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Molecular Oncology, Markers, Clinical Correlates |
Gene Aberrations as Predictive Markers in Node-positive Breast Cancer Patients Randomly Treated Either with an Anthracycline-based Therapy or with Cyclophosphamide, Methotrexate, and 5-Fluorouracil1
Translational Research Unit, Jules Bordet Institute-Brussels, 1000 Brussels, Belgium [A. D. L., D. G., D. L., G. R., S. D., J-Y. L., M. J. P.]; Jules Bordet Institute Data Center, 1000 Brussels, Belgium [M. P.]; and Laboratory of Cancer Biology, Institute of Medical Technology [M. T., J. I.], Department of Surgery and Institute of Medical Technology [T. J.], Tampere, 33101 Finland
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(topo II) as candidates for predicting the activity of anthracyclines in the adjuvant treatment of breast cancer patients.
Experimental Design: In this study, we evaluated HER-2 and topo II gene aberrations by fluorescence in situ hybridization in a series of 430 primary breast cancer samples. Samples came from node-positive breast cancer patients randomly treated either with one of two anthracycline-based regimens [full-dose epirubicin-cyclophosphamide (HEC) and moderate-dose epirubicin-cyclophosphamide (EC)] or with cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) in the context of a Phase III adjuvant therapy trial. Event-free survival comparisons were performed between the three study arms in the subgroups of HER-2-amplified and nonamplified tumors. An explorative analysis was also performed to evaluate the predictive value of topo II in the cohort of HER-2-amplified patients.
Results: HER-2 amplification was observed in 73 of the 354 evaluable samples (21%), whereas topo II amplification was found in 23 of the 61 evaluable HER-2-amplified tumors (38%). The three event-free survival comparisons were CMF versus HEC, CMF versus EC, and EC versus HEC. Hazard ratios (HRs) and 95% confidence intervals (CIs) were as follows: (a) CMF versus HEC, HR = 1.42 for HER-2-amplified tumors (95% CI, 0.54–3.76; P = 0.48) and 0.84 for HER-2-nonamplified tumors (95% CI, 0.49–1.44; P = 0.53); (b) CMF versus EC, HR = 1.65 for HER-2-amplified tumors (95% CI, 0.66–4.13; P = 0.29) and 0.66 for HER-2-nonamplified tumors (95% CI, 0.39–1.10; P = 0.11); and (c) EC versus HEC, HR = 0.93 for HER-2-amplified tumors (95% CI, 0.31–2.77, P = 0.90) and 1.33 for HER-2-nonamplified tumors (95% CI, 0.82–2.14; P = 0.25). Observed HRs suggest that the anthracycline-based therapy could be more effective than CMF in the subgroup of HER-2-amplified patients, whereas treatments could be equally active in the HER-2-nonamplified cohort. topo II evaluation suggests that the superiority of anthracyclines over CMF in HER-2-amplified patients could be confined to the subgroup of topo II-amplified tumors.
Conclusions: HER-2 could have a predictive value for the activity of anthracycline-based regimens in the adjuvant therapy of breast cancer patients. The predictive value of HER-2 would most likely be related to the concomitant amplification of the topo II gene.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In these retrospective studies, HER-2 has been evaluated mainly by IHC. Our group has reported that the interaction between HER-2 status and anthracycline efficacy may change radically according to the anti-HER-2 antibody used in the IHC assay (11) . Moreover, when we evaluated the degree of interlaboratory agreement with IHC, we observed that 61 of 109 HER-2-positive samples in one center were classified as HER-2 negative in a second laboratory (14) .
Because of the existing difficulties in standardization of the IHC procedures, we have decided to address the issue of HER-2 as a predictive marker by using FISH to evaluate the HER-2 status.
FISH is an expensive and time-consuming technique whose accuracy in selecting HER-2-positive patients has been documented (15 , 16) . Accordingly, we gathered primary breast cancer samples from node-positive breast cancer patients randomly treated with either an anthracycline-based adjuvant therapy or CMF in the context of a multicenter Phase III trial, and we evaluated HER-2 amplification by FISH. The primary study aim was to investigate the interaction between anthracycline efficacy and HER-2 status when the latter is evaluated by FISH.
A second exploratory analysis was carried out in the present study because of some data suggesting that: (a) the HER-2 status does not interfere in vitro with the degree of sensitivity to anthracyclines (17
, 18)
; and (b) the presumed predictive value of HER-2 would be related mainly to the concomitant amplification of the topo II gene, which occurs in about 40% of HER-2-amplified breast cancer patients (19)
. Moreover, it has been recently reported that in breast cancer cell lines, HER-2 activation increases the topo II enzyme activity directly, thus leading to increased sensitivity to topo II inhibitors (20)
.
topo II is the molecular target of anthracyclines. Recently, it has been reported that HER-2-amplified, topo II-amplified breast cancer cells are extremely sensitive to anthracyclines, whereas HER-2-amplified, topo II-nonamplified cells show an intermediate degree of sensitivity to the same drugs (21)
.
