ARTICLE

HER-2/neu Gene Amplification and Response to Paclitaxel in Patients With Metastatic Breast Cancer

Gottfried E. Konecny, Christoph Thomssen, Hans Joachim Lück, Michael Untch, He-Jing Wang, Walter Kuhn, Holger Eidtmann, Andreas du Bois, Sigrid Olbricht, Dieter Steinfeld, Volker Möbus, Gunter von Minckwitz, Suganda Dandekar, Lillian Ramos, Giovanni Pauletti, Mark D. Pegram, Fritz Jänicke, Dennis J. Slamon

Affiliations of authors: Division of Hematology–Oncology, Department of Medicine, David Geffen School of Medicine, University of California at Los Angeles, and Jonsson Comprehensive Cancer Center, Los Angeles, CA (GEK, SD, LR, GP, MDP, DJS); Department of Biomathematics, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles (HJW); Breast Cancer Study Group, Arbeitsgemeinschaft für Gynäkologische Onkologie, Germany (CT, HJL, MU, WK, HE, ADB, SO, DS, VM, GVM, FJ)

Correspondence to: Dennis J. Slamon, MD, PhD, University of California, Los Angeles, Peter Ueberroth Bldg., 3360B, 10945 Le Conte Ave., Los Angeles, CA 90095-7077 (e-mail: dslamon{at}mednet.ucla.edu)


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Background: HER-2/neu overexpressionappears to be associated with improved response to anthracycline-based chemotherapy, but its association with response to taxane-based chemotherapy is unclear. In this retrospective subset analysis of patients with metastatic breast cancer enrolled in a randomized treatment trial, we investigated the response of patients with known HER-2/neu status to treatment with taxane-based epirubicin–paclitaxel (ET) chemotherapy compared with treatment with epirubicin–cyclophosphamide (EC) chemotherapy. Methods: HER-2/neu status (positive [i.e., HER-2/neu amplification] or negative [i.e., no HER-2/neu amplification]) of archival specimens of primary tumors from 297 patients with metastatic breast cancer was determined by use of fluorescence in situ hybridization. Associations between HER-2/neu status and the efficacy of randomly assigned chemotherapy (ET versus EC) were investigated. All statistical tests were two-sided. Results: Patients with HER-2/neu–positive tumors had a statistically significantly greater objective response rate than patients with HER-2/neu–negative tumors to treatment with ET (76% versus 50%, respectively; P = .005) but not to treatment with EC (46% versus 33%; P = .130). The objective response rate associated with ET was greater than that associated with EC for both HER-2/neu–positive tumors (76% versus 46%; P = .004) and HER-2/neu–negative tumors (50% versus 33%; P = .002). However, the improvement in the objective response rate associated with ET, compared with that associated with EC, was greater for patients with HER-2/neu–positive tumors (adjusted odds ratio [OR] = 3.64, 95% confidence interval [CI] = 1.48 to 8.92; P= .005) than for patients with HER-2/neu–negative tumors (adjusted OR = 1.92, 95% CI = 1.01 to 3.64; P= .046). Among patients with HER-2/neu–positive tumors, those who received ET had better progression-free survival and overall survival than those who received EC (for progression-free survival, adjusted relative risk [RR] = 0.65, 95% CI = 0.42 to 1.02; P= .062; for overall survival, adjusted RR = 0.60, 95% CI = 0.36 to 1.02; P= .059). However, among patients with HER-2/neu–negative tumors, those who received ET and those who received EC had similar progression-free survival and overall survival. Conclusions: HER-2/neu amplification does not adversely influence response to first-line chemotherapy with either ET or EC. Furthermore, a taxane-containing regimen such as ET may provide a preferential benefit to patients with HER-2/neu–positive tumors.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Overexpression of HER-2/neu that results from amplification of the HER-2/neu gene is associated with a poor clinical outcome in 20%–25% of all primary breast cancer patients (13). Results from early clinical trials suggest that the unfavorable prognosis associated with HER-2/neu overexpression results, in part, from resistance to chemotherapy, especially to the combination regimen of cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) (4,5). However, recent reports comparing the efficacy of CMF in a treated group with that in an untreated control group have not supported this finding and have found instead that both patients with HER-2/neu–negative and HER-2/neu–positive tumors benefited from CMF treatment (6,7). Results from other studies indicate that patients with HER-2/neu–positive tumors may receive more benefit from anthracycline-containing adjuvant regimens than from regimens not containing an anthracycline (8,9), but more recent clinical data indicate that the benefit from an anthracycline-based regimen is only marginally better than that from the CMF regimen (10). Consequently, the question of whether HER-2/neu is a marker of chemotherapy resistance or sensitivity is still debated. In addition, little clinical information is available on the association between HER-2/neu status and response to taxane-based regimens. Preliminary results from smaller uncontrolled phase II studies in patients with metastatic breast cancer (11,12), investigating the interaction between HER-2/neu status and the response to treatment with paclitaxel or docetaxel (11) or with a combination of paclitaxel and doxorubicin (12), showed better response rates in patients with HER-2/neu–positive tumors than in patients with HER-2/neu–negative tumors. However, these studies do not provide insights on whether the increased response rates are a taxane-specific effect or whether a HER-2/neu–positive status is generally associated with improved response rates to cytotoxic agents.

