Cytotoxic and antiproliferative activity of the single agent epirubicin versus epirubicin plus tamoxifen as primary chemotherapy in human br
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《肿瘤与内分泌》
Breast Unit and Anatomia Patologica Azienda Ospedaliera Istituti Ospitalieri, Cremona, Dipartimento di Scienze Cliniche e Biologiche, Universitàdi Torino, Oncologia Medica, Azienda Ospedaliera San Luigi, Orbassano, Italy
Abstract
This study was designed to address whether simultaneous primary chemo-hormonal therapy provides additional activity compared with chemotherapy alone in breast cancer patients with operable or locally advanced disease. Between January 1997 and January 2002, 211 consecutive patients with T2–4, N0–1, M0 breast cancer were randomized to receive either epirubicin alone (EPI) or epirubicin plus tamoxifen (EPI-TAM). Ki67 expression was evaluated immunohistochemically in tumor specimens obtained before chemotherapy by incision biopsy and at definitive surgery. Tumor shrinkage of >50% was obtained in 76% of patients randomized in the EPI arm and 81.9% of patients randomized in the EPI-TAM arm (not significant). The corresponding rates of clinical and pathological complete response were 20.2 and 21.9% (not significant), and 4.8 and 6.7% (not significant), respectively. Pathologically complete response was more frequently observed in estrogen receptor (ER)-negative (ER–) tumors (P=0.04) and correlated with elevated baseline Ki67 expression (P<0.01). Both EPI and EPI-TAM treatments resulted in a significant reduction in Ki67 expression, either in overall patients (P=0.000) or in patients with ER+ breast cancer (P=0.000). The reduction in Ki67 immunostaining in the EPI-TAM arm was greater than in the EPI arm, leading to a lower Ki67 expression at post-operative residual histology (P=0.0041). The addition of tamoxifen to epirubicin chemotherapy did not improve the response rate but led to a significantly higher reduction in the Ki67 expression. Baseline elevated Ki67 expression and the ER– status were both associated with a greater chance of obtaining a pathological complete response at residual histology.
Introduction
Chemotherapy and endocrine therapy are known to be efficacious in the management of breast cancer. When given as adjuvant systemic therapy shortly after surgery for primary disease, both treatment strategies have been found to increase either the disease-free survival or the overall survival (Early Breast Cancer Trialists’ Collaborative Group 1998a, 1998b).
Among the chemotherapeutic agents available, anthracyclines are the most efficacious (Early Breast Cancer Trialists’ Collaborative Group 1998a), while tamoxifen is the most-used endocrine therapy and still remains the treatment of choice in the adjuvant setting (Early Breast Cancer Trialists’ Collaborative Group 1998b). There is no doubt that the combination of tamoxifen with chemotherapy yields greater benefits over the administration of either treatment alone (Early Breast Cancer Trialists’ Collaborative Group 1998b); however, the optimal association of these two treatments represents an intriguing issue (Goldirsch et al. 2002).
Since endocrine therapy and chemotherapy exhibit different mechanisms of action and different patterns of toxicity, the simultaneous administration of the two treatment modalities might be advantageous. There are however theoretical arguments suggesting that simultaneous chemo-hormonal therapy may be detrimental. Endocrine therapies are cytostatic and may decrease cell turnover, whereas chemotherapeutic agents require DNA synthesis to exert their full efficacy (Osborne et al. 1989). In addition, tamoxifen may alter membrane lipids and antagonizes both calmodulin and Ca2+ channels, thereby changing diffusion rates of certain drugs and potentially altering the drug uptake (Greensberg et al. 1987). On the other hand, in vitro studies have demonstrated that tamoxifen is able to overcome the multidrug resistance mediated by the p-170 glycoprotein, suggesting a rationale for combining this agent with anthracyclines (Berman et al. 1991).
In patients with metastatic disease a systematic review of published randomized trials of chemotherapy versus chemotherapy plus tamoxifen (involving a total of 1294 cases recruited in seven trials) did not show a significant advantage for the combination treatment in terms of survival, although the response rate was significantly superior in the chemo-hormonal arm (Fossati et al. 1998). Similar results have been obtained in an additional study recently published in which chemo-hormonal therapy improved the time to treatment failure but not overall survival (Sledge et al. 2000). The results of a 10-year update from the North American Intergroup Trial 0100 comparing tamoxifen alone with tamoxifen commenced at the same time as CAF (cyclophosphamide, doxorubicin, 5-fluorouracil) chemotherapy with tamoxifen commenced upon completion of the same regimen (Albain et al. 2002), showed that tamoxifen conferred a slight but significant disadvantage in disease-free survival and overall survival if administered during chemotherapy rather than sequentially. Similar results have been obtained in a randomized European study (Pico et al. 2004).
The administration of primary chemotherapy in breast cancer patients permits in vivo assessment of tumour chemosensitivity and evaluation of treatment-induced changes in tumour biology. This treatment modality provides an unique opportunity to derive biological information related to tumor response in order to understand mechanisms of action of systemic antineoplastic treatments as well as mechanisms of their eventual interaction (Cleator et al. 2002).
This article presents data of a randomized trial conducted in a single institution that compared the activity of the single agent epirubicin versus epirubicin plus tamoxifen as primary chemotherapy in human breast cancer. The secondary aim was the reduction in proliferation activity as assessed by expression of the Ki67 antigen.
