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Preliminary results from a pilot trial on trastuzumab's mechanism of action against operable breast tumors overexpressing Her2 suggested a role for antibody-dependent cell cytotoxicity (ADCC). To examine factors affecting ADCC intensity and variability, we extended this study to the phenotypic and functional analysis of circulating mononuclear cells in 18 patients. ADCC was induced by trastuzumab therapy in 15 of 18 patients (83%). Inability to develop ADCC in three patients did not depend on inadequate levels of trastuzumab because further increase in its concentration in vitro was ineffective. Rather, susceptibility to develop ADCC was fairly predicted by test with trastuzumab before therapy and was correlated to the number of lymphocytes coexpressing CD16 and CD56. Phenotypic analysis at the end of ADCC evaluating down-regulation of CD16, and up-regulation of CD69 and CD107a, confirmed that natural killer (NK) cells and CD56(+) T cells were involved in productive engagement of trastuzumab. Also, the killing efficiency of CD16(+) lymphocytes was influenced by 158 V/F polymorphism of Fc gamma RIII (CD16), whereas variations of CD247 on NK cells were consistent with trends between ADCC before and after therapy. Complete pathologic response was observed in one patient showing ADCC of outstanding intensity, whereas four cases of partial response showed intermediate ADCC; none of the three patients unable to mount ADCC had significant tumor regression. These data indicate that quantity and lytic efficiency of CD16(+) lymphocytes are major factors for ADCC induction by trastuzumab, and confirm that breast cancer responses to short-term trastuzumab monotherapy may depend on involvement of the ADCC mechanism.

Purpose: To elucidate the mechanism by which trastuzumab, a humanized monoclonal antibody against HER2 with proven survival benefit in women with HER2-positive metastatic breast cancer, mediates its antitumor activity., Experimental Design: A pilot study including 11 patients with HER2-positive tumors treated in a neo-adjuvant setting with trastuzumab was performed. Trastuzumab was administered i.v. at a dose of 4 mg/kg followed by three weekly i.v. doses of 2 mg/kg. The primary tumor was surgically removed 7 days after the last treatment. Surgical samples, tumor biopsies, and lymphocytes from these patients were collected for biological studies., Result: Clinical data indicated one complete pathological remission and four partial remissions using RECIST (Response Evaluation Criteria in Solid Tumors). Trastuzumab was well tolerated and neither serious adverse events nor changes in cardiac function were observed during this short-term treatment and after surgery. The biological data showed that, independent of response, (a) all patients showed high levels of circulating trastuzumab; (b) saturating level of trastuzumab was present in all of the tumors; (c) no down-modulation of HER2 was observed in any tumors; (d) no changes in vessel diameter was observed in any tumors; (e) no changes in proliferation was observed in any tumors; and (f) a strong infiltration by lymphoid cells was observed in all cases. Patients with complete remission or partial remission were found to have a higher in situ infiltration of leukocytes and a higher capability to mediate in vitro antibody-dependent cellular cytotoxicity activity., Conclusions: The results of this pilot study argue against trastuzumab activity in patients through down-modulation of HER2 but in favor of antibody-dependent cellular cytotoxicity guiding efforts to optimize the use of trastuzumab in breast cancer patients.

Dendritic cells (DCs) are fundamental for immunity. We investigated reconstitution of plasmacytoid DC (PDC) and myeloid DC (My-DC) precursors in the first 2 months after allogeneic hematopoietic stem cell transplantation (Allo-HSCT). Circulating DCs were monitored from the earliest phase of hematopoietic reconstitution in 43 children given standard therapy to prevent graft-versus-host disease (GVHD) and either treated or untreated with granulocyte colony-stimulating factor (G-CSF) after HSCT. In patients without GVHD, both My-DCs and PDCs reached consistently high absolute values during the initial phase. Time of engraftment did not differ between My-DCs and PDCs, regardless of administration of G-CSF. Treatment with G-CSF (1) accelerated early recovery of My-DC absolute numbers; (2) was associated with lower numbers of both My-DCs and PDCs in the later phase; and (3) significantly reduced the proportion of interleukin-12 (IL-12)-secreting cells. In some patients who developed acute GVHD, we found high numbers of circulating DC precursors during the early phase of this complication. However, treatment with steroids invariably induced rapid decrease of PDCs. Altogether, these data provide an evaluation of DC release after Allo-HSCT, indicate that postgrafting administration of G-CSF impairs the appearance of IL-12-producing DCs, and suggest that DC homeostasis may be disrupted at onset of GVHD.

