1 Department of Medicine, 2 Division of Pathology and Laboratory Medicine and University of Milan School of Medicine, 3 Division of Senology, 4 Division of Epidemiology, European Institute of Oncology, Milan, Italy
Received 23 January 2003; revised 31 March 2003; accepted 17 June 2003
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Abstract |
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Experimental data on perioperative chemotherapy (PeCT) indicate that its initiation might be most useful if administered as close as possible to the time of first disturbance of the tumour. Regimens including 5-fluorouracil (5-FU) as continuous infusion are commonly used in the preoperative setting, especially for large tumours and locally advanced disease. We therefore evaluated the role of PeCT with 5-FU as continuous infusion after preoperative chemotherapy (PreCT), covering the surgical phase and acute wound healing period, in patients with breast cancer too large to attempt breast-conserving surgery upon diagnosis.
Patients and methods:
Breast cancer patients, clinical stages T2T3, N0N2, M0, and Ki-67 labelling index 20%, were treated every 3 weeks with a maximum of six courses of vinorelbine 20 mg total dose intravenously (i.v.) on days 1 and 3, cisplatin 60 mg/ m2 i.v. on day 1 and 5-FU 200 mg/m2/day as a continuous infusion (ViFuP regimen). Patients who achieved a clinical and radiological objective remission with PreCT were also treated with perioperative 5-FU that was continued until 30 min before, and restarted immediately after surgery, prolonging infusion until 15 days after surgery.
Results:
Following preoperative treatment, 39 of 49 evaluable patients [80%; 95% confidence interval (CI) 70% to 90%] had an objective response. Pathological complete remission (pCR) was achieved in 14 (29%) patients. No relevant clinical or haematological toxicity due to PeCT was observed. In 36 patients submitted to PeCT the rate of pCR was 33% (95% CI 18% to 48%). The highest response of the primary tumour to PreCT and PeCT was observed in women with tumours not expressing estrogen and progesterone receptors (pCR 46%; 95% CI 19% to 73%).
Conclusions:
Preoperative therapy can be protracted into the surgical (and wound healing) period without significant additional short-term toxicity. Proper selection of patients according to biological features might improve the therapeutic yield of preoperative therapies.
Key words: fluorouracil, operable breast cancer, perioperative chemotherapy
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Introduction |
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Preoperative chemotherapy (PreCT) has been demonstrated to be beneficial for patients with breast cancer, allowing breast-conserving surgery in some of the patients [8]. One of the most effective regimens used in this setting is the combination of continuous infusion of 5-fluorouracil (5-FU), epirubicin and cisplatin (ECF). Smith et al. reported high complete remission rates in 50 patients with large operable breast cancer treated with this regimen. Ninety-eight per cent of these patients had significant tumour shrinkage, 66% had a complete clinical remission, and 27% a pathological complete response [9]. Surgery was performed after completion of 6 months of chemotherapy. The same investigators recently compared preoperative ECF with adriamycin and cyclophosphamide (AC) in a phase III randomised trial involving 426 patients with invasive operable breast cancer. The interim analysis showed a significant survival benefit for continuous infusional 5-FU-containing chemotherapy over conventional AC [10].
The aims of the current study were to assess the feasibility and the biological effects on the tumour proliferative fraction (Ki-67 labelling index) of PeCT with 5-FU administered as a continuous infusion after primary chemotherapy protracted into the surgical (and wound healing) period. We also aimed to evaluate the effects of PreCT in a cohort of patients with high chance of response to chemotherapy (high baseline Ki-67 labelling index), based upon results of a previous study [11]. We selected a non-anthracycline-containing regimen (vinorelbine, cisplatin, fluorouracil as continuous infusion; ViFuP regimen) as PreCT, based upon results of a recent phase II trial in advanced breast cancer [12].
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Patients and methods |
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Patients with cardiac disease (congestive heart failure, history of myocardial infarction within the previous 3 months), severe vascular disease or uncontrolled concomitant infections were excluded. Patients had baseline liver and renal function tests, electrolyte studies and complete blood count performed within 2 weeks of inclusion in the study. Also, bilateral mammography and breast ultrasound, chest X-ray, abdominal ultrasound, bone scan, serum CA 15.3 determination, and electrocardiography were performed within 2 weeks from the treatment start. The study was carried out with ethical committee approval, and written informed consent from all patients was obtained.