Patients and methods
Patients
Breast cancer patients with operable or locally advanced disease (T2–4, N0–1, M0) were considered eligible. They were required to have an Eastern Cooperative Oncology Group performance status of <2, adequate bone marrow reserve (white blood cell count, >3.5 x 109/l; platelets, >100 x 109/l; hemoglobin, >10 g/dl), hepatic function (aspartate amino-transferase (AST) alanine aminotransferase (ALT), bilirubin and alkaline phosphatase levels <1.25 times the upper limit of the normal value) and renal function (serum creatinine <1.25 the upper limit of normal value). Patients with non-malignant systemic disease that precluded them from receiving study therapy (e.g. active infection, any clinically significant cardiac arrhythmia, congestive heart failure, complete bundle brand block, functional New York Heart Association class III or worse, or pregnancy) and patients with second primary malignancies (except in situ carcinoma of the cervix or adequately treated basal cell carcinoma of the skin) were not eligible. The study was approved by the local ethical committee. Written informed consent was obtained from all patients before randomization.
Treatment schedule
Patients were randomized to receive epirubicin alone (EPI arm), or epirubicin and tamoxifen (EPI-TAM arm). Patients in the EPI arm received epirubicin (Farmorubicina; Pharmacia, Milan, Italy) 60 mg/m2 by slow i.v. push on days 1 and 2; patients in the EPI-TAM arm received epirubicin 60 mg/m2 by slow i.v. push on days 1 and 2 and tamoxifen (Kessar; Pharmacia) 30 mg daily. Epirubicin infusions in both arms were repeated every 21 days for three or four cycles before definitive surgery, whereas tamoxifen was given continuously until the end of the cytotoxic treatment. From then, patients with estrogen receptor-positive (ER+) primary tumor in both arms received tamoxifen (20 mg) up to progression or for a maximum of 5 years.
Recommended treatment modifications for hematologic toxicities were as follows: 1-week delay was introduced if granulocyte counts were less than 1.5 x 109/l and or platelet counts were less than 100 x 109/l on day 21; after 1 week, if hematologic parameters did not recover, a dose reduction of 50% was applied when granulocyte counts ranged between 1.0 x 109 and 1.5 x 109/l and/or platelet counts ranged between 75 x 109 and 100 x 109/l. In case of lower hematological values the following week, a further 1-week delay was allowed. Patients went off-study if the delay exceeded 4 weeks. Delays and dose reductions were also performed for stomatitis of grade >3. Supportive care could include blood transfusion and the administration of antiemetics, analgesics and growth factors as appropriate.
Treatment evaluation
On first presentation an incision biopsy was performed on each patient and a small wedge of tissue (0.5–0.8 cm) was removed. Chemotherapy was started within 1 or 2 days of diagnosis. The primary objective of the study was to compare the response rate of patients randomized to receive epirubicin versus those randomized to received epirubicin plus tamoxifen. The secondary objective was to compare changes in Ki67 expression in tumor samples obtained before treatment and at definitive surgery.
The following tests were evaluated before entry into the study: medical history and physical examination, performance status, weight, hemoglobin and hematocrit levels, white blood cell and platelet counts, alkaline phosphatase, AST, ALT and bilirubin levels; serum electrolytes, serum creatinine and serum CA 15-3 levels; electrocardiogram, echocardiography; chest radiograph; mammography and breast echography, isotope bone scan with radiograph of abnormal areas; abdominal ultrasonography; and computed tomography scan of the chest and abdomen if necessary. Physical examination and hematological profile were repeated before each course of chemotherapy, whereas a complete blood cell count was recommended at the nadir of leukocytes and platelets (10–12 days from epirubicin administration).
Each month the size of the primary tumor and the size of the axillary lymph nodes, when appreciable, were measured using callipers by the same clinician. Response was assessed by the clinical measurement of the changes in the product of the two largest diameters recorded in two successive evaluations. According to criteria of the World Health Organization (1978) tumor progression (PD) was defined as an increase of at least 25% in tumor size; stable disease (SD) as an increase of less than 25% or a reduction of less than 50%; partial response (PR) as a tumor shrinkage greater than 50%; and complete response (CR) as the complete disappearance of all clinical signs of disease. Pathological complete response was defined as the absence of neoplastic cells either in the breast or in the axillary lymph nodes.
Surgery was planned after full clinical reassessment. Quadrantectomy or modified radical mastectomy were performed when indicated in association with full axillary node dissection. All patients subjected to quadrantectomy underwent irradiation of the residual breast (60 Gy delivered over 6 weeks).
Histopathologic grade and immunohistochemistry
The degree of malignancy was assessed according to the Elston and Ellis grading system which classifies tumors into grade I (well differentiated), grade II (moderately differentiated) and grade III (poorly differentiated; Elston & Ellis 1991).
The immunohistochemical methodology used in this study is fully described elsewhere (Bottini et al. 2000). Briefly, an antigen-retrieval step was performed by heating a tissue section in a citrate buffer. The primary antibodies applied were: ER (mouse monoclonal 6F11 (Novocastra Lab); dilution 1 : 50), progesterone receptor (PgR; mouse monoclonal 1A6 (Novocastra Lab); dilution 1 : 20) and Ki67 (mouse monoclonal Mib-1 (Dako, Glostrup, Denmark); dilution 1 : 30). All required 1 h incubation at room temperature.
Biotinylated horse anti-mouse IgG and avidin–biotin–peroxidase complex were applied as a staining method (Vectastatin ABC kit; Vector Laboratories, Burlingame, CA, USA). A solution containing H2O2 (0.06%, v/v) and diaminobenzidine-4-HCl (0.05%, v/v) was used as a chromogen.