Recovery of total T cell numbers after in vivo T-cell depletion in humans is accompanied by complex perturbation within the CD8+ subset. We aimed to elucidate the reconstitution of CD8+ T cells by separate analysis of putative naïve CD95- CD28+, memory CD95+ CD28+ and CD28- T cell compartments after acute maximal depletion by high-dose chemotherapy (HD-ChT) in women with high-risk breast cancer. We found that recovery of putative naïve CD8+ CD95- CD28+ and CD4+ CD95- CD28+ T cells, was compatible with a thymus-dependent regenerative pathway since their recovery was slow and time-dependent, their values were tightly related to each other, and their reconstitution patterns were inversely related to age. By analysing non-naïve T cells, a striking diversion between putative memory T cells and CD28- T cells was found. These latter increased early well beyond normal values, thus playing a pivotal role in total T-cell homeostasis, and contributed to reduce the CD4 : CD8 ratio. In contrast, putative memory T cells returned to values not significantly different from those seen in patients at diagnosis, indicating that this compartment may recover after HD-ChT. At 3-5 years after treatment, naïve T cells persisted at low levels, with expansion of CD28- T cells, suggesting that such alterations may extend further. These findings indicate that CD28- T cells were responsible for 'blind' T-cell homeostasis, but support the notion that memory and naïve T cells are regulated separately. Given their distinct dynamics, quantitative evaluation of T-cell pools in patients undergoing chemotherapy should take into account separate analysis of naïve, memory and CD28- T cells.

Background: Mitomycin C (MMC), an antitumoral antibiotic, has been described inhibiting the proliferation of different cell types in vitro. Since irradiation is commonly used to stop the cell growth of adherent cells in several experimental models, we aimed to define the optimal dose and incubation time of MMC capable of inhibiting the growth of murine fibroblasts, used as an adherent feeder layer in long-term hematopoietic culture assay., Methods: M2 10B4 (both parental and engineered to produce human IL-3 and G-CSF) and Sl/Sl (engineered to produce human IL-3 and steel factor) murine fibroblast cell-lines, frequently used in LTC-IC assay, were incubated with increasing doses of MMC for either a short (3 h) or a long (16 h) period. The efficiency of MMC in stopping the cell growth was evaluated for 5 days following MMC removal. The effects of MMC treatment on human hematopoietic cells were studied using both LTC-IC and limiting dilution (CAFC) assays., Results: The growth of M2 10B4 cells was stopped at 3 and 16 h in the presence of 20 microg/mL and 2 microg/mL of MMC, respectively while Sl/Sl fibroblasts required a lower dose of drug (2 and 0.2 microg/mL, respectively). No significant difference was found between the number of LTC-IC or CAFC obtained from cultures containing irradiated or MMC-treated feeder cells., Discussion: MMC inhibits the growth of murine fibroblasts used as adherent feeder cells in long-term culture assays, without interfering with the subsequent growth of co-cultured hemopoietic cells. Different cell types might present a different sensitivity to MMC and therefore a dose-response curve to MMC has to be obtained for each cell type of interest.

