Mol Cancer Ther. 2004;3:1207-1214
© 2004 American Association for Cancer Research
Topoisomerase-II
expression as a predictive marker in a population of advanced breast cancer patients randomly treated either with single-agent doxorubicin or single-agent docetaxel
Virginie Durbecq1,
Marianne Paesmans1,
Fatima Cardoso1,
Christine Desmedt1,
Angelo Di Leo1,
Stephen Chan2,
Kay Friedrichs3,
Tamas Pinter4,
Simon Van Belle5,
Elizabeth Murray6,
István Bodrogi7,
Euan Walpole8,
Bernard Lesperance9,
Stefan Korec10,
John Crown11,
Peter Simmonds12,
Thimothy J. Perren13,
Jean-Yves Leroy1,
Ghizlane Rouas1,
Christos Sotiriou1,
Martine Piccart1 and
Denis Larsimont1
1 Jules Bordet Institute, Brussels, Belgium; 2 Nottingham City Hospital, Nottingham, United Kingdom; 3 Universitäts-Frauenklinik und Poliklinic, Hamburg, Germany; 4 Petz Aladar Country Hospital, Gyor, Hungary; 5 UZ Gent, Gent, Belgium; 6 Groote Schuur Hospital, Cape Town, South Africa; 7 National Institute of Oncology, Budapest, Hungary; 8 Princess Alexandra Hospital, Brisbane, Queensland, Australia; 9 Hôpital du Sacré-Coeur de Montréal, Montréal, Quebec, Canada; 10 National Cancer Institute, Bratislava, Slovakia; 11 St. Vincent's Hospital, Dublin, Ireland; 12 Southampton General Hospital, Southampton, United Kingdom; and 13 St. James University Hospital, Leeds, United Kingdom
Requests for reprints: Denis Larsimont, Department of Pathology, Jules Bordet Institute, 125 Boulevard de Waterloo, 1000 Brussels, Belgium. Phone: 32-2-5413115; Fax: 32-2-5413281/32-2-5413090. E-mail: denis.larsimont{at}bordet.be
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Abstract
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Purpose: The predictive value of topoisomerase-II
(topo-II) has been evaluated in advanced breast cancer patients randomly treated with single-agent doxorubicin or docetaxel. Experimental design: Primary tumor samples from patients enrolled in a randomized, phase III clinical trial comparing single-agent doxorubicin (75 mg/m2 q3wks) with docetaxel (100 mg/m2 q3wks) were collected and topo-II status was evaluated by immunohistochemistry (clone KiS1). Results: Topo-II status was evaluated in 108 samples, 55 (51%) in the doxorubicin arm and 53 (49%) in the docetaxel arm. An increment of 10% in cells expressing topo-II is associated with a statistically significant odds ratio (OR; 95% confidence interval) of 1.09 (1.03–1.15; P = 0.002) for overall response to doxorubicin versus 1.002 (0.94–1.07; P = 0.95) in the docetaxel arm. With increasing topo-II, the favorable OR for overall response to docetaxel compared with doxorubicin decreases to become not significant in patients with topo-II tumor content >10%. In a multivariate analysis, (a) HER-2 status seems positively correlated with overall response to chemotherapy (OR, 2.34; 95% confidence interval, 0.87–6.27; P = 0.09). (b) Overall response to doxorubicin is significantly lower than overall response to docetaxel (OR, 0.17; 95% confidence interval, 0.04–0.64; P = 0.009) but with a significant interaction term for doxorubicin-treated patients with topo-II tumor content >10% (OR, 8.31; 95% confidence interval, 1.86–37.03; P = 0.05). Conclusions: (a) Topo-II overexpression confers a higher probability of response in the doxorubicin arm only. (b) Despite being a small retrospective study, this study is in line with previously reported studies and the hypotheses raised are now being tested in a prospective neoadjuvant trial.