All samples had a negative control slide (no primary antibody) of an adjacent section to assess the degree of non-specific staining. Positive controls included breast carcinomas known to exhibit high levels of each marker. All staining was scored by counting the number of positively stained cells and expressed as a percentage of the total tumor cells (at least 1000) counted across several representative fields of the section using a standard light microscope equipped with a 10 x 10 square graticule. Reproducibility of counting was assessed by a second investigator rescoring 10 slides.
The relative intensity of ER and PgR staining was assessed in a semi-quantitative fashion as previously described by McCarty et al. (1985), incorporating both the intensity and distribution of specific staining. A value (HSCORE) was derived from the sum of the percentages of positively stained epithelial cells multiplied by the weighted intensity of staining. Specimens were deemed receptor-positive if the HSCORE was greater than 100. The immunohistochemical evaluation at definitive surgery was performed by the same pathologists, who remained blinded to the disease response and the score assessed at first biopsy.
Statistical methodology
The trial was designed to detect a difference in response rate between the EPI arm and the EPI-TAM arm based on clinical measurements. On the basis of a previous experience by our group epirubicin alone yielded to a clinical response rate of 67% (Bottini et al. 2000). A sample size of 196 patients was considered adequate to detect an improvement in response rate from 67 to 85% with the addition of tamoxifen to epirubicin at a 5% significance level (two-sided) and 80% power. The 2 test was used to perform comparisons of proportions, whereas the Mann–Whitney U test was performed to compare continuous variables. The independent role of ER and PgR status (dichotomous variables), menopausal status (dichotomous variable), T stage (categorized variable), axillary lymph-node status (dichotomous variable) and Ki67 expression (continuous variable) in predicting for a clinically complete response or pathologically complete response was assessed by multivariate logistic regression analysis. Ki67 had a marked left-skewed distribution, and was modeled using log tranformation. All P values reported were two-sided; P values of <0.05 were considered statistically significant. The statistical computations were performed using the SPSS for Windows software (Statsoft, Tulsa, OK, USA).
Results
January 1997 to January 2002, 211 consecutive patients meeting the inclusion criteria entered the study. 105 were randomized to receive epirubicin alone (EPI) and 106 were randomized to receive epirubicin plus tamoxifen (EPI-TAM). Tumor and patient characteristics are outlined in Table 1. ER+ and ER– tumors were uniformly distributed between the two treatment arms, whereas a slightly higher expression of PgR was observed in tumours of patients randomized in the EPI-TAM arm. The remaining tumor and patient characteristics were well balanced between the two treatment arms.
Treatment received and toxicity
144 patients received three epirubicin cycles and 65 patients received four epirubicin cycles without difference between the two treatment arms. Toxicity did not differ from that expected with epirubicin at 120 mg/m2 reported previously (Dogliotti et al. 1996). Leukopenia was the most relevant side effect, with about 50% of patients experiencing grade III–IV toxicity in both arms. Nevertheless, only a few fever episodes were reported. Platelet toxicity was uncommon and mild. Grade II–III hemoglobin was observed in 15% of patients. Diarrhoea and mucositis from mild to moderate occurred in 10% of patients. Acute cardiac toxicity was absent in both arms. All these side effects, attributable to epirubicin, were equally distributed in both arms. Overall, 198 patients received the planned chemotherapy dose (99 patients (94.2%) in the EPI arm and 99 patients (93.4%) in the EPI-TAM arm). 13 patients received 75% of the planned dose (six in the EPI arm and seven in the EPI-TAM arm) due to toxicity. No patients reduced tamoxifen assumption. Two patients, one from each treatment arm, refused to continue the treatment after the first cycle and were referred for immediate surgery. These patients were not evaluable for disease response.
Treatment response and loco-regional treatment
The best clinical responses recorded for each patient are listed in Table 2. There was no difference in overall response rate between the two treatment arms (76 versus 82% in the EPI and EPI-TAM arms respectively). Pathological complete response was documented in 12 patients, whereas nine additional patients had residual in situ carcinoma. Pathological complete responses were equally distributed in the two arms, while residual in situ carcinoma was more frequently observed in the EPI-TAM arm, even though the difference failed to attain the statistical significance. Clinical complete response, pathological complete responses and in situ carcinoma at residual histology were more frequently observed in patients with ER– primary disease than their ER+ counterparts (Figure 1).
Patients destined to undergo clinical complete response had greater baseline Ki67 expression than those failing to obtain a clinical complete response (median 23.5% (range 7–90%) versus 16.0% (range 1–90%); P<0.01). Similarly, patients destined to undergo pathological complete response also had greater Ki67 expression at baseline conditions than their counterpart (median 30.0% (range 7–90%) versus 18.0% (range 1–90%); P<0.01). On dividing patients according to their steroid hormone-receptor status, there was no difference in overall response rate, clinical complete response rate or pathological complete response rate between the two treatment arms in ER+ and ER– breast cancer patients. The frequency of residual in situ carcinoma between the two arms did not differ in patients with ER+ breast carcinoma, whereas it was significantly higher in patients randomized to receive the combination regimen in ER– patient subgroup (Table 2).
209 patients underwent surgery after chemotherapy. Conservative surgery (quadrantectomy) was performed in 44 patients out of 104 randomized in the EPI arm (42.3%) and in 53 patients out of 105 randomized in EPI-TAM arm (50.4%). Mastectomy was performed in the remaining patients.