Accordingly, in the present study, we have also evaluated topo II gene aberrations by FISH in the subgroup of patients with HER-2 gene amplification. The aim of this second analysis was to explore in vivo the concept that HER-2 amplification would be indirectly implicated in the degree of sensitivity to anthracyclines through the association with topo II gene amplification. No topo II gene evaluation was carried out in HER-2-nonamplified tumors because of previous data suggesting that in this subgroup of patients, no topo II gene aberrations are observed (19)
.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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70 years) with radically resected, histologically confirmed, node-positive breast cancer were randomly allocated to one of the following treatment arms: (a) CMF every 4 weeks x 6 cycles (oral cyclophosphamide version); (b) EC x 8 cycles [epirubicin (60 mg/m2 day 1) and cyclophosphamide (500 mg/m2 i.v. day 1), q 3 weeks]; and (c) HEC x 8 cycles [epirubicin (100 mg/m2 day 1) and cyclophosphamide (830 mg/m2 i.v. day 1), q 3 weeks]. Patients were stratified according to the participating institution, number of histologically positive axillary nodes, and menopausal status. After the end of chemotherapy, postmenopausal patients with estrogen receptor-positive tumors or tumors with unknown estrogen receptor status received tamoxifen (40 mg/day) for 5 years. Locoregional radiotherapy was performed at the end of chemotherapy in all patients treated with breast-conserving surgery and in mastectomy-treated patients according to specific radiotherapy guidelines from each participating center.
Predictive Marker Study
Tumor Sample Collection
A list of all patients entered into the clinical study was submitted to each participating center. Each center had to provide the operational office with one paraffin-embedded sample of the primary tumor. Samples were sent by the participating center to the operational office by conventional mail. Once samples were received, they were classified and stored at room temperature until the predictive markers analysis was carried out.
HER-2 Evaluation by FISH
The evaluation of HER-2 status by FISH was carried out at the pathology department of the Jules Bordet Institute.
Tissue Specimens and Slide Preparation.
Four-µm sections were cut from archival tissue that had been fixed in buffered formalin and embedded in paraffin. Sections were placed on Superfrost Plus slides (Menzel-Gläser, Germany). Slide-mounted tissue sections were then baked overnight at 65°C and deparaffinized in xylene three times for 10 min each, followed by immersion in 100% ethanol twice for 5 min each. Air-dried tissue sections were subsequently treated in 0.2 M HCl for 20 min at room temperature, washed twice in 2x SSC [1x SSC = 0.15 M NaCl and 0.015 M sodium citrate (pH 7.0)] for 5 min each, and then treated in a solution of 1 M NaSCN for 30 min at 80°C. Slides were then washed twice in 2x SSC and treated in a solution of pepsin (0.5 mg/ml) in 0.9% NaCl (pH 2.0) for 10–20 min at 37°C. After pepsin digestion, tissue sections were washed in 2x SSC as described above and dehydrated in graded alcohol (70%, 90%, and 100% ethanol).
In Situ Hybridization (According to the Vysis Protocol).
Slide-mounted tissue sections were denatured in 70% formamide 2x SSC (pH 7.0) at 74°C for 5 min and then immersed in 70% ethanol at room temperature. After dehydration through graded alcohols, slides were air dried and warmed at 45°C. The dual color probe Spectrum Orange HER-2/Spectrum Green CEP17 (PathVysion, catalogue number 30-161060; Vysis, Downers Grove, IL) was then applied (10 µl) on a selected area of the sample (the invasive part of the tumor), and sections were coverslipped and sealed with rubber cement. Hybridization was carried out overnight at 37°C in a moist chamber. Washes were performed at 74°C for 2 min in 0.2x SSC 0.3% NP40. After drying, sections were counterstained with a DAPI (Boehringer Mannheim, Mannheim, Germany) antifade solution and analyzed.
Fluorescence Microscopy and Scoring Criteria.
A Leica DMRB epifluorescence microscope equipped with a 100 W mercury-arc lamp and x40 and x100 objectives was used in combination with a Triple bandpass filter (DAPI/FITC/tetramethylrhodamine isothiocyanate). Scanned at high magnification (x100), at least 60 hybridized nuclei from invasive parts of tissue sections were counted. Amplification was defined as a ratio of the number of HER-2 signals:centromeric 17 signals of >2. H&E-stained slides were read by one pathologist (D. L.) to select the areas with invasive cancer. Then the hybridized slides were read, and the spots were counted by another observer (D. G.), who was blinded with regard to the correspondent treatment arm and clinical outcome for each evaluated sample.
topo II Evaluation by FISH
This marker was evaluated by the laboratory of Cancer Biology, University of Tampere (Tampere, Finland).