Previous studies of the association between HER-2/neu status and the response to chemotherapy have been challenged because of the use of inappropriate control groups or inaccurate methods to detect the alteration of the HER-2/neu gene. To avoid such controversies, we analyzed HER-2/neu status in the present study with fluorescence in situ hybridization (FISH), as this method allows the most accurate detection of the HER-2/neu alteration in formalin-fixed paraffin-embedded tissue (1315). Furthermore, we studied the association between HER-2/neu amplification and response to a taxane in the context of a randomized controlled study investigating the use of a taxane in patients with metastatic breast cancer. This approach allowed us to assess the objective response rate (as a direct measure of chemosensitivity), progression-free survival, and overall survival and enabled us to separate taxane-specific effects from effects generally associated with chemotherapy. We obtained data for this retrospective subset analysis from a randomized trial of the Arbeitsgemeinschaft für Gynäkologische Onkologie (AGO) that compared an epirubicin–cyclophosphamide (EC) regimen with an epirubicin–paclitaxel (ET) regimen as first-line therapies for patients with metastatic breast cancer. Preliminary findings from the first analysis of 429 patients, which were reported previously (16), indicated that the two treatment arms were not demonstrably different in terms of overall efficacy, with comparable objective response rates for ET and EC (46% and 41%, respectively) and comparable median progression-free survivals for both ET and EC (39 and 33 weeks, respectively;P = .089) (16). In this retrospective subset analysis, we investigated whether HER-2/neu amplification is associated with an improved response to a taxane-based ET regimen compared with an EC regimen.


    PATIENTS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
The study group consisted of patients with metastatic breast cancer enrolled between October 1, 1996, and December 31, 1999, in a prospective randomized multicenter trial of the AGO (16). For the original study, 579 patients were recruited from 71 centers in Germany. Twenty (3.4%) of these 579 patients enrolled from noncompliant participating centers were excluded from the analysis. Of the 559 remaining patients, 34 (6.1%) did not meet the eligibility criteria (first detected at in-house monitoring sessions) because of nonmeasurable disease at randomization (n = 13), concomitant other therapy (n = 8), or other reasons (n = 10). Of these 559 patients, 516 patients were enrolled in active treatment arms and had received at least one cycle of chemotherapy. All women aged 18–75 years had evidence of progressive, radiographically assessable, metastatic breast cancer and had not received prior chemotherapy for metastatic disease. Only one preceding hormonal therapy was allowed for treatment of metastatic breast cancer before study entry (i.e., tamoxifen). All patients were required to have a Karnofsky performance status of more than 60%, a life expectancy of 3 months or more, normal serum calcium levels, and normal cardiac, renal, hepatic, pulmonary, hematologic, and coagulation functions. Patients were excluded for active infections, pregnancy or lactation, clinically significant cardiac disease, known hemorrhagic diseases, metastasis to the central nervous system, metastasis to bone only, prior treatment with epirubicin at a dose of more than 400 mg/m2 of body surface area, or concomitant use of any investigational agent. After stratification (no hormonal therapy versus one preceding hormonal therapy for treatment of metastatic breast cancer), patients were randomly assigned to receive either epirubicin (60 mg/m2) and cyclophosphamide (600 mg/m2) or epirubicin (60 mg/m2) and paclitaxel (175 mg/m2) over a 3-hour period for a maximum of 10 cycles—with each combination therapy given once every 3 weeks. No further treatment was allowed in the protocol without documented evidence of treatment failure. All patients signed a written internal review board–approved informed consent document before study entry. Tumor samples and clinical information were obtained under institutional review board approval; this study is a retrospective statistical analysis of results obtained from the AGO study.

Before entry into the study, all patients underwent a complete physical examination, chest x-rays, an ultrasonographic examination of the liver, and a bone scan. Computed tomography examinations of the chest and abdomen were performed when clinically indicated. Response was determined at 12-week intervals. A complete response was defined as the disappearance of all radiographically and/or visually apparent tumor tissue. A partial response was defined as a greater than 50% decrease (two-dimensional measurements) of all measurable lesions. Disease progression was defined as a greater than 25% increase in any measurable lesion or the appearance of any new lesion. Objective responses were not routinely confirmed by a later evaluation. The objective response rate was defined as the percentage of patients achieving a complete or partial response. Progression-free survival was defined as the time from the date of randomization to the date of documented disease progression. The occurrence of a secondary malignancy or death from any cause was also considered to be disease progression. Overall survival was defined as the time from the date of randomization to the date of death from any cause or to the date of loss to follow-up.

Specimens

Archived primary tumor specimens from all 516 patients were requested from 71 institutions participating in the randomized AGO study of first-line treatment for patients with metastatic breast cancer. However, because the primary surgery was often performed at institutions other than those participating in the clinical AGO trial, tissue blocks of primary breast cancer biopsy samples were available for HER-2/neu analysis from 297 (58%) of the 516 patients. Each tissue block was sectioned for histologic analysis, and five blocks lacking residual invasive carcinoma were omitted, leaving a total of 292 samples. Of these 292 samples, 275 (94%) were technically satisfactory for analysis of HER-2/neu gene amplification.