Multivariate logistic regression analysis of factors predicting of clinical complete response and pathological complete response
The relative impact of proliferative activity expressed as Ki67 immunostaining and the estrogen receptor status in predicting complete clinical response and pathological complete response was evaluated in a multivariate analysis adjusting for menopausal status, T stage, axillary lymph-node status, tumor grade, progesterone receptor status and treatment arm. As outlined in Table 3, Ki67 immunostaining was the only independent variable predictive for both clinical and pathological complete response, while ER status did not enter the model.
Changes in Ki67 expression
Both EPI and EPI-TAM treatments resulted in a significant reduction in Ki67 expression, for overall and patients with ER+ breast cancer (Table 4). The reduction in Ki67 immunostaining, however, was significantly greater in the EPI-TAM arm than in the EPI arm, leading to a lower Ki67 expression at postoperative residual histology (Table 4). In the patient subset with ER– breast cancer, Ki67 immunostaining significantly diminished after treatment in the EPI-TAM arm but not in the EPI arm. The Ki67 expression in the residual tumor in ER–patients did not differ between the two arms.
Discussion
The scenario of primary chemotherapy is attractive for making comparisons of both biological and clinical end points. This study describes a randomized comparison of chemotherapy and chemotherapy plus tamoxifen as primary systemic treatment in patients with operable breast cancer or locally advanced disease. To our knowledge there have been no equivalent randomized trials addressing this issue. In order to avoid interferences of multiple drugs, the single agent epirubicin administered at full doses was preferred. In order to obtain full explorative information on possible synergisms of tamoxifen with epirubicin, patients with ER– primary malignancy were also included.
The present study showed that the addition of tamoxifen to epirubicin led to a trend of overall response rate and pathological response rate in favour of the combined regimen. These data are consistent with previous reports on advanced disease showing a slightly higher activity of chemo-hormonal therapy over chemotherapy alone (Fossati et al. 1998). The distribution of clinical and pathological response was more frequently observed in patients with ER– primary breast cancer as well as in those with elevated Ki67 expression at baseline. This is a subgroup analysis and by definition underpowered; these data anyway confirm the results of a number of published studies showing that steroid receptor negativity (Bonadonna et al. 1990, MacGrogan et al. 1996, Colleoni et al. 1999) and elevated proliferation activity (Bonetti et al. 1996, Amadori et al. 1997, Daidone et al. 1999) are both predictors for chemo-sensitivity. The relationship between proliferation activity and chemotherapy response is expected since the cellular growth rate is traditionally thought to be important in determining the sensitivity of human cancer to chemotherapy. However the underlying mechanisms by which a lack of ER sensitizes cells to chemotherapy cytotoxicity is not fully established. The proliferative activity is notoriously greater in ER– primary breast cancer than in ER+ breast cancer (Campiglio et al. 2003) and this relationship could offer a possible explanation. The multivariate analysis performed in the present study provided support to this assumption, since ER status failed to enter the model in predicting both clinical and pathological complete response when adjusting for baseline Ki67 immunostaining.
There is good reason to believe that patients with ER+ tumors may benefit less from chemotherapy than those with ER– tumors. It is also established that hormone therapy is highly active in ER+ tumors (Clarke et al. 1993). In the present study, however, the addition of tamoxifen to epirubicin did not lead to greater pathological CR in ER+ cases. It may be that hormone therapy takes longer to see a response than the 3–4-month duration of this study.
In the patient subset with ER– tumors, a greater proportion of patients with residual in situ carcinoma was found in the combination arm. This observation is difficult to explain, and caution should be adopted in interpreting data coming from subgroup analyses.
Reduction in proliferation activity is a very well recognized phenomenon after either chemotherapy (Fisher et al. 1998, 2001) or tamoxifen therapy (Daidone et al. 1991). In a phase II study conducted by our group with CMF (cyclophosphamide, methotrexate, 5-flurouracil) administered in association with tamoxifen in the patient subset with ER+ primary tumors, we observed that reduction in proliferation activity was mainly confined to ER+ neoplasms (Bottini et al. 1998, Bottini et al. 2001). Whether this phenomenon was linked or not to the tamoxifen administration was uncertain, since this study was unable to separate the effect of chemotherapy from that of tamoxifen. In this prospective randomized trial a greater reduction in proliferation activity was obtained with concomitant administration of both epirubicin and tamoxifen as opposed to the administration of epirubicin alone. Some investigators hypothesized, in the early 1980s, that a biological mechanism of a possible antagonistic interaction between tamoxifen and chemotherapy might be due to the alteration in tumor cell kinetics induced by tamoxifen (Osborne et al. 1989). The results of this study provide support to this hypothesis, thus suggesting a possible mechanism of reduced long-term efficacy of anthracyline-containing schemes (Albain et al. 2002). A greater reduction in Ki67 expression in favour of the EPI-TAM arm was found in the ER– patient subset. Again these data should be interpreted with great caution since the number of ER– patients is very small and serial Ki67 could only be assessed in 75% of them (25% had a pathological complete response).
To conclude, this small randomized trial of epirubicin versus epirubicin plus tamoxifen as primary chemotherapy in breast cancer patients substantially confirms the notion of an absence of additional activity of concomitant chemo-hormonal therapy as opposed to chemotherapy alone. Concomitant epirubicin and tamoxifen led to a significantly higher reduction in the Ki67 expression than epirubicin alone. Since the chemotherapy efficacy is dependent upon proliferative activity, the increased reduction in proliferation activity in the combination arm suggests a mechanism of possible antagonistic interaction between tamoxifen and chemotherapy.