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Introduction
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Anthracyclines are some of the most powerful agents available for the treatment of breast cancer. Retrospective studies have suggested that the benefits of anthracycline-based adjuvant therapy in breast cancer could be restricted to or at least more evident in the Her-2-positive tumors (1–5). Anthracyclines were found to be more effective than cyclophosphamide, methotrexate, and 5-fluorouracil–like regimens in terms of disease-free survival only in Her-2-positive subset of breast cancer patients. Further marker analysis revealed that the superiority of the drug is associated with the coamplification or overexpression of the topoisomerase-II (topo-II), the target of anthracyclines (6, 7). In vitro studies have established that sensitivity to anthracyclines is dependent on the expression level of topo-II protein in target cancer cells (8, 9). In the same way, preoperative doxorubicin chemotherapy was found to be highly effective in breast cancers that have coamplification of Her-2 and topo-II genes (10). Due to the close location of topo-II and Her-2 genes on chromosome 17, topo-II gene aberrations (either amplification or deletion) are mainly associated with Her-2 gene amplification (9, 11). These facts have led to the hypothesis that Her-2 amplification is only a surrogate marker and the topo-II amplification/overexpression could be the real predictive marker of response to anthracycline-based chemotherapy.
A retrospective study conducted in the population of the TAX 303 clinical trial—single-agent doxorubicin versus docetaxel administrated as first-line or second-line treatment for advanced breast cancer—suggested that docetaxel might be more effective than doxorubicin in Her-2 amplified tumors, whereas the two treatments seem to have a similar efficacy in patients in which Her-2 is not amplified (12). The use of Her-2 as a predictive marker of response to anthracyclines is therefore debatable, because Her-2 amplification alone has not been able to select the subset of patients that respond better to anthracyclines than to taxanes. Moreover, Her-2 gene amplification did not influence the response to anthracycline-based chemotherapy in a study of locally advanced/metastatic breast cancer (13).
Because topo-II protein is the target of anthracyclines and Her-2 status alone seems to be unable to identify patients that derive the higher benefit from anthracycline-based chemotherapy, we evaluated the potential predictive value of topo-II protein status regarding response to anthracycline using the population of the TAX 303 clinical trial.
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Patients and Methods
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Clinical Trial
The patients and methods of the TAX 303 clinical trial have already been published (14) and summarized in our previous retrospective study done in this population (12). In summary, this randomized, prospective, phase III trial compared single-agent doxorubicin (75 mg/m2 q3wks) with single-agent docetaxel (100 mg/m2 q3wks), both drugs administered at full doses, as first-line or second-line treatment of advanced breast cancer patients after previous treatment with alkylating agents. Of the 326 patients entered in this clinical study, 161 were randomized to receive docetaxel and 165 doxorubicin. The main patient characteristics were well balanced between the two study arms.
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Translational Research Study
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Tumor Sample Collection.
A paraffin-embedded sample representative of the primary tumor of 183 patients were collected at the Jules Bordet Institute (Brussels, Belgium), which served as the coordinating center for the biological retrospective studies. Collected samples correspond to 56% of the clinical trial population, 89 (49%) in the docetaxel arm and 94 (51%) in the doxorubicin arm.
Topo-II Protein Evaluation by Immunohistochemistry.
Topo-II protein status was evaluated at the Jules Bordet Institute in all collected samples as described previously (7). Negative controls comprised serial sections incubated with buffer alone instead of primary antibody. As no cutoff for positivity has been validated yet to define topo-II overexpression, we primarily analyzed this variable as a continuous one. An additional analysis was also done based on an arbitrary cutoff of >10%, which corresponds to the median percentage of cells with positive staining in this population and has been used previously in other studies (7, 15). Staining intensity was assessed and ranged from 0 to 3 (with 0 for no staining and 3 for the highest intensity). When topo-II evaluation was done, the pathologist (D.L.) was blinded with regard to the treatment and clinical outcome of the patient.
Statistical Analysis.
The primary binary outcome for the present study was objective response, which was defined, as reported previously (12), as complete or partial response. Secondary outcomes were time to progression, calculated from the date of randomization to the date of progression or death for any cause (death being considered as an event, censored observations correspond to patients alive and without evidence of progression at the time of analysis) or to the date of last follow-up, and overall survival, calculated from the date of randomization to the date of death for any reason or to the date of last follow-up (censored event).
