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Randomized Clinical Trial of Adjuvant Fluorouracil, Epirubicin, and Cyclophosphamide Chemotherapy for Patients With Fast-Proliferating, Node-Negative Breast Cancer

From the Clinical Experimental Oncology Laboratory, Senology Unit, Histopathology Service, and Medical Oncology Unit, National Oncology Institute, Bari, Italy.

Address reprint requests to Angelo Paradiso, MD, Clinical Experimental Oncology Laboratory, National Oncology Institute, Via Amendola, 209, 70126 Bari, Italy; email: anpara{at}tin.it

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: The prospective applicability of new biologic tumor information to personalize adjuvant treatment of women with operable breast cancer remains to be demonstrated. The aim of the present study was to investigate whether patients with fast-proliferating, node-negative breast cancer could benefit from adjuvant chemotherapy with fluorouracil, epirubicin, and cyclophosphamide (FEC).

PATIENTS AND METHODS: Beginning in November 1989, we analyzed the proliferative activity of primary tumors in a consecutive series of women with node-negative breast cancer to identify subgroups of patients with a worse prognosis and who were therefore suitable candidates for adjuvant systemic therapy. Proliferative activity was determined by means of the [³H]-thymidine incorporation assay using an autoradiographic technique. Women with fast-proliferating breast cancer ([³H]-thymidine labeling index, > 2.3%) were randomized to receive either six cycles of adjuvant FEC or no adjuvant therapy until disease progression.

RESULTS: One-hundred twenty-five and 123 patients treated with radical surgery for pT1 to T2, N0, M0 breast cancer were randomized to the FEC and control arms, respectively. After a median follow-up of 70 months, 27 events (21.6%) were observed in the FEC arm and 39 (32.2%) in the control arm, with a significantly lower number of locoregional relapses in the FEC group. Five-year disease-free survival (DFS) was 81% in the FEC group and 69% in the control group (P < .02 by log-rank test). Cox multivariate analysis described the impact of adjuvant therapy with FEC on DFS as independent of the patients’ main clinical-pathologic characteristics.

CONCLUSION: FEC adjuvant polychemotherapy seems able to significantly improve the clinical outcome of patients with fast-proliferating, node-negative breast cancer.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
DESPITE THE LOW risk of relapse generally reported for patients with node-negative breast cancer, according to updated criteria, up to 90% of all node-negative patients are nowadays candidates for some form of adjuvant systemic therapy.¹ This obvious discrepancy has engendered the need to verify whether it is possible to select specific subgroups of women with node-negative breast cancer with a potentially worse prognosis or higher sensitivity to adjuvant approaches.

It has repeatedly been demonstrated that several biologic tumor characteristics are capable of identifying subgroups of node-negative breast cancer patients bearing significantly different prognoses; however, only few examples of the prospective applications of biologic prognostic factors in the decision-making process of postsurgical therapy have been provided to date.

First-generation clinical trials that used biologic information in a prospective randomized design were planned in the early 1980s; they relied on estrogen receptor status and tumor size as prognostic factors to identify patients to be treated with adjuvant therapy.^2-6 In these trials, the receptor status was also important in directing the choice of the systemic therapy to be adopted: polychemotherapy with cyclophosphamide, methotrexate, and fluorouracil (CMF) was generally decided on for estrogen receptor (ER)–negative tumors, whereas the antiestrogen tamoxifen was the treatment of choice for ER-positive tumors.^3,5,7 All these trials showed that a significant benefit could be achieved with adjuvant therapy by treating subgroups of node-negative women with different risks of relapse.

New-generation trials that prospectively used cell kinetics variables were planned only in the early 1990s. In a Netherlands study, Van Diest et al⁸ evaluated whether adjuvant CMF was beneficial for node-negative premenopausal women with a high mitotic index. This trial started in 1990, but its clinical results are still not available.