At the time of topo II evaluation, investigators in Tampere were blinded with regard to the correspondent treatment arm and clinical outcome for each evaluated sample. Two-color FISH of topo II was carried out with a previously characterized PAC probe and a chromosome 17 pericentromeric probe (p17H8), which was used as a reference to determine the overall copy number of chromosome 17 (19
, 21)
. In brief, the sections were deparaffinized and incubated in 0.1 M Tris-HCl (pH 7.3) in a temperature-controlled microwave oven (92°C for 15 min). Enzymatic digestion was carried out with FFPE digestion enzyme (Zymed; 10–30 min at room temperature). The slides were washed with PBS and dehydrated in graded dilutions of ethanol. The probe mixture consisting of topo II and 17 centromere probes (30 ng and 10 ng of probe DNA, respectively) was applied to the slides (5–10 µl/slide) under coverslips. The slides were codenatured on a hot plate (94°C for 3 min), followed by overnight hybridization at 37°C. After hybridization, the slides were stringency washed with 0.5x SSC (5 min at 75°C), followed by three washes in PBS 0.2% Tween 20. The topo II probe was detected with anti-digoxigenin rhodamine (diluted 1:300; Roche-Boehringer, Mannheim, Germany). Nuclei were counterstained with 0.1 µM DAPI in an antifade solution (Vectashield; Vector Laboratories, Burlingame, CA). FISH was evaluated using an Olympus BX50 epifluorescence microscope equipped as described previously (19
, 21)
. Signals from at least 50–500 nonoverlapping nuclei with intact morphology were evaluated to determine the mean number of signals/cell for each probe. Both absolute copy numbers and the relative copy number ratio (ratio between mean number of topo II signals and the mean number of chromosome 17 centromere signals) were determined. Control hybridizations to nonmalignant breast tissue and to normal peripheral blood lymphocytes were carried out to ascertain equal hybridization efficiencies of topo II and the chromosome 17 centromere. Amplification of topo II was defined as a relative copy number ratio of 
1.5, and deletion of topo II was defined as a ratio of 0.8.
Statistical Analysis
EFS was the outcome used to assess treatment efficacy and was defined as the interval elapsed between the date of randomization and the date of documented disease relapse, second primary tumor, or death. EFS distribution was estimated by the Kaplan-Meier method or by using Cox modeling. No OS analysis was done in the subgroup of patients identified by the investigated biological markers because of the small number of events observed in each treatment group.
The patients evaluated in the present retrospective study (predictive markers study) were a subgroup of the study population entered into the prospective clinical trial. To evaluate the representativeness of this subgroup, main patient and tumor characteristics, including those factors with prognostic significance in the univariate analysis performed on the 777 patients entered into the clinical study, were compared in the two distinct groups (i.e., the 347 patients not evaluated in the present study because of the lack of a formalin-fixed tumor sample and the 430 patients entered into the present study). Patient and tumor characteristics were all categorical, and 
2 tests for heterogeneity were used to compare both groups.
The different cohorts of patients identified by HER-2 were examined with regard to differences in the magnitude of treatment effect on patient outcome by estimating HRs and the corresponding 95% CIs and p-values for no effect. HRs were calculated for the following comparisons: (a) CMF versus HEC (comparison 1); (b) CMF versus EC (comparison 2); and (c) EC versus HEC (comparison 3). For comparisons 1 and 2, a HR of >1 has to be interpreted as a poorer EFS distribution for patients treated with CMF, whereas for comparison 3, a HR of >1 has to be interpreted as a poorer EFS distribution for patients treated with EC. To test the main study hypothesis that the therapeutic benefit from the anthracycline-based therapy compared with CMF was greater in HER-2-positive patients than in HER-2-negative patients, an interaction term combining treatment arm and HER-2 status was added to the Cox proportional hazards model. Its significance was tested using a likelihood ratio test.
The Cox proportional hazards model also included those factors with prognostic significance (P < 0.05) in the univariate analysis performed on 777 patients entered into the clinical trial. The following list of putative prognostic factors for EFS was evaluated in the univariate analysis: age, menopausal status, surgery type, histological grade, histological type, tumor clinical size, tumor pathological size, number of involved ipsilateral axillary nodes, and estrogen and progesterone receptor status. Surgery type (mastectomy versus breast-conserving surgery) and the number of involved axillary nodes (1–3 or 4) were the only factors with prognostic significance in the univariate analysis. Accordingly, these two factors were introduced into the Cox proportional hazards model. Moreover, a third factor (i.e., use of adjuvant tamoxifen, yes or no) was introduced in the Cox model because of previous data suggesting a negative interaction between tamoxifen treatment and HER-2 overexpression (23)
. All reported p-values are two tailed.
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RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The main patient characteristics were well balanced between the three study arms, and no statistically significant differences in their distributions were found. The median relative dose intensities were 0.89, 0.95, and 0.90 for CMF, EC, and HEC, respectively. The median number of cycles delivered was six for CMF and eight for both the EC and HEC treatment groups.
