Abstract
Background: Poor prognosis is associated with estrogen- and/or progesterone receptor-positive (ER+, PGR+) premenopausal breast cancer (PM-BC) with high Ki-67 labeling index and extensive axillary lymph node involvement. The role of adjuvant chemotherapy (CT) and hormonal therapy have not yet been established in these patients. Patients and Methods: Twenty-five PM-BC patients received, in sequence, leuprorelin, taxane-anthracycline induction chemotherapy, radiation therapy, a platinum-based intensification high-dose CT, followed by leuprorelin and anastrazole for five years. Vascular endothelial growth factor (VEGF) levels were measured as the primary end-point; secondary end-points were 10-year relapse-free survival (RFS) and overall survival (OS) rates. Results: The median patient age was 44 years, and the mean number of positive axillary nodes was 14. All patients were ER+ and/or PGR+, with a median Ki-67 index of 33%. Five patients were Cerb-B2 positive. Grade 4 hematologic toxicity was observed in all patients, no patient showed a decrease of cardiac ejection fraction and hot flashes and arthralgias were of moderate intensity. After a median follow-up of 70 months, VEGF levels significantly decreased (p<0.001); 10-year RFS and OS were 76% and 78%, respectively. Conclusion: Total estrogen blockade and high-dose CT in PM-BC patients is feasible, has moderate toxicity, significantly reduces VEGF levels, and seems to improve the expected RFS and OS.
Patients with early breast cancer and more than 10 involved axillary nodes have a grim prognosis: 10-year overall survival (OS) rates with and without adjuvant chemotherapy have been reported as 30% and 20%, respectively (1). Young age and positivity of estrogen receptors (ER+) worsens long-term prognosis (2) due to a continued risk of relapse for many years after initial breast cancer diagnosis (3). Chemotherapy, which has been shown to improve the disease-free survival of patients with high-risk ER+ early breast cancer (4), in premenopausal women, may serve a double function: cytotoxicity and late-induced amenorrhea, associated with improved outcomes in premenopausal ER+ patients (5). In addition, ovarian suppression with a luteinizing hormone-releasing hormone (LH-RH) significantly reduces circulating estrogens and improves relapse-free survival (RFS) and OS in premenopausal women with early breast cancer (6).
Estrogens, besides playing an important role in the initiation and progressive growth of human breast cancer (7), have two additional important functions: induction of angiogenesis through vascular endothelial growth factor (VEGF) (8) and regulation of peripheral development of CD4+CD25+ T-regulatory cells (T-regs) (9).
Firstly VEGF, the key mediator of angiogenesis (10), is associated with poor prognosis when overexpressed in breast cancer (11) and changes in circulating VEGF levels have been related to clinical responses to chemotherapy in metastatic breast cancer (12). Secondly estrogens, by regulating T-reg numbers, modulate the immune response against tumor antigens, and, therefore, have a fundamental role in the progression of breast cancer (13-15). Both these properties could be exploited for therapeutical intervention: reducing plasma estrogen, we might decrease both plasma VEGF and T-regs and thus, improve the prognosis. Hence, measurement of plasma VEGF could be an important intermediate end-point.
Aromatase inhibitors have been shown to be superior to tamoxifen in postmenopausal patients (16-18). However, the use of aromatase inhibitors in premenopause may lead to increased gonadotropin secretion due to the reduced estrogen feedback to the hypothalamus and pituitary, with subsequent stimulation of ovarian activity (19). Therefore, ovarian suppression with an LH-RH agonist would permit the administration of an aromatase inhibitor shown to induce more intense estrogen suppression than tamoxifen in premenopausal patients with early breast cancer (20).
The primary end-point of the present study was to investigate whether administration of an LH-RH analog followed by chemotherapy, radiation therapy and an aromatase inhibitor would reduce VEGF levels with respect to baseline values. Secondary endpoints were the evaluation of RFS and OS.