A prospective application of the S-phase fraction as analyzed by flow cytometry has been planned by the Intergroup (Intergroup study 0102) to identify high-risk node-negative women eligible for adjuvant polychemotherapy with or without an anthracycline. The preliminary results indicated a slightly superior benefit of cyclophosphamide, doxorubicin, and fluorouracil with respect to CMF in women with high–S-phase tumors.⁹

Several studies have validated the importance of the [³H]-thymidine labeling index (TLI) as an independent and consistent prognostic marker in the long term.¹⁰ Moreover, studies on experimental tumors have shown a direct relationship between cell proliferation and response to antitumor drugs.¹¹ In clinical tumors, a major benefit has been observed for slowly proliferating advanced breast cancers treated with hormone therapy^12,13 and for rapidly proliferating breast cancers after polychemotherapy,^14-17 but information on operable breast cancer patients is limited to only one retrospective study on a small series of node-negative, ER-negative breast cancer patients.⁶

The use of TLI in prospective randomized studies was focused on almost concomitantly by two research groups in Italy. In the late 1980s, Amadori et al planned a prospective clinical trial to assess whether women with high-TLI node-negative breast cancer and a high risk of relapse would benefit from an adjuvant CMF regimen. The study has been concluded, and the results indicate a clear advantage of adjuvant CMF both in terms of local and distant metastases.¹⁸

The aim of our twin trial was to investigate whether and to what extent patients with fast-proliferating node-negative breast cancer benefit from an anthracycline-containing regimen. The feasibility of such a study has previously been reported.¹⁹ The design of our trial did not include the addition of tamoxifen in consideration of the fact that it has been reported that rapidly proliferating tumors, even if ER-positive, may have a high probability of escaping hormonal control in terms of objective response rate^12,13,20,21 and relapse,²² although these results remain highly controversial.²³

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
From November 1989 to April 1994, all the women treated at the Breast Unit of the National Oncology Institute of Bari (Bari, Italy) with a modified radical mastectomy or conservative surgery and radiotherapy (quadrantectomy + complete axillary dissection + breast irradiation) for unilateral pT1 to T2, N0, M0 operable breast cancer were considered for inclusion in two prospective clinical trials after being selected on the basis of high or low primary tumor proliferative activity. Women with slowly proliferating tumors were randomized to receive either no further therapy after primary radical surgery or tamoxifen for 5 years. Those with fast-proliferating breast cancer were randomized to receive either no further therapy after primary radical surgery or adjuvant polychemotherapy with the fluorouracil, epirubicin, and cyclophosphamide (FEC) regimen. In this article, we report the results from the latter clinical trial.

Women were considered pre- and perimenopausal if they had regular menstrual cycles or if they were within 2 years from their last menses. Tumors were classified according to the pathologic tumor-node-metastasis classification of the International Union Against Cancer.²⁴ During the period of study activity, 1,331 new breast cancers were diagnosed at the National Oncology Institute of Bari; 5% (n = 67) of them presented with stage IV disease, and the remaining ones (n = 1,264) were classified as operable disease. After surgical therapy, 612 of the latter cases (46% of the overall series) had no pathologically involved axillary lymph nodes.

Eligibility Criteria
The eligibility criteria were as follows: radical resection for monolateral invasive breast cancer; absence of pathologically involved axillary lymph nodes (at least 10 lymph nodes examined); absence of distant metastases; age 70 years; and available TLI and ER status information. Patients were considered ineligible if they were pregnant, had a previous history of cancer, had undergone prior systemic therapy for cancer, or had any nonmalignant systemic disease (including a history of heart disease) precluding their receiving the treatment option. Patients also had to satisfy the following requirements: peripheral WBC count 3,500/mL, platelet count 100,000/mL, creatinine level 1.5 mg/dL, bilirubin level 1.5 mg/dL, and transaminase levels twice the normal maximum limit.

Patients were required to be accessible for the follow-up, and their informed consent was obtained before assignment to a treatment arm. The protocol design was fully approved by the local ethical committee of the National Oncology Institute of Bari and by the Scientific Committee of the National Research Council of Italy.