Acknowledgements
Presented in part at the 39th Annual Meeting of ASCO, Chicago, IL, USA, 31 May–3 June 2003. Supported in part by the Association ‘Amici dell’Ospedale di Cremona’ and a grant from the Consiglio Nazionale Ricerche (CNR), Rome, Italy. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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Abstract
This study was designed to address whether simultaneous primary chemo-hormonal therapy provides additional activity compared with chemotherapy alone in breast cancer patients with operable or locally advanced disease. Between January 1997 and January 2002, 211 consecutive patients with T2–4, N0–1, M0 breast cancer were randomized to receive either epirubicin alone (EPI) or epirubicin plus tamoxifen (EPI-TAM). Ki67 expression was evaluated immunohistochemically in tumor specimens obtained before chemotherapy by incision biopsy and at definitive surgery. Tumor shrinkage of >50% was obtained in 76% of patients randomized in the EPI arm and 81.9% of patients randomized in the EPI-TAM arm (not significant). The corresponding rates of clinical and pathological complete response were 20.2 and 21.9% (not significant), and 4.8 and 6.7% (not significant), respectively. Pathologically complete response was more frequently observed in estrogen receptor (ER)-negative (ER–) tumors (P=0.04) and correlated with elevated baseline Ki67 expression (P<0.01). Both EPI and EPI-TAM treatments resulted in a significant reduction in Ki67 expression, either in overall patients (P=0.000) or in patients with ER+ breast cancer (P=0.000). The reduction in Ki67 immunostaining in the EPI-TAM arm was greater than in the EPI arm, leading to a lower Ki67 expression at post-operative residual histology (P=0.0041). The addition of tamoxifen to epirubicin chemotherapy did not improve the response rate but led to a significantly higher reduction in the Ki67 expression. Baseline elevated Ki67 expression and the ER– status were both associated with a greater chance of obtaining a pathological complete response at residual histology.
Introduction
Chemotherapy and endocrine therapy are known to be efficacious in the management of breast cancer. When given as adjuvant systemic therapy shortly after surgery for primary disease, both treatment strategies have been found to increase either the disease-free survival or the overall survival (Early Breast Cancer Trialists’ Collaborative Group 1998a, 1998b).
Among the chemotherapeutic agents available, anthracyclines are the most efficacious (Early Breast Cancer Trialists’ Collaborative Group 1998a), while tamoxifen is the most-used endocrine therapy and still remains the treatment of choice in the adjuvant setting (Early Breast Cancer Trialists’ Collaborative Group 1998b). There is no doubt that the combination of tamoxifen with chemotherapy yields greater benefits over the administration of either treatment alone (Early Breast Cancer Trialists’ Collaborative Group 1998b); however, the optimal association of these two treatments represents an intriguing issue (Goldirsch et al. 2002).
Since endocrine therapy and chemotherapy exhibit different mechanisms of action and different patterns of toxicity, the simultaneous administration of the two treatment modalities might be advantageous. There are however theoretical arguments suggesting that simultaneous chemo-hormonal therapy may be detrimental. Endocrine therapies are cytostatic and may decrease cell turnover, whereas chemotherapeutic agents require DNA synthesis to exert their full efficacy (Osborne et al. 1989). In addition, tamoxifen may alter membrane lipids and antagonizes both calmodulin and Ca2+ channels, thereby changing diffusion rates of certain drugs and potentially altering the drug uptake (Greensberg et al. 1987). On the other hand, in vitro studies have demonstrated that tamoxifen is able to overcome the multidrug resistance mediated by the p-170 glycoprotein, suggesting a rationale for combining this agent with anthracyclines (Berman et al. 1991).
In patients with metastatic disease a systematic review of published randomized trials of chemotherapy versus chemotherapy plus tamoxifen (involving a total of 1294 cases recruited in seven trials) did not show a significant advantage for the combination treatment in terms of survival, although the response rate was significantly superior in the chemo-hormonal arm (Fossati et al. 1998). Similar results have been obtained in an additional study recently published in which chemo-hormonal therapy improved the time to treatment failure but not overall survival (Sledge et al. 2000). The results of a 10-year update from the North American Intergroup Trial 0100 comparing tamoxifen alone with tamoxifen commenced at the same time as CAF (cyclophosphamide, doxorubicin, 5-fluorouracil) chemotherapy with tamoxifen commenced upon completion of the same regimen (Albain et al. 2002), showed that tamoxifen conferred a slight but significant disadvantage in disease-free survival and overall survival if administered during chemotherapy rather than sequentially. Similar results have been obtained in a randomized European study (Pico et al. 2004).
The administration of primary chemotherapy in breast cancer patients permits in vivo assessment of tumour chemosensitivity and evaluation of treatment-induced changes in tumour biology. This treatment modality provides an unique opportunity to derive biological information related to tumor response in order to understand mechanisms of action of systemic antineoplastic treatments as well as mechanisms of their eventual interaction (Cleator et al. 2002).
This article presents data of a randomized trial conducted in a single institution that compared the activity of the single agent epirubicin versus epirubicin plus tamoxifen as primary chemotherapy in human breast cancer. The secondary aim was the reduction in proliferation activity as assessed by expression of the Ki67 antigen.