Treatment
After insertion of a central venous cathether (CVC; Dome PortTM, Bard®), patients were treated with vinorelbine 20 mg total dose intravenous (i.v.) bolus on days 1 and 3, cisplatin 60 mg/m2 i.v. on day 1 over 1 h with appropriate saline hydration and 5-FU 200 mg/m2 i.v. daily as continuous infusion provided by a Portable Elastomeric Infusion System (Baxter®). Patients were instructed to change their own infusion bag every week. Courses were repeated every 3 weeks. Antiemetic prophylaxis included granisetron 3 mg i.v. and dexamethasone 8 mg i.v. PeCT consisted of infusion of 5-FU continued until 30 min before surgery and restarted immediately after surgery (15 days of 5-FU infusion after surgery were required). Patients received antibiotic prophylaxis with cefazoline 2 g i.v. immediately before surgery followed by 1 g twice a day intramuscularly for 3 days.
Tumour response was evaluated after each cycle, and surgery was planned in patients with clinically progressing disease. Response after each cycle was evaluated by clinical measurement of the two largest tumour diameters. After three cycles, patients also had mammography and ultrasound breast examination to assess response. Patients with stable disease were submitted to surgery. Patients with partial remission or complete remission were candidate to three more courses of chemotherapy and PeCT.
Toxicity and dose modifications
Toxicity was evaluated according to NCIC-CTG criteria by clinical and laboratory evaluations at day 21 of each cycle. Dose adjustments were performed according to the following criteria: if creatinine/blood urea nitrogen was up to 1.5x the upper normal value cisplatin was omitted. In cases of neuroconstipation, mucositis or diarrhoea (grade >2), the next cycle of chemotherapy was postponed by 1 or 2 weeks until recovery and then administered at 75% of the initial dose.
The treatment was postponed by 1 week if the blood count on day 21 showed a neutrophil count <1000/mm3 and/or platelet count <100 000 mm3. The treatment was re-administered on day 28 if the blood count on the same day showed a neutrophil count >1000/mm3 and a platelet count >100 000 mm3. In the opposite case, treatment was postponed by another week. If, after 2 weeks of treatment delay (on day 35), haematological recovery (neutrophils >1000 mm3 and platelets >100 000/mm3) was not obtained, treatment was discontinued because of marrow toxicity.
During PeCT, patients had weekly white blood cell evaluation, and every 2 weeks evaluation of liver and renal function tests, electrolyte studies and a visit.
Response criteria
Responses were evaluated according to both radiological (breast ultrasound plus mammography) and clinical evaluation and graded according to standard World Health Organization criteria. A complete response was defined as the disappearance of all parameters of disease by two observations not <4 weeks apart. A partial response was defined as a 50% reduction in the products of the perpendicular diameters of the lesion without any evidence of new lesions. Stabilisation of disease was defined as a <50% reduction or <25% increase in the products of the perpendicular diameters of the lesion without any evidence of new lesions. Progressive disease was defined as a >25% increase or the appearance of new lesions.
Pathology and immunohistochemistry
Surgical specimens were extensively sampled for the evaluation of residual tumour after primary chemotherapy. In cases of no macroscopic evidence of tumour the quadrantectomy specimens were entirely blocked in paraffin and examined histologically, as were the tumour-bearing quadrants of the mastectomies. In the latter cases, the other quadrants were also thoroughly evaluated with the examination of at least three tissue blocks.
Immunostaining experiments for the localisation of estrogen (ER) and progesterone (PgR) receptors, Her2/neu protein and Ki-67 antigen were performed on consecutive tissue sections of the core biopsies obtained before primary treatment, and of the residual tumour after surgery, as reported previously [11].
All immunoreactions were performed with an automated immunostainer (Autostainer; Dako, Glostrup, Denmark) using an avidinbiotin detection system. Briefly, the slides were dewaxed, rehydrated and immersed in boiling citrate buffer (pH 6) in a microwave oven at 650 W, two changes for 5 min each. Slides were then allowed to cool at room temperature for 20 min, and treated with 3% hydrogen peroxide in distilled water to inhibit endogenous peroxidase activity. After washing, the slides were subsequently incubated with: specific monoclonal antibodies, overnight, at 4°C; 1:200 dilution of a biotinylated rabbit antiserum to mouse immunoglobulin for 30 min; and 1:100 dilution of the streptavidin-biotinylated peroxidase complex for 30 min. Peroxidase activity was detected using diaminobenzidine as the chromogenic substrate.