Randomization and Data Management
Within 5 weeks of surgery, patients who fulfilled all the eligibility criteria were prospectively stratified according to tumor size and ER status using a cutoff value of 10 fmol/mg protein and randomized to receive either six courses of FEC or no therapy until progression. A system of random permutated blocks within the strata was used with a block size of eight. The randomization was performed by the Data Management Center of the Clinical Experimental Oncology Laboratory of the National Cancer Institute of Bari.

Treatment and follow-up data were collected directly into the clinical portfolio of each patient. An ad hoc database was updated with follow-up information every 6 months.

Treatment and Follow-Up
After randomization, patients were allocated to different arms of the study. The FEC regimen consisted of fluorouracil 500 mg/m² on days 1 and 8, epirubicin 50 mg/m² on day 1, and cyclophosphamide 500 mg/m² on day 1. All drugs were administered intravenously (IV) every 3 weeks for a total of six cycles. Information on dates, doses of chemotherapy, and chemotherapy-induced side effects was recorded. Toxicity and complete blood cell counts were evaluated on the first day of each cycle of therapy according to World Health Organization (WHO) criteria. Treatment following disease recurrence varied on the basis of several factors (length of disease-free interval, steroid receptor status, site of relapse, menopausal status, and so on). Dose-modification procedures in the case of hematologic toxicity consisted of a delay of 1 to 2 weeks when the WBC count was less than 3,500/mL on the first day of therapy or when the platelet count was less than 100,000/mL. If values had not returned to normal within 1 to 2 weeks, dose reduction was recommended.

In women who underwent breast-conserving surgery, the remaining area of the breast was irradiated (50 Gy plus a boost of 10 Gy over 5 to 6 weeks) starting from the 8th week after surgery and concomitantly with polychemotherapy in the patients allocated to the FEC arm.

All patients received a periodic laboratory assessment, including complete blood counts, electrolyte profiles, and liver and kidney function tests. During the first year, liver scans and chest x-rays were taken every 6 months; during the second year, chest x-rays were taken every 6 months and liver scans were taken only once in the 12th month. Subsequently, both chest x-rays and liver scans were taken once a year. From the time of randomization, all patients underwent an annual mammography and bone scan.

Biologic Determination
The removed breast tissue was immediately delivered to the Histopathology Unit, where the pathologist set aside two contiguous tumor samples for standard histologic analyses and for biologic determinations. Subsequently, the histologic subtype, axillary lymph node status, and cytohistologic grade according to Fisher’s modified criteria²⁵ were determined.

Determination of Tumor Proliferative Activity
Fresh tumor samples, transferred to the laboratory on ice, were immediately incubated in a culture medium containing [³H]-thymidine for 1 hour at 37°C and fixed in formalin. An autoradiographic procedure (photographic emulsion Ilford K5; Ilford Photographics, London, United Kingdom) was used to count using a light microscope the percentage of cells actively incorporating [³H]-thymidine according to the previously reported method.¹² Samples were stained with hematoxylin and eosin at 4°C. TLI, expressed as the ratio between thymidine-labeled cells and the total number of tumor cells, was determined independently by two observers, with 2,000 to 3,000 cells scored from different fragments of the same tumor. Quality control procedures were repeated periodically in compliance with the National Quality Control Program promoted by the Italian Society of Basic and Applied Cell Kinetics.²⁶

Fast-proliferating tumors were defined on the basis of a 2.3% cutoff, which represented the median value of the overall series of node-negative breast cancers recruited from the beginning of the TLI determination at the Institute of Bari.

Analysis of Steroid Receptor Content
Fresh tumor tissue to be used for steroid receptor analysis was frozen at -80°C until the in vitro biochemical assay was carried out. ERs and progesterone receptors (PgRs) were assayed by the dextran-coated charcoal method according to the European Organization for Research and Treatment of Cancer.²⁷ Quality control procedures for hormone receptor dosage were coordinated by the Italian ad hoc committee. In the present study, a cutoff of 10 fmol/mg protein for both ER and PgR was adopted to classify positive and negative cases.