HER-2/neu Analysis

HER-2/neu gene copy number was analyzed by use of FISH on 4-µm sections of paraffin-embedded and formalin-fixed tumor tissue. Specimen preparation, hybridization, and microscopy were performed as previously described (14). A SpectrumOrange-labeled HER-2/neu probe (Vysis, Downers Grove, IL) was used to determine gene copy number (number of gene signals per cell). In addition, to account for increased HER-2/neu gene copy number because of chromosome 17 polysomy (increased copy number of the entire chromosome, which is frequently seen in breast cancer), we also hybridized specimens with a probe for the chromosome 17 centromere labeled with the SpectrumGreen fluorochrome (PathVision 30-161060; Vysis). Data were expressed as the number of HER-2/neu genes per chromosome 17 centromere; we used two HER-2/neu genes per chromosome 17 centromere as the biological cutoff to distinguish tumors with and without HER-2/neu amplification, respectively (14).

FISH Scoring Criteria

FISH scores were analyzed as previously described (14). In brief, slides were initially scanned for the HER-2/neu signals and sorted into the following three groups: 1) tumors with obvious gene amplification (i.e., signal clusters of more than 10–15 signals per cell), 2) tumors readily classified as single copy (i.e., no cells with more than four HER-2/neu signals detectable), and 3) an intermediate group with low-level copy number increases. In the intermediate group, 100 randomly selected nuclei were scored for both HER-2/neu and chromosome 17 centromere signals, and HER-2/neu amplification was defined as the mean ratio of the number of HER-2/neu signals to the number of chromosome 17 centromere signals. Tumors with a ratio of more than 2 were classified as HER-2/neu positive, and tumors with a ratio of 2 or less were classified as HER-2/neu negative.

Statistical Analysis

Baseline prognostic and patient characteristics between the 275 patients with FISH data available and the 241 patients without such data were compared by use of the chi-square test to exclude the possibility of selection bias. Baseline prognostic and patient characteristics were also compared between treatment groups (ET and EC) for patients with a known HER-2/neu status by use of the chi-square test. We explored associations between the frequency of HER-2/neu gene amplification, prognostic factors, and patient characteristics, including age, histology (ductal, lobular, or other), nuclear grade (1, 2, or 3), estrogen or progesterone receptor status of tumor, location of metastases (liver, lung, or other), number of metastatic sites (1, 2, or ≥3), and whether prior palliative hormonal therapy or prior adjuvant chemotherapy was received. The association of HER-2/neu gene amplification with each of these characteristics was tested individually by use of the chi-square test. The prognostic relevance of baseline factors for progression-free survival and overall survival was assessed with a univariate Cox model.

To determine whether HER-2/neu gene amplification was associated with therapeutic efficacy, we calculated objective response rates for both treatment regimens and patient subsets according to HER-2/neu status. Progression-free survival and overall survival were estimated with the Kaplan–Meier method. Of the 275 patients with evaluable FISH results, 258 (94%) had radiographically measurable disease at a later evaluation, and information on progression-free survival and overall survival was available for all 275 patients. Progression-free survival and overall survival were compared by using the log-rank test. We estimated the odds ratio (OR) for the objective response rate and the relative risks (RRs) for progression-free survival and overall survival by use of a Cox univariate model. In multivariable analysis, a logistic regression model (objective response rate) and a Cox proportional hazard regression model (progression-free survival and overall survival; the assumptions of proportionality for the Cox regression were verified by residuals plots. Martingale [or deviance] residuals were plotted against survival time or covariates. For logistic regression, goodness-of-fit statistics was used for model checking) were used to investigate associations between the probability of response and age, histology (ductal, lobular, or other), localization of metastasis (liver, lung, or other), number of metastatic sites (1, 2, or ≥3), whether palliative endocrine therapy or adjuvant chemotherapy had been received, nuclear grade (1, 2, or 3), and estrogen and progesterone receptor status determined at the primary diagnosis. A stepwise procedure was used for covariate selection. All statistical tests were two-sided. Data from patients whose tumors were HER-2/neu negative and patients whose tumors were HER-2/neu positive were tested for equality by adding an interaction term to the logistic regression or the Cox proportional hazards model and by testing its statistical significance with the Wald test.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Characterization of Study Cohort

Comparison of characteristics of the cohort of eligible patients with and without known HER-2/neu status detected no statistically significant differences between the group whose HER-2/neu status was known and the group whose HER-2/neu status was unknown (Table 1), indicating that our study sample was representative of the overall patient population. Among the 275 patients whose HER-2/neu status could be determined, 137 had been randomly assigned to receive EC and 138 had been randomly assigned to receive ET (Table 2). Patients in both treatment groups received a median of six cycles of chemotherapy. As indicated in Table 2, patient and disease characteristics were well balanced across the two treatment arms. Of the 275 evaluable primary tumor specimens, HER-2/neu gene amplification was detected in 97 (35%) tumors, 48 from the EC arm and 49 from the ET arm. The prognostic relevance of baseline factors for progression-free survival and overall survival for patients with known HER-2/neu status is shown in Table 3.


