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Disease-Free Survival Advantage of Adjuvant Cyclophosphamide, Methotrexate, and Fluorouracil in Patients With Node-Negative, Rapidly Proliferating Breast Cancer: A Randomized Multicenter Study

By Dino Amadori, Oriana Nanni, Maurizio Marangolo, Paolo Pacini, Alberto Ravaioli, Andrea Rossi, Angelo Gambi, Giuseppina Catalano, Davide Perroni, Emanuela Scarpi, Donata Casadei Giunchi, Amelia Tienghi, Aldo Becciolini, Annalisa Volpi

From the Department of Oncology, Pierantoni Hospital; Istituto Oncologico Romagnolo, Forlì; Department of Oncology, S Maria delle Croci Hospital, Ravenna; Oncology Day Hospital, Radiotherapy Unit, Careggi Hospital; Department of Clinical Physiopathology, University of Florence, Florence; Department of Oncology, Degli Infermi Hospital, Rimini; Oncology Unit, Bufalini Hospital, Cesena; Oncology Unit, Degli Infermi Hospital, Faenza; Department of Oncology, S. Salvatore Hospital, Pesaro; and Department of Oncology, S Croce e Carle Hospital, Cuneo, Italy.
Deceased.

Address reprint requests to Dino Amadori, MD, Department of Medical Oncology, Pierantoni Hospital, Via Forlanini 34, 47100 Forlì, Italy; email divonco{at}ausl.fo.it or i.o.r@fo.nettuno.it.

	ABSTRACT

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PURPOSE: According to one of the most recent key scientific questions concerning the use of biomarkers in clinical trials, we investigated whether node-negative breast cancer patients, defined as high-risk cases on the basis of tumor cell proliferation, could benefit from cyclophosphamide, methotrexate, and fluorouracil (CMF) adjuvant therapy.

PATIENTS AND METHODS: Two hundred eighty-one patients with negative nodes and rapidly proliferating tumors, defined according to thymidine labeling index (TLI), were randomized to receive six cycles of CMF or no further treatment after surgery ± radiotherapy.

RESULTS: The 5-year disease-free survival (DFS) was 83% for patients treated with CMF compared with 72% in the control group (P = .028). Adjuvant treatment reduced both locoregional and distant metastases. When clinical outcome was analyzed in cell kinetic subgroups characterized according to tertile criteria, compared with patients in the control arm, 5-year DFS was significantly higher after adjuvant CMF in patients with TLI values in the second (78% v 88%, respectively; P = .037) and third tertiles (58% v 78%, respectively; P = .024).

CONCLUSION: The results from this randomized clinical study indicate that patients with node-negative, rapidly proliferating tumors significantly benefit from adjuvant CMF.

	INTRODUCTION

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SEVERAL CLINICAL studies on large case series have consistently shown a risk of relapse at 5 and 10 years of approximately 20% and 30%, respectively, for patients with node-negative operable breast cancer.^1-4 This finding has led to the search for biologic markers capable of identifying patients at risk and predicting response to treatment.

The first and most widely investigated biologic marker is steroid receptor status. In particular, estrogen receptors (ERs) have been analyzed as a prognostic variable and, subsequently, as a predictor of response to systemic hormonal or chemical therapy. A direct relation between steroid receptor content and response to hormone therapy, especially tamoxifen, has repeatedly been reported.⁵ Moreover, some important randomized adjuvant clinical studies on node-negative, ER-negative breast cancer patients have shown a high benefit both in terms of disease-free survival (DFS) and overall survival from different types of chemotherapy compared with locoregional treatment alone.^6-12

More recently, cell proliferation has been repeatedly investigated to define its prognostic and predictive role.¹³ In clinical tumors, no general conclusions can be drawn because the different approaches used to determine cell kinetics require separate analyses. However, the studies performed using the proliferation index, based on [³H]-thymidine incorporation, have validated its relevance as an independent and, over time, consistent prognostic marker.^14,15 Moreover, studies on experimental tumors have shown a direct relation between cell proliferation and response to antitumor drugs.¹⁶ In clinical tumors, a major benefit has been observed for rapidly proliferating advanced breast cancers after polychemotherapy including antimetabolites.^17-21 Information on operable breast cancer patients is available from only one study on a small case series of node-negative, ER-negative breast cancer patients.¹¹ The prognostic and predictive relevance of oncogenes, p53 and c-erbB-2, or apoptosis-related markers, bcl-2 and bax, have also been investigated, but the results are not always consistent.^22-32