The patients evaluated in the present study were a subgroup of the study population entered into the prospective clinical trial. To evaluate the representativeness of this subgroup, the main patient characteristics as well as study treatment efficacy were compared between the cohort of patients with available tumor samples who participated in the present study and the cohort of patients who were not eligible due to unavailable tumor samples, insufficient tumor material, or technical problems. To this end, the nonparametric Kruskal-Wallis test was used for age (continuous variable) and the 
2 test for homogeneity was used for all the other variables, which were reported as binary variables.
Association between topo-II staining, assessed as a percentage, and clinical variables was assessed using the nonparametric Mann-Whitney test, because the distribution of topo-II was found to be significantly different from a normal distribution.
The predictive value of topo-II on response was assessed using logistic regression models with different modelizations of topo-II as covariate. Two representations were used: as the measured percentage of cells showing expression and as a binary variable using the median value for dichotomization. To assess interaction between treatment and topo-II, interaction terms were added into the models with the categorical assessments of topo-II.
The subpopulation treatment effect pattern plot analysis (16) was done to evaluate the impact of increasing topo-II expression level on treatment effect estimate; subpopulation treatment effect pattern plot depicted odds ratios (OR) for women treated with anthracyclines versus taxanes according to subgroups of patients constructed to have increasing percentages of topo-II-positive cells. Each subpopulation contained 43 or 44 patients, and subsequent subpopulation was formed by, moving from left to right, dropping 22 patients with the lowest values for topo-II and adding 22 patients (21 for the last step) with the next higher values of topo-II.
The distributions of time to progression and overall survival were estimated by the Kaplan-Meier method or by using Cox regression models. Hazard ratios and 95% confidence intervals (95% CI) were calculated in the two cohorts of topo-II-positive and topo-II-negative patients. An interaction term combining treatment arm and topo-II status was added to the Cox proportional hazards models.
All reported Pvalues are two tailed.
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Results
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Clinical Trial
The results of the TAX303 trial have already been published extensively (12, 14). A total of 326 patients were recruited to the study, 161 (49%) in the docetaxel arm and 165 (51%) in the doxorubicin arm. There were no statistically significant differences in the pretreatment characteristics of the patients randomized to each group. All patients had metastatic disease, and the most important negative prognostic factors (age <50 years, visceral and liver involvement, involvement of three or more organs, previous adjuvant chemotherapy, and resistance to previous chemotherapy) were well balanced between the two groups. The results showed that the overall response rate was significantly higher for the docetaxel arm than doxorubicin, but no statistically significant difference was found between the two treatment arms regarding time to progression and overall survival.
Translational Research Study
Representativeness of the Biological Study Population.
Paraffin-embedded samples from 183 primary tumors (56%) were collected and assessed for topo-II protein evaluation by immunohistochemistry (IHC). In 75 tumors, topo-II status could not be evaluated by IHC due to insufficient tumor material (45 cases, 25%) or technical reasons (30 cases, 16%; inappropriate tissue fixation). The present biological study population is therefore composed of the remaining 108 (59%) samples for which an IHC result was obtained, 55 (51%) in the doxorubicin arm and 53 (49%) in the docetaxel arm. Response rate, defined as the percentage of patients that achieved a complete or partial response, was 24% (13 of 55) for doxorubicin and 42% (22 of 53) for docetaxel.
Table 1 compares the main patients' characteristics and study treatment efficacy between the subgroup of patients who participated to the present biological study and the subgroup who did not (tumor samples unavailable, insufficient tumor material, or technical problems). No statistically significant differences were found between these two subgroups concerning the patients' characteristics. This shows that our population is representative of the overall clinical study population in terms of baseline characteristics.
However, a close to statistically significant difference of overall response was observed for the patients treated with doxorubicin, with response to doxorubicin being lower in the biological study population compared with the remaining clinical population (P = 0.06). Multivariate analyses were done to explain the influence of sample availability on overall response, thanks to the impact of possible confounding factors that include age, performance index, number of metastatic sites, presence of visceral involvement, and treatment arm. However, even after adjustment for age and treatment arm (older age and doxorubicin treatment were found to have a negative impact on overall response), inclusion in the present study was still found as a predictive factor negatively associated with overall response.