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Table 1. Baseline characteristics of patients with known and unknown HER-2/neu status*

 

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Table 2. Baseline characteristics of patients treated with an ET regimen compared with those of patients treated with an EC regimen*

 

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Table 3. Prognostic relevance of baseline factors for progression-free survival and overall survival in univariate analysis (Cox model)*

 
Association Between HER-2/neu and Baseline Characteristics

HER-2/neu amplification was inversely associated with estrogen receptor status, in that 46 (52%) of the 89 HER-2/neu–positive tumors were classified as estrogen receptor positive and 110 (67%) of the 163 HER-2/neu–negative tumors were classified as estrogen receptor positive (P = .014). HER-2/neu amplification was also positively associated with a higher proportion of patients who had received prior adjuvant chemotherapy, in that 48 (49%) of the 97 patients with HER-2/neu–positive tumors had received adjuvant chemotherapy compared with 52 (30%) of the 175 patients with HER-2/neu–negative tumors (P = .001). HER-2/neu gene amplification, however, was not associated with the progesterone receptor status, number or site of metastatic lesions, the type of histology, the nuclear grade of the primary tumor, patient age, or the application of a prior palliative hormone therapy (data not shown).

Association Between HER-2/neu Status and Treatment Outcome

We initially investigated whether HER-2/neu amplification was associated with treatment outcomes in the entire study cohort of 275 patients. Patients with HER-2/neu–positive tumors had a statistically significantly higher objective response rate (60% [95% CI = 51% to 70%] versus 41% [95 CI = 34% to 49%]; {chi}2 P = .004; logistic regression OR = 2.19 [95% CI = 1.31 to 3.65]; P = .003) than patients with HER-2/neu–negative tumors. Despite an increased primary response, patients with HER-2/neu–positive tumors, however, had a poorer prognosis than patients with HER-2/neu–negative tumors. Statistically significantly shorter progression-free survival was observed among the 97 patients with HER-2/neu–positive tumors (median progression-free survival = 8.4 months, 95% CI = 7.3 to 10.3 months) than among the 178 patients with HER-2/neu–negative tumors (median progression-free survival = 10.3 months, 95% CI = 7.6 to 11.7 months) (P = .020). In addition, statistically significantly shorter overall survival was observed among the 97 patients with HER-2/neu–positive tumors (median overall survival = 19.0 months, 95% CI = 15.1 to 22.1 months) than among the 178 patients with HER-2/neu–negative tumors (28.4 months, 95% CI = 20.9 to 35.2 months) (P = .023).

We next investigated the association between HER-2/neu status and treatment outcome separately for patients treated with EC and for patients treated with ET. Among those treated with EC, patients with HER-2/neu–positive tumors and HER-2/neu–negative tumors had comparable objective response rates (46% [95% CI = 32% to 60%] versus 33% [95% CI = 22% to 43%]; P = .130; Fig. 1). However, patients with HER-2/neu–positive tumors had statistically significantly shorter progression-free survival (median progression-free survival = 7.1 months, 95% CI = 4.1 to 9.3 months) than patients with HER-2/neu–negative tumors (median progression-free survival = 10.4 months, 95% CI = 6.9 to 14.9 months) (P = .010) (Fig. 2, A). Likewise, patients with HER-2/neu–positive tumors had statistically significantly shorter overall survival (median overall survival = 16.4 months, 95% CI = 12.1 to 20.1 months) than patients with HER-2/neu–negative tumors (median overall survival = 33.1 months, 95% CI = 20.9 to 50.6 months) (P = .010) (Fig. 2, C).



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Fig. 1. Objective response rates. Patients with HER-2/neu–positive tumors who were treated with epirubicin–cyclophosphamide (EC) showed an objective response rate that was similar to that of HER-2/neu–negative patients (46% versus 33%; P = .130). In contrast, patients with HER-2/neu–positive tumors who were treated with epirubicin–paclitaxel (ET) showed a statistically significantly higher objective response rate than patients with HER-2/neu–negative tumors (76% versus 50%; P = .005). ET, compared with EC, was associated with a higher objective response rate in both patients with HER-2/neu–positive tumors (76% versus 46%; P = .004) and patients with HER-2/neu–negative tumors (50% versus 33%; P = .002). However, after adjustment, the difference in response associated with ET, compared with that associated with EC, was greater in patients with HER-2/neu–positive tumors (odds ratio [OR] = 3.64, 95% confidence interval [CI] = 1.48 to 8.92; P = .005) than in patients with HER-2/neu–negative tumors (OR = 1.92, 95% CI = 1.01 to 3.64; P = .046) (see Table 4). Objective response rates were calculated for both treatment regimens and patient subsets according to HER-2/neu status. Error bars = 95% confidence intervals; solid bars = ET; open bars = EC. All statistical tests were two-sided.

 


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Fig. 2. Survival by HER-2/neu status. A and B) Progression-free survival (PFS) for patients with HER-2/neu–positive tumors and patients with HER-2/neu–negative tumors in the epirubicin–cyclophosphamide (EC) treatment arm (A) and in the epirubicin–paclitaxel (ET) treatment arm (B). Median progression-free survival is shown on each plot with 95% confidence intervals (CIs). C and D) Overall survival (OS) for patients with HER-2/neu–positive tumors and for patients with HER-2/neu–negative tumors in the EC treatment arm (C) and in the ET treatment arm (D). Median overall survival is shown on each plot with 95% CIs.