The main aim of the present study was to investigate whether node-negative breast cancer patients, defined as high-risk cases on the basis of tumor cell proliferation, could benefit from adjuvant chemotherapy in terms of DFS. For this purpose, in 1989, we activated a prospective multicenter clinical protocol in which patients with rapidly proliferating tumors, regardless of steroid receptor status, were randomized to receive locoregional therapy (mastectomy or quadrantectomy plus radiotherapy) alone or followed by cyclophosphamide, methotrexate, and fluorouracil (CMF), considered as the most effective drug combination at that time.

	PATIENTS AND METHODS

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Clinical Protocol
Patients with slowly proliferating tumors received only locoregional therapy (surgery with or without radiotherapy) until early relapse, whereas patients with rapidly proliferating tumors (as defined below) were randomized to receive locoregional treatment alone or followed by adjuvant chemotherapy.

Patients were eligible for the study if they met the following criteria: female 70 years of age who underwent radical resection (mastectomy or quadrantectomy plus radiotherapy) for invasive breast cancer, with histologic and clinical confirmation of no axillary lymph node involvement (at least 10 lymph nodes examined) or distant metastases, and with thymidine labeling index (TLI) and ER status information. Patients also had to satisfy the following requirements: peripheral WBC count 4,000/mL, platelet count 100,000/mL, creatinine level 1.5 mg/dL, bilirubin level 1.5 mg/dL, and good hepatic function (-glutamyltransferase less than twice the normal maximum limit).

Pregnant patients, patients with a previous malignancy or with bilateral breast cancer, patients who had received prior systemic therapy for cancer, or those with any nonmalignant systemic disease (including a history of heart disease) that would preclude their receiving the treatment option or prevent prolonged follow-up evaluation were not eligible for the study. The patients were required to be accessible for follow-up, and their informed consent was obtained before assignment to treatment. The study protocol was approved by the institutional review boards of each participating center.

Within 6 weeks of surgery, patients eligible for the randomized study were allocated either to receive no systemic treatment or six courses of CMF. Before randomization, patients were stratified prospectively within each center on the basis of ER content (< 10 fmol/mg, 10 to 49 fmol/mg, and > 49 fmol/mg). Randomization was carried out via telephone by the Forlì data center, which collected all documentation and handled the data management of all patients. A system of random permuted blocks within the strata was used with a block size of four.

A clinical, hematologic, and biochemical assessment for each patient was performed at 3-month intervals for the first 2 years, every 6 months during years 3, 4, and 5, and then once a year up to year 10. During the year 1, a biannual liver scan and chest x-ray were carried out; during year 2, a chest x-ray was performed at the 6 and 12 months and a liver scan at the 12 months. Subsequently, both examinations were carried out once a year. Moreover, from the time of randomization, all patients underwent an annual mammography and bone scan.

Routine follow-up evaluation was carried out, when possible, by an oncologist or by the patient’s general practitioner if the patient no longer attended the participating oncology center. Initially, treatment and follow-up data were collected on case record forms in no carbon required booklets. Data were then recorded on a computerized database designed specifically for the management of clinical trial data.

Feasibility information and reasons for study exclusion are reported in Fig 1 for six of the nine centers where clinical, pathologic, and biologic information was recorded for all breast cancer patients diagnosed during the recruitment period. In particular, the first cause of study exclusion was the 24% TLI nonassessability rate, which has now decreased to approximately 5%, and, subsequently, to some objective and insurmountable reasons.

View larger version (19K): [in this window] [in a new window]   Fig 1. Feasibility profile for 6 of the 9 centers involved in the multicenter clinical protocol.

