Randomized trial of sucrosomial iron supplementation in patients with chemotherapy-related anemia treated with ESA.

BACKGROUND: Iron supplementation improves the erythropoiesis-stimulating agents' (ESAs) response in chemotherapy-related anemia. The primary aim of our study is to assess the efficacy of sucrosomial iron, a new oral iron formulation, in cancer patients with chemotherapy-induced anemia treated with ESAs. The secondary objectives included the efficacy into two subgroups of patients (iron replete and functional iron deficiency) between the two study arms, safety and the effect on transfusion need. METHODS: In this randomized, multicentre, open-label, phase III clinical trial, 60 cancer patients were enrolled. Each patient was randomly assigned (1:1) to receive 12 weeks of oral sucrosomial iron at the dose of 30 mg daily in combination with ESAs or no supplementation to ESA treatment. The endpoint considered for efficacy was the proportion of patients achieving complete hematological response at 12 weeks (increase in Hb > 2 g/dL from baseline, without RBC transfusions in the previous 28 days or achieving Hb ≥ 12 g/dL). RESULTS: There was a statistically significant association between oral sucrosomial iron supplementation in combination with ESAs and the achievement of a complete hematological response. This response was achieved within 12 weeks by 31% of patients in the control group and by 52% of patients supplemented with oral sucrosomial iron. A trend of greater response in sucrosomial iron arm was found in both subgroups. No difference was observed about safety and transfusion need. CONCLUSIONS: Sucrosomial iron is well tolerated and its combination with ESAs improves the hematological response in cancer patients with chemotherapy-related anemia. TRIAL REGISTRATION NUMBER AND DATE OF REGISTRATION: This study has been reviewed by the Institutional Ethics Committee of the IRCCS Policlinico San Matteo Foundation, Pavia, Italy (28/04/2015; prot. N. 20,150,002,059), and by the Institutional Ethics Committee of the other Italian oncological centers involved in this study.

The heterogeneity of cancer endothelium: The relevance of angiogenesis and endothelial progenitor cells in cancer microenvironment.

Tumor-associated vessels constitution is the result of angiogenesis, the hallmark of cancer essential for tumor to develop in dimension and to spread throughout the organism. Tumor endothelium is configured as an active functioning organ capable of determine interaction with the immune response and all the other components of the variegate cancer microenvironment, determining reciprocal influence. Angiogenesis is here analyzed in its molecular and cellular mechanisms, multiple mediators and principal players, represented by Endothelial Cells. It is discussed the striking heterogeneity of cancer endothelium, due to morphological and molecular aberrations that it often presents and its multiple origin. Among the cells that participate to the composition of tumor vasculature, Endothelial Progenitor Cells represent an important source for physical sustain and paracrine signaling in the process of angiogenesis. Treatment options are reviewed, with particular focus on novel therapeutic strategies for overcoming tumor resistance to anti-angiogenic agents.

An eight-colour flow cytometric method for the detection of reference values of lymphocyte subsets in selected healthy donors.

Determination of immunoregulatory cells in peripheral blood is important in the management of disease or in the therapeutic approaches that involve alterations in lymphocyte subpopulations. The aims of the present study were (1) to develop a standard multiparametric flow cytometric method for phenotypic detection and enumeration of lymphocyte subsets so as to reduce the variability in both sample preparation methodology and flow cytometric operations; (2) to furnish reference values of lymphocytes by using a selected healthy population; and (3) to examine the influence of age and sex on the distribution of lymphocytes expressing surface markers. Eighty healthy donors were analysed, and ten-parameter, eight-colour analytical procedure was performed. We furnished a panel to detect and to enumerate lymphocyte subpopulations by a multiparametric flow cytometric method to set the reference values to a selected healthy population. These values showed statistically but not clinically significant differences in T lymphocyte subsets and natural killer cells. Furthermore, significant age-related correlations in T lymphocyte and natural killer cells were observed. Lastly, males and females in relation to age showed a significant different trend in T and B lymphocyte subsets. We confirmed that this study provides a rapid and accurate method for the detection and quantification of lymphocyte subsets that could be utilized in the clinical settings. The definition of reference values in the healthy selected population could be helpful also to better define the disease status and to evaluate the treatment efficacy during clinical trials.