At a median study follow-up of 74 months, no differences between CMF and HEC were observed with regard to EFS and OS [EFS, HR = 1.08 (95% CI, 0.81–1.44), P = 0.61; OS, HR = 1.07 (95% CI, 0.75–1.51), P = 0.72]. No differences with regard to EFS were observed between CMF and HEC in the subgroups of patients identified according to the following factors: (a) age (<50 or 50 years); (b) number of positive axillary nodes (1–3 or 4); (c) tumor size (pT1 or >pT1); (d) estrogen receptor and progesterone receptor status (positive or negative); and (e) histological grade (1, 2, or 3).
As far as the CMF versus EC comparison is concerned, the following results were observed for EFS and OS: (a) EFS, HR = 0.84 (95% CI, 0.64–1.12), P = 0.24; and (b) OS, HR = 0.82 (95% CI, 0.59–1.14), P = 0.24.
The results of the EC versus HEC comparison suggest an increased benefit associated with the use of the latter [EFS, HR = 1.28 (95% CI, 0.96–1.69), P = 0.09; OS, HR = 1.31 (95% CI, 0.93–1.83), P = 0.12].
Predictive Marker Study
Collection of Tumor Samples
A total of 625 paraffin-embedded samples of primary tumors were collected between September 1996 and December 1998, corresponding to 80% of the clinical study population (777 eligible patients). The 625 blocks were collected from 12 of the 14 centers participating in the clinical study; all centers that entered at least 10 patients into the clinical study contributed to the collection of tumor samples. Of the 625 blocks, 430 were fixed in formalin and appropriate for FISH evaluation. The remaining 195 samples were fixed in bouin, making the evaluation of HER-2 by FISH not feasible. Of the 430 patients with available formalin-fixed samples, representing 55% of the clinical trial population, 148 were treated with CMF, 137 were treated with HEC, and 145 were treated with EC.
To explore whether the subgroup of patients evaluated in the present study was representative of the original clinical trial population, main patient and tumor characteristics were compared between this subgroup (n = 430) and the subgroup without available samples (n = 347).
According to the results shown in Table 1
, it can be concluded that no detectable selection bias occurred, although an increased but nonstatistically significant prevalence of tumors with 1–3 positive nodes were found in the subgroup of patients with available tumor samples. When EFS was compared between the two groups of patients with and without available tumor samples, no statistically significant difference was found (P = 0.26, log-rank test), with 2- and 5-year EFS rates of 85% and 70%, respectively, for the cohort with available samples and 86% and 65%, respectively, for the other cohort.
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summarizes the results related to the HER-2 status of the 430 patients with available formalin-fixed samples.
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In the remaining 354 patients, no statistically significant difference was observed in the prevalence of HER-2-amplified tumors among the three study arms (P = 0.45, 2 test for homogeneity).
Interaction between HER-2 Amplification and Treatment Efficacy
CMF versus HEC.
Fig. 1
summarizes the results of the EFS comparison between CMF and HEC in the two cohorts of HER-2-amplified and -nonamplified patients. It can be observed that although the number of evaluated patients is modest, the difference in point estimates of the HRs suggests that in HER-2-amplified patients, HEC is more effective than CMF (HR = 1.42), whereas in the HER-2-nonamplified cohort, the superiority of the anthracycline-based regimen seems to be lost (HR = 0.84). However, statistical significance is not reached in either of the two subgroups, and the CIs of the two HRs overlap.
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reports the results of the EFS comparison between CMF and EC in the two distinct subgroups of HER-2-amplified and -nonamplified patients.
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EC versus HEC.
The results of the EFS comparison between EC and HEC are reported in Fig. 3
. The analysis has been performed in the two distinct cohorts of HER-2-amplified and -nonamplified patients. Point estimates of the HRs are more homogeneous than those for the other two comparisons. In HER-2-amplified tumors, the two anthracycline-based regimens seem to be almost equally effective (HR = 0.93), whereas in HER-2-nonamplified tumors, HEC is associated with a better observed efficacy than EC (HR = 1.33), as reported in the prospective clinical trial. It must be emphasized that a limited number of patients have been evaluated, and no statistical significance has been reached in the two subgroups of patients analyzed.
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Amplifications and Treatment Efficacy in HER-2-amplified Tumors gene aberrations were evaluated only in HER-2-amplified tumors because in a published study, no aberrations were found in a series of >100 HER-2-nonamplified tumors (19)
.4
Of the 73 HER-2-amplified tumors, 61 were evaluable for topo II amplification by FISH. topo II amplification was found in 23 of the 61 tumors (38%; CMF = 8 cases, HEC = 5 cases, and EC = 10 cases). No topo II amplification was found in the remaining 38 HER-2-amplified tumors (CMF = 15 cases, HEC = 13 cases, and EC = 10 cases). In the 38 tumors without topo II amplification, topo II deletion was found in 8 cases (CMF = 4 cases, HEC = 3 cases, and EC = 1 case).