The following primary antibodies were used: the monoclonal antibody (mAb) to ER (Dako; at 1/100 dilution), the mAb to PgR (Dako; at 1/800 dilution), the MIB-1 mAb to the Ki-67 antigen (Immunotech, Marseille, France; at 1/1200 dilution) and the polyclonal antiserum (Dako; at 1/3200 dilution) to the Her2/neu protein.
The immunostained slides were evaluated independently by two of the authors. Only nuclear reactivity was taken into account for ER, PgR and Ki-67 antigen, whereas only an intense and complete membrane staining of the tumour cells was taken as evidence of Her2/neu overexpression. The results were recorded as the percentage of immunoreactive cells over at least 2000 neoplastic cells. The value of Ki-67 labelling index (20%) was used as a cut-off in distinguishing tumours with low and high proliferative fractions. The value of 20% was selected based on previous data from our group indicating that this threshold may identify tumours with a higher chance of response to PreCT [11].
Biological study
In a subgroup of patients treated with PeCT, blood samples were drawn the day before surgery (day 1), immediately after surgery (day 0) and the day after surgery (day +1). Blood was allowed to clot for 2 h at room temperature, then centrifuged at 1080 g for 10 min. Serum was separated and stored at 30°C until the analysis was performed. Vascular endothelial growth factor (VEGF) assay was performed by means of a commercial quantitative sandwich enzyme immunoassay technique using Quantikine Human VEGF Immunoassay (R&D Systems, Inc., Minneapolis, MN, USA). A monoclonal antibody specific for VEGF was pre-coated onto a 96-well microplate. Standards and samples were dispensed into the wells and any VEGF present was bound by the immobilised antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for VEGF was added to the wells. Following washing to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells and colour developed in proportion to the amount of VEGF bound in the initial step. The colour development was stopped and the optical density was determined on a microtiter plate reader at 450 nm. The minimal amount of serum VEGF detectable by this kit is 9.0 pg/ml and the maximum undiluted is 2000 pg/ml. All samples were measured in duplicate. Intra-assay and inter-assay coefficients of variation were <7% and 9%, respectively.
Statistical analysis
The study was designed to investigate whether the mean percentage decrease in Ki-67 between the values at biopsy and at surgery was significantly greater in the new therapy than the 41% (standard deviation 30%) observed with previous PreCT [11]. We considered that the proposed treatment with PeCT would be of biological interest with a mean decrease in Ki-67 of 55%. A sample size of 36 allowed us to test the hypothesis with a 90% power and a 5% significance level using a one-sided test. A one-sample t-test was used to test this hypothesis. As a secondary research objective, we were interested in testing whether the proportion of complete responses (pCR) with the new therapy was greater than the
13% observed with established therapies [13]. The minimum proportion of patients with pCR that we considered of clinical importance for the proposed therapy was 30%. These 36 patients allowed us to test the secondary hypothesis with a power of 79% and a significance level of 5% using a one-sided exact test. The exact test was used also to compare pCR rate within subgroups. The non-parametric Wilcoxon signed rank sum test (two-sided) was used to test whether serum VEGF concentrations were different before and after surgery.
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Results |
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Sixteen patients treated with PeCT had serum VEGF measured the day before (day 1), the day of surgery immediately after surgery (day 0) and the day after surgery (day +1). The median VEGF level for this group of patients changed from 208.8 pg/ml (day 1) to 182.5 pg/ml (day 0) to 211.6 pg/ml (day +1). There was no significant difference in the values observed (P = 0.6).
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Discussion |
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There are some reports on the possible interference of the extent of surgery, and metastatic growth or tumour progression. Stimulation of cellular growth [3, 6, 19], enhancement of metabolic activity of tumour cells [20], and reduced immunological competence of the host to suppress transformed cells [21] were mechanisms implicated in these phenomena. Moreover, breast cancer progression and growth-related stimulation or inhibition are often influenced by endocrine mechanisms. Interactions between surgery, tumour growth (prognosis) and endocrine features were hypothesised in relationship with the timing of breast surgery and the phase of menstrual cycle in premenopausal women [22, 23]. Other mechanisms involved in the progression of micrometastatic disease are related to growth factors and neoangiogenesis [24].