Background: Recurrence after PBSC transplantation in breast cancer (BC) patients may be related to the reinfusion of tumor cells contaminating the graft. We have developed a liquid culture (LC) method for the identification of viable epithelial tumor cells in PBSC collections., Methods: Mononuclear fraction from PBSC harvests of BC patients undergoing high dose chemotherapy (HDC) (adjuvant setting n = 60, metastatic disease n = 30) were seeded in petri dishes containing round cover slips. Cells were cultured for 3 weeks, then cover slips were stained with the pan-cytokeratin A45-B/B3 mAb and scored under a light microscope. Samples were considered positive when more than one adherent cell or a cluster of cells staining bright red was present. Results were compared with those obtained on cytospins prepared directly from the PBSC harvest. Specificity of the method was tested on lymphoma patients, collections: all were negative. The sensitivity, evaluated by serial dilutions of CG5 BC cell line, was 1 epithelial cell in 10(6) mononuclear cells., Results: The percentage of positivity was superimposable in the two groups (adjuvant 25%, metastatic 24%). However, a significantly higher proportion of positive samples from metastatic vs adjuvant patients has shown the presence of tumor clusters (86% vs 33%, p = 0.063). In 21% of all samples a discrepancy with the results obtained by immunocytochemical analysis (ICC) was found, mostly due to liquid-culture-positive/ICC-negative PBSCs., Discussion: Our data suggest that LC assay may enhance the identification of viable disseminated epithelial tumor cells in PBSC grafts and might provide insights about their growth capacity.

Background and Objective: Based on our preliminary experience, we have further evaluated the capacity of the paclitaxel/epirubicin combination (at the dose of 175 and 90 mg/m(2), respectively) plus G-CSF to mobilize hematopoietic progenitors into the circulation., Design and Methods: The study was conducted in a homogeneous cohort of 25 stage IV breast cancer patients showing response to three cycles of the same chemotherapy regimen and who were included in a high-dose chemotherapy program., Results: In most cases (68%) more than 5_10(6) CD34+ cells/kg b.w. (the threshold fixed in our study) were collected by a single leukapheresis, 28% and 4% of patients requiring 2 and 3 procedures, respectively. Based on the CD34+ cell count in the peripheral blood, most of the leukaphereses (53%) were performed on day 11 after chemotherapy. More than 50 CD34+ cells/mL along with a preleukapheresis WBC count between 10 and 20_10(9)/L predicted that only a single harvest would be required in 100% of cases. The evaluation of the clonogenic potential of collected cells showed that a large number of committed colony-forming cells (CFCs) and more primitive long-term culture-initiating cell (LTC-IC) hematopoietic progenitors were present in 20 harvests studied., Interpretation and Conclusions: These data demonstrate that the epirubicin/paclitaxel combination followed by G-CSF, besides being a very active regimen in MBC, is effective in releasing large amounts of progenitor cells into the circulation which can then be safely employed to support myeloablative regimens.

Background and Objective: Concerns about the risk of transfusion therapy are driving towards new strategies which are designed to minimize exposure to allogeneic blood products. We aimed to find out whether it is possible to support the phase of thrombocytopenia following high-dose chemotherapy (HDC) and circulating progenitor cells (CPC) transplantation by autologous platelet concentrates (PC)., Design and Methods: PC were collected from 32 patients undergoing HDC and CPC transplantation for stage II/III breast cancer. A single plateletpheresis was performed at rebound after high-dose cyclophosphamide, when platelet count exceeded 250 x 10(9)/L. PC were cryopreserved in 5% DMSO after controlled-rate freezing and stored in liquid nitrogen. In vitro studies of cryopreserved platelets (aggregation, ATP release and change of mean platelet volume induced by EDTA) were performed. When platelet counts dropped below 20 x 10(9)/L following HDC (thiotepa 600 mg/m2, L-PAM 160 mg/m2) and CPC transplant (CD34+ cells > 5 x 10(6)/kg), PC were thawed in a 37 degrees C water bath, centrifuged to remove DMSO, resuspended in autologous plasma and reinfused within one hour., Results: Large quantities of platelets were harvested in all patients (median 6.6 x 10(11), range 4.8-12.2). In vitro studies showed preserved platelet function as compared to both fresh platelets and standard PC. Twenty-eight out of 32 patients received autologous PC. At the time of transfusion most of the patients were febrile (> 38 degrees C) and had mucositis > G2. The median number of platelets reinfused was 3.8 x 10(11) (range 2.0-8.1) with a median loss during the freeze-thaw-wash procedure of 37%. Autotransfusion was able to maintain platelet count above 20 x 10(9)/L in most patients, with a corrected count increment > 7.5 in 20 cases. Four patients required one additional allogeneic transfusion, two because of a poor increment and two due to a late-occurring epistaxis. No side effects related to PC infusion were recorded. Sixteen control patients who received the same HDC and a similar number of CD34+ cells required a total of 17 allogeneic PC units (1 patient did not require platelet transfusion)., Interpretation and Conclusions: Our data demonstrate that large doses of autologous platelets can easily be collected and safely administered to support the period of thrombocytopenia in patients undergoing HDC and CPC transplantation. Autologous PC in these patients can abrogate the risks deriving from allogeneic platelet transfusion.