Topo-II Evaluation by IHC
No statistical difference was observed between the two treatments arms regarding the percentage of topo-II-stained cells (P = 0.11). No association was found between the percentage of cells expressing topo-II and the clinicopathologic variables (P's = 0.51 for age, 0.15 for Karnofsky (
80), 0.15 for viscera involvement, 0.21 for estrogen receptor, and 0.95 for HER-2 [IHC confirmed by fluorescence in situ hybridization (FISH)]), with the exception of metastatic sites; patients with less than three metastatic sites presented a higher percentage of cells with topo-II expression in their primary tumors (P = 0.01). The intensity of staining has been evaluated, and a median intensity of 3 was observed for responders in the doxorubicin arm, whereas the remaining patients presented a median intensity of 2.
- (a) Topo-II Status Analysis as a Continuous Variable
As no universal cutoff has been defined for topo-II positivity, a bubble plot was used to analyze the repartition of tumors in terms of response to treatment and content of positively stained cells (Fig. 1). In each treatment arm, "responders" are defined as patients where a complete or partial response was obtained and "nonresponders" as patients where a stable or progressive disease was seen. The repartition of tumors regarding the content of positively stained cells is similar between the two subgroups (responders and nonresponders) of the docetaxel arm and the nonresponders of the doxorubicin arm, the great majority of the tumors (73%, 64%, and 61%, respectively) presenting 
10% of positively stained cells. On the contrary, the responders subgroup of the doxorubicin arm presents a higher content of positively stained cells with 77% (10 of 13 patients) of the tumors presenting >10% of positively stained cells.
Median percentage of topo-II-stained cells according to response to each treatment revealed that the impact of topo-II is dependent on treatment arm, as the responders subgroup in the doxorubicin arm presents a median percentage of topo-II higher than all the other groups (Table 2).Considering the percentage of topo-II-positive cells, an increment of 10% in cells expressing topo-II is associated with a statistically significant OR (95% CI) of 1.09 (1.03–1.15; P = 0.002) for response to doxorubicin, whereas the corresponding OR (95% CI) in the docetaxel arm is 1.002 (0.94–1.07; P = 0.95), leading to the conclusion that topo-II expression is associated with higher chances of response in the doxorubicin arm but not in the docetaxel arm.
A subpopulation treatment effect pattern plot analysis was done to evaluate the impact of increasing topo-II expression level on treatment effect estimate. As depicted in Fig. 2, overall response to doxorubicin increases with increasing percentage of topo-II-positive cells. It is interesting to notice that overall response to doxorubicin is lower than overall response to docetaxel for the two first steps (population with a topo-II level content 10%), whereas overall response to doxorubicin becomes similar to overall response to docetaxel for the two last steps (population with a topo-II level content >10%). Nevertheless, no statistically significant interaction was found between topo-II percentage of stained cells and treatment when time to progression and overall survival were evaluated. Median times to progression [hazard ratio (95% CI)] were 1.08 (0.86–1.35; P = 0.52) in the doxorubicin arm and 1.02 (0.69–1.51; P = 0.91) in the docetaxel arm. Estimated median survival times [hazard ratio (95% CI)] were 0.89 (0.67–1.16; P = 0.39) in the doxorubicin arm and 0.85 (0.56–1.29; P = 0.44) in the docetaxel arm.
- (b) Topo-II Overexpression Defined as >10% of Positively Stained Cells
To further explore the correlation between topo-II overexpression and response to doxorubicin treatment, we defined a cutoff for topo-II positivity as >10% of stained cells, which corresponds to the median of positively stained cells for all samples (see Patients and Methods). Moreover, previous results showed that tumors with <10% of topo-II-positive cells were not responsive to doxorubicin and that the impact of topo-II content on the overall response to doxorubicin is only detectable for a percentage of >10%.With this binary representation, topo-II positivity was observed in 43 of 108 (40%) evaluable patients with no significant imbalance between treatment arms: 17 (16%) in the docetaxel arm and 26 (24%) in the doxorubicin arm. Among the 13 responders in the anthracycline arm, 10 (76.9%) had a tumor defined as topo-II positive. Fifteen of 39 (38.5%) patients with a stable or progressive disease to the same type of chemotherapy also had a topo-II-positive tumor. In the taxane arm, topo-II positivity was seen in 6 of 22 (27.3%) patients presenting a complete or partial response and in 10 of 28 (35.7%) patients with a progressive disease.