Information on dates and doses of chemotherapy as well as on chemotherapy-induced side effects was recorded. Toxicity and complete blood cell count were evaluated according to World Health Organization (WHO) criteria on the first day of each cycle of therapy. Dose modification procedures in the case of hematologic toxicity were outlined in the protocol. When WBC count was less than 3,500/mL or platelet count less than 100,000/mL on the first day of therapy, treatment was delayed for a maximum of 2 weeks. If normal values had not recovered after this time, a 25% dose reduction was recommended for a WBC count of 3,000 to 3,499/mL and/or a platelet count of 75,000 to 99,999/mL, and a 50% reduction was recommended for a WBC count of 2,500 to 2,999 and a platelet count of 75,000/mL. Treatment was suspended for a WBC count of less than 2,500/mL or a platelet count of less than 75,000/mL. In the event of disease relapse, therapy was at the discretion of the participating centers.

Biologic Determinations
Fresh tumor samples were incubated in culture medium containing [³H]-thymidine for 1 hour at 37°C and then fixed in formalin.³³ The recent availability of a commercial kit (Euroframe, Asti, Italy) enabled all the clinical participating centers to easily perform this first step of in vitro tumor labeling with [³H]-thymidine. Samples from all centers were then sent to Forlì for autoradiographic procedures and TLI determination, with the exception of the centers in Cuneo and Florence, Italy, which performed their own determinations. Histologic sections were dipped in a photographic emulsion (Ilford K5; Ilford Photographics, London, United Kingdom) and exposed in the dark for 3 days at 4°C. Autoradiograms were developed in Ilford Phenprint for 6 minutes at 19°C and fixed in Hypam compound (both from Ilford) for 10 minutes. Samples were stained with hematoxylin and eosin at 4°C. When the specimen was small enough to allow the radioactive precursor to penetrate completely, labeled cells were counted throughout the whole section; if not, counting was limited to the periphery of the section (up to 80 µm in depth). 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 5,000 cells scored from different fragments of the same tumor. Quality control procedures were periodically repeated within the context of a National Quality Control Program initiated in the late 1980s by the Italian Society of Basic and Applied Cell Kinetics.³³

Rapidly proliferating tumors were defined on the basis of a 3.1% cutoff value, which represents the median value of the overall series of 360 patients recruited from the beginning of TLI determinations in the Forlì laboratory (January 1987) up to the activation of this study (June 1989). This median value is similar to that observed by other groups.^30,34

ER and progesterone receptors (PgR) were assayed by the dextran-coated charcoal method according to the European Organisation for Research and Treatment of Cancer.³⁵ Quality control procedures for hormone receptor dosage were coordinated by the Italian ad hoc committee. Quantitative biochemical analysis was adopted to allow the use of different cutoff values and to identify different steroid receptor content subgroups for future basic and clinical analyses. In the present study, ER content was classified into three categories (< 10 fmol/mg, 10 to 49 fmol/mg, and > 49 fmol/mg).

Statistical Analysis
Sample size was determined a priori during the planning of the study, with DFS considered as the primary clinical end point. Starting from an assumed 70% 5-year DFS from diagnosis for patients with rapidly proliferating, node-negative tumors treated with locoregional therapy alone (control arm)³⁴ and hypothesising an absolute increase of 13% in patients treated with chemotherapy (5% error fixed for a two-sided test and a power of 80%), a recruitment of 270 patients over 3 years was required. No interim analysis was planned.

DFS was considered as the time elapsed from the date of surgery to the first documented evidence of new disease manifestation in locoregional or distant sites or in the contralateral breast. Owing to the difficulty of distinguishing between a second breast carcinoma and contralateral recurrence, the latter lesion was considered as an event. In the case of a second primary cancer in a non-breast site, disease-free follow-up data were censored at the time of the diagnosis of the second malignancy. All new disease manifestations were assessed by clinical, radiologic, and when feasible, histologic examination. DFS probability and the 95% confidence interval (CI) were computed by the Kaplan-Meier product-limit method.³⁶ The null hypothesis concerning the differential effect of treatment in univariate analysis was tested by a stratified (according to center and ER content: < 10 fmol/mg, 10 to 49 fmol/mg, and > 49 fmol/mg) log-rank test.³⁷

Analysis for TLI as well as for other clinical, pathologic, and biologic subgroups was planned in advance as a secondary aim with explorative intent. Results were analyzed according to the intent-to-treat principle, ie, patients were evaluated on the basis of their assigned therapy.