Patients and methods
Patients
Breast cancer patients with operable or locally advanced disease (T2–4, N0–1, M0) were considered eligible. They were required to have an Eastern Cooperative Oncology Group performance status of <2, adequate bone marrow reserve (white blood cell count, >3.5 x 109/l; platelets, >100 x 109/l; hemoglobin, >10 g/dl), hepatic function (aspartate amino-transferase (AST) alanine aminotransferase (ALT), bilirubin and alkaline phosphatase levels <1.25 times the upper limit of the normal value) and renal function (serum creatinine <1.25 the upper limit of normal value). Patients with non-malignant systemic disease that precluded them from receiving study therapy (e.g. active infection, any clinically significant cardiac arrhythmia, congestive heart failure, complete bundle brand block, functional New York Heart Association class III or worse, or pregnancy) and patients with second primary malignancies (except in situ carcinoma of the cervix or adequately treated basal cell carcinoma of the skin) were not eligible. The study was approved by the local ethical committee. Written informed consent was obtained from all patients before randomization.
Treatment schedule
Patients were randomized to receive epirubicin alone (EPI arm), or epirubicin and tamoxifen (EPI-TAM arm). Patients in the EPI arm received epirubicin (Farmorubicina; Pharmacia, Milan, Italy) 60 mg/m2 by slow i.v. push on days 1 and 2; patients in the EPI-TAM arm received epirubicin 60 mg/m2 by slow i.v. push on days 1 and 2 and tamoxifen (Kessar; Pharmacia) 30 mg daily. Epirubicin infusions in both arms were repeated every 21 days for three or four cycles before definitive surgery, whereas tamoxifen was given continuously until the end of the cytotoxic treatment. From then, patients with estrogen receptor-positive (ER+) primary tumor in both arms received tamoxifen (20 mg) up to progression or for a maximum of 5 years.
Recommended treatment modifications for hematologic toxicities were as follows: 1-week delay was introduced if granulocyte counts were less than 1.5 x 109/l and or platelet counts were less than 100 x 109/l on day 21; after 1 week, if hematologic parameters did not recover, a dose reduction of 50% was applied when granulocyte counts ranged between 1.0 x 109 and 1.5 x 109/l and/or platelet counts ranged between 75 x 109 and 100 x 109/l. In case of lower hematological values the following week, a further 1-week delay was allowed. Patients went off-study if the delay exceeded 4 weeks. Delays and dose reductions were also performed for stomatitis of grade >3. Supportive care could include blood transfusion and the administration of antiemetics, analgesics and growth factors as appropriate.
Treatment evaluation
On first presentation an incision biopsy was performed on each patient and a small wedge of tissue (0.5–0.8 cm) was removed. Chemotherapy was started within 1 or 2 days of diagnosis. The primary objective of the study was to compare the response rate of patients randomized to receive epirubicin versus those randomized to received epirubicin plus tamoxifen. The secondary objective was to compare changes in Ki67 expression in tumor samples obtained before treatment and at definitive surgery.
The following tests were evaluated before entry into the study: medical history and physical examination, performance status, weight, hemoglobin and hematocrit levels, white blood cell and platelet counts, alkaline phosphatase, AST, ALT and bilirubin levels; serum electrolytes, serum creatinine and serum CA 15-3 levels; electrocardiogram, echocardiography; chest radiograph; mammography and breast echography, isotope bone scan with radiograph of abnormal areas; abdominal ultrasonography; and computed tomography scan of the chest and abdomen if necessary. Physical examination and hematological profile were repeated before each course of chemotherapy, whereas a complete blood cell count was recommended at the nadir of leukocytes and platelets (10–12 days from epirubicin administration).
Each month the size of the primary tumor and the size of the axillary lymph nodes, when appreciable, were measured using callipers by the same clinician. Response was assessed by the clinical measurement of the changes in the product of the two largest diameters recorded in two successive evaluations. According to criteria of the World Health Organization (1978) tumor progression (PD) was defined as an increase of at least 25% in tumor size; stable disease (SD) as an increase of less than 25% or a reduction of less than 50%; partial response (PR) as a tumor shrinkage greater than 50%; and complete response (CR) as the complete disappearance of all clinical signs of disease. Pathological complete response was defined as the absence of neoplastic cells either in the breast or in the axillary lymph nodes.
Surgery was planned after full clinical reassessment. Quadrantectomy or modified radical mastectomy were performed when indicated in association with full axillary node dissection. All patients subjected to quadrantectomy underwent irradiation of the residual breast (60 Gy delivered over 6 weeks).
Histopathologic grade and immunohistochemistry
The degree of malignancy was assessed according to the Elston and Ellis grading system which classifies tumors into grade I (well differentiated), grade II (moderately differentiated) and grade III (poorly differentiated; Elston & Ellis 1991).
The immunohistochemical methodology used in this study is fully described elsewhere (Bottini et al. 2000). Briefly, an antigen-retrieval step was performed by heating a tissue section in a citrate buffer. The primary antibodies applied were: ER (mouse monoclonal 6F11 (Novocastra Lab); dilution 1 : 50), progesterone receptor (PgR; mouse monoclonal 1A6 (Novocastra Lab); dilution 1 : 20) and Ki67 (mouse monoclonal Mib-1 (Dako, Glostrup, Denmark); dilution 1 : 30). All required 1 h incubation at room temperature.
Biotinylated horse anti-mouse IgG and avidin–biotin–peroxidase complex were applied as a staining method (Vectastatin ABC kit; Vector Laboratories, Burlingame, CA, USA). A solution containing H2O2 (0.06%, v/v) and diaminobenzidine-4-HCl (0.05%, v/v) was used as a chromogen.
All samples had a negative control slide (no primary antibody) of an adjacent section to assess the degree of non-specific staining. Positive controls included breast carcinomas known to exhibit high levels of each marker. All staining was scored by counting the number of positively stained cells and expressed as a percentage of the total tumor cells (at least 1000) counted across several representative fields of the section using a standard light microscope equipped with a 10 x 10 square graticule. Reproducibility of counting was assessed by a second investigator rescoring 10 slides.