Statistical Analysis
Sample size was determined during the planning of the study assuming a 5-year disease-free survival (DFS) rate from diagnosis of 68% for patients with fast-proliferating node-negative breast cancer treated only with local therapy²⁸ and an expected absolute reduction of 14% in patients treated with chemotherapy (5% error fixed for a two-sided test and a power of 80%). An accrual of 250 patients over a 3-year period was planned.

DFS was defined as the time from randomization to documented evidence of disease recurrence in locoregional and/or distant sites, the manifestation of a contralateral breast cancer, a second primary cancer in a nonbreast site, or death from other causes. All new disease manifestations were assessed by clinical, radiologic, and, when feasible, histologic examination. Overall survival was defined as the time from randomization to documented death from any cause. The clinical outcome of patients was analyzed according to the intention to treat principle, ie, patients were evaluated on the basis of their assigned therapy.

DFS probability (and 95% confidence interval [CI]) were computed by the Kaplan-Meier product-limit method.²⁹ The null hypothesis concerning the differential effect of treatment in univariate analysis was tested by the log-rank test.³⁰ Estimated hazard ratios (the ratio of the treated group to the control group), their 95% CIs, and P values were calculated from proportional hazard regression models stratified according to tumor size and ER content. The multivariate model was used to investigate potential confounding factors.³¹

Analysis of clinical outcome according to TLI and other clinical, pathologic, and biologic variables was planned in advance as a secondary aim of the study with an exploratory intent. All P values were based on two-sided testing, and statistical analyses were carried out with SPSS statistical software (SPSS, Inc, Chicago, IL).³²

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
During the study activation period, 612 node-negative breast cancers were diagnosed. TLI information was available for 565 (92%) of these cases. In 31 cases, a TLI assay was not performed because of insufficient pathologic tissue. In 16 cases, the TLI assay result was not assessable for various reasons (poor tumor cellularity in 12 cases; various technical reasons in four cases).

Of the 565 node-negative patients with available TLI information (Table 1), 287 patients (50.8%) were classified as having rapidly proliferating tumors. Two hundred forty-eight of these patients were eligible for the present study. The reasons for patient exclusion were age more than 70 years in 16 cases, important concomitant diseases in seven cases, bilateral breast cancer in two cases, and nonattendance of our Institute’s outpatient clinic in 14 cases.

The clinical and biologic characteristics of the randomized patients are reported in Table 2. Overall, median patient age was 53 years (range, 28 to 70 years), and 42% were pre- or perimenopausal. Fifty-two percent of patients underwent conservative surgery. A mean number of 16.9 axillary lymph nodes for each patient were examined by the pathologist (range, 10 to 42); 59% of the patients had a primary T1 tumor, and 76% had an infiltrating ductal histologic diagnosis. Fifty-six percent of the tumors were ER-positive, and 51% were PgR-positive. Finally, 50% of the tumors were grade 3. All these clinical and biologic characteristics were well balanced between the two study arms, with the exception of a higher percentage of T2 (46% v 35%) and G3 (57% v 41%) tumors in FEC-treated patients.

A total of 66 events (26.8%) were observed in the 246 patients assessable for follow-up: 15 relapses (6.1%) were in locoregional areas; 12 (4.9%) were in the contralateral breast; 24 (9.7%) were in distant sites; and nine (3.6%) were in multiple sites. Finally, five second primary cancers (2%) and one death (0.4%) from unrelated cancer causes were also recorded. The events for the two study arms are reported separately in Table 3. A total of 27 and 39 events were observed in the FEC and control arms, respectively. FEC-treated patients experienced a significantly lower number of local relapses than did patients in the control arm (three v 12; P < .01); a somewhat lower number of contralateral breast cancers (five v seven, respectively) and bone relapses (five v eight, respectively) was also noted.