 
In contrast, among patients treated with ET, patients with HER-2/neu–positive tumors had a statistically significantly higher objective response rate (76% [95% CI = 63% to 88%]) than patients with HER-2/neu–negative tumors (50% [95% CI = 39% to 61%]; P = .005; Fig. 1). Progression-free survival of patients with HER-2/neu–positive tumors (median progression-free survival = 10.5 months, 95% CI = 8.1 to 11.9 months) was similar to that of patients with HER-2/neu–negative tumors (median progression-free survival = 9.6 months, 95% CI = 7.5 to 11.3 months) (P = .584) (Fig. 2, B). Overall survival of patients with HER-2/neu–positive tumors (median overall survival = 21.4 months, 95% CI = 15.3 to 27.3 months) was similar to that of patients with HER-2/neu–negative tumors (median overall survival = 27.5 months, 95% CI = 17.1 to 35.2 months) (P = .463) (Fig. 2, D).

Association Between HER-2/neu Status and Response to Paclitaxel

The central hypothesis tested in the present study was whether the ET regimen is more effective than the EC regimen among patients with HER-2/neu–positive tumors than in patients with HER-2/neu–negative tumors. We found that the objective response rate associated with the ET regimen was better than that associated with the EC regimen among patients with HER-2/neu–positive tumors (76% [95% CI = 63% to 88%] versus 46% [95% CI = 32% to 60%]; P = .004) and among patients with HER-2/neu–negative tumors (50% [95% CI = 39% to 61%] versus 33% [95% CI = 22% to 43%]; P = .002; Fig. 1). The difference in objective response rates associated with the ET and EC regimens, however, was greater for patients with HER-2/neu–positive tumors (adjusted OR = 3.64, 95% CI = 1.48 to 8.92; P = .005) than for patients with HER-2/neu–negative tumors (adjusted OR = 1.92, 95% CI = 1.01 to 3.64; P = .046) (Table 4).


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Table 4. Risks to patients treated with ET relative to those treated with EC according to HER-2/neu status*

 
Furthermore, in patients with HER-2/neu–positive tumors, longer progression-free survival (median progression-free survival = 10.5 months [95% CI = 8.1 to 11.9 months] versus 7.1 months [95% CI = 4.1 to 9.3 months]; P = .116) (Fig. 3, B) and longer overall survival (median overall survival = 21.4 months [95% CI = 15.3 to 27.3 months] versus 16.4 months [95% CI = 12.1 to 20.4 months]; P = .319) (Fig. 3, D) were associated with the ET regimen than with the EC regimen, although these differences were not statistically significant. After adjusting for the effects of other statistically significant prognostic variables in a multivariable analysis (such as localization of metastases, number of sites, estrogen receptor status, and prior adjuvant chemotherapy), the relative risk of failure for the ET regimen compared with that for the EC regimen in patients with HER-2/neu–positive tumors was 0.65 for progression-free survival (95% CI = 0.42 to 1.02; P =.062) and 0.60 for overall survival (95% CI = 0.36 to 1.02; P = .059) (Table 4), where a relative risk greater than 1 favors the EC regimen and a relative risk less than 1 favors the ET regimen. Among patients with HER-2/neu–negative tumors, both progression-free survival (median progression-free survival = 9.6 months [95% CI = 7.5 to 11.3 months] versus 10.4 months [95% CI = 6.9 to 14.9 months];P = .350) (Fig. 3, A) and overall survival (median overall survival = 27.5 months [95% CI = 17.1 to 35.2 months] versus 33.1 months [95% CI = 20.9 to 50.6 months]; P = .292) (Fig. 3, C) were similar for patients treated with the EC regimen and for patients treated with the ET regimen. The adjusted relative risk of failure for the ET regimen compared with that for the EC regimen in patients with HER-2/neu–negative patients was 1.10 for progression-free survival (95% CI = 0.77 to 1.57; P = .590) and 1.10 for overall survival (95% CI = 0.70 to 1.72; P = .680). Statistical tests for the interaction between treatment and HER-2/neu status were suggestive of such interactions but were not statistically significant (objective response rate, unadjusted P for interaction = .308 and adjusted P = .256; progression-free survival, unadjusted P = .090 and adjusted P = .109; overall survival, unadjusted P = .152 and adjusted P = .136; Table 4 and Fig. 4).



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Fig. 3. Survival by treatment arm. A and B) Progression-free survival (PFS) associated with the epirubicin–cyclophosphamide (EC) and epirubicin–paclitaxel (ET) treatment arms among patients with HER-2/neu–negative tumors (A) and among patients with HER-2/neu–positive tumors (B). Median progression-free survival is shown on each plot with 95% confidence intervals (CIs). Relative risk (RR) of failure and corresponding P value shown on each plot are adjusted (by use of the Cox model) for localization of metastasis (liver, lung, or other), number of sites (1, 2, or ≥3), estrogen receptor status, and prior adjuvant chemotherapy. C and D) Overall survival (OS) associated with EC and ET treatment arms among patients with HER-2/neu–negative tumors (C) and among patients with HER-2/neu–positive tumors (D). Median overall survival is shown on each plot with 95% CIs. Relative risk (RR) of failure and corresponding P value shown on each plot are adjusted (by use of the Cox model) for localization of metastasis (liver, lung, or other), number of sites (1, 2, or ≥3), estrogen receptor status, and prior adjuvant chemotherapy. All statistical tests were two-sided.

 


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Fig. 4. Association between objective response rates (ORRs), progression-free survival (PFS), or overall survival (OS) and treatment according to HER-2/neu status. Data for the objective response rate were adjusted (by use of the logistic model) for prior adjuvant chemotherapy. Relative risks (RRs) of failure were adjusted (by use of the Cox model) for localization of metastasis (liver, lung, or other), number of metastatic sites (1, 2, or ≥3), estrogen receptor status, and prior adjuvant chemotherapy. Bars = 95% confidence intervals. All statistical tests were two-sided.