In the doxorubicin arm, topo-II status was found to have a predictive value on response with a significant OR (95% CI) of 2.40 (1.37–4.20; P = 0.002) for patients with topo-II-positive tumor, whereas the corresponding OR (95% CI) was 1.02 (0.54–1.91; P = 0.95) in the docetaxel arm. An interaction term between topo-II and treatment was found significant (P = 0.05).
As mentioned previously, no statistically significant interaction was found between topo-II status and treatment effect when time to progression and overall survival were evaluated. Median times to progression were 5.2 and 4.8 months for doxorubicin and docetaxel, respectively, in the group of topo-II-positive tumors (hazard ratio for doxorubicin compared with docetaxel, 0.97; 95% CI, 0.51–1.87; P = 0.94). In the group of topo-II-negative tumors, times to progression were 7.7 and 5.2 months for doxorubicin and docetaxel, respectively (hazard ratio, 0.63; 95% CI, 0.36–1.11; P = 0.78). Estimated median survival times were 14.5 and 18.5 months for doxorubicin and docetaxel, respectively, in the group of topo-II-positive tumors (hazard ratio, 1.19; 95% CI, 0.59–2.41; P = 0.62) and 11.6 and 12.4 months for doxorubicin and docetaxel, respectively, in the group of topo-II-negative tumors (hazard ratio, 0.92; 95% CI, 0.51–1.66; P = 0.78).
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Figure 1. Repartition of tumors in terms of response to treatment and content of positively stained cells. Responders are defined as patients where a complete response (CR) or partial response (PR) was obtained and nonresponders as patients where a stable or progressive disease (PD) was observed. A, docetaxel; B, doxorubicin.
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Figure 2. A subpopulation treatment effect pattern plot showing OR according to topo-II-positive cells values for women treated with a single-agent docetaxel or doxorubicin. For this sliding-window subpopulation treatment effect pattern plot analysis, each subpopulation contained 43 or 44 patients and each subsequent subpopulation was formed by, moving from left to right, dropping 22 patients with the lowest values for topo-II and adding 22 patients (21 for the last step) with the next higher values of topo-II. Subpopulations: S1 (43 patients): OR, 0.16; 95% CI, 0.03–0.89; topo-II % = 5.0; S2 (44 patients): OR, 0.06; 95% CI, 0.01–0.49; topo-II % = 10.0; S3 (44 patients): OR, 0.30; 95% CI, 0.07–1.31; topo-II % = 12.5; S4 (43 patients): OR, 1.08; 95% CI, 0.30–3.82; topo-II % = 20.0. Patients with topo-II-positive cells > 10% are included only in steps 3 and 4.
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Combination of Topo-II and HER-2 Status
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HER-2 status was assessed previously (12) by IHC and confirmed by FISH; we observed that docetaxel is probably more effective than doxorubicin in HER-2-positive breast cancer patients (overall response to docetaxel = 66.7% versus overall response to doxorubicin = 16.7%). Topo-II overexpression (defined as >10% of positively stained cells) was observed in 37% and 38% of the HER-2-positive and HER-2-negative tumors, respectively.
A combination of the topo-II and HER-2 status allowed the definition of four subgroups for each treatment arm (see Table 3). In the doxorubicin arm, we saw a correlation between response rate and the biological characteristics of the tumors: good responders to doxorubicin present a topo-II-positive tumor and the response rate to doxorubicin is higher in HER-2-negative tumors; in the HER-2-negative/topo-II-positive tumors, where the median topo-II expression is 40%, the response rate to doxorubicin is 50%.
On the contrary, good responders to docetaxel present opposite biological characteristics with generally a HER-2-positive and/or topo-II-negative tumor. For the HER-2-positive/topo-II-negative tumors, the overall response to docetaxel was significantly higher than the overall response to doxorubicin (P = 0.02).