Assuming that no topo II amplifications existed in HER-2-nonamplified tumors, the prevalence of topo II amplification in a population of 354 node-positive breast cancer patients is estimated to be 6%.
Figs. 4
5
6
report the EFS curves of HER-2-amplified patients for the following comparisons: (a) CMF versus HEC; (b) CMF versus EC; and (c) EC versus HEC, respectively. EFS is shown by treatment arm and by topo II status (amplified versus nonamplified).
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-amplified tumors (Fig. 4)
. Of the five patients with HER-2-amplified, topo II-amplified tumors receiving HEC, four patients remain disease free, whereas one has relapsed after 60 months from the date of randomization (data not shown in the curves of Fig. 4
, which describe the first 4 years of follow-up). In Fig. 5
, it can be observed that EC seems to be better than CMF only in the subgroup of HER-2-amplified, topo II-amplified tumors, whereas CMF seems to be better than EC in the cohort of HER-2-amplified, topo II-nonamplified patients. Fig. 7
reports the EFS curves of HER-2-amplified patients for the comparison of CMF versus anthracycline-based therapy. In this analysis, EFS for patients receiving anthracyclines has been calculated independently of the epirubicin dose delivered. This analysis allows us to increase the number of patients evaluated in the anthracycline-based arm and supports the concept that topo II, rather than HER-2, could be the true candidate to predict the efficacy of anthracyclines in early breast cancer patients receiving adjuvant therapy.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IHC has been the most widely used technique in these retrospective studies to evaluate HER-2 on archival samples of the primary tumor (7, 8, 9, 10, 11, 12, 13) .
Because of some known problems existing with the evaluation of HER-2 by IHC (14 , 24, 25, 26) , we decided to investigate the predictive value of HER-2 by FISH, which has been demonstrated to be a reliable technique for HER-2 evaluation (15 , 16 , 27) . Moreover, it has been reported recently that in an unselected population of 900 stage I–III breast cancer patients, IHC does not consistently discriminate cases likely to have a poor prognosis, whereas FISH provides superior prognostic information in distinguishing high-risk from low-risk patients (15) .
This study, to the best of our knowledge, is the first to investigate the predictive value of HER-2 by FISH in a population of early breast cancer patients randomly treated with either an anthracycline-based therapy or CMF. Its results seem to support the concept of an interaction between HER-2 status and anthracycline efficacy.
In particular, in the cohort of HER-2-amplified patients, results suggest that the anthracycline-based adjuvant therapy is more effective than CMF, whereas the latter seems to be at least as effective as anthracyclines in HER-2-nonamplified patients. It is worth noting that even when a suboptimally dosed anthracycline-based regimen such as EC [epirubicin (60 mg/m2) q 3 weeks] was compared with CMF in the subgroup of HER-2-amplified tumors, the anthracycline-based regimen appeared to be the most effective, although in the whole study population entered into the clinical trial, there was a trend favoring CMF over EC.
Nevertheless, because of the limited number of patients analyzed in the present study, no firm conclusions can be drawn regarding the predictive value of HER-2 in the adjuvant therapy of breast cancer.
An additional aim of this study was to explore the potential value of topo II gene amplification as a marker of hypersensitivity to anthracyclines. There are several in vitro experiments as well as some recently reported clinical data supporting the investigation of topo II in this specific context (11
, 19, 20, 21
, 28
, 29)
.
topo II is the target enzyme for topo II inhibitors such as anthracyclines. Some years ago, in vitro data demonstrated a direct correlation between the intranuclear topo II levels and the degree of sensitivity to anthracyclines (28)
. Moreover, it has been reported that topo II gene aberrations (i.e., amplification, deletion, or both) can be found in 50–80% of HER-2-amplified breast cancer samples, whereas no topo II gene aberrations are found in HER-2-nonamplified tumors (19)
. Interestingly, HER-2-amplified, topo II-amplified cells were shown to be highly sensitive to anthracyclines, whereas HER-2-amplified, topo II-deleted cells displayed a poor degree of sensitivity to anthracyclines (21)
.
In the clinical setting, only a few experiences have been reported with regard to the predictive value of topo II in breast cancer patients (11
, 29)
. In our previous study, in which topo II was evaluated by IHC in the same series of patients as the one reported in the present study, results suggested that topo II-positive tumors (i.e., >10% of cells with positive staining) derived the highest benefit from the anthracycline-based adjuvant therapy (11)
. In a second study correlating the efficacy of anthracyclines in metastatic breast cancer with topo II status as evaluated by FISH, it was found that all cases experiencing a clinical or radiological complete response (seven patients) had topo II gene amplification and that tumors with topo II gene deletion were the least responsive to a first-line therapy with anthracyclines (17% response rate; Ref. 29
).
The results of the present study support the hypothesis that topo II could be the most attractive candidate for predicting the efficacy of an anthracycline-based regimen and that the predictive value of HER-2 could be most likely be related to the concomitant amplification of these two genes located on the same arm of chromosome 17. Nevertheless, our present results must be seen as hypothesis-generating results because of the fairly limited number of patients in which both HER-2 and topo II gene amplifications could be evaluated.
