Thirty women with advanced breast cancer undergoing chemotherapy with single agent Paclitaxel or Docetaxel, were evaluable for the present study and had blood samples collected. Patients were separated randomly into two groups; the Paclitaxel Group and the Docetaxel Group. The clinical characteristics of the patients are summarised in Table 1Table 1. Histologic type and tumour grade were assessed according to the WHO classification (Miller et al, 1981).

Prior to entering the study, patients were clinically evaluated by physical examination, ECG, chest X-ray and abdominal CT scans, blood cell count, platelet count, serum biochemical analyses and urine analyses. Patients were thus assessed routinely every 4 weeks throughout the duration of the study. The study was approved by the Scientific-Ethics committee of our Institution.

All patients completed six chemotherapy courses with taxanes every 21 days, without the use of G-CSF or GM-CSF. Other eligibility criteria included measurable disease, response to chemotherapy, performance status (Karnofsky) (PS) equal or >80, life expectancy >3 months, absence of brain metastases unless controlled after brain irradiation and off steroids, active ischaemic cardiac disease, normal haematological, renal or hepatic function tests unless the abnormalities had resulted from direct tumour invasion. A histological documentation of measurable metastatic disease was obtained whenever possible. Patient characteristics are shown in Table 1. Informed consent was obtained from all patients according to our Institutional policy.

Patients were randomised to receive Paclitaxel or Docetaxel, and were allocated into the treatment groups by simple randomisation (closed envelopes). Age, gender, performance status (Karnofsky) (PS) and sites of metastases were the same in both arms as indicated in Table 1.

Treatment was carried-out in the day clinic. Two groups of patients were formed according to their therapeutic schedule (Table 1).

Before each treatment cycle every patient had a complete blood count, SMA-12, EKG, chest roentgenography, and abdominal CT scan. Between the treatment cycles CBC's were performed weekly. Patients were evaluated for response between the treatment cycles during the 2-week rest period. Responses were categorised as follows: (1) Complete response (CR) which was defined as a complete disappearrance of all clinically and radiographically evident disease. (2) Partial response (PR) was defined as a decrease of more than 50% in the sum of the products of the largest perpedicular diameters of the measurable lesions. (3) A 25–50% decrease, without satisfying the criteria of a PR was defined as minor response (MR).

Toxicity was recorded according to WHO criteria (Miller et al, 1981).

The control group consisted of healthy volunteers from our hospital personnel. Twenty healthy individuals who ranged in age from 35–63 years (median 46) were studied. Normal donors were always studied concurrently with the cancer patients.

Peripheral blood from all patients treated with either paclitaxel and docetaxel was collected in heparinised tubes, initially before the first chemotherapy cycle and subsequently 4 weeks after the sixth treatment cycle. Blood from age and sex matched hospital staff volunteers was collected in parallel as for a control. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation over Ficoll-Hypaque density gradient (Pharmacia, Fine Chemicals, Uppsala, Sweden) as previously described (Baxevanis et al, 1994). Autologous serum collected from the top of the Ficoll was aliquoted and stored at −70°C until assayed for cytokine levels.

For each PBMC sample, T lymphocytes (CD3+) and monocytes (CD14+) were isolated using MACS CD3 Microbeads and the Monocyte Isolation Kit (Miltenyi, Biotec, Bergisch Gladbach, Germany) respectively, according to the manufacturer's instructions and as described (Baxevanis et al, 2000). Upon purification, cells were washed once and resuspended in RPMI-1640 culture medium (Gibco, Grand Island, NY, USA), supplemented with 10% foetal calf serum (FCS; Gibco), 2 mM L-glutamine (Sigma Chemical Company, St. Louis, MO, USA), 10 mM HEPES (Gibco) and 100 μg ml⁻¹ gentamycin (referred to thereafter as complete medium). The purity of the isolated cell populations was tested by flow cytometry on a FACScan (Becton Dickinson, Mountain View, CA, USA), using anti-CD3 and anti-CD14 mAbs conjugated with PE (Pharmingen, San Diego, CA, USA). In all cases, isolated CD3+ or CD14+ cells represented >98% of the relative fraction.

This was performed as described (Baxevanis et al, 1992). Briefly, T lymphocytes (10×10⁴/well) were plated in a 96-well U-bottomed plate (Greiner, Kirheim u. Teck, Germany) and irradiated (3000 rad) autologous monocytes (5×10⁴/well) were added to a final volume of 200 μl complete medium/well. Cultures were maintained for 6 days at 37°C, in a CO₂ incubator. Eighteen hours prior to harvesting, cultures were pulsed with 1 μCi/well [³H] TdR (Amersham, UK), harvested and radioactivity incorporation was measured in a β-counter (Packard, Cowners Grove, IL, USA). Cell cultures were set up in triplicates. For each sample AMLR was set up similarly using as effector cells autologous blasts generated by incubating T lymphocytes (1×10⁶ ml) for 3 days in complete medium supplemented with 10 μg ml⁻¹ PHA in 25 cm² flasks.