When considering the impact on response of HER-2 combined with topo-II in a model testing HER-2, topo-II, and treatment arm, (a) HER-2 status seems positively correlated with overall response to chemotherapy (OR, 2.34; 95% CI, 0.87–6.27; P = 0.09). (b) Overall response of the study population to doxorubicin is significantly lower than overall response to docetaxel (OR, 0.17; 95% CI, 0.04–0.64; P = 0.009). (c) Response to doxorubicin increased significantly in topo-II-positive tumors after adjustment for HER-2 status (OR, 8.31; 95% CI, 1.86–37.03; P = 0.05).
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Discussion
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The present retrospective study reports data regarding the topo-II status evaluated in tumor samples from patients previously enrolled in a randomized, phase III clinical trial, which compared single-agent doxorubicin with single-agent docetaxel as first-line and second-line chemotherapy for advanced breast cancer. The topo-II protein status was evaluated by IHC in 33% (108 of 326 patients) of the clinical trial population and revealed that topo-II protein overexpression confers a higher probability of response in the doxorubicin arm but not in the docetaxel arm.
No statistically significant difference was observed for the main patients' characteristics between those who participated in the present study and those who did not, which indicates that the group of patients evaluated is representative of the entire clinical trial population. Nevertheless, we observed a close to statistically significant reduced overall response to doxorubicin in the biological study population compared with the remaining clinical population; therefore, the observed results regarding doxorubicin treatment efficacy could be somewhat underestimated.
The present study reports data regarding the topo-II status on the protein level. Topo-II amplification, which is present in 
5% of the total population (one third of Her-2 amplified tumors), is not presented in this study, because the number of topo-II amplified samples (n = 12) is too small to be statistically relevant or even hypothesis generating. Because the target for anthracycline is the topo-II protein and not the gene, regarding prediction of response to anthracycline, biologically, it makes sense to analyze the protein status. Moreover, FISH may miss some tumors with biologically significant topo-II overexpression but without topo-II gene amplification (no topo-II gene amplification or CEP17 polysomy). Indeed, previous studies revealed that, contrary to Her-2, where gene amplification is almost always correlated with protein overexpression, topo-II gene amplification apparently does not always lead to protein overexpression (17, 18). Moreover, a detectable difference in the distribution of topo-II protein level according to topo-II gene amplification was observed only in proliferating tumors (defined as Ki-67-positive cases; ref. 17).
An interaction test has been carried out previously between some clinical variables (such as the performance status, the number of metastatic sites, and the presence or not of visceral involvement) and the treatment efficacy and suggests that docetaxel might be more efficient in patients with visceral involvement (51% of overall response for docetaxel versus 29% for doxorubicin), whereas doxorubicin seems to be more efficient in patients without visceral involvement (47% of overall response for doxorubicin versus 30% for docetaxel; ref. 12). In the present study, no association was observed between the topo-II expression level and the clinicopathologic variables, except regarding metastatic sites: patients with less than three metastatic sites presented the higher percentage of topo-II expression. In the literature, a high discrepancy was observed regarding the association of topo-II overexpression and clinicopathologic variables (19–24). No association between HER-2 status (evaluated by IHC and confirmed by FISH) and topo-II status (evaluated by IHC) was observed, which is in accordance with the fact that topo-II gene status, evaluated by FISH and present in Her-2 amplified cases, is weakly correlated with topo-II protein status (17). Moreover, Her-2 amplification can determine not only amplification but also deletion of topo-II gene; Her-2 amplification could then be related with a reduced copy number of the topo-II gene and in turn a reduced protein level.
Statistical analysis of the correlation between the percentage of topo-II-positive cells and the response to treatment revealed that topo-II expression is associated with higher chances of response in the doxorubicin arm but not in the docetaxel arm. These results were in agreement with a recent report by MacGrogan et al. (24), which revealed that high expression of topo-II protein (>15%) was associated with tumor regression (50%), including complete tumor regression (P = 0.004) after six courses of neoadjuvant chemotherapy, including epirubicin. The topo-II staining intensity, also assessed in the present study, seems to be correlated with response rate in the patients treated with doxorubicin. A median intensity of 3 was observed for responders in the doxorubicin arm, whereas the remaining patients presented a median intensity of 2. The definition of a score that takes into account both the percentage of positively stained cells and the intensity of staining could then be useful to better select patients that respond to anthracycline-based treatment.