View larger version (14K): [in this window] [in a new window]   Fig 1. DFS of patients with fast-proliferating node-negative breast cancer treated (n = 125) or not treated (n = 121) with adjuvant FEC polychemotherapy.

With regard to overall survival, 31 deaths were observed, 19 in the control arm and 12 in the FEC arm. Five-year overall survival was 85% in the control arm and 91% in FEC arm (P = .089).

Treatment Compliance and Toxicity
One hundred twelve (91%) of the 123 eligible patients assigned to receive the adjuvant FEC completed the six planned courses of FEC. Of the 11 remaining patients, two refused to start the treatment and subsequently received antiestrogen therapy for 3 years and nine refused to continue the chemotherapy after varying numbers of cycles for psychologic reasons (five patients quit after one cycle) or because of toxicity (gastrointestinal grade 3 WHO toxicity after two, three, and four cycles; grade 3 leukopenia after three cycles).

Side effects associated with FEC chemotherapy are reported in Table 5. WHO grade 4 toxicity was never observed. Alopecia was a frequently observed side effect, but hair regrowth was documented in all patients. Transient or permanent amenorrhea was evident in 40% of premenopausal women, and it was still present in 25% of patients 1 year after the end of chemotherapy. Permanent amenorrhea was evident mainly in women over 40 years of age. Twenty-five percent of the FEC-treated patients had nausea/vomiting symptoms classified as WHO grade 2 and 24% had grade 3 nausea and vomiting. Nine percent and 3% of women experienced WHO grade 2 and 3 leukopenia, respectively. Finally, grade 2 local skin reaction was observed in 9% of women who received concurrent administration of FEC and radiation therapies. In this latter subgroup of women, no other particular side effects were reported.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
In our study, the choice of TLI as a marker of tumor proliferative activity was based on previously mentioned scientific evidence and on some additional technical considerations. First, our group had a long-standing laboratory experience of this biologic variable, whose clinical cutoff value was defined by retrospectively analyzing a large series of node-negative breast cancer patients and then applied prospectively.²⁸ Furthermore, our laboratory had already been part of a national quality control program for approximately 5 years, and this assured the long-term reproducibility of TLI determinations.²⁶

In this study, TLI analysis was performed in a large number of consecutive patients. In the end, only approximately 20% of the newly diagnosed breast cancers satisfied the eligibility criteria of the present study. It is also worth noting that our study was monoinstitutional, which eliminated any problems of interlaboratory reproducibility of biologic marker determinations and clinical treatment that may occur in multicenter clinical-biologic trials.

This was one of the first trials to use an adjuvant systemic treatment including an anthracycline for high-risk, node-negative breast cancer patients. Consequently, we feared we would meet with low patient compliance with a new therapeutic approach. In an analysis performed in Canada, compliance did not exceed 77% among patients treated according to practice guidelines in use in 1991 for delivering polychemotherapy to women with high-risk node-negative breast cancer.³³ In our study, more than 90% of the randomized patients completed the planned chemotherapy.

The FEC schedule we used has already been reported to be very effective in the adjuvant therapy of node-positive breast cancer patients. We administered epirubicin 17 mg/m²/wk to a total dose of 300 mg, which is similar to the drug dose used by Brémond et al³⁴ and higher than that used by Coombes et al³⁵ and in Cancer and Leukemia Group B trial 8541.³⁶ As regards toxicity, WHO grade 2/3 nausea/vomiting was observed in approximately half of all the cases and alopecia in almost all the cases. Amenorrhea proved to be less frequent with a transient/permanent interruption of mensis in 40% of the premenopausal patients. With the exception of a relatively higher percentage of local skin reaction, the concurrent administration of FEC and radiotherapy did not increase toxicity.