NK cell activity was tested in the total PBMC population against the NK-sensitive K562 tumour targets. For testing LAK cell activity, PBMC were cultured for 7 days with 1000 U ml human recombinant IL-2 (Cetus Corp. CA, USA). Lytic activity of these effectors were tested against the NK-resistant Daudi tumour targets. Cytotoxicity assays for assessing NK- or LAK-activity were performed essentially as recently reported (Baxevanis et al, 1999). Briefly, effector PBMC, freshly isolated or cultured with IL-2, were plated in 100 μl aliquots (2×10⁶ cells ml in complete medium) in 96-well V-bottomed microtiter plates (Costar, Cambridge MA, USA). Tumour targets (10⁷ cells) were incubated with 100–150 uCi of sodium [⁵¹Cr] chromate (Amersham) for 60 min at 37°C washed twice to remove excess isotope and a quantity amounting to 5×10³ cells/well was added to the effector cells, to assess an effector to target (E/T) ratio of 40 to 1. After 18 h incubation at 37°C in a CO₂ incubator, 100 μl of supernatant were removed from each well for isotope counting in a γ-counter (Packard, Downers Grove, IL, USA). Spontaneous and maximum release were estimated by incubating targets in medium alone and with 1 M of HCl (Merck), respectively. Spontaneous release did not exceed 15% of the maximum release. In all cases, cultures were set up in triplicate and% specific target cell lysis was calculated as follows:

Sera from patients and normal donors were assayed for the determination of IL-2, IL-1β, IFN-γ, GM-CSF, IL-6, TNF-α, and PGE₂ using commercially available ELISA kits; for human IL-2, IL-1β, IL-6, and TNF-α were obtained from R&D Systems (Europe), for IFN-γ and GM-CSF from Endogen (Boston, MA, USA), and for PGE₂ from Advance Magnetics Inc (Cambridge, MA, USA). All determinations were performed in duplicate.

The analysis that follows aims at comparing the effect of treatment with Paclitaxel to the effects of treatments with Docetaxel on a number of parameters. For this reason 30 patients participated in the study (equally distributed between treatments), measurements were collected for nine parameters; for each parameter, measurements were carried out before treatment and 4 weeks after the last treatment cycle.

After the administration of six doses of taxanes, data were collected for all participants in the two time points (before the administration of treatment and after six doses). The parameters under study are presented in Table 2Table 2. The data analysis was conducted in three steps. The first step evaluated pre-treatment differences between the three groups of patients. The second step evaluated the post-treatment differences between groups taking into account the initial measurements. Pre-treatment differences between the two groups were tested via one-way analysis of variance (ANOVA) and differences in treatment effects were tested via one-way analysis of covariance (ANCOVA).

Variables were compared by means of the χ² test with Yate's correction as appropriate; continuous variables were compared by Student's t-test or a non-parametric test (Mann–Whitney) for intergroup comparisons. For matched pairs, Student's t-test or Wilcoxon's signed-rank test were applied. Logarithmic transformation was applied as appropriate in order to correct for distribution (Dixon and Massey, 1983; Machin and Campbell, 1987). Differences between mean values of the time-points were assessed through repeated measurement analysis of variance, using the statistical package SPSS (Ver. 8.0). The accepted level of significance was P<0.05.

A total of 30 evaluable patients were enrolled onto this study, with no difference in the basic clinical and laboratory parameters (Table 1). The clinical characteristics (age, menopausal status, Karnofsky index and site of metastases and extension of the disease) of our patients were equally balanced between the study groups. In order to find patients who received six cycles of taxane therapy, only those with at least stable disease were evaluable in the present study. No delays were noticed in the administration of treatment because of toxicity or other reasons. Significant differences were observed in the following immune parameters between the control group of healthy blood donors and the pretreatment values of both taxane groups; IL-2, GM-CSF, IFN-γ levels and NK and LAK cell cytotoxicity were depressed, whereas TNF-α and IL-6 levels were raised in breast cancer patients before treatment compared to controls. Table 2 demonstrates the P-values calculated for the comparison of the initial measurements between the two treatment arms, docetaxel and paclitaxel, and the healthy normal blood donors along with the basic statistical measures for each subgroup. There were no significant differences between groups with taxanes regarding any of the parameters studied. In comparison to normal control group, the groups of taxanes presented statistical significant differences in all the examined immunological parameters, before the initiation of chemotherapy. All the examined parameters, in taxane treated groups, were found decreased in comparison to normal control group (P<0.007), except PGE2 and TNF-α (P<0.0001) which were found decreased. No difference was found between the taxane groups (Table 2, Figure 1Figure 1

Comparison of the percentages of differences, between normal control group (NCG), Docetaxel (DCT), and Paclitaxel (PCT), before the administration of taxanes.

Significant differences (P<0.0001) were observed between post-treatment values of the examined parameters and the values after the administration of six courses of each taxane, either paclitaxel or docetaxel (Table 3Table 3). Differences between immunological parameter values before the initiation of chemotherapy and after six courses of chemotherapy were significant, both in the Docetaxel-treated group (P<0.0001) and also for Paclitaxel (P<0.0001) (Table 3).

Both drugs led to increases in MLR values, NK and LAK cell cytotoxicity, and IL-6, GM-CSF, IFN-γ levels, and decreases for IL-1, TNF, and PGE2 levels (Figure 2Figure 2

Comparison of the percentages of differences, before and after the administration (after six cycles) of both taxanes; Docetaxel and Paclitaxel (P<0.0001).