1Division of Medical Oncology, 2Division of Laboratory Medicine and Pathology, 3Division of Epidemiology and Biostatistics, European Institute of Oncology, Milan, Italy
Received 29 March 2001; revised 26 June 2001; accepted 5 July 2001.
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Abstract |
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Anticancer chemotherapy is thought to be effective by means of direct cytotoxicity on tumor cells. Alternative mechanisms of efficacy have been ascribed to several common anticancer agents, including cyclophosphamide (CTX), methotrexate (MTX), anthracyclines and taxanes, postulating an antiangiogenic activity.
Patients and methods
We evaluated the clinical efficacy and impact on serum vascular endothelial growth factor (VEGF) levels of low-dose oral MTX and CTX in patients with metastatic breast cancer. MTX was administered 2.5 mg bd on days 1 and 2 each week and CTX 50 mg/day administered continuously.
Results
Sixty-four patients were enrolled, 63 were evaluable: Eastern Cooperative Oncology Group (ECOG) performance status 01, 2 sites of metastatic disease (n = 50 patients), progressive disease at study entry (n = 51), 1 regimen for metastatic disease (n = 32) and
2 regimens (n = 20). Among the 63 evaluable patients, there were two complete remissions (CR), 10 partial remissions (PR) for an overall response rate of 19.0% (95% CI 10.2% to 30.9%) and an overall clinical benefit (CR+ PR+ stable disease >24 weeks) of 31.7% (95% CI 20.6% to 44.7%). Grade
2 leucopenia was registered in only 13 patients. The median serum VEGF level for the subgroup of patients on treatment for at least 2 months decreased with treatment from 315 pg/ml (95% CI 245 to 435) at baseline to 248 pg/ml (95% CI 205 to 311) at 2 months (P <0.001). Both responders and non-responders showed similar reductions in serum VEGF (P = 0.78). After 6 months patients still on treatment had a median VEGF level of 195 pg/ml (95% CI 96 to 355), which was significantly lower than the median baseline values (P = 0.001).
Conclusions
Continuously low-dose CTX and MTX is minimally toxic and effective in heavily pretreated breast cancer patients. A drop in VEGF was associated with the treatment and so alternative hypotheses, other than that of direct toxicity on tumor cells, must be favored when trying to explain the anticancer effect.
Key words: angiogenesis, breast cancer, chemotherapy, cyclophosphamide, methotrexate
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Introduction |
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There is evidence that several common anticancer agents including cyclophosphamide (CTX), doxorubicin (Adriamycin) and paclitaxel (Taxol) have antiangiogenic activity in the animal model [8, 10, 11]. Low-dose methotrexate (MTX) inhibits endothelial cell proliferation in vitro, and blocks endothelial cell growth factor-induced neovascularization in the rabbit cornea assay [10]. Cytotoxic agents including CTX and MTX [11] markedly inhibited the growth of chick embryo. It is therefore possible that conventional cytotoxic agents may exert a tumor suppressive effect through an antiangiogenic mechanism. Antiangiogenic therapy appears to be improved when administered over a long period of time without treatment interruptions. Therefore, oral therapy in doses, which would be tolerable on a long run, should be considered. Among orally available drugs for the treatment of breast cancer, significant bioavailability was demonstrated for both CTX and MTX [12, 13].
Vascular endothelial growth factor (VEGF) is an angiogenic polypeptide that is detected in a large variety of malignant human tumors including breast, brain, lung and gastrointestinal tract cancers [14, 15]. Overexpression of VEGF increased tumor growth, angiogenesis and metastases, whereas anti-VEGF antibodies inhibited the growth of tumor xenografts [16, 17]. Moreover, growth inhibition of glioma was observed in nude mice bearing this tumor derived from cells transfected with an antisense VEGF sequence [18]. Values of VEGF demonstrated a correlation with tumor bulk. Patients with disseminated breast cancer had higher serum VEGF concentrations than those with localized cancer. In particular, VEGF was found to be abnormally elevated in the serum of more than 70% of patients with advanced breast cancer [14]. VEGF content in tumor cytosols is a predictor of relapse free survival and overall survival in primary breast cancer and it might also predict outcome after adjuvant endocrine treatment [19].
We therefore evaluated the clinical efficacy and tolerance of low-dose, oral CTX and MTX in metastatic breast cancer patients and also the impact of this regimen upon serum VEGF values.
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Patients and methods |
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Study evaluation and treatment
Baseline evaluation included clinical examination, chest X-ray, liver ultrasound or CT scan, bone nuclear scan (plus segmental bone radiographs when bone scans were positive), ECG, complete biochemical and hematological tests. Complete blood count was then repeated every 14 days and biochemical tests every 28 days.