Previous experiences and the results of this study support further investigation of topo II as a predictive marker for the activity of anthracyclines in breast cancer.
A serious problem in running future research programs evaluating the predictive value of topo II is the low prevalence of topo II gene amplification or deletion in a population of breast cancer patients (<10% and <5% of patients, respectively). The low prevalence of topo II amplification can explain the modest advantage of anthracyclines over CMF in an unselected population of breast cancer patients. On the other hand, it also makes the interpretation of results coming from individual studies difficult because of the extremely modest number of patients in whom a topo II gene aberration is found.
Accordingly, only a meta-analysis of individual studies can address the potential predictive value of this marker in breast cancer patients. Such a meta-analysis is now planned and will hopefully collect data on >3000 early breast cancer patients randomly treated with either CMF or an anthracycline-based therapy. In this project, a meta-analysis of clinical data will be performed, whereas HER-2 and topo II evaluation will most likely be centralized. Because of the cost and time needed to perform FISH evaluation on nearly 3000 cases, it seems logical to screen all cases for HER-2 by IHC and to perform HER-2 evaluation by FISH only in cases with immunostaining (1% of positive cells). Moreover, because of the data suggesting that no topo II amplifications are observed in HER-2-nonamplified tumors (19)
, topo II evaluation will be performed only in HER-2-amplified cases.
In conclusion, we have investigated the predictive value of HER-2, as evaluated by FISH, in a population of node-positive breast cancer patients randomly treated with either an anthracycline-based therapy or CMF. Our results, although not conclusive, support the concept that an interaction exists between anthracycline efficacy and HER-2 status. Moreover, the present study, in which topo II gene aberrations have also been evaluated, highlights that the interaction between HER-2 and anthracycline could most likely be explained by a concomitant topo II gene amplification in patients with HER-2-amplified tumors.
Because of the low prevalence of HER-2 and topo II genes aberrations, only a meta-analysis of individual studies comparing an anthracycline-based therapy with CMF in early breast cancer patients will fully address the potential predictive value of these two biological markers.
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FOOTNOTES |
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1 Supported by two research grants from Les Amis de l’Institut Bordet and Pharmacia-Upjohn Belgium. T. J. was supported by a grant from the Finnish Cancer Institute, Tampere University Hospital Research Fund, Pirkanmaa Cultural Foundation, and Finnish Medical Association. 
2 To whom requests for reprints should be addressed, at Jules Bordet Institute, Translational Research Unit, Department of Chemotherapy, Boulevard de Waterloo 125, 1000 Brussels, Belgium. Phone: 32-2-5413180; Fax: 32-2-5413090; E-mail: Angelo.Dileo{at}bordet.be
3 The abbreviations used are: CMF, cyclophosphamide, methotrexate, and 5-fluorouracil; OS, overall survival; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; topo II, topoisomerase II; EFS, event-free survival; HEC, full-dose epirubicin-cyclophosphamide; EC, moderate-dose epirubicin-cyclophosphamide; DAPI, 4',6-diamidino-2-phenylindole-dihydrochloride; HR, hazard ratio; CI, confidence interval.
4 J. Isola, personal communication.
Received 10/31/01; revised 1/31/02; accepted 2/ 6/02.
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E. de Azambuja, M. Paesmans, M. Beauduin, A. Vindevoghel, N. Cornez, C. Finet, F. Ries, M. T. Closon-Dejardin, J. Kerger, P. Gobert, et al. Long-Term Benefit of High-Dose Epirubicin in Adjuvant Chemotherapy for Node-Positive Breast Cancer: 15-Year Efficacy Results of the Belgian Multicentre Study J. Clin. Oncol., February 10, 2009; 27(5): 720 - 725. [Abstract] [Full Text] [PDF]
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J. M.S. Bartlett, A. Munro, D. A. Cameron, J. Thomas, R. Prescott, and C. J. Twelves Type 1 Receptor Tyrosine Kinase Profiles Identify Patients With Enhanced Benefit From Anthracyclines in the BR9601 Adjuvant Breast Cancer Chemotherapy Trial J. Clin. Oncol., November 1, 2008; 26(31): 5027 - 5035. [Abstract] [Full Text] [PDF]