Estimated hazard ratios (the ratio of the treated group to the control group), their 95% CI, and P values were calculated from proportional hazards regression models stratified according to center and ER content (< 10 fmol/mg, 10 to 49 fmol/mg, > 49 fmol/mg). The multivariate model was used to investigate potential confounding factors.³⁸ All P values were based on two-sided testing, and statistical analyses were carried out with SAS Statistical software (SAS/STAT User’s Guide, Version 6; SAS Institute, Cary, NC, 1990).

	RESULTS

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Between 1989 and 1993, a total of 281 patients were entered onto the randomized trial by nine participating Italian centers (see Appendix). Of these, 138 were allocated to receive postsurgical adjuvant CMF, and 143 were to receive locoregional therapy alone. Three patients (1.1%) were found to be ineligible after randomization (two patients in the control group, one for liver metastases and one for bilateral breast carcinoma, and one patient in the CMF group because of -glutamyltransferase > twice the normal maximum limit). The results presented refer to 278 eligible randomized patients analyzed according to the intent-to-treat principle.

Clinical and biologic characteristics of the case series are listed in Table 1. Overall, median patient age was 52 years (range, 26 to 69 years), and approximately 42% of patients were premenopausal. Sixty-four percent of patients had a tumor 2 cm in size, and approximately 75% were of ductal type. A median of 16 lymph nodes were examined (range, 10 to 37 nodes); in only 25% of cases, the number of lymph nodes examined was from 10 to 12. In regard to receptor content, 65% of patients had positive ER disease ( 10 fmol/mg), and approximately 50% had positive PgR tumors ( 25 fmol/mg). Fifty-eight percent of patients had conservative surgery plus radiotherapy. All the clinical, pathologic, and biologic characteristics were well-balanced in the control and CMF adjuvant subgroups.

The 5-year DFS was 83% (95% CI, 77% to 89%) for patients treated with adjuvant CMF compared with 72% (95% CI, 65% to 79%) in the control group (log-rank = 4.82, P = .028) (Fig 2). There were 47 relapses or deaths in the control group and 28 in the CMF group; relapse sites are listed in Table 2. Compared with the group treated only with surgery, in the surgery plus CMF group, distant metastases were reduced from 30 to 17, respectively, and locoregional recurrences were reduced from nine to four, respectively.

View larger version (18K): [in this window] [in a new window]   Fig 2. DFS in control patients and CMF-treated patients (log-rank = 4.82, P = .028).

View larger version (19K): [in this window] [in a new window]   Fig 3. DFS according to TLI value in control patients and CMF-treated patients: (A) TLI value 3.1% to 4.4% (log-rank = 0.086, P = .769); (B) TLI value 4.5% to 6.8% (log-rank = 4.37, P = .037); and (C) TLI value > 6.8% (log-rank = 5.07, P = .024).

The unadjusted DFS estimate (hazard ratio = 0.59; 95% CI, 0.36 to 0.95) was similar to that adjusted by age, histology, tumor size, number of examined lymph nodes, type of surgery, TLI, and PgR content (hazard ratio = 0.55; 95% CI, 0.33 to 0.91). In regard to the effect of chemotherapy on survival, up to now, 23 deaths in the control group and 19 deaths in the CMF group have been registered, and the 5-year overall survival for both groups is 89% (95% CI, 83% to 94%) and 91% (95% CI, 86% to 96%), respectively.

Treatment Compliance and Toxicity
Of 137 eligible patients assigned to the CMF arm, 123 (89.8%) completed the six planned courses of CMF. Two patients stopped after the fifth cycle, one for worsened health conditions (uncontrollable hyperglycaemia) and the other for infection and prolonged WHO grade 4 leukopenia (according to planned dose modification criteria). One patient stopped after the fourth cycle, one after the third, one after the second, and three after only one cycle because they refused further treatment. Six patients (4.4%) refused to begin treatment.