Patients and Methods
Patient selection. Premenopausal patients with histologically confirmed ER+ and/or progesterone receptor (PGR)+ primary breast cancer were screened for the following inclusion criteria: stage pT1-3 or pT4b, pN greater than nine, M0, and Ki-67 labeling index >30%. Women were considered premenopausal if they had normal menses with plasma estradiol levels >40 pg/ml. Patients were enrolled either at the Civilian Hospital of Avezzano or at the University of L'Aquila and the Carlo Ferri Foundation, Monterotondo, Rome, Italy. The study was conducted after approval was obtained from the local Ethics Committee of the participating institutions and each patient provided written informed consent. The primary local treatment consisted of mastectomy or breast-conserving surgery with axillary dissection, with at least 10 identifiable and involved lymph nodes in the specimen. The following ranges for laboratory parameters served as additional inclusion criteria: creatinine <2 mg/dl, bilirubin <2 mg/dl, hemoglobin >10 g/dl, platelets >100,000/dl, and neutrophils >2,000/dl. Participants were excluded if they had heart disease or reduced lung function, if they had received any prior hormonal treatment, if they were pregnant or lactating, if they had an active infection, or if they had a history of other active malignancies in the past five years except in situ cervical carcinoma or non-melanoma skin cancer.
Treatment. Three weeks after surgery, 11.25 mg leuprorelin was administered and repeated every 84 days, during chemotherapy and afterwards, for five years, to maintain serum estradiol levels below 40 pg/ml. Six courses of 90 mg/m2 epirubicin and 75 mg/m2 docetaxel on day 1 (D1) were administered every 21 days as induction chemotherapy. After the sixth course, granulocyte colony-stimulating factor (7 μg/kg) was administered from D2 to D10 and peripheral blood progenitor cells (PBPC) were collected by apheresis. Radiation therapy was delivered at 50 Gy, 2 Gy/fraction, five fractions per week, to the chest wall after mastectomy or to the residual breast after breast-conserving surgery, at the apex of the axillal and supraclavicular lymph nodes. A boost of 10 Gy was delivered to the tumor bed. One to two months after the end of radiation therapy, when recovery from the toxicity was complete, patients received an intensification high-dose chemotherapy (HDCT) regimen consisting of carboplatin at an area under the curve (AUC) of 7, 9000 mg/m2 ifosfamide, and 300 mg/m2 etoposide, intravenously over three consecutive days, followed by PBPC transplantation and by hematopoietic growth factors. One month after HDCT, endocrine therapy with leuprorelin and 1 mg/day anastrazole was administered to all patients for five years. During this study period, patients received psychological support, vitamin D, and monthly intravenous bisphosphonates.
Characteristics of patients.
Follow-up. At the beginning of the study, participants underwent history and physical examinations and assessment of performance status. Laboratory studies included: complete blood counts (CBCs), liver and renal function tests, estradiol, progesterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), VEGF, CD4+/CD8+ ratio, carcino-embryonic antigen (CEA), and carbohydrate antigen CA 15-3. Bone scanning and computed tomography of the chest and liver were also performed. During the administration of chemotherapy and radiation therapy, CBC was repeated weekly and (SMA-14) monthly. One month after HDCT, serum VEGF levels, CD4+/CD8+ ratios, and natural killer cell levels (NK) were measured. During hormonal therapy, laboratory studies were repeated every four months and included plasma estradiol, progesterone, FSH, LH, VEGF, CD4+/CD8+ ratio, CEA, and CA 15-3. After January 2004, measurements of T-reg levels became available; the last ten patients entered into the trial were monitored for this parameter during follow-up. Mammograms and computed tomography of the chest and liver were repeated yearly.
Biological parameters measured during the course of the study.