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A. Di Leo and E. Moretti Anthracyclines: The First Generation of Cytotoxic Targeted Agents? A Possible Dream J. Clin. Oncol., November 1, 2008; 26(31): 5011 - 5013. [Full Text] [PDF]
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M. P. DiGiovanna, D. F. Stern, S. Edgerton, G. Broadwater, L. G. Dressler, D. R. Budman, I. C. Henderson, L. Norton, E. T. Liu, H. B. Muss, et al. Influence of Activation State of ErbB-2 (HER-2) on Response to Adjuvant Cyclophosphamide, Doxorubicin, and Fluorouracil for Stage II, Node-Positive Breast Cancer: Study 8541 From the Cancer and Leukemia Group B J. Clin. Oncol., May 10, 2008; 26(14): 2364 - 2372. [Abstract] [Full Text] [PDF]
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A. Gennari, M. P. Sormani, P. Pronzato, and P. Bruzzi Response:Re: HER2 Status and Efficacy of Adjuvant Anthracyclines in Early Breast Cancer: A Pooled Analysis of Randomized Trials J Natl Cancer Inst, May 7, 2008; 100(9): 680 - 681. [Full Text] [PDF]
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F. Cardoso, M. Saghatchian, A. Thompson, E. Rutgers, and for the TRANSBIG Consortium Steering Committee Inconsistent Criteria Used in American Society of Clinical Oncology 2007 Update of Recommendations for the Use of Tumor Markers in Breast Cancer J. Clin. Oncol., April 20, 2008; 26(12): 2058 - 2059. [Full Text] [PDF]
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L. N. Harris, D. F. Hayes, and R. C. Bast In Reply J. Clin. Oncol., April 20, 2008; 26(12): 2060 - 2061. [Full Text] [PDF]
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K. I. Pritchard, H. Messersmith, L. Elavathil, M. Trudeau, F. O'Malley, and B. Dhesy-Thind HER-2 and Topoisomerase II As Predictors of Response to Chemotherapy J. Clin. Oncol., February 10, 2008; 26(5): 736 - 744. [Abstract] [Full Text] [PDF]
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A. Gennari, M. P. Sormani, P. Pronzato, M. Puntoni, M. Colozza, U. Pfeffer, and P. Bruzzi HER2 Status and Efficacy of Adjuvant Anthracyclines in Early Breast Cancer: A Pooled Analysis of Randomized Trials J Natl Cancer Inst, January 2, 2008; 100(1): 14 - 20. [Abstract] [Full Text] [PDF]
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 A. Di Leo, S. Licitra, W. Claudino, and L. Biganzoli Molecular Predictors of Response to Anthracyclines ASCO Educational Book, January 1, 2008; 2008(1): 3 - 7. [Abstract] [Full Text] [PDF]
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L. Harris, H. Fritsche, R. Mennel, L. Norton, P. Ravdin, S. Taube, M. R. Somerfield, D. F. Hayes, and R. C. Bast Jr American Society of Clinical Oncology 2007 Update of Recommendations for the Use of Tumor Markers in Breast Cancer J. Clin. Oncol., November 20, 2007; 25(33): 5287 - 5312. [Abstract] [Full Text] [PDF]
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P. Lonning, S Knappskog, V Staalesen, R Chrisanthar, and J. Lillehaug Breast cancer prognostication and prediction in the postgenomic era Ann. Onc., August 1, 2007; 18(8): 1293 - 1306. [Abstract] [Full Text] [PDF]
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J. T. Jorgensen, K. V. Nielsen, and B. Ejlertsen Pharmacodiagnostics and Targeted Therapies--A Rational Approach for Individualizing Medical Anticancer Therapy in Breast Cancer Oncologist, April 1, 2007; 12(4): 397 - 405. [Abstract] [Full Text] [PDF]
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 A. J. Murphy, C. A. Hughes, C. Barrett, H. Magee, B. Loftus, J. J. O'Leary, and O. Sheils Low-Level TOP2A Amplification in Prostate Cancer Is Associated with HER2 Duplication, Androgen Resistance, and Decreased Survival Cancer Res., March 15, 2007; 67(6): 2893 - 2898. [Abstract] [Full Text] [PDF]
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B. Ejlertsen, H. T. Mouridsen, M.-B. Jensen, N.-O. Bengtsson, J. Bergh, S. Cold, P. Edlund, M. Ewertz, P. W. de Graaf, C. Kamby, et al. Similar Efficacy for Ovarian Ablation Compared With Cyclophosphamide, Methotrexate, and Fluorouracil: From a Randomized Comparison of Premenopausal Patients With Node-Positive, Hormone Receptor-Positive Breast Cancer J. Clin. Oncol., November 1, 2006; 24(31): 4956 - 4962. [Abstract] [Full Text] [PDF]
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 Y. Tang, H. Akbulut, J. Maynard, L. Petersen, X. Fang, W.-W. Zhang, X. Xia, J. Koziol, P.-J. Linton, and A. Deisseroth Vector Prime/Protein Boost Vaccine That Overcomes Defects Acquired during Aging and Cancer J. Immunol., October 15, 2006; 177(8): 5697 - 5707. [Abstract] [Full Text] [PDF]