 

    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
In this study, we found that patients with HER-2/neu–positive tumors had a better response to first-line chemotherapy than patients with HER-2/neu–negative tumors. However, when we used progression-free survival or overall survival as end points for treatment outcome, the poorer prognosis for patients with HER-2/neu–positive tumors, compared with patients with HER-2/neu–negative tumors, did not appear to concur with sensitivity toward chemotherapy. A possible explanation for this discordance could be based on the observation that tumor cells with HER-2/neu overexpression proliferate more rapidly than HER-2/neu–negative cells (17,18), so that patients with HER-2/neu–positive tumors might relapse sooner (unless their tumors are completely eradicated) than patients with HER-2/neu–negative tumors. As previously described, HER-2/neu overexpression results in increased cell proliferation in preclinical xenograft models, and this increased proliferation can lead to a more rapid recovery from the cytotoxic effects of chemotherapeutic agents, despite the tumor's initial chemosensitivity (1720). Our results from this clinical study confirm these preclinical results; i.e., patients with HER-2/neu–positive tumors relapsed more quickly after chemotherapy than patients with HER-2/neu–negative tumors, despite the higher initial response rate in patients with HER-2/neu–positive tumors.

Although patients with HER-2/neu–positive tumors have a poor prognosis, our results underscore the fact that HER-2/neu amplification is not an intrinsic marker of chemoresistance to either the EC regimen or the ET regimen. When response rates of patients with HER-2/neu–positive and patients with HER-2/neu–negative tumors were compared separately by treatment arm, chemotherapeutic sensitivity in patients with HER-2/neu–positive tumors, compared with patients with HER-2/neu–negative tumors, appeared to be particularly pronounced after treatment with the ET regimen. Among patients treated with the ET regimen, but not among those treated with the EC regimen, HER-2/neu amplification was statistically significantly associated with improved response rates. These findings support the following two hypotheses: 1) that a HER-2/neu–positive status does not appear to confer unique clinical chemoresistance to the EC or ET regimen and 2) that patients with HER-2/neu–positive tumors might be particularly sensitive to the paclitaxel-containing regimen.

For further validation, we compared the outcomes after treatment with the ET regimen and after the EC regimen separately in patients with HER-2/neu–positive tumors and in patients with HER-2/neu–negative tumors. In patients with HER-2/neu–positive tumors, substitution of paclitaxel for cyclophosphamide resulted in a statistically significantly increased objective response rate (adjusted OR = 3.64, 95% CI = 1.48 to 8.92; P = .005), which was more pronounced than the increase observed in HER-2/neu–negative patients (adjusted OR = 1.92, 95% CI = 1.01 to 3.64; P = .046). We also found evidence for better progression-free survival and better overall survival after treatment with the ET regimen than after treatment with the EC regimen in patients with HER-2/neu–positive tumors than in patients with HER-2/neu–negative tumors. Although these differences in progression-free survival and overall survival did not reach statistical significance, they may be clinically meaningful because the relative risk for disease progression associated with the ET regimen, compared with that of the EC regimen, was reduced by 35% (adjusted RR = 0.65, 95% CI = 0.42 to 1.02; P =.062), but not statistically significantly so. However, we believe that this association should be studied further in a larger study with more statistical power. Similar evidence of an improved overall survival after treatment with the ET regimen among patients with HER-2/neu–positive tumors was also observed (adjusted RR = 0.60, 95% CI = 0.36 to 1.02; P = .059), which is remarkable in view of the fact that many of the patients had received taxane-based second-line chemotherapy after EC treatment failure. Although we could not confirm a statistically significant interaction between treatment and HER-2/neu status, the data are suggestive for such an interaction for progression-free survival (P = .109) and for overall survival (P = .136). The estimated power for detecting a statistically significant interaction in the present study cohort, however, was only 40% for progression-free survival and 25% for overall survival; thus, this study was not large enough to allow meaningful interaction studies between treatment and HER-2/neu status.

Previous studies reported higher response rates after taxane-based therapies for patients with HER-2/neu–positive tumors than for patients with HER-2/neu–negative tumors (11,12). However, these earlier studies did not have appropriate control cohorts that would allow separate analyses of taxane-specific treatment effects and of general treatment effects associated with chemotherapy. A more recent study conducted by the Breast Cancer International Research Group (BCIRG) provides more comprehensive data in support of our findings. The BCIRG Study 001, in which 1491 patients with lymph node–positive primary breast cancer were randomly assigned to an adjuvant chemotherapy regimen of docetaxel–doxorubicin–cyclophosphamide or a regimen of fluorouracil–doxorubicin–cyclophosphamide, provided an important control group to investigate taxane-specific effects. Results of the first analysis of this study are consistent with our results because they indicate that a better outcome was associated with the docetaxel–doxorubicin–cyclophosphamide regimen than with the fluorouracil–doxorubicin–cyclophosphamide regimen, especially among patients with HER-2/neu–positive tumors (for disease-free survival, hazard ratio [HR] = 0.60; P = .009) compared with patients with HER-2/neu–negative tumors (for disease-free survival, HR = 0.76; P = .046) (21).