Treatment was administered as follows: MTX orally at a dose of 2.5 mg bd (10 am, 5 pm) on day 1 and 2 every week and CTX orally at a dose of 50 mg/day (9 am). No antiemetic treatment was prescribed. Serum VEGF was determined at baseline and every month thereafter. Fifteen milliliters of peripheral blood were collected partly into a citrate tube, for plasmatic VEGF determination, and partly into a clot-activator tube for serum VEGF determination. As the value of serum VEGF increased during clot formation and reached a plateau after 60 min [20] the samples were allowed to clot at room temperature for 2 h and then centrifuged for 10 min at 1900g. The aliquoted samples were then stored at 30°C until analysis was performed. VEGF was assessed by an ELISA kit (Quantikine®, R&D Systems, Minneapolis, USA). All samples and standards were assayed in duplicate. The serum was stocked in aliquots at 30°C until analysis was performed. The samples were analyzed in duplicate by a sandwich enzyme-linked immunosorbent assay (ELISA), using a monoclonal antibody specific for VEGF165 (Quantikine®). In order to evaluate possible differences in VEGF values between serum and plasma, 29 patients had a control value of VEGF in both serum and plasma for each sampling.
Side effects and response
Toxicity was evaluated according to NCICTG criteria by clinical and laboratory investigations. Treatment was withheld and delayed for 1 week in case of a neutrophil count <1000 mm3 and/or platelet count <75000 mm3. A 50% dose reduction in the total amount of drug administered in each cycle was prescribed after hematological recovery. In the case of a neutrophil count <1500 mm3 but >1000 mm3 and/or platelet count <100000 mm3 but >75000 mm3, therapy was administered with a 50% dose reduction in the total amount of drug administered in each cycle. Re-escalation of drug doses was attempted if close monitoring was possible.
In the event of grade 2 anorexia, nausea, vomiting, diarrhea, stomatitis, dryness of the mouth, epigastric pain or increase in transaminases, all therapy was postponed until symptoms subsided. A 50% reduction of combined CTX and MTX therapy was performed for the next cycle, with subsequent re-escalation to full dosage if tolerated. Any other non-hematological grade 3 toxicity was managed by a 50% reduction of dosage in the next cycle, which was not commenced until full recovery had occurred.
Assessment of response was performed according to WHO criteria after every 8 weeks of therapy. Complete remission (CR) was defined as the disappearance of all known lesions on two separate measurements at least 4 weeks apart. Partial remission (PR) was defined as a reduction of each lesion by at least 50%. Stable disease (SD) was defined as a decrease of <50% or an increase of <25% with no new lesions, and progressive disease as an increase of >25% or appearance of new lesions. Overall success rate was defined as the proportion of patients who achieved CRs, PRs or SDs for at least 24 weeks. Case report forms were reviewed by an independent panel composed of two medical oncologists.
Statistical analysis
The aim of the study was to obtain at least a 25% overall success rate. With 63 patients the power to detect a difference in response rate from 10% to 30% is 98% and from 10% to 25% is 91%. Estimated curves of survival and time to progression were plotted from the first day of treatment using the KaplanMeier method; response duration was measured from the date of achievement of response. Confidence intervals (CI) for the response rates were calculated using exact binomial methods. Given the non-normality of VEGF distribution in our sample (ShapiroWilk test), the non-parametric Wilcoxon matched pairs test was applied to compare VEGF values at different assay times, while the MannWhitney U test was performed to compare responders and non-responders with respect to their baseline VEGF levels. Relative changes in VEGF levels were calculated as ratio of the values measured at 2 months and at baseline and compared between responders and non-responders by the MannWhitney U test. Confidence intervals for the medians were obtained using 2000 bootstrap samples. Log transformed VEGF and platelet values were used in the correlation analysis as well as to estimate the effect of VEGF in the prediction of a response using logistic regression.
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Results |
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In 29 patients for whom measurements of VEGF values in both serum and plasma for each sample were taken, a good correlation between plasma and serum levels was observed at baseline as indicated by the correlation coefficient of 0.51 (P <0.01). At baseline the median plasma level of VEGF was 29.2 pg/ml (95% CI 23.8 to 40.9) while at 2 months it was 34.8 pg/ml (95% CI 24.3 to 39.5); there was no evidence of a change in plasma VEGF from baseline to 2 months (P = 0.94) nor that there was a difference between responders and non-responders (P = 0.17).