Adjuvant CMF was generally well-tolerated. Nausea and vomiting were frequent symptoms, but only 11 patients (8.4%) had WHO grade 3 nausea-vomiting, and only one patient (0.8%) reported grade 4 nausea-vomiting. There were other episodes of WHO grade 3 toxicity, which included diarrhea (one patient), conjunctivitis (one patient), stomatitis (one patient), cystitis (two patients), gastrointestinal problems (two patients), and liver problems (one patient). Alopecia was frequent, but hair regrowth was documented in all patients. According to protocol criteria, treatment was suspended in the presence of myelosuppression in an attempt to administer full drug doses. Of an overall 760 cycles, hematologic toxicity required a delay of 1 week in 153 cycles (20.1%) and of 2 weeks in 33 cycles (4.3%). In patients treated with the six planned cycles, approximately 90% had no dose reduction, nine patients (7%) had a 10% to 25% dose reduction, and six patients (5%) had more than a 25% dose reduction. No toxic death occurred.

	DISCUSSION

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The important results achieved by adjuvant chemotherapy in the 1970s have not progressed over time. Different lines of research are currently being pursued in an attempt to further improve the management of cancer patients. Among these, great effort has been put into the search for new and non–cross-resistant drugs and regimens and, recently, for novel approaches to biologic therapy. At present, one of the possible ways to improve therapeutic results lies in the use of biologic markers for planning clinical protocols, in agreement with the recommendations of Consensus Conferences and the National Cancer Institute Breast Cancer Progress Review Group.^39,40

Up to now, steroid receptor status has been used in some randomized studies to select node-negative patients to be treated with adjuvant chemotherapy.^6-12 With regard to cell proliferation, a recent study used flow cytometric S-phase cell fraction, together with steroid receptors, to identify high-risk node-negative breast cancer patients who were candidates for adjuvant chemotherapy.⁴¹

To our knowledge, the study we conducted represents one of the first adjuvant trials prospectively planned on a biologic rationale that uses cell kinetics as a prognostic tool for defining high-risk node-negative breast cancer patients. This approach is in agreement with one of the most recent key scientific questions concerning the use of biomarkers that could predict clinical outcome and, possibly, response to treatment in an adjuvant setting.⁴⁰ In particular, cell proliferation could help to answer the question of who needs and will benefit from chemotherapy and who does not require further therapy after locoregional treatment.

Among the various methods currently used to determine cell proliferation, we chose TLI because experimental and clinical studies have demonstrated its relevance as an indicator of risk and a predictor of response to antitumor drugs.¹⁵ One of the major drawbacks to the translation of cell proliferation variables into clinical practice is the lack of standardization among laboratories that measure these markers. In our experience, this problem has largely been overcome through a National Quality Control Program, which ensures intra- and interlaboratory reproducibility of results.³³

To perform this study, we were faced with a huge amount of work to determine TLI in a large consecutive number of patients. In fact, to randomize 281 patients who satisfied eligibility criteria onto the trial, more than 2,000 newly diagnosed breast cancers were consecutively evaluated for the biologic variable. Despite this difficulty, the number of patients necessary for the study was recruited within the planned period.⁴²

The results of this study indicate that patients with node-negative, rapidly proliferating tumors benefit from adjuvant CMF with respect to only locoregional treatment, with a reduction in the annual relapse risk of approximately 40% associated with an 11% absolute benefit for DFS at 5 years. The benefit we observed is comparable with that reported in other similar randomized clinical studies in which the worth of adjuvant chemotherapy was analyzed in specific biologic subgroups, ER-negative^6,7,10 or ER-negative and ER-positive tumors of 3 cm or more in diameter.^8,9

In our study’s untreated control group, DFS at 4 years was 75% compared with 73% in the Ludwig study,¹⁰ and at 5 years, it was 72% compared with the extrapolated data of approximately 68% by Fisher et al,⁷ 65% by the Intergroup Study,⁹ and the lower 50% by Zambetti et al.^11,12 However, it must be pointed out that the case series considered in the various studies are different in regard to biologic and pathologic eligibility criteria and duration of follow-up. Moreover, as literature data report an advantage in terms of OS for the chemotherapy-treated arm starting from a median follow-up of at least 8 years,^7,9 with the exception of Zambetti’s study,¹¹ a definitive analysis of the effect of treatment on survival will be performed at a longer follow-up.