Experimental results regarding HER-2/neu overexpression and the response to taxane-based regimens are contradictory, with some investigators reporting unique resistance to such regimens (22,23) and others reporting the lack of such resistance (17). In a previous study investigating the association of HER-2/neu overexpression with taxane sensitivity, we stably transfected six human breast and ovarian cancer cell lines with a full-length human HER-2/neu cDNA (17) to directly compare drug sensitivity in parent and daughter cells that differed genetically because one member of the pair overexpressed the human HER-2/neu gene. In three of six human breast or ovarian cancer cell lines, HER-2/neu overexpression resulted in a statistically significantly increased sensitivity toward paclitaxel; this result led us to conclude that HER-2/neu overexpression was most likely not associated with in vitro resistance to paclitaxel (17).

Previous results indicated an interaction between HER-2/neu overexpression and the dose of an anthracycline-based combination chemotherapy (24), in which a statistically significantly better outcome was found for patients with HER-2/neu–positive primary breast cancer treated with standard doses of fluorouracil–doxorubicin–cyclophosphamide than for patients treated with lower doses of these drugs. In our study, however, the ET and EC regimens were given at standard doses, indicating that the observed treatment outcomes most likely did not result from an interaction between HER-2/neu amplification and the dose level of chemotherapeutic agents.

Because preceding adjuvant chemotherapy reduces the response to first-line chemotherapy, it is noteworthy that patients with HER-2/neu–positive tumors (who received more adjuvant chemotherapy than patients with HER-2/neu–negative tumors) still responded better than patients with HER-2/neu–negative tumors to first-line chemotherapy for metastatic breast cancer. However, exposure to an anthracycline-based adjuvant chemotherapy might increase sensitivity of metastatic cancer to a non–cross-resistant taxane-containing regimen and lead to a better response to the ET regimen than to the EC regimen. Nevertheless, the independence of the observed treatment effects from other potentially confounding variables in multivariable analysis (such as a negative estrogen receptor status, localization and number of metastatic sites, or the prior application of adjuvant chemotherapy) does strengthen our hypothesis that HER-2/neu amplification might be associated with increased response to taxane-based first-line chemotherapy.

The results of the present study, nonetheless, should be interpreted cautiously because the study was based on a retrospective subgroup analysis whose objective was not part of the original randomized study. Although our results are not yet useful for determining the best treatment for patients with metastatic breast cancer, they do suggest that there may be an interesting biological question regarding the role of HER-2/neu and response to treatment with a taxane-based regimen. The best way to evaluate the association between treatment and HER-2/neu status would be a prospective controlled study; however, it is unlikely that such a study could be conducted now because chemotherapy alone as a first-line treatment has been replaced with a combination of chemotherapy and trastuzumab, which is associated with a statistically significant survival advantage for patients with HER-2/neu–positive metastatic breast cancer (25). Therefore, confirmatory studies including sufficiently powered retrospective analysis are needed and would be of great interest to those for whom the efficacy of taxane-based regimens is currently a matter of debate.


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Supported by Bristol-Myers Squibb (Munich, Germany) and the Revlon/UCLA Women's Cancer Research Program. We thank Adelheit Klein and Heidi Spicher for their invaluable assistance in collecting the tissue samples, Wendy Aft for her excellent preparation of the manuscript, and Hans Bochtler for his efforts during the study.

Presented in part at the Thirty-Seventh Annual Meeting of the American Society of Clinical Oncology, San Francisco, CA, May 12–15, 2001


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

1 Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification on HER-2/neu oncogene. Science 1987;235:177–82.[ISI][Medline]

2 Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:707–12.[ISI][Medline]

3 Pauletti G, Dandekar S, Rong H, Ramos L, Peng H, Seshadri R, et al. Assessment of methods for tissue-based detection of the HER-2/neu alteration in human breast cancer: a direct comparison of fluorescence in situ hybridization and immunohistochemistry. J Clin Oncol 2000;18:3651–64.[Abstract/Free Full Text]

4 Allred DC, Clark GM, Tandon AK, Molina R, Tormey DC, Osborne CK, et al. HER-2/neu in node-negative breast cancer: prognostic significance of overexpression influenced by the presence of in situ carcinoma. J Clin Oncol 1992;10:599–605.[Abstract]

5 Gusterson BA, Gelber RD, Goldhirsch A, Price KN, Save-Soderborgh J, Anbazhagan R, et al. Prognostic importance of c-erbB-2 expression in breast cancer. International (Ludwig) Breast Cancer Study Group. J Clin Oncol 1992;10:1049–56.[Abstract]

6 Miles DW, Harris WH, Gillett CE, Smith P, Barnes DM. Effect of c-erbB(2) and estrogen receptor status on survival of women with primary breast cancer treated with adjuvant cyclophosphamide/methotrexate/fluorouracil. Int J Cancer 1999;84:354–9.[CrossRef][ISI][Medline]

7 Menard S, Valagussa P, Pilotti S, Gianni L, Biganzoli E, Boracchi P, et al. Response to cyclophosphamide, methotrexate, and fluorouracil in lymph node-positive breast cancer according to HER-2 overexpression and other tumor biologic variables. J Clin Oncol 2001;19:329–35.[Abstract/Free Full Text]

8 Paik S, Bryant J, Park C, Fisher B, Tan-Chiu E, Hyams D, et al. erbB-2 and response to doxorubicin in patients with axillary lymph node-positive, hormone receptor-negative breast cancer. J Natl Cancer Inst 1998;90:1361–70.[Abstract/Free Full Text]

9 Pritchard K, O'Malley FA, Andrulis I, Shepherd LE, Tu D, Levine MN, et al. Prognostic and predictive value of HER-2/neu in a randomized trial comparing CMF to CEF in premenopausal women with axillary lymph node positive breast cancer (NCIC CTG MA.5) [abstract 165]. Proc ASCO 2002;21:42a.