Circulating endothelial cells and their apoptotic fraction are mutually independent predictive biomarkers in Bevacizumab-based treatment for advanced colorectal cancer.

BACKGROUND: Bevacizumab has shown consistent clinical efficacy in metastatic colorectal cancer (mCRC), but some patients respond better than others. Thus, it is crucial to identify biomarkers that permit the recognition of potentially responsive subjects and to spare toxicity in those who are unlikely benefit from treatment. METHODS: In 24 mCRC patients undergoing Bevacizumab-based first-line treatment, we assessed by multiparameter flow cytometry changes in circulating endothelial cell (CEC) number, their apoptotic fraction (APO-CEC) and their mutual relationship. Data were compared with those from a group of 21 healthy subjects. RESULTS: CECs and APO-CECs were higher in patients versus controls (p = 0.01 and p > 0.05, respectively). The increase in CECs at the 3rd cycle in complete response (CR) patients was statistically significant (p = 0.048). A better progression-free survival was evidenced in patients that showed an increase in CECs at the 6th cycle (p = 0.009). Regarding the changes in CECs and APO-CECs, a strong correlation was evidenced, at baseline, both in the global population (0.002; r: 0.53) and in the CR subgroup (p: 0.02; r: 0.77). In the partial response + stable and progression disease (SD + PD) subgroup, this correlation was highly significant at the 6th cycle (p: 0.001; r: 0.83). CONCLUSIONS: We confirmed the predictive role of an increase in CECs in mCRC patients treated with Bevacizumab-based therapy and showed that modifications in CECs and APO-CECs are independent factors. This underlines the relevance of a simultaneous quantitative and functional evaluation of these biomarkers in view of their possible diagnostic utility.

Lymphocyte subpopulation and dendritic cell phenotyping during antineoplastic therapy in human solid tumors.

Patients with cancer show variable levels of immunosuppression at the time of the presentation, and cytotoxic antineoplastic therapy is the primary contributor to the clinical immunodeficiency often observed during the course of the disease. In both hematological and solid tumors, this phenomenon is primarily related to the T-cell depletion associated with inhibition of dendritic cell ability to induce both primary and secondary T- and B-cell responses. Complete restoration of immunocompetence following antineoplastic therapy implicates the progressive recovery of various cell subpopulations, and it is a complex process that also depends on the type, the dose, the scheduling, and the associations of the employed drugs. In the era of target therapies, several antiangiogenic drugs are increasingly used in combination with standard chemotherapy in the treatment of advanced solid tumors. Their clinical efficacy has been recently related not only to the specific antiangiogenic properties but also to an indirect hypothetical effect on the host immune system. In the present work, we have reviewed the most recent information regarding (1) the capacity of standard antineoplastic therapy to induce and maintain an immunodeficiency in patients with solid tumors and (2) the influence of the antiangiogenic treatment in association with standard chemotherapy on lymphocyte and dendritic cell subsets and the possible resulting additional antitumor mechanism.

Flow cytometric analysis of peripheral blood dendritic cells in patients with severe sepsis.

BACKGROUND: Dendritic cells (DC) play a key role in cell-mediated immunity. We aimed to analyse the number and function of peripheral blood (PB) myeloid and plasmacytoid DC (mDC/pDC) in patients with severe sepsis. METHODS: Twenty-six septic patients, 20 surgical patients (abdominal aortic aneurysm) and 20 healthy controls were enrolled in this prospective study. RESULTS: At day 1 (enrollment in the study), septic patients showed in comparison with healthy controls, decreased mDC (P < 0.001) and increased pDC (P = 0.03), resulting in a reduction of the mDC/pDC ratio (P < 0.001). Surgery induced a decrease in both mDCs and pDCs level, without modification of mDC/pDC ratio. Septic patients included 15 survivors and 11 nonsurvivors. At day 1 no significant difference was found in mDC between the two groups, while pDCs were significantly higher in nonsurvivors (P = 0.03). At the outcome, mDC were selectively increased with respect to day 1 in survivors (P = 0.001), while no significant differences were observed as far as pDC count is concerned in both groups. Sorted DC from septic patients showed in comparison with healthy controls, reduced levels of HLA DR (P < 0.001), CD11c (P < 0.001), CD83 (P = 0.006) and of costimulatory molecule CD86 (P = 0.003); an up-regulation of chemokine receptor CXCR4 (P = 0.031) and increased apoptosis (P < 0.001). CONCLUSIONS: Sepsis has a profound effect on PB DC compartment. These alterations appear to be sepsis-specific. Increased apoptosis and alteration of migration and trafficking mechanism may be involved in DC compartment modification. DC alterations could contribute to sepsis-mediated immunoparalysis.

