Circulating Vascular Endothelial Growth Factor, C ...

Melichar B. et al.: Dose-dense Chemotherapy

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Pteridines Vol. 19, 2008, pp. 65 - 71

Circulating Vascular Endothelial Growth Factor, C-reactive Protein and Urinary Neopterin Concentrations During dose-dense Chemotherapy Bohuslav Melichar1, Anna Balloková2, Eva Malirová2, Lubor Urbánek3, 5, Lenka Krcmová3, 5, Radomir Hyspler3, Helena Hornychová4, Ales Ryska4, Dagmar Solichová3 Departments of 1Oncology & Radiotherapy, 2Nuclear Medicine, 3Gerontology & Metabolic Care, and 4Pathology, Charles University Medical School & Teaching Hospital, and 5Department of Analytical Chemistry, Charles University School of Pharmacy, Hradec Kralove, and 6Department of Oncology, Palacky University Medical School, Olomouc, Czech Republic Abstract Angiogenesis plays a crucial role in tumor progression. Prominent among angiogenic factors is vascular endothelial growth factor (VEGF). VEGF is produced by cancer cells as well as by the cells infiltrating tumor stroma, mainly macrophages. Macrophage activation may be assessed by measuring serum or urinary neopterin. Systemic inflammatory response could be evaluated by measuring serum C-reactive protein concentrations. The aim of the present study was to examine the effect of a regimen combining dose dense combination of doxorubicin and cyclophosphamide with sequential weekly paclitaxel on plasma VEGF, urinary neopterin and serum C-reactive protein concentrations. Thirty-four female patients with histologically verified breast carcinoma treated with dose-dense regimen combining doxorubicin and cyclophosphamide with sequential weekly paclitaxel administration were studied. Plasma VEGF was measured by enzyme-linked immunosorbent assay. Urinary neopterin was determined by high performance liquid chromatography. Compared to baseline plasma VEGF was significantly decreased one week after the start of therapy. VEGF concentrations subsequently increased, but this increase was significant compared to baseline only at week 16. Urinary neopterin was significantly increased compared to baseline at every visit with the exception of visits 12 and 20 at which the significance was borderline. Serum C-reactive protein was increased compared to baseline only at visits 4, 6 and 8. A positive correlation was observed between plasma VEGF and serum C-reactive protein at baseline and at visits 5 and 19. Significant correlations were observed between serum C-reactive protein and urinary neopterin at visits 6, 7, 9, 11, 14, 15 and 17. In conclusion, only minor changes in plasma VEGF and serum C-reactive protein were observed during dose-dense chemotherapy. In contrast, urinary neopterin was increased throughout the course of treatment. Correlations between VEGF and C-reactive protein and between Creactive protein and urinary neopterin were observed only at some time points. Key words:

breast carcinoma, C-reactive protein, cyclophosphamide, doxorubicin, neopterin, paclitaxel, vascular endothelial growth factor

Introduction Breast carcinoma is the most common malignant disease of women in the Western world (1). The remarkable progress accomplished in the treatment of breast cancer over the last decades is now reflected in improved survival. There is now strong evidence indicating that, in addition to early diagnosis, much of the improvement of the prognosis of women with breast

cancer results from the use of systemic therapy, such as hormonal treatment and chemotherapy (2). Chemotherapy may be administered either before (primary or neoadjuvant chemotherapy) or after (adjuvant chemotherapy) definitive surgery. Neoadjuvant chemotherapy is the treatment of choice in locally advanced inoperable breast carcinoma. In addition, neoadjuvant chemotherapy is used in patients with operable breast carcinoma in whom adjuvant chemo-

Correspondence to: Prof. MUDr. Bohuslav Melichar Ph.D., Onkologická klinika, Lékarská fakulta Univerzity Palackeho a Fakultni nemocnice, I.P. Pavlova 6,775 20 Olomouc, Czech Republic. Tel +420-583444295; fax +420-588442522; e-mail: [email protected] Pteridines/Vol. 19/No. 3