10 Paik S, Bryant J, Tan-Chiu E, Yothers G, Park C, Wickerham DL, et al. HER-2 and choice of adjuvant chemotherapy for invasive breast cancer: National Surgical Adjuvant Breast and Bowel Project Protocol B-15. J Natl Cancer Inst 2000;92:1991–8.[Abstract/Free Full Text]

11 Seidman AD, Baselga J, Yao TJ, Gilewski T, Rosen PP, Norton L. HER-2/neu overexpression and clinical taxane sensitivity: a multivariate analysis in patients with metastatic breast cancer (MCB) [abstract 80]. Proc ASCO 1996;15:104.

12 Gianni L, Capri G, Mezzelani A, Valagussa P, Greco M, Bertuzzi A, et al. HER-2/neu (HER-2) amplification and response to doxorubicin/paclitaxel (AT) in women with metastatic breast cancer [abstract 491]. Proc ASCO 1997;16:139a.

13 Kallioniemi OP, Kallioniemi A, Kurisu W, Thor A, Chen LC, Smith HS, et al. ERBB2 amplification in breast cancer analyzed by fluorescence in situ hybridization. Proc Natl Acad Sci U S A 1992;89:5321–5.[Abstract]

14 Pauletti G, Godolphin W, Press MF, Slamon DJ. Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization. Oncogene 1996;13:63–72.[ISI][Medline]

15 Press MF, Slamon DJ, Flom KJ, Park J, Zhou JY, Bernstein L. Evaluation of HER-2/neu gene amplification and overexpression: comparison of frequently used assay methods in a molecularly characterized cohort of breast cancer specimens. J Clin Oncol 2002;20:3095–105.[Abstract/Free Full Text]

16 Luck HJ, Thomssen C, Untch M, Kuhn W, Eidtmann H, duBois A, et al. Multicenter phase III study in first line treatment of advanced metastatic breast cancer (ABC). Epirubicin/paclitaxel (ET) vs epirubicin/cyclophosphamide (EC) [abstract 280]. A study of the AGO Breast Cancer Group. Proc ASCO 2000;19:73a.

17 Pegram MD, Finn RS, Arzoo K, Beryt M, Pietras RJ, Slamon DJ. The effect of HER-2/neu overexpression on chemotherapeutic drug sensitivity in human breast and ovarian cancer cells. Oncogene 1997;15:537–47.[CrossRef][ISI][Medline]

18 Pegram M, Hsu S, Lewis G, Pietras R, Beryt M, Sliwkowski M, et al. Inhibitory effects of combinations of HER-2/neu antibody and chemotherapeutic agents used for treatment of human breast cancers. Oncogene 1999;18:2241–51.[CrossRef][ISI][Medline]

19 Pietras RJ, Fendly B, Chazin VR, Pegram MD, Howell SB, Slamon DJ. Antibody to HER-2/neu receptor blocks DNA repair after cisplatin in human breast and ovarian carcinoma cells. Oncogene 1994;9:1829–38.[ISI][Medline]

20 Pietras RJ, Pegram MD, Finn RS, Maneval DA, Slamon DJ. Remission of human breast cancer xenografts on therapy with humanized monoclonal antibody to HER-2 receptor and DNA-reactive drugs. Oncogene 1998;17:2235–49.[CrossRef][ISI][Medline]

21 Martin M, Pienkowski T, Mackey J, Pawlicki M, Guastalla JP, Weaver C, et al. TAC improves disease free survival and overall survival over FAC in node positive early breast cancer patients, BCIRG 001: 55 months follow-up [abstract 43]. Breast Cancer Res Treat 2003;21:36a.

22 Yu D, Jing T, Liu B, Yao J, Tan M, McDonnell TJ, et al. Overexpression of ErbB2 blocks Taxol-induced apoptosis by upregulation of p21Cip1, which inhibits p34Cdc2 kinase. Mol Cell 1998;2:581–91.[ISI][Medline]

23 Zhang L, Lau YK, Xia W, Hortobagyi GN, Hung MC. Tyrosine kinase inhibitor emodin suppresses growth of HER-2/neu-overexpressing breast cancer cells in athymic mice and sensitizes these cells to the inhibitory effect of paclitaxel. Clin Cancer Res 1999;5:343–53.[Abstract/Free Full Text]

24 Thor AD, Berry DA, Budman DR, Muss HB, Kute T, Henderson IC, et al. erbB-2, p53, and efficacy of adjuvant therapy in lymph node-positive breast cancer. J Natl Cancer Inst 1998;90:1346–60.[Abstract/Free Full Text]

25 Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, et al. Use of chemotherapy plus a monoclonal antibody against HER-2 for metastatic breast cancer that overexpresses HER-2. N Engl J Med 2001;344:783–92.[Abstract/Free Full Text]

Manuscript received January 7, 2004; revised May 19, 2004; accepted May 20, 2004.


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