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M. Tanner, J. Isola, T. Wiklund, B. Erikstein, P. Kellokumpu-Lehtinen, P. Malmstrom, N. Wilking, J. Nilsson, and J. Bergh Topoisomerase II{alpha} Gene Amplification Predicts Favorable Treatment Response to Tailored and Dose-Escalated Anthracycline-Based Adjuvant Chemotherapy in HER-2/neu-Amplified Breast Cancer: Scandinavian Breast Group Trial 9401 J. Clin. Oncol., June 1, 2006; 24(16): 2428 - 2436. [Abstract] [Full Text] [PDF]
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 K. I. Pritchard, L. E. Shepherd, F. P. O'Malley, I. L. Andrulis, D. Tu, V. H. Bramwell, M. N. Levine, and the National Cancer Institute of Canada Clinical T HER2 and Responsiveness of Breast Cancer to Adjuvant Chemotherapy N. Engl. J. Med., May 18, 2006; 354(20): 2103 - 2111. [Abstract] [Full Text] [PDF]
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M. J. Piccart-Gebhart Anthracyclines and the Tailoring of Treatment for Early Breast Cancer N. Engl. J. Med., May 18, 2006; 354(20): 2177 - 2179. [Full Text] [PDF]
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S. Rodenhuis, M. Bontenbal, Q. G. C. M. van Hoesel, W. M. Smit, M. A. Nooij, E. E. Voest, E. van der Wall, P. Hupperets, H. van Tinteren, J. L. Peterse, et al. Efficacy of high-dose alkylating chemotherapy in HER2/neu-negative breast cancer Ann. Onc., April 1, 2006; 17(4): 588 - 596. [Abstract] [Full Text] [PDF]
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M. Colozza, E. Azambuja, F. Cardoso, C. Sotiriou, D. Larsimont, and M. J. Piccart Proliferative markers as prognostic and predictive tools in early breast cancer: where are we now? Ann. Onc., November 1, 2005; 16(11): 1723 - 1739. [Abstract] [Full Text] [PDF]
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A. S. Knoop, H. Knudsen, E. Balslev, B. B. Rasmussen, J. Overgaard, K. V. Nielsen, A. Schonau, K. Gunnarsdottir, K. E. Olsen, H. Mouridsen, et al. Retrospective Analysis of Topoisomerase IIa Amplifications and Deletions As Predictive Markers in Primary Breast Cancer Patients Randomly Assigned to Cyclophosphamide, Methotrexate, and Fluorouracil or Cyclophosphamide, Epirubicin, and Fluorouracil: Danish Breast Cancer Cooperative Group J. Clin. Oncol., October 20, 2005; 23(30): 7483 - 7490. [Abstract] [Full Text] [PDF]
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H. M. Khandwala, D. Mirchandani, R. Chibbar, and V. Chow Uncommon Presentations of Cancer Patients: CASE 1. Metastatic Breast Cancer Presenting With Panhypopituitarism J. Clin. Oncol., December 1, 2004; 22(23): 4851 - 4853. [Full Text] [PDF]
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 V. Durbecq, M. Paesmans, F. Cardoso, C. Desmedt, A. Di Leo, S. Chan, K. Friedrichs, T. Pinter, S. Van Belle, E. Murray, et al. Topoisomerase-II{alpha} expression as a predictive marker in a population of advanced breast cancer patients randomly treated either with single-agent doxorubicin or single-agent docetaxel Mol. Cancer Ther., October 1, 2004; 3(10): 1207 - 1214. [Abstract] [Full Text] [PDF]
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 J. S. Ross, J. A. Fletcher, K. J. Bloom, G. P. Linette, J. Stec, W. F. Symmans, L. Pusztai, and G. N. Hortobagyi Targeted Therapy in Breast Cancer: The HER-2/neu Gene and Protein Mol. Cell. Proteomics, April 1, 2004; 3(4): 379 - 398. [Abstract] [Full Text] [PDF]
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S. Menard, E. Biganzoli, P. Valagussa, P. Boracchi, and A. Moliterni In Reply: J. Clin. Oncol., August 15, 2003; 21(16): 3180 - 3180. [Full Text] [PDF]
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J. S. Ross, J. A. Fletcher, G. P. Linette, J. Stec, E. Clark, M. Ayers, W. F. Symmans, L. Pusztai, and K. J. Bloom The HER-2/neu Gene and Protein in Breast Cancer 2003: Biomarker and Target of Therapy Oncologist, August 1, 2003; 8(4): 307 - 325. [Abstract] [Full Text] [PDF]
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 D. E. Hansel, A. Rahman, M. Hidalgo, P. J. Thuluvath, K. D. Lillemoe, R. Shulick, J.-L. Ku, J.-G. Park, K. Miyazaki, R. Ashfaq, et al. Identification of Novel Cellular Targets in Biliary Tract Cancers Using Global Gene Expression Technology Am. J. Pathol., July 1, 2003; 163(1): 217 - 229. [Abstract] [Full Text] [PDF]
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) and HEC (
) in HER-2-amplified and -nonamplified patients. 95% CIs are reported in parentheses.
) in HER-2-amplified and -nonamplified patients. 95% CIs are reported in parentheses.
) therapy in HER-2-amplified, topo II