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therapy would be indicated (3, 4). The results of randomized clinical trials as well as the meta-analysis of these trials indicate that the neoadjuvant and adjuvant strategies of chemotherapy administration are equivalent in terms of overall survival (5-8). Currently, there is no single universally accepted regimen of adjuvant or neoadjuvant chemotherapy of breast carcinoma (3, 4). A survival benefit has been demonstrated for the administration of anthracyclines (2), dose-dense approach (9), and the addition of taxanes (10). There is a consensus that, unless contraindicated, neoadjuvant chemotherapy regimen should include an anthracycline, and most currently used regimens of primary systemic chemotherapy comprise anthracycline-based combinations with or without taxanes. Based on earlier randomized clinical trials, the standard regimen used in our center for neoadjuvant chemotherapy or adjuvant treatment of high-risk patients combines the dose-dense administration of the combination of doxorubicin and cyclophosphamide (9) as well as sequential weekly paclitaxel administration (11). Angiogenesis plays a crucial role in tumor progression. Tumor angiogenesis is driven by host-derived humoral factors. Prominent among these factors is vascular endothelial growth factor (VEGF) (12). Several agents currently used in cancer therapy or under development target the VEGF pathway. Moreover, metronomic regimens, similar to the dose dense regimen with weekly paclitaxel, are known to inhibit tumor angiogenesis (13). VEGF is produced by cancer cells as well as by stromal cells, mainly macrophages (14, 15). Macrophages are prominent among leukocyte populations infiltrating tumors, including breast carcinoma. Macrophage activation may be assessed by measuring neopterin. The production of neopterin by macrophages is induced by interferon-γ, and serum or urinary neopterin concentrations are considered to represent an indicator of systemic immune activation (16). Systemic inflammatory activity may be assessed by measuring serum concentrations of C-reactive protein (17, 18). The aim of the present study was to evaluate the effect of a regimen combining dose-dense combination of doxorubicin and cyclophosphamide with sequential weekly paclitaxel on plasma VEGF concentrations. The relation between VEGF levels and pathological response, urinary neopterin or serum C-reactive protein was also investigated. Patients and Methods Thirty-four female patients, aged (mean ± standard deviation) 47 ± 9, range 29 - 68 years, with histologically verified stage II - IV breast carcinoma were Pteridines/Vol. 19/No. 3


included in the present study. Thirty-one patients were treated before surgery (neoadjuvant chemotherapy) and 3 patients were treated in the adjuvant setting after surgery. All patients were treated with dose-dense regimen combining doxorubicin and cyclophosphamide (9) with sequential weekly paclitaxel administration (11). The regimen consisted of 4 doses of doxorubicin (60 mg/m2) and cyclophosphamide (600 mg/m2) administered in 14-day intervals followed by 12 doses of paclitaxel (90 mg/m2) administered in weekly intervals. Granulocyte colony-stimulating factor (480 µg subcutaneously for 2 - 5 doses) was administered after each dose of doxorubicin and cyclophosphamide and in case of neutropenia also during the weekly paclitaxel administration. Pathological response was evaluated based on criteria described by Chevallier et al. (19). Plasma samples were taken before the therapy and then at weekly intervals during the treatment, processed immediately, frozen and stored at - 40°C until analysis. The study protocol was approved by local ethical committee, and the patients signed informed consent. Plasma VEGF was measured by enzyme-linked immunosorbent assay using a commercial kit (Immuno-Biological Laboratories, Gunma, Japan). The assay was performed according the manufacturer's instructions. The limit of detection was 10 pg/ml. C-reactive protein was determined by latex enhanced immunoturbidimetry using Modular analyzer (Roche, Basel, Switzerland) with commercial kits according the manufacturer's instructions. Urinary neopterin was determined as described (20). Briefly, urine sample were collected and stored at - 20°C until analysis. After centrifugation (5 minutes, 1300 g) and diluting 100 µl of urine specimens with 1.0 ml of mobile phase containing 2 g of disodiumEDTA per liter, samples were injected onto a column, and neopterin was determined using high performance liquid chromatography system Prominence LC20 (Shimadzu, Kyoto, Japan). Neopterin was identified by its native fluorescence (353 nm excitation, 438 nm emission wavelenghts) and quantified by external standard method. Creatinine was determined by Jaffé reaction after dilution of the sample 1:50 on a Modular analyzer (Roche, Basel, Switzerland) using a commercial kit according to the manufacturer's instructions, and neopterin concentrations were expressed as neopterin to creatinine ratio (µmol/mol creatinine). Plasma VEGF, urinary neopterin and serum C-reactive protein concentrations were compared according to the pathological complete response that was defined as complete disappearance of tumor cells (Chevallier 1) by Mann-Whitney U test. The values before and during the treatment were compared by Wilcoxon signed rank test. Correlations were studied using Spearman's rank correlation. The decision on statistical