An analysis of relapse sites showed that adjuvant treatment reduced both locoregional recurrences (from nine [6.4%] in the control group to four [2.9%] in the CMF group) and distant metastases (from 30 [21.3%] in the control group to 17 [12.4%] in the CMF group). An equivalent decrease in the rate of local relapse but a lower benefit regarding distant relapse have been reported in other studies.^7-10 Moreover, the frequency of relapse in different sites was similar in the treatment and control groups (19% v 14% locoregional recurrences, 64% v 61% distant metastases, and 15% v 21% contralateral cancers, respectively). Furthermore, a breakdown analysis of different pathologic, clinical, and biologic subgroups was conducted to explore one of the main study objectives, that is, the identification of a suitable treatment for patients selected on the basis of clinical and biologic characteristics. Classifying patients with respect to menopausal status, our results suggest a greater benefit for pre- rather than postmenopausal patients, in agreement with those from other studies.^8-10 The analysis carried out on the group of patients with small tumors ( 2 cm) revealed an increase in DFS for those treated with CMF (87% compared with 74% for the control group). This result is in contrast to that of the Intergroup Study,^8,9 which did not report a significant advantage for chemotherapy-treated patients whose tumors measured less than 3 cm in size with respect to the control group. It must be stressed that the two patient populations are not comparable because in the Intergroup Study, patients with less than 3 cm tumors were eligible only if they were ER negative, regardless of cell kinetic characteristics. Furthermore, in our case series of rapidly proliferating lesions including both ER-negative and ER-positive tumors, for the first time, a higher benefit was observed in patients with high PgR–level tumors.

It must be emphasized that a significant reduction in the hazard ratio was observed only for the subsets of patients with higher proliferating tumors. However, by considering that the number of events was similar in the three cell kinetic subgroups treated with adjuvant CMF, the lack of a significant effect in patients with slowly proliferating tumors may be attributed to a lower number of events in this control group at 5 years. Therefore, it cannot be excluded that, at a longer follow-up and even in this relatively less aggressive tumor subgroup, an increase in the number of events could result in a significant impact of CMF.

Overall, results show that TLI is capable of identifying high-risk patients who will benefit from adjuvant chemotherapy. Nevertheless, in all these subgroups, six cycles of CMF did not prevent relapse in approximately 20% of patients. Therefore, a new trial has recently been started to evaluate the efficacy of CMF alone versus CMF plus anthracyclines given in different sequences in node-negative and one to three node-positive patients with biologically aggressive tumors, defined on the basis of tumor proliferative activity.

On the basis of the data reported in our and other trials, there is general agreement on the advisability of treating the majority of node-negative breast cancer patients, but at the same time, it is necessary to carry out a detailed analysis of the cost/benefit ratio for each patient in relation to tumor characteristics and, consequently, to the different risk rates of relapse and death. For this reason, it is vital to identify those patients who are candidates for adjuvant therapy and, subsequently, to plan treatment that will bring about the greatest advantages.

In conclusion, our study highlights a significant improvement in DFS for the CMF group with respect to the control group. Therefore, patients with node-negative highly aggressive tumors, as defined by cell kinetics, really do benefit from and must be treated with adjuvant chemotherapy.

	APPENDIX Participating Investigators and Institutions

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	ACKNOWLEDGMENTS

We thank Rosella Silvestrini, PhD, for her invaluable scientific contribution and Gráinne Tierney for editing the manuscript.

	REFERENCES

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Submitted November 29, 1999; accepted May 1, 2000.

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