The combined analysis of topo-II and HER-2 status showed that HER-2-positive tumors are highly responsive to doxorubicin only when topo-II overexpression is present. This result is in accordance with the results of Campiglio et al. (25) who identified tumor proliferation rate as the major biological variable associated with response to anthracycline in breast cancer cells, because HER-2 positivity was associated with drug sensitivity only when it identified highly proliferating tumors. As topo-II protein is a good marker of cell proliferation (26), our observations reinforce the hypothesis that HER-2 positivity is associated with drug sensitivity only in highly proliferating tumors (i.e., when topo-II is overexpressed), independent of topo-II gene status. Indeed, the impact of cell proliferation overcame the topo-II gene amplification status, because a detectable difference in the distribution of topo-II protein level according to topo-II gene amplification was observed only in the proliferating subgroup of tumors (17). Tumors presenting the higher response rate to doxorubicin belong to the HER-2-negative/topo-II-positive subgroup, highlighting the fact that topo-II status determined by FISH could be too restrictive because it excludes patients with highly proliferating tumors but with no topo-II amplification.
As mentioned previously (12), docetaxel seems to be more effective than doxorubicin in HER-2-positive patients, especially when topo-II is absent. Additionally, even in the subset of patients highly responsive to doxorubicin (HER-2-negative/topo-II-positive subgroup), overall response to doxorubicin did not reach statistically significant difference from overall response to docetaxel. Nevertheless, it should be kept in mind that overall response to doxorubicin may be underestimated in the present study population.
The fact that HER-2-negative/topo-II-positive subgroup presents a higher response to doxorubicin than HER-2-positive/topo-II-positive subgroup could be explained by the presence of mutation(s) in the p53 gene, present in 70% of HER-2-positive tumors, which can induce anthracycline resistance without modification of taxane sensitivity (27–30). This result highlights the importance of the evaluation of p53 status in addition to topo-II for the prediction of response to chemotherapy; on the contrary, Her-2 status evaluation is secondary as Her-2 is only a surrogate of the two other markers. To confirm, in a clinical setting, the preclinical data suggesting that p53 gene mutations predict resistance to doxorubicin but not to docetaxel, p53 gene mutations are currently evaluated in exons 5 to 9 by denaturing high-performance liquid chromatography in 127 (64 doxorubicin, 63 docetaxel) archival primary tumor samples from patients enrolled in the TAX 303 trial.
The hypotheses generated in our retrospective studies are currently being evaluated in two important projects that will, hopefully, enable us to achieve a definite conclusion regarding the predictive value of topo-II gene/protein status.
One of the projects is a meta-analysis of four phase III trials comparing anthracycline-based regimens with cyclophosphamide, methotrexate, and 5-fluorouracil as adjuvant treatment of early breast cancer, with a total of 4,500 patients. A centralized analysis of Her-2 and topo-II gene status will be done by an independent laboratory and then correlated with response to treatment.
The second project is a prospective neoadjuvant study, also known as the Trial of Principle, involving 400 patients with hormone receptor–negative tumors >2 cm. All patients are treated with four cycles of epirubicin (120 mg/m2) as neoadjuvant treatment, followed by breast cancer surgery, and four cycles of Taxotere (100 mg/m2) as adjuvant chemotherapy. This ongoing trial is a multicentric European effort under the coordination of the Jules Bordet Institute.
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Summary
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The present retrospective study has investigated the predictive value of topo-II status in a population of advanced breast cancer patients randomly treated with single-agent doxorubicin or single-agent docetaxel. The interaction between topo-II status and treatment effect, evaluated in terms of response rate, reaches statistical significance.
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Acknowledgments
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We thank May Alakl (Aventis) for his contribution when the study was activated.
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Footnotes
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Grant support: Aventis Research and Development.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4/19/04;
revised 7/26/04;
accepted 8/17/04.
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Abstract
Introduction