Statistical analyses. The primary end-point of the study was to assess whether this therapy regimen produced a decrease in VEGF levels, with respect to baseline values. The number of patients required for the study was calculated according to a Simon minimax design (21). The first stage required at least one of 15 patients to exhibit a confirmed VEGF decrease to rule out an undesirably low response probability of 0.1 (P0) for a desirable probability of 0.30 (P1), with a 5% probability of accepting a poor agent (α=0.05) and a 20% probability of rejecting a good agent (β=0.20). In the second stage, a total of 10 assessable patients were to be added if five or more patients showed a confirmed VEGF decrease, meeting the primary end-point. The results of the immunological laboratory tests are expressed as the mean±standard deviation of four determinations, and the differences were determined using a repeated-measure analysis of variance. Secondary end-points were RFS and OS. The RFS was defined as the time between the start of HDCT with PBPC transplantation to any relapse and the appearance of a second primary cancer or death, whichever occurred first. The OS was measured from study entry to death, or May 2010 for censored patients. Statistical analysis of RFS and OS was performed using the Kaplan-Meier method (22). All comparisons were performed using Pearson's χ2 contingency table analysis. Patients were evaluated for toxicity at each study visit according to the National Cancer Institute Common Toxicity Criteria Version 2.0.11. Statistical analysis was performed with SAS statistical software (version 8.12, 2000; SAS Institute Inc, Cary, NC, USA).
Results
Patient characteristics. Patient characteristics are listed in Table I. Between May 2000 and May 2005, a total of 25 patients received the therapy regimen described above. Twenty patients were both ER+ and PGR+, while five patients were ER+ and PGR−. Five patients had Cerb-B2 overexpressed or amplified disease. Twenty patients exhibited ductal-infiltrating carcinoma and five had lobular-infiltrating carcinoma. Modified radical mastectomies were performed in 14 patients and partial mastectomies in 11 patients. The median patient age was 44 years (range 34-49 years). The mean Ki-67 index was 39%, while the mean value of serum VEGF before chemotherapy was 508±69 pg/mm3. The median duration of treatment from initiation of leuprorelin and anastrazole was five years.
Variations in serum estradiol: baseline, after 6 months and after 12 months.
Efficacy. After one year of hormonal therapy, VEGF levels significantly decreased with respect to the values recorded after PBPC transplantation: in fact, the mean value of serum VEGF levels of 421±75 pg/mm3, decreased, progressively, to 134±29 pg/mm3 after one year, 88±12 pg/mm3 after three years, and 55±21 pg/mm3 after five years. In parallel, we observed a progressive increase of the CD4+/CD8+ ratio, an increase in the levels of NK cells, and a decrease in T-regs levels in 10 patients (Table II). In particular, T-regs decreased by 30% after one year of therapy, with further decreases after three (54%) and five years (62%).
Estradiol levels were evaluated at various time points to assess the hormonal status. The dose of 11.25 mg leuprorelin administered every 84 days was not sufficient in three patients to keep castration levels of estrogen (Figure 1). In two of these patients, the dose of leuprorelin had to be administered every 56 days and, in one patient, every 28 days. The 10-year RFS and OS rates were 76%, and 78%, respectively (Figure 2). Two patients suffered local regional failures after a median period of 29 months, three patients had distant failures after a median period of 19 months, and two patients displayed a second cancer (small-cell lung cancer and cervical cancer) after a median period of 44 months. No patient had disease relapse after five years. All six patients with disease recurrence had PGR - tumors, confirming a possible role of PRG expression in determining tumor endocrine responsiveness. CEA, measured before the first cycle of chemotherapy, was elevated in 19/25 patients, while 10/25 patients had elevated CA15-3 levels (>25 U/ml).
Relapse free survival (RFS) and overall survival (OS) of all patients.
Safety. Toxicity frequencies and grades are shown in Table III. Grade 4 hematological toxicity was observed in all patients after HDCT. The median time from transplantation to engraftment of leukocytes at a value greater than 1,000/dl was 10 days (range 7 to 13 days). The median time to engraftment of platelets at a value greater than 20,000/dl was 10 days (range 5 to 15 days). One episode of fever was observed in eight patients during the aplastic period following transplantation. Infection was diagnosed in three patients (12%). No patient died due to treatment-related causes. No statistically significant decrease of the baseline values of ejection fraction was observed after the end of treatment. Grade 2 skin toxicity was observed in three patients after radiation therapy. The most frequent toxicities of hormonal therapy were hot flashes (45%), arthralgia (20%), and fatigue (15%).