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Discussion |
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In view of the goal of palliative treatment in this setting, it is reasonable to consider disease stabilization (i.e. NC for 24 weeks or longer) as a clinical outcome [30]. The overall success rate (CR + PR + NC for 24 weeks or longer) of 32% suggests that a meaningful percentage of patients in our study benefited from treatment with low-dose chemotherapy. The observation of a 19% response rate, using a regimen with CTX and MTX at a very low-dose level, indicates that cytotoxicity cannot possibly explain entirely the efficacy of this regimen. In fact, direct cytotoxicity of anticancer agents is expected to cause bone marrow suppression, hair loss and altered mucosal repair. Conversely, this regimen demonstrated no significant hematological side effects or hair loss. Only 21% of the patients presented a grade >1 leucopenia; 12%, grade >1 neutropenia; and 8% had some hair loss. Moreover, no significant correlation was detected between the tumor shrinkage and the most commonly used surrogate indicator of cytotoxicity, i.e. leucopenia. The absence of significant bone marrow suppression, mucositis or hair loss, usually observed with standard dose CTX (± MTX), leads to a hypothetical alternative target. One plausible explanation for cell-growth inhibition might relate to the antiangiogenic effects of CTX (and also doxorubicin but not 5-fluorouracil) as postulated by Folkman et al., based upon a neovascularization model in the mouse cornea [9]. Also, low-dose MTX inhibited endothelial cell proliferation in vitro, and inhibited neovascularization by endothelial cell growth factor in the rabbit cornea assay [10].
We assessed a possible correlation between angiogenic pathways and efficacy of the regimen by repeated determination of VEGF concentrations in the serum of patients during treatment. Table 4 illustrates the association between VEGF levels and response. Values were elevated in all patients before treatment and no significant difference in baseline values was detected between patients who subsequently responded to the treatment and patients that subsequently failed to respond.
Recently published results indicate that VEGF in the bloodstream is transported by blood cells, including leukocytes and platelets [31, 33], and a correlation between VEGF value and platelet number has been observed [33]. The blood cells of cancer patients contain greatly elevated amounts of this major angiogenic growth factor [32, 33]. Therefore changes in VEGF during chemotherapy might be related with chemotherapy-induced thrombocytopenia and a subsequent rebound increase in platelet numbers rather than the persistent decrease in tumor-derived VEGF [34]. Moreover the finding of lower VEGF in patients undergoing chemotherapy compared with those untreated may reflect effects that not only have an influence on platelet numbers but also on platelet volume [35]. Although a possible influence of platelet volume on VEGF values cannot be excluded in the present study, no significant change in the platelet numbers was observed and serum VEGF significantly decreased after 2 months of treatment. Conversely, in a subgroup of patients where we tested both serum and plasma concentrations of VEGF, we showed that although there is a good correlation between serum and plasmatic values, there was no evidence of a change in plasma VEGF from baseline to 2 months. The role of platelets and particularly of platelet-derived VEGF in tumor biology is obscure, but not negligible. Cancer patients frequently have elevated platelet counts and an increased platelet consumption compared with healthy individuals. Moreover, thrombocytosis is a negative prognostic factor in some cancers, and antitumor effects and improvements in terms of survival have been observed with anticoagulation therapy [36]. Other factors may influence serum VEGF concentrations other than tumor and platelets and therefore might have influenced the results observed. Studies have found variable correlations with hormones and have variably reported changes in the levels of VEGF concentration according to the phase of the menstrual cycle [3739]. Finally, VEGF concentrations in peripheral blood mononuclear cells have been reported in cancer patients [40].
Important aspects such as the chronic use of CTX (and MTX) should be raised for the sake of discussion, especially because the response duration might last for several months. Cyclophosphamide, given together with MTX and fluorouracil has been reported to slightly increase the incidence of leukemia [41]. Dealing with leukemic risk while discussing treatment for advanced breast cancer might be futile. However, the possible use of this regimen in the adjuvant setting might require a careful estimation of such risk. Even a year of treatment, using a cumulative CTX dose of 18 g will produce only a small increase in the risk of leukemia [41]. This figure is lower than that observed with anthracyclines, which are commonly used in the adjuvant setting [42]. Also, changes of transitional epithelium [43] and immunosuppression [44, 45] are associated with prolonged use of CTX. While instructions for an abundant fluid intake evade the former, the latter side effect might require a pause in the admin-istration of the drug(s). We encountered neither of these.
A side issue, which is worth mentioning, relates to the personal and economical costs of this regimen. Increased attention to patients quality of life favors the use of an active oral treatment [46]. Advanced breast cancer responsive to chemotherapy might require a treatment duration of several months in responders [47]. Subjective toxicity and frequent visits to care providers represents a significant burden in terms of personal costs to the patient. Furthermore, the utility of a treatment regimen which has an economic cost of about US$10 per month is obvious.
In conclusion, low-dose, oral CTX and MTX demonstrated significant efficacy in pre-treated metastatic breast cancer. Theoretically, treatments aimed at inhibiting angiogenesis should be chronically administered for a prolonged period. These treatments might have particular usefulness for subsets of patients with limited tumor burden, such as early breast cancer [48]. Moreover, combinations of therapy that inhibit angiogenesis plus cytotoxic therapy may be more effective than either type of therapy alone. Use of oral low-dose CTX and MTX should be investigated further as a strategy against tumor progression after standard chemotherapy in the adjuvant setting.
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Footnotes |
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