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apy (visit 1). VEGF concentrations subsequently increased, but this increase was significant compared to baseline only at visit (week) 16 (Figure 1). In contrast, urinary neopterin was significantly increased compared to baseline at every visit with the exception of visit 12 and 20 at which the significance was borderline (p = 0.06 and p = 0.07, respectively; Figure 2). Serum C-reactive protein was significantly increased compared to baseline only at visits (weeks) 4, 6 and 8 (p = 0.01, 0.006 and 0.03, respectively; Figure 3).

70 60

VEGF (pg/ml)

50 40 30 20 10 0

9 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

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Figure 1. Plasma VEGF during dose-dense chemotherapy. Shown are the medians of plasma VEGF concentrations obtained in weekly intervals during the chemotherapy with 95 % confidence intervals indicated by the dashed line. Compared to baseline (visit 0) plasma VEGF was significantly (p < 0.05) decreased at visit (week) 1 and increased at visit 16.

C-reactive protein (mg/l)

visit (weeks)

7 6 5 4 3 2 1 0

250

µ neopterin( η(µ mol/molcreatinine) creatinine) neopterin mol/mol

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 visit (weeks)

200

Figure 3. Serum C-reactive protein during dose-dense chemotherapy. Shown are the medians of serum Creactive protein concentrations obtained in weekly intervals during the chemotherapy with 95 % confidence intervals indicated by the dashed line. Serum Creactive protein was significantly increased compared to baseline only at visits (weeks) 4, 6 and 8.

150

100

50

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 visit (weeks)

Figure 2. Urinary neopterin during dose-dense chemotherapy. Shown are the medians of urinary neopterin concentrations obtained in weekly intervals during the chemotherapy with 95 % confidence intervals indicated by the dashed line. Compared to baseline, urinary neopterin was significantly (p < 0.05) increased at every subsequent visit with the exception of visit 12 and 20 at which the significance was borderline (p = 0.06 and p = 0.07, respectively). significance was based on p = 0.05 level. The analyses were performed using NCSS software (Number Cruncher Statistical Systems, Kaysville, Utah, USA). Results Compared to baseline (visit 0) plasma VEGF was significantly decreased one week after the start of ther-

No significant differences in plasma VEGF concentrations were observed at any time point between patients with (n = 13) or without pathological complete response (n = 18) except of significantly higher VEGF concentrations of in patients with pathological complete response at visit 12 (median 34.7, 95 % confidence interval 16.6 - 51.8 vs. 14.2, 0 - 28.6 pg/ml). Similarly, no significant differences were observed according the pathological response in urinary neopterin and serum C-reactive protein concentrations. A positive correlation was observed between plasma VEGF and serum C-reactive protein at baseline (rs = 0.34; p = 0.05) and at visits (weeks) 5 (rs = 0.44; p = 0.03) and 19 (rs = 0.55; p = 0.005). Significant correlations were observed between serum C-reactive protein and urinary neopterin at visits (weeks) 6 (rs = 0.34; p = 0.05), 7 (rs = 0.41; p = 0.04), 9 (rs = 0.44; p = 0.03), 11 (rs = 0.43; p = 0.03), 14 (rs = 0.52; p = 0.01), 15 (rs = 0.62; p = 0.002) and 17 (rs = 0.44; p = 0.04).