Darbepoetin alfa for the treatment of anemia associated with myelodysplastic syndromes: efficacy and quality of life.

To evaluate efficacy, safety, changes in biological features, and quality of life (QoL) in low-risk anemic patients with MDS treated with darbepoetin alfa (DPO), 41 patients received DPO 150 microg weekly for 24 weeks. The dose was increased to 300 microg weekly in non-responsive patients. During treatment, 10/17 (59%) transfusion-dependent (TD) and 13/23 (56%) transfusion-free (TF) patients responded. In TF patients, Hb increased from 9.2 +/- 0.9 g/dL to 10.3 +/- 1.4 g/dL by 24 weeks (p = 0.004). The mean response duration was 22 weeks (95% CI: 19.7-24.0) in TF patients compared with 15.1 weeks (95% CI: 13.3-17.5) in TD patients. Response to treatment was associated with increases in QoL. Decreases in the percentage of apoptotic progenitor cells (p = 0.007) and CD34+ cells (p = 0.005) were observed. These results confirm previous studies demonstrating the safety and efficacy of DPO in anemic patients with MDS. Biological changes and improvement in QoL were associated with response. Adequate dosing is to be determined.

Chemotherapy-induced anemia in breast cancer patients treated with pegfilgrastim-supported dose-dense regimens.

The primary use of recombinant granulocyte colony-stimulating factors has reduced the incidence of febrile neutropenia during dose-dense adjuvant/neoadjuvant chemotherapy programs for breast cancer. Otherwise, in this population, filgrastim seems to worse chemotherapy-induced anemia, especially when administered with prolonged schedules that induced leukocytosis. No exhaustive data are available about the effect of long-lasting formulation of filgrastim (pegfilgrastim) on hemoglobin levels. We retrospectively analyzed the data regarding hemoglobin level and leukocyte count of 38 breast cancer patients treated with dose-dense anthracycline and/or taxane-based chemotherapy with pegfilgrastim support, both in adjuvant and in neoadjuvant settings. Mean hemoglobin levels progressively decreased throughout the treatment (without correlation with both the schedule of chemotherapy and the patient's age) but only two patients developed mild anemia. No significant correlation was found between the degree of leukocytosis and the hemoglobin decrease. These data suggest that pegfilgrastim, per se, doesn't seem to worse chemotherapy-induced anemia. This fact may be at least in part explains by its "balanced" impact on hematopoietic recovery during dose-dense chemotherapy.

Bone marrow stem cell damage after three different chemotherapy regimens for advanced Hodgkin's lymphoma.

The aim of this study was to evaluate the apoptotic damage to bone marrow cells caused by three chemotherapy regimens for advanced Hodgkin's lymphoma, ABVD, COPPEBVCAD and BEACOPP, which were randomly administered in the HD 2000 GISL trial. Bone marrow mononuclear cells (BMMCs) stained with anti-CD34 antibody and Annexin V, were evaluated by flow cytometry before starting chemotherapy, 30 days after completing chemotherapy and after 6 months. Results are expressed as the percentages of BMMCs positive to anti-CD34, to Annexin V or to both. Fourteen patients treated with ABVD, 11 with COPPEBVCAD and 13 with BEACOPP were evaluated before and 30 days after treatment. Late assessments were made in 6, 7 and 8 of them, respectively. No differences were found among the pretherapeutic flow cytometry findings in relation to the staging characteristics (marrow involvement included). All the regimens increased the apoptotic fraction of the whole mononuclear bone marrow cells (COPPEBVCAD did so significantly) and increased the CD34+ compartment (with significant early differences after ABVD and BEACOPP, tending to late persistence for ABVD, only). All the regimens increased the apoptotic CD34+ cells within the whole BMMC population (significantly after BEACOPP), although with a general trend to decrease in their percentage within the CD34+ compartment over time, even after the most dose-dense regimens. Based on the variations induced in the apoptotic fraction of all mononuclear and CD34+ cells, ABVD was the least toxic regimen and COPPEBVCAD the most toxic one.