Discussion
Following treatment of 25 ER+ breast cancer patients with a combined multimodality approach, we observed 10-year RFS and OS rates of 76% and 78%, respectively with acceptable toxicity profiles.
Chemotherapy with docetaxel and epirubicin, followed by radiation therapy, was delivered sequentially over approximately 18 weeks in order to avoid the possible development of resistance (23). Subsequently, a course of HDCT with carboplatin, ifosfamide and etoposide was administered when the tumor burden was low. These non-crossresistant drugs were delivered at high doses to maximize the probability of eradicating micrometastatic disease after standard chemotherapy and radiation therapy.
After HDCT and PBPC transplantation, a depressed immune function was observed, with an elevated VEGF level, low CD4+/CD8+ ratio, and low level of NK cells (Table II). These observations agree with previous reports; NK levels and T-cell function decreased following adjuvant therapy for early breast cancer (24), and breast cancer patients exhibited markedly reduced blood dendritic cell counts at diagnosis (25). After chemotherapy, we observed a progressive improvement of the immune function that paralleled the decrease in VEGF levels. Previously, VEGF was shown to cause a deficiency in the number and functional maturation of dendritic cells from progenitors (26), and was demonstrated to act as an important mediator of tumor-associated immunodeficiency (27).
Toxicities experienced from therapy.
The long-term efficacy of the aromatase inhibitor anastrozole as a postsurgical adjuvant treatment for postmenopausal women with hormone-sensitive tumors has been clearly established (16). In a separate study, premenopausal node-positive women received chemotherapy followed by randomization to tamoxifen or placebo for five years. The patients with ER+ tumors receiving tamoxifen exhibited improved RFS at 5 years (75% versus 62%; hazard ratio (HR) 0.59; p<0.0001). However, for patients with ER+ disease, chemotherapy-induced amenorrhea was significantly correlated with improved RFS, with the magnitude of the benefit being independent of the use of tamoxifen (4).
Trial 15-95 randomized young high-risk breast cancer patients to standard-dose chemotherapy or to dose-intensive chemotherapy, followed by tamoxifen treatment. After a median follow-up period of 99 months, the patients randomized to dose-intensive chemotherapy demonstrated significantly improved RFS (47% versus 37%, p=0.05); however, this effect was observed only in patients with ER+ disease, suggesting that the efficacy of dose-intensive chemotherapy may be related to its endocrine effects (28).
After chemotherapy, we treated our patients for five years with concurrent anastrazole and leuprorelin, producing a total estrogen blockade. Recently, a group of premenopausal women with ER+ and PGR+ locally advanced operable breast cancers were treated with total estrogen blockade. A response rate of 50% (95% confidence interval 32-68%) was obtained, a rate higher than the response rates reported in postmenopausal women (24-35%) treated with preoperative endocrine therapy with aromatase inhibitors (29). However, a trend to an improved response rate was observed with longer therapy duration in these selected populations of premenopausal patients with endocrine-responsive tumors.
We observed no statistically significant difference (p<0.9) between the Ki-67 labeling index of patients with disease progression (35±4%) and the index of disease-free patients (35.7±5%). In five patients with high body mass index, the dose of leuprorelin was increased to maintain estrogen levels <40 pg/ml, in accordance with previous investigations (30).
The results of this study indicate that for ER+ premenopausal patients with high Ki-67 index and extensive axillary node involvement, measurement of VEGF may be considered as an acceptable intermediate endpoint and treatment with total estrogen blockade and dose intensive sequential multimodality treatment is feasible, safe, and seems to improve the expected RFS and OS rates.
- Received November 2, 2010.
- Revision received December 27, 2010.
- Accepted January 4, 2011.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