Although the time period immediately before, during and after surgery represents a window of opportunity for the assessment of treatment efficacy interference with tumour growth and with micrometastatic progression, it was rarely clinically tested in the past. PreCT has been evaluated by completing drug administration a few weeks before surgery to allow bone marrow and mucous membrane recovery [810, 25, 26], and postoperative chemotherapy has been delayed a few weeks after surgery to allow wound healing and scar formation [27, 28].
Two important aspects of the herein reported study should be highlighted. First, the continuation of 5-FU infusion during the surgical phase was feasible, without any apparent obstacle for wound healing or any significant side-effects, which could be expected from interaction with anaesthesia.
Secondly, we observed a remarkable remission rate with a non-anthracycline and/or taxane-based regime. The introduction in the preoperative setting of taxanes to a standard anthracycline-containing regimen, provided encouraging results in two recently presented trials. In the National Surgical Adjuvant Breast Project (NSABP) B-27 Study the addition of four cycles of taxotere after four cycles of AC significantly improved pCR from 9.8% to18.7% [29]. In the recently published trial from Smith et al. [30], patients with large or locally advanced breast cancer received four courses of CVAP (cyclophosphamide 1000 mg/m2, doxorubicin 50 mg/m2, vincristine 1.5 mg/m2 and prednisolone 40 mg for 5 days). Those who responded (complete response or partial response) were randomised to receive a further four cycles of CVAP or four cycles of taxotere. In patients who received eight cycles of chemotherapy, the clinical response (94% versus 66%) and pCR (34% versus 16%) rates were higher (P = 0.001 and 0.04) in those who received further taxotere. However, an evaluation of the magnitude of the treatment effect in selected populations according to baseline biological features is at present not available for these trials.
In the present study, 33% (95% CI 18% to 48%) of pCR were observed in a prospectively defined cohort of 36 patients with tumours showing a high proliferation fraction (inclusion criteria of Ki-67 labelling index 20%), who received PreCT and PeCT. The largest proportion of pathologically complete disappearance of the primary tumour was observed in cases of absence of steroid hormone receptor immunoreactivity [six out of 13 patients (46%; 95% CI 19% to 73%)]. Although the study was not designed to detect a difference between ER- and PgR-absent tumours and tumours with some expression of steroid hormone receptors, the finding is consistent with a previous study including 117 patients treated with PreCT [11]. We found a significant difference in the response rate to PreCT, not only in highly proliferating tumours showing a Ki-67 labelling index
20% (74% versus 40%; P = 0.0004), but also for patients with tumours that did not express steroid hormone receptors compared with those expressing some (ER absent and PgR absent, 82% versus 56%, respectively; P = 0.03) [9]. Pathological complete remission rates were also different in the two groups (22% versus 7%, respectively; P = 0.04) [11]. One might argue that ER-positive tumours might require more time to respond and that in the present study the submission to surgery of patients with stable disease after three courses of chemotherapy might have in fact excluded these patients from prolonged chemotherapy and subsequent possible response. However, there is evidence in the literature of high response to PreCT after only three courses of chemotherapy [31] and in the present study only highly proliferating tumours, theoretically requiring a short time in order to achieve a response, were included. The possible increase in responsiveness to PreCT and PeCT for selected subgroups according to biological features has a clinical relevance and deserves further investigation, since degree of response (pCR) predicted outcome in terms of disease freedom and overall survival in previous randomised studies [8].
There is evidence in the literature that surgery might increase levels of VEGF in the first postoperative day, supporting an angiogenic burst following surgical trauma [32]. An exploratory analysis conducted in the present study on the biological effects of PeCT, indicated that in patients receiving PeCT, VEGF values were substantially unchanged in the perioperative period, thus supporting a possible favourable effect of PeCT on growth factors. We are now conducting a randomised study in order to further characterise the biological effects of PeCT and confirm these preliminary data.
We conclude that the proposed combination of PreCT and PeCT is highly effective in patients with chemotherapy-responsive tumours. The perioperative infusional therapy for patients already receiving PreCT seems convenient and safe, and might be a reasonable option for patients who have chemotherapy-responsive tumours.
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Footnotes |
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References |
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