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Discussion The concentrations of plasma VEGF observed in the present study were similar to levels previously reported in the literature (21). Similarly to the results reported by Kümmel et al. (22), only minor changes have been observed in the present study during the therapy. With a single exception that could be due to chance, plasma VEGF during the therapy was similar in patients with or without pathological complete response. These data indicate that circulating VEGF in a group of breast cancer patients mostly with stage II or III disease may be derived predominantly from sources other than the tumor. Fluctuations of serum Creactive protein concentrations were observed with significantly increased concentrations observed two weeks after the second, third and fourth dose of doxorubicin/cyclophosphamide. In contrast, urinary neopterin was increased throughout the course of therapy. Surprisingly, only limited correlation was observed between C-reactive protein and neopterin, indicating that these two markers of systemic inflammatory and immune response behave differently in patients with breast carcinoma treated with chemotherapy. The term pathological complete response denotes complete disappearance of tumor cells in the operative specimen obtained at definitive surgery after primary chemotherapy (19). Pathological complete response is important as it represents the most significant prognostic parameter in women undergoing primary chemotherapy (23-26). Based on demonstration of benefit of taxane administration in adjuvant setting (10, 27), there is a tendency to incorporate paclitaxel or docetaxel into regimens of neoadjuvant chemotherapy. Sequential regimens seem to be superior to the concomitant administration of taxanes and anthracyclines, and, in case of paclitaxel, the weekly administration was shown to be superior to the administration every 3 weeks (11). Weekly paclitaxel represents a dose-dense regimen. In adjuvant setting, a survival benefit has been demonstrated for dose-dense administration of doxorubicin with cyclophosphamide followed by sequential paclitaxel supported by the administration of granulocyte colony-stimulating factor (9). Metronomic regimens that are similar to this dosedense regimen are known to inhibit tumor angiogenesis (13). We originally hypothesized that plasma VEGF would be lower in patients who experience pathological complete response. However, a decrease in plasma VEGF was observed only one week after the start of chemotherapy and an opposite trend for higher VEGF was observed during most of the treatment. No differences according the pathological complete response were observed in serum C-reactive protein and urinary Pteridines/Vol. 19/No. 3


neopterin during the course of treatment. VEGF has multiple roles in tumor progression and is responsible not only for tumor angiogenesis (12), but also for tumor hypertension (28) and immune suppression associated with cancer (29). However, present data indicate that plasma VEGF concentrations in patients with breast carcinoma treated with dose-dense chemotherapy are, in general, low, and changes in the levels of this cytokine may be of limited significance in the present setting. Large quantity of VEGF is transported in platelets (30), and serum VEGF concentrations are therefore higher compared to plasma, but plasma concentrations were reported to be of more importance than serum VEGF levels (31). Concentrations of VEGF in the tumor microenvironment may be of more significance (32, 33), but changes of local levels of this cytokine may not be reflected at systemic level. An increase in serum or urinary neopterin concentrations in cancer patients has been amply documented (34, 35), but neopterin concentrations are increased only in a minority of patients with breast carcinoma (34, 36). The present observation of increased urinary neopterin during dose-dense chemotherapy is in agreement with the reports of increased serum concentrations of pro-inflammatory cytokines after the administration of taxanes (also in breast carcinoma in neoadjuvant setting) (37, 38). The administration of filgrastim may have also contributed to the observed increase of urinary neopterin concentrations. In an earlier study, we have described an increase in urinary neopterin that was associated with changes peripheral blood lymphocyte phenotype associated with immune activation, e.g. increase of CD3+CD69+ lymphocytes (39). Neopterin is produced from guanosine triphosphate (GTP) by activated macrophages in a reaction catalyzed by the enzyme GTP cyclohydrolase I. The activity of GTP cyclohydrolase I is induced by interferon-γ (40). Neopterin may be measured not only in serum, but also in urine, and both serum and urinary neopterin concentrations have been validated as indicators of systemic immune and inflammatory response in disorders ranging from cancer to viral infections, transplant rejection, or atherosclerosis (16). Urinary neopterin concentrations are relatively stable in cancer patients in the absence of complications (41). In contrast, the production of C-reactive protein is induced by interleukin-6, and serum concentrations of C-reactive protein are indicative of systemic inflammatory response (17). It has been demonstrated that an increase of C-reactive protein in patients with breast carcinoma indicates the presence of metastatic disease (42) and a decrease of this acute phase protein is associated with response to therapy (43). In many instances, systemic immune and inflammatory


responses overlap, and C-reactive protein and neopterin concentrations correlate. However, in the present study, C-reactive protein and neopterin followed different dynamics and only limited correlation was observed. A correlation between C-reactive protein and VEGF is in agreement with earlier observations in cancer patients (44) as well as with experimental data demonstrating the effect of C-reactive protein on VEGF production (45). In conclusion, low levels of plasma VEGF were observed in the present cohort of breast carcinoma patients and only minor changes in the concentration of this cytokine were noted during dose-dense chemotherapy. Limited changes were also observed in the concentrations of C-reactive protein, but urinary neopterin was increased throughout the course of therapy. Correlations between VEGF and C-reactive protein and between C-reactive protein and urinary neopterin were observed only at some time points. Acknowledgement

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