Effect of peg-filgrastim-supported dose-dense adjuvant chemotherapy on the peripheral blood leukocyte phenotype in breast cancer patients.

The aim of this study was to evaluate the effect of dose-dense adjuvant chemotherapy regimens with peg-filgrastim support on the phenotype of peripheral blood leukocytes in breast cancer patients. We evaluated the leukocyte phenotype of 14 patients aged 46-67 years undergoing 4 courses of chemotherapy with either epirubucin/cyclophosphamide (n=7) or 5-fluorouracil/epirubucin/cyclophosphamide (n=7) followed by 4 courses of taxol supported by peg-filgrastim (6 mg) administered 72 h after each chemotherapy course. The overall leukocyte number significantly increased from the first treatment course, while total lymphocytes tended to decrease with a negative peak following the 6th course (p=0.03). B (CD19+, CD20+) and early B lymphocyte subsets (CD20+/CD38+) significantly decreased during treatment (p<0.05), while T lymphocyte subsets did not show significant changes, except a decrease in T helper (CD4+) cells. Immature T lymphocytes (CD4+/CD8+ subset), dendritic cells (CD11c+) and NK cells (CD56+) increased with respect to the baseline. Our results suggest that dose-dense chemotherapy programs with the support of peg-filgrastim did not significantly impair the immune system of breast cancer patients and allowed for a rapid restoration of most immune competent cells. These observations may have important clinical implications with a view to vaccination or other immunotherapeutic approaches to solid tumours.

Immunological stress in kidney cancer patients undergoing either open nephrectomy or nephron-sparing surgery: an immunophenotypic study of lymphocyte subpopulations and circulating dendritic cells.

Immunosuppression is a characteristic hallmark of renal cell carcinoma (RCC), with several complex immune defects, almost solely at the level of cell-mediated immune function, well evident even in patients at first diagnosis. The main circulating lymphocyte subsets and the total number of circulating dendritic cells were quantified in 47 RCC patients at diagnosis (T0), 12 h (T1), 24 h (T2) and 8 days following either radical nephrectomy or nephron-sparing surgery using flow cytometry. RCC patients presented, at baseline, (T0) a profound state of immunosuppression involving naïve T-cells, memory T-cells, CD16+ NK and total circulating dendritic cells, that worsened after 12 (T1) and 24 h (T2) from surgery, involving the majority of the analysed subsets; after 8 days (T3) from surgical removal of tumor, however, there was a return of all the analyzed parameters to the basal state. In conclusion, surgery causes transient but relevant immune suppression in RCC patients; even though, by day +8, this tends to return to baseline, immunostimulatory therapies could be considered in the peri-operative setting with the aim of reducing immunosuppression and, hopefully, also disease recurrence.

Flow cytometric detection of circulating dendritic cells in healthy subjects.