The relative intensity of ER and PgR staining was assessed in a semi-quantitative fashion as previously described by McCarty et al. (1985), incorporating both the intensity and distribution of specific staining. A value (HSCORE) was derived from the sum of the percentages of positively stained epithelial cells multiplied by the weighted intensity of staining. Specimens were deemed receptor-positive if the HSCORE was greater than 100. The immunohistochemical evaluation at definitive surgery was performed by the same pathologists, who remained blinded to the disease response and the score assessed at first biopsy.
Statistical methodology
The trial was designed to detect a difference in response rate between the EPI arm and the EPI-TAM arm based on clinical measurements. On the basis of a previous experience by our group epirubicin alone yielded to a clinical response rate of 67% (Bottini et al. 2000). A sample size of 196 patients was considered adequate to detect an improvement in response rate from 67 to 85% with the addition of tamoxifen to epirubicin at a 5% significance level (two-sided) and 80% power. The 2 test was used to perform comparisons of proportions, whereas the Mann–Whitney U test was performed to compare continuous variables. The independent role of ER and PgR status (dichotomous variables), menopausal status (dichotomous variable), T stage (categorized variable), axillary lymph-node status (dichotomous variable) and Ki67 expression (continuous variable) in predicting for a clinically complete response or pathologically complete response was assessed by multivariate logistic regression analysis. Ki67 had a marked left-skewed distribution, and was modeled using log tranformation. All P values reported were two-sided; P values of <0.05 were considered statistically significant. The statistical computations were performed using the SPSS for Windows software (Statsoft, Tulsa, OK, USA).
Results
January 1997 to January 2002, 211 consecutive patients meeting the inclusion criteria entered the study. 105 were randomized to receive epirubicin alone (EPI) and 106 were randomized to receive epirubicin plus tamoxifen (EPI-TAM). Tumor and patient characteristics are outlined in Table 1. ER+ and ER– tumors were uniformly distributed between the two treatment arms, whereas a slightly higher expression of PgR was observed in tumours of patients randomized in the EPI-TAM arm. The remaining tumor and patient characteristics were well balanced between the two treatment arms.
Treatment received and toxicity
144 patients received three epirubicin cycles and 65 patients received four epirubicin cycles without difference between the two treatment arms. Toxicity did not differ from that expected with epirubicin at 120 mg/m2 reported previously (Dogliotti et al. 1996). Leukopenia was the most relevant side effect, with about 50% of patients experiencing grade III–IV toxicity in both arms. Nevertheless, only a few fever episodes were reported. Platelet toxicity was uncommon and mild. Grade II–III hemoglobin was observed in 15% of patients. Diarrhoea and mucositis from mild to moderate occurred in 10% of patients. Acute cardiac toxicity was absent in both arms. All these side effects, attributable to epirubicin, were equally distributed in both arms. Overall, 198 patients received the planned chemotherapy dose (99 patients (94.2%) in the EPI arm and 99 patients (93.4%) in the EPI-TAM arm). 13 patients received 75% of the planned dose (six in the EPI arm and seven in the EPI-TAM arm) due to toxicity. No patients reduced tamoxifen assumption. Two patients, one from each treatment arm, refused to continue the treatment after the first cycle and were referred for immediate surgery. These patients were not evaluable for disease response.
Treatment response and loco-regional treatment
The best clinical responses recorded for each patient are listed in Table 2. There was no difference in overall response rate between the two treatment arms (76 versus 82% in the EPI and EPI-TAM arms respectively). Pathological complete response was documented in 12 patients, whereas nine additional patients had residual in situ carcinoma. Pathological complete responses were equally distributed in the two arms, while residual in situ carcinoma was more frequently observed in the EPI-TAM arm, even though the difference failed to attain the statistical significance. Clinical complete response, pathological complete responses and in situ carcinoma at residual histology were more frequently observed in patients with ER– primary disease than their ER+ counterparts (Figure 1).
Patients destined to undergo clinical complete response had greater baseline Ki67 expression than those failing to obtain a clinical complete response (median 23.5% (range 7–90%) versus 16.0% (range 1–90%); P<0.01). Similarly, patients destined to undergo pathological complete response also had greater Ki67 expression at baseline conditions than their counterpart (median 30.0% (range 7–90%) versus 18.0% (range 1–90%); P<0.01). On dividing patients according to their steroid hormone-receptor status, there was no difference in overall response rate, clinical complete response rate or pathological complete response rate between the two treatment arms in ER+ and ER– breast cancer patients. The frequency of residual in situ carcinoma between the two arms did not differ in patients with ER+ breast carcinoma, whereas it was significantly higher in patients randomized to receive the combination regimen in ER– patient subgroup (Table 2).
209 patients underwent surgery after chemotherapy. Conservative surgery (quadrantectomy) was performed in 44 patients out of 104 randomized in the EPI arm (42.3%) and in 53 patients out of 105 randomized in EPI-TAM arm (50.4%). Mastectomy was performed in the remaining patients.
Multivariate logistic regression analysis of factors predicting of clinical complete response and pathological complete response
The relative impact of proliferative activity expressed as Ki67 immunostaining and the estrogen receptor status in predicting complete clinical response and pathological complete response was evaluated in a multivariate analysis adjusting for menopausal status, T stage, axillary lymph-node status, tumor grade, progesterone receptor status and treatment arm. As outlined in Table 3, Ki67 immunostaining was the only independent variable predictive for both clinical and pathological complete response, while ER status did not enter the model.