The clinical results observed in this study clearly indicate that women with rapidly proliferating node-negative breast cancer benefit from adjuvant FEC, with a reduction in the annual risk of relapse of approximately 2.5% and a 12% absolute benefit in 5-year DFS. The benefit is comparable to that reported in other similar randomized clinical studies in which the efficacy of adjuvant chemotherapy was analyzed in specific biologic subgroups, that is, in patients with ER-negative tumors^2,3,5 or tumors larger than 3 cm.^4,7 Unfortunately, our trial offers no information about the potential extra benefit that patients with fast-proliferating ER-positive breast cancer could achieve by adding adjuvant tamoxifen after FEC chemotherapy. In fact, considering that our trial was designed when the results of the National Surgical Adjuvant Breast and Bowel Project B-14 trial³⁷ and 1998 Oxford meta-analysis²³ were not yet available, a further adjuvant tamoxifen treatment for women with ER-positive breast cancers was not planned.

Analysis of the relapse sites showed that adjuvant treatment significantly reduced all locoregional relapses and also led to a decrease in the number of distant metastases and contralateral breast cancers, albeit not significantly. A similar decrease in local relapses was reported in other trials.^3,5

The clinical benefit of the FEC adjuvant chemotherapy became evident on the DFS curves only after the first few years of follow-up. A possible explanation for this evidence could be that the adjuvant chemotherapy was more effective against the slower-growing tumors, thus leading to the appearance of a delayed benefit.

The present study was activated in parallel with another Italian trial¹⁸ similar in its biologic rational and clinical design, in an attempt to prospectively define whether node-negative rapidly proliferating tumors benefit from adjuvant chemotherapy. The only difference lay in the type of polychemotherapy administered: CMF was used in the study by Amadori et al, and FEC (ie, the substitution of the antimetabolite methotrexate with an anthracycline) was used in our study. In the CMF adjuvant study, a significant reduction in both locoregional and distant metastases was observed, in agreement with the results from a retrospective analysis on a small series of node-negative, ER-negative tumors.⁶ Moreover, in Amadori et al’s study, the benefit from CMF seemed directly and significantly related to the TLI value. Conversely, in our study, we observed that the anthracycline-containing FEC regimen produced an overall reduction of relapses that was significant only for locoregional recurrences. Furthermore, the benefit from chemotherapy did not seem to be directly related to tumor proliferative activity. This latter finding is in agreement with previous reports that did not highlight any relation between cell proliferation and clinical response to anthracycline-containing regimens.^14,38

Even though cautious interpretation of the results, obtained by analyzing small subsets of patients, is recommended, there is some biologic data to support the possibility that CMF and FEC polychemotherapies may achieve a different clinical benefit in women with tumors of different proliferative activity.

Data from in vitro experiments provide evidence to justify a specific cytokinetic basis; in fact, anthracycline causes a G2/M block,³⁹ which may compromise and reduce the efficacy of antimetabolites administered concurrently. Recently, Silvestrini et al⁴⁰ observed that while node-positive women with rapidly proliferating tumors benefited more from CMF adjuvant polychemotherapy, the sequence of doxorubicin followed by CMF was more effective in women with slowly proliferating tumors.

Alternatively, we can hypothesize that biologic cell characteristics other than proliferative activity may be predictive of resistance or sensitivity to anthracycline. A recent study by Paik et al supports this last hypothesis, demonstrating that HER2 tumor status is relevant in determining a preferential benefit from anthracycline-containing or CMF regimens in women with node-positive breast cancer.⁴¹

In conclusion, it would seem that the analysis of proliferative activity and HER2 status of primary breast cancer could provide different information that is potentially important for selecting the most effective adjuvant polychemotherapy regimen.

	ACKNOWLEDGMENTS

 
Funded in part by the Consiglio Nazionale delle Ricerche Project PF39 grant nos. 92.02342, 93.02321, 94.01285, 95.00523, 96.00719, and 94.ST97, by Associazione Italiana Ricerca sul Cancro, and by Ministry of Health of Italian Gouvernement grant no. PF92/157.

We thank Professor Rosella Silvestrini for her crucial scientific help during all phases of the study and manuscript preparation.

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Submitted August 29, 2000; accepted June 1, 2001.

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