Dendritic cells (DCs) are the key antigen-presenting cells controlling the initiation of the T cell- dependent immune response. Currently, two peripheral blood DC subsets have been identified on the basis of their CD11c expression. The CD11c-negative (CD11c-) DCs (expressing high levels of CD123) are designated as lymphoid-derived DCs (DC2), whereas the CD11c+/CD123- cells, do identify the myeloid-derived DCs (DC1). A growing number of studies have been conducted in recent years on both the quantitative and functional alterations of DCs and their subsets in different pathological conditions. In the present study we assessed, using two different flow cytometric (FCM) techniques, the normal profile of blood DCs in 50 italian adult healthy subjects (M/F: 25/25, median age 42.5 years, range 20-65). The percentage and the absolute number of DCs and their subsets, were obtained starting from whole blood samples in two ways: 1) by calculating the number of DCs when gated as lineage-negative/ HLA-DR+ and identifing the two subsets as CD11c+ (DC1) and CD123+ (DC2) and 2) by using three specific markers: BDCA.1 (CD11c+ high/CD123+ low, myeloid DCs); BDCA.2 (CD11c-/ CD123+high, lymphoid DCs); BDCA.3 (CD11c+low /CD123-, myeloid DCs). Six parameters, 4-color FCM analysis were perfomed with a BD FACSCanto equipment. The mean values of the percentage and of the absolute number were: 0.5+/-0.2% and 30+/-11 cells/microL for DCs; 0.2+/-0.1% and 15+/-6 cells/microL for DC1; 0.2+/-0.1% and 15+/-7 cells/microL for DC2. The same values were: 0.2+/-0.1% and 16+/-7 cells/microL for BDCA.1; 0.2+/-0.1% and 12+/-7 cells/microL for BDCA.2; 0.02+/-0.01% and 2+/-1 cells/microL for BDCA.3, respectively. Our study confirmes that the two types of FCM analysis are able to identify the DC population. We also provides the first reference values on normal rates and counts of blood DCs in italian adult healthy subjects.

In vivo biological effects of pegfilgrastim after myelosuppressive chemotherapy in breast cancer.

BACKGROUND: No exhaustive data are available on the in vivo biological effects of pegfilgrastim utilized in dose-dense chemotherapy (CT). The cytokinetic effects exerted in a multicyclic CT program by this cytokine on CD34+/38+ peripheral blood (PB) progenitor cells was the focus of this study. PATIENTS AND METHODS: PB samples from 19 breast cancer patients treated with 4 courses of docetaxel and epirubicin followed by pegfilgrastim were studied. The absolute number of CD34+/38+ circulating progenitor cells (CPCs) along with the percentage undergoing GO/G1, S and G2-M phases of the cell cycle or showing apoptotic features, were evaluated at baseline, after the first and before the fourth CT course using a dedicated flow cytometric technique. RESULTS AND CONCLUSION: Pegfilgrastim, after CT, exerted stimulatory effects on the cell cycle status of PB CD34+/38+ CPCs, at the same time protecting them from apoptosis. This was particularly evident 7 days after administration and tended to decrease one week later, without additional cytokinetic changes during the subsequent CT courses.

Renal cell carcinoma-induced immunosuppression: an immunophenotypic study of lymphocyte subpopulations and circulating dendritic cells.

BACKGROUND: Renal cell carcinoma (RCC)-induced immune dysfunction in patients at first diagnosis was investigated. PATIENTS AND METHODS: The main circulating lymphocyte subsets, the total number of circulating and intratumor dendritic cells and the titers of circulating VEGF were quantified in 47 RCC patients, using flow cytometric, immunohistochemical and ELISA assays. RESULTS: Despite a significant activation of CD3/HLA-DR+ lymphocytes and of the CD56+ NK subset, RCC patients presented a marked immunosuppression of CD4/CD45RA naïve T-cells, CD4/CD45RO memory T-cells, CD16+ NK-cells, and total circulating dendritic cells, as well as a significant increase of lymphocytes co-expressing the CD4 and CD8 antigens. Finally, CD16+/CD56+ NK and DCs were poorly represented in tumor specimens. CONCLUSION: The complex immunological dysfunctions demonstrated involve different levels of immunocompetence and indicate a pattern of major disturbance of the immune system.

Dendritic cells and vascular endothelial growth factor in colorectal cancer: correlations with clinicobiological findings.