Changes in Ki67 expression
Both EPI and EPI-TAM treatments resulted in a significant reduction in Ki67 expression, for overall and patients with ER+ breast cancer (Table 4). The reduction in Ki67 immunostaining, however, was significantly greater in the EPI-TAM arm than in the EPI arm, leading to a lower Ki67 expression at postoperative residual histology (Table 4). In the patient subset with ER– breast cancer, Ki67 immunostaining significantly diminished after treatment in the EPI-TAM arm but not in the EPI arm. The Ki67 expression in the residual tumor in ER–patients did not differ between the two arms.
Discussion
The scenario of primary chemotherapy is attractive for making comparisons of both biological and clinical end points. This study describes a randomized comparison of chemotherapy and chemotherapy plus tamoxifen as primary systemic treatment in patients with operable breast cancer or locally advanced disease. To our knowledge there have been no equivalent randomized trials addressing this issue. In order to avoid interferences of multiple drugs, the single agent epirubicin administered at full doses was preferred. In order to obtain full explorative information on possible synergisms of tamoxifen with epirubicin, patients with ER– primary malignancy were also included.
The present study showed that the addition of tamoxifen to epirubicin led to a trend of overall response rate and pathological response rate in favour of the combined regimen. These data are consistent with previous reports on advanced disease showing a slightly higher activity of chemo-hormonal therapy over chemotherapy alone (Fossati et al. 1998). The distribution of clinical and pathological response was more frequently observed in patients with ER– primary breast cancer as well as in those with elevated Ki67 expression at baseline. This is a subgroup analysis and by definition underpowered; these data anyway confirm the results of a number of published studies showing that steroid receptor negativity (Bonadonna et al. 1990, MacGrogan et al. 1996, Colleoni et al. 1999) and elevated proliferation activity (Bonetti et al. 1996, Amadori et al. 1997, Daidone et al. 1999) are both predictors for chemo-sensitivity. The relationship between proliferation activity and chemotherapy response is expected since the cellular growth rate is traditionally thought to be important in determining the sensitivity of human cancer to chemotherapy. However the underlying mechanisms by which a lack of ER sensitizes cells to chemotherapy cytotoxicity is not fully established. The proliferative activity is notoriously greater in ER– primary breast cancer than in ER+ breast cancer (Campiglio et al. 2003) and this relationship could offer a possible explanation. The multivariate analysis performed in the present study provided support to this assumption, since ER status failed to enter the model in predicting both clinical and pathological complete response when adjusting for baseline Ki67 immunostaining.
There is good reason to believe that patients with ER+ tumors may benefit less from chemotherapy than those with ER– tumors. It is also established that hormone therapy is highly active in ER+ tumors (Clarke et al. 1993). In the present study, however, the addition of tamoxifen to epirubicin did not lead to greater pathological CR in ER+ cases. It may be that hormone therapy takes longer to see a response than the 3–4-month duration of this study.
In the patient subset with ER– tumors, a greater proportion of patients with residual in situ carcinoma was found in the combination arm. This observation is difficult to explain, and caution should be adopted in interpreting data coming from subgroup analyses.
Reduction in proliferation activity is a very well recognized phenomenon after either chemotherapy (Fisher et al. 1998, 2001) or tamoxifen therapy (Daidone et al. 1991). In a phase II study conducted by our group with CMF (cyclophosphamide, methotrexate, 5-flurouracil) administered in association with tamoxifen in the patient subset with ER+ primary tumors, we observed that reduction in proliferation activity was mainly confined to ER+ neoplasms (Bottini et al. 1998, Bottini et al. 2001). Whether this phenomenon was linked or not to the tamoxifen administration was uncertain, since this study was unable to separate the effect of chemotherapy from that of tamoxifen. In this prospective randomized trial a greater reduction in proliferation activity was obtained with concomitant administration of both epirubicin and tamoxifen as opposed to the administration of epirubicin alone. Some investigators hypothesized, in the early 1980s, that a biological mechanism of a possible antagonistic interaction between tamoxifen and chemotherapy might be due to the alteration in tumor cell kinetics induced by tamoxifen (Osborne et al. 1989). The results of this study provide support to this hypothesis, thus suggesting a possible mechanism of reduced long-term efficacy of anthracyline-containing schemes (Albain et al. 2002). A greater reduction in Ki67 expression in favour of the EPI-TAM arm was found in the ER– patient subset. Again these data should be interpreted with great caution since the number of ER– patients is very small and serial Ki67 could only be assessed in 75% of them (25% had a pathological complete response).
To conclude, this small randomized trial of epirubicin versus epirubicin plus tamoxifen as primary chemotherapy in breast cancer patients substantially confirms the notion of an absence of additional activity of concomitant chemo-hormonal therapy as opposed to chemotherapy alone. Concomitant epirubicin and tamoxifen led to a significantly higher reduction in the Ki67 expression than epirubicin alone. Since the chemotherapy efficacy is dependent upon proliferative activity, the increased reduction in proliferation activity in the combination arm suggests a mechanism of possible antagonistic interaction between tamoxifen and chemotherapy.
Acknowledgements
Presented in part at the 39th Annual Meeting of ASCO, Chicago, IL, USA, 31 May–3 June 2003. Supported in part by the Association ‘Amici dell’Ospedale di Cremona’ and a grant from the Consiglio Nazionale Ricerche (CNR), Rome, Italy. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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