-2 due to low power in this subgroup (Table 4). Lymph node–positive patients. In node-positive cases, the best-fit baseline model included the log of the number of positive lymph nodes, tumor size, and ER status for DFS. The addition of ErbB-2 as either a continuous or dichotomized variable significantly improved the prediction of survival (Table 4). Other cut points for ErbB-2 were also significantly associated with DFS (data not shown). Both the continuous and dichotomized variables for P-ErbB-2 were also significantly associated with DFS (Table 4). The addition of either P-ErbB-2 or ErbB-2 did not improve survival prediction over the use of only one factor for DFS.
The best-fit model for DSS in node-positive cases included the log of the number of positive lymph nodes, tumor size, histologic grade, ER status, and patient age. The addition of ErbB-2 as either a continuous variable or dichotomized (< 80% v
Kaplan-Meier curves show the relationship between ErbB-2–negative, ErbB-2–positive/P-ErbB-2–negative, and ErbB-2–positive/P-ErbB-2–positive patients and survival. As shown in Figs 3A and 3B, P-ErbB-2 provided additional significant survival information for ErbB-2–positive breast cancer patients when any positivity was considered positive. Survival (DFS or DSS) for ErbB-2–negative patients was only marginally better than ErbB-2–positive P-ErbB-2–negative patients (statistical significance between the groups was not achieved). Figures 3C and 3D show similar (but not identical) results for survival if ErbB-2–positive tumors were separated into high and low expressors (based on the distribution shown in Fig 1; ErbB-2 < 80% v ErbB-2
The value of ErbB-2 (HER-2/neu) analysis for prognosis or prediction has been controversial. Our goal was to determine whether ErbB-2 signaling (as reflected by site-specific phosphorylation; P-ErbB-2) improved the prognostic power of ErbB-2 analyses. We have previously shown the antibody PN2A, which detects P-ErbB-2, is specific for the Tyr1248-phosphorylated receptor in both fixed and unfixed tissues using a variety of methods.3 This site is one of possibly five sites of phosphorylation and therefore may not recognize all activated forms of the receptor. However, studies have demonstrated that it recognizes a major functional site as well as epidermal growth factor–activated ErbB-2.36 We have also shown that P-ErbB-2 is detectable in only in a minority of ErbB-2–overexpressing breast cancers.3,32
In the present study, we found site-specific receptor phosphorylation to be more prevalent among ErbB-2–positive/node-positive cases (18%) than among ErbB-2–positive/node-negative cases (5%). In the entire study population and in the node-positive cases in particular, P-ErbB-2 data provided more valuable prognostic information (illustrated by the value of the change in For these patients, PN2A staining identified a subset of ErbB-2–positive patients with a worse DFS and overall survival that was statistically significant by both univariate and multivariate methods. Of interest, PN2A-positive cases also showed significantly higher positivity for p53, as compared with ErbB-2–negative or ErbB-2–positive/P-ErbB-2–negative cases. The biologic significance of this finding is unknown. Of possible relevance is the recent Cancer and Leukemia Group B (CALGB) Trial 8541, which showed enhanced response to CAF in ErbB-2–positive/p53-positive cases.41 Our study population was not part of any particular clinical protocol and they were not uniformly treated. This excludes analyses of interactions between P-ErbB-2 and therapy. We are currently performing P-ErbB-2 analyses on ErbB-2–positive tumors from CALGB 8541, a three-arm trial of dose/dose-intensity of CAF on stage II breast cancer patients.45,46 Prior correlative studies of these patients have documented ErbB-2 alterations using immunohistochemical, molecular, and fluorescence in situ methods.37,47 Using these patients and all testing methods for ErbB-2, prognostic and predictive significance has been demonstrated. This study will test the hypothesis that P-ErbB-2 may provide greater predictive value than ErbB-2 testing alone for patients treated with CAF. In more than 500 patients who were ErbB-2 negative, P-ErbB-2 immunopositivity was not observed. Cases with low-level overexpression of ErbB-2 and P-ErbB-2 negativity had similar biologic and outcome characteristics to ErbB-2–negative cases. Those cases with highly overexpressed ErbB-2 or P-ErbB-2 positivity were associated with a worse outcome and more adverse biologic features (high grade, larger size, higher number of lymph nodes positive, ER negativity, p53 positivity).
Because of the expected relationship of phosphorylation/P-ErbB-2 positivity with high-level overexpression in general,16,32 we considered that P-ErbB-2 positivity could simply be a surrogate for high-level overexpression. To address this directly, P-ErbB-2 analysis was compared with a high cut-point of ErbB-2 overexpression (
ErbB-2 data from this large patient cohort confirmed the bimodal distribution of ErbB-2 immunohistochemical data. If ErbB-2 is reported as a continuous variable (as recommended by the recent guidelines of the College of American Pathologists48 ), then identification of the patients with the highest immunopositivity ( ErbB-2 testing is most commonly applied because of its value as a predictive factor, either for consideration for trastuzumab treatment or because of interactions between ErbB-2 and other chemotherapeutic agents. We are currently evaluating P-ErbB-2 to determine whether it is predictive of response to trastuzumab, doxorubicin, or tamoxifen.
Supported by grant nos. CA44768 (A.D.T.), CA45708 (D.F.S.), and DAMD17–97-1–7065 (M.P.D.).
D.F.S. has negotiated with a diagnostic company regarding the licensing of the antibody PN2A. The content of the information contained herein does not necessarily reflect the position or the policy of the United States Government, and no official endorsement should be inferred. D.F.S. and M.P.D contributed equally to this work.
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 statistic, equal to twice the difference in the log likelihood between the baseline model with and without the factor of focused interest, was used to determine statistical significance of ErbB-2 and P-ErbB-2 in predicting time to failure or death. 
, ErbB-2–negative; 
, ErbB-2–positive and P-ErbB-2–negative; and 
, ErbB-2–positive and P-ErbB-2–positive. (C) DFS and (D) DSS separated into three groups by ErbB-2: 