OBJECTIVE: Dendritic cells (DC) are central to the development of immune system responses. In a cohort of 54 patients affected by colorectal cancer, we prospectively investigated the number of peripheral blood (PB) DC type 1 (DC1) and type 2 (DC2) and correlated their counts and functionality to the stage of the disease and to vascular endothelial growth factor (VEGF) levels. RESULTS: At diagnosis, compared with healthy controls, patients presented reduced PBDC1 and PBDC2 numbers (p < 0.001). Moreover, in cancer patients, PBDC showed low levels of DC-associated antigens (HLA DR, p = 0.004; CD11c, p < 0.001; CD83, p = 0.01; CD86, p = 0.007 and Mannose receptor, p = 0.029), an upregulation of CXCR4 (p = 0.017) and a reduced T cell stimulation capability (p < 0.001). DC1 and DC2 loss was higher in stage D versus stage ABC patients (p = 0.003 and p = 0.002, respectively); surgery and chemotherapy appeared to attenuate a DC defect, although the restoration of normal PBDC levels is completed only at 6 and 12 months after diagnosis, respectively. In this series of patients, PBDC1 and PBDC2 numbers inversely correlated with VEGF serum levels (p < 0.001), suggesting a possible effect of this cytokine on DC compartment. In culture, the exposure of monocyte-derived DC to VEGF produced a dramatic alteration of DC differentiation by (1) induction of apoptosis, (2) alteration of DC immunophenotypic profile and (3) increased CXCR4 expression. Exposure to anti-VEGF blocking antibodies reversed VEGF inhibitory effects in all cases. CONCLUSIONS: These findings suggest that in colorectal cancer patients there is a numerical and functional impairment of PBDC compartment possibly related to the stage of the disease and to VEGF levels.

Flow cytometric analysis of circulating dendritic cell subsets and intracellular cytokine production in advanced breast cancer patients.

Patients with advanced cancer are known to have dysfunctions of the immune system. Dendritic cells (DCs) are potent antigen-presenting cells that play a crucial role in antitumor immune response. At least two peripheral blood DC subsets have been described: myeloid-derived CD11c+CD123- DCs (DC1) and lymphoid-derived CD11c-CD123+ DCs (DC2). Upon interaction with T cells, DC2 seemed to support the generation of a Th2 response, while DC1 predominantly prime a Th1 response. Our study was aimed at investigating the number of circulating DCs, and their subsets and functions in 32 patients with advanced breast cancer that achieved an objective response after a standard-dose sequential chemotherapy (CT), compared to 40 healthy controls. Circulating DC subsets and intracellular cytokine production in CD4+ and CD8+ subsets were analyzed using a tri-color flow cytometry assay. DC subsets were identified in peripheral blood, calculating their percentage gated as lin- HLA-DR+ and using BDCA-1, BDCA-2 and BDCA-3 specific markers, as DC1 and DC2 according to expression of CD11c and CD123, respectively. Intracellular cytokines were evaluated in CD4+(Th1 and Th2) and CD8+ (Tc1 and Tc2) T lymphocytes. The mean percentage of BDCA-1+BDCA-2+BDCA-3 was similar to that of DC1+DC2 (p=ns). The mean percentage of DCs and DC1/DC2 ratio were slightly decreased before CT in cancer patients compared with healthy controls (p=ns). After CT, the percent-age of DC1 further decreased (p=0.02). The production of IFN-gamma (Th1 and Tc1) significantly decreased (p<0.03) while that of IL-4 (Th2 and Tc2) increased (p=0.04), thus confirming a shift toward a Th2 CD4 and Tc2 CD8 phenotype and the predominance of type 2 DCs. Our results could help clarify the mechanisms of the immune response or immune status of patients with advanced breast cancer that undergo cytotoxic CT and contribute to improve the selection of potential candidates for active immunotherapy trials.

Thalidomide treatment reduces apoptosis levels in bone marrow cells from patients with myelodysplastic syndromes.

We treated with thalidomide seven patients with primary MDS and observed reduction of the transfusion requirement in three cases and reduction of bone marrow blasts in one case. The apoptotic rate of bone marrow cells diminished significantly from a mean of 43.8% to a mean of 17.5%, whereas the proliferative activity did not change. Plasma TNF-alpha, bFGF, IL-1beta levels decreased variably, whereas VEGF levels tended to increase. Matrix metalloproteinases 2 and 9 expression decreased in bone marrow cells of responders. A reduction of CD4 cells and an increase of NK cells was observed in the peripheral blood. Thus, thalidomide may produce a fairly good hematological improvement in erythroid series in MDS, with complex biological mechanisms.

Lack of dendritic cell mobilization into the peripheral blood of cancer patients following standard- or high-dose chemotherapy plus granulocyte-colony stimulating factor.

BACKGROUND: Dendritic cells (DC), the most specialized antigen-presenting cells, can be detected in the peripheral blood (PB) and divided into two subsets of populations, DC1 and DC2, endowed with different functions. The aim of this study was to evaluate the effect on DC release and on their subsets of three regimens utilized to mobilize CD34+ cells into the PB in cancer patients and in normal CD34+ cell donors. PATIENTS AND METHODS: The mobilizing sequences were: standard-dose epirubicin+taxol+granulocyte-colony-stimulating factor (G-CSF; 15 patients with advanced breast cancer), high-dose cyclophosphamide (CTX)+G-CSF (10 patients with breast cancer patients and 7 with non-Hodgkin's lymphoma, NHL), and G-CSF alone (5 normal donors of CD34+ cells for allogeneic transplantation). Comparative data were obtained from the steady-state PB of 20 healthy volunteers. For flow cytometric analysis, DC were gated as negative for specific lineage markers (CD3, CD11b, CD14, CD16, CD56, CD19, CD20, CD34) and positive for HLA-DR. The DC1 and DC2 subsets were defined as CD11c and CDw123 positive, respectively. RESULTS: The percentages of DC at baseline and the time of CD34+ cell peak were: 0.48 and 0.51 for standard-dose chemotherapy (CT); 0.55 and 0.63 for breast cancer after high-dose CTX+G-CSF; 0.53 and 0.71 for NHL after high-dose CTX+G-CSF; and 0.51 and 0.54 for normal donors of CD34+ cells after G-CSF alone (all p=n.s.). Mean DC1/DC2 ratios in each study group at the time of CD34+ cell peak were 0.10, 0.12, and 0.18, respectively. Finally, in the group of healthy volunteers, the percentage of circulating DC was 0.95 and the mean DC1/DC2 ratio was 1.28. CONCLUSION: To our knowledge, this is the first report that demonstrates that both standard-dose or high-dose CT, when utilized together with G-CSF, do not induce DC mobilization into the PB, whereas a reversed DC1/DC2 ratio is observed. Furthermore, a lack of significant DC mobilization after G-CSF alone was also seen, in contrast to what was previously observed by others. These data should be taken in account when evaluating clinical correlations between DC number and CPC engraftment in both the transplantation setting, when monitoring the effects on the immune system of combinations of new drugs and/or cytokines, and when high numbers of DC are required for both experimental and clinical applications.

Impact of topotecan-based chemotherapy on the immune system of advanced ovarian cancer patients: an immunophenotypic study.

The effects of topotecan-based chemotherapy (CT) on peripheral blood lymphocyte (PBL) subsets were evaluated in ovarian cancer patients. Fourteen patients with epithelial ovarian cancer, at the diagnosis or relapsed after platinum-based CT, were treated with: a) topotecan in association with carboplatin and taxanes as first line CT; b) topotecan alone or c) topotecan in association with carboplatin both as second line of treatment after platinum. The phenotype of PBL was determined before starting treatment and immediately before each CT course by flow cytometric analysis. Before starting CT, the absolute number of lymphocytes and the CD2+, CD3+, CD4+ subsets were significantly lower in pre-treated patients and not significantly altered in CT-naive patients with respect to a cohort of 20 healthy donors utilized as control. Lymphocytes co-expressing CD4+/CD8+ were significantly higher in both subgroups of patients than in normal donors. CD4+/CD45RA+ and CD4+/CD45RO+ subsets were significantly decreased in pre-treated patients and normal in CT-naive patients. CD3+/HLA-DR+ T cell population significantly increased in CT-naive patients at baseline. During CT and after its discontinuation, no relevant changes were recorded for both subgroups of patients with respect to the baseline in lymphocyte absolute count, CD2+, CD3+, CD4+, CD4+/CD45RO+ subsets, while CD4+/CD45RA+ subpopulation was significantly decreased in CT-naive patients. CD8+, CD19+, CD20+, CD16+, CD56+, CD2+/CD25+ subsets did not differ statistically comparing to normal donors both at baseline and during CT. The treatment was well tolerated and no patient developed non-neutropenic infection. Topotecan-based therapy does not have a negative impact on PBL in either CT-naive or in pretreated ovarian cancer patients. This information should be considered when utilizing topotecan with other anticancer drugs in the adjuvant setting as well as when dose-intensification of topotecan with stem cell support is planned.