Background: We compared hematopoietic progenitor cell (HPC) collection and neoplastic cell contamination in breast cancer patients given cyclophosphamide (CTX) plus granulocyte-colony stimulating factor (G-CSF) or G-CSF alone for mobilization., Patients and Methods: In 57 stage II-III breast cancer patients, CD34+ cells, colony-forming units-granulocyte macrophage (CFU-GM), early HPC and breast cancer cells were counted in HPC collections obtained after CTX plus G-CSF (n = 27) or G-CSF-alone mobilization (n = 30)., Results: The CD34+ cell collection was about two-fold greater after CTX plus G-CSF mobilization (11.0 +/- 7.9 vs. 5.8 +/- 3.5 x 10(6)/kg, P < 0.001). Similarly, the total number of CFU-GM, CD34+CD38- cells and of week-5 cobblestone area forming cells (CAFC) collected was significantly higher in patients mobilized with CTX plus G-CSF. Breast cancer cells were found in the apheresis products of 22% of patients mobilized with CTX plus G-CSF and in 10% of patients mobilized with G-CSF alone (P = 0.36). Of seven patients who failed G-CSF-alone mobilization and eventually underwent chemotherapy plus G-CSF mobilization, none had cytokeratin-positive cells after G-CSF mobilization, whereas four out of seven had cytokeratin-positive cells after chemotherapy plus G-CSF (P = 0.07 by chi 2 test)., Conclusion: The CTX plus G-CSF mobilization protocol was associated with a significantly higher HPC collection. However, this benefit was not accompanied by a reduction in the incidence of tumor-contaminated HPC graft.

Peripheral blood progenitor cells (PBPCs) are increasingly used instead of bone marrow for autologous or allogeneic transplantation. In this study PBPCs mobilized in cancer patients by chemotherapy and granulocyte-colony stimulating factor were collected by apheresis and first enriched by immunoaffinity removal of lineage positive cells. When these cells were exposed to both cyclophosphamide and taxol or cultured for 7 days in the presence of 5-fluorouracil, stem cell factor, and interleukin-3, 88% to 93% of the enriched PBPCs were killed and short-term clonogenic capacity in methylcellulose assays was lost, but week-5 cobblestone area-forming cell (CAFC) enrichment was higher than 10-fold in comparison to enriched PBPCs and higher than 700-fold in comparison to unmanipulated apheresis cells. After drug exposure, most of the progenitors displayed a CD34+, CD38-, multidrug-resistance (MDR+), Rhodamine 123 low, Hoechst 33342 low phenotype, and as few as 180 of these drug-resistant cells were able to generate a stable multilineage human hematopoiesis in sublethally irradiated immunodeficient mice. In these animals, the level of human hematopoietic engraftment was significantly increased by cotransplantation of irradiated cells from the human L87/4 stromal cell line. These observations are consistent with the functional isolation of a population of very early hematopoietic progenitors and might help to design new protocols for the removal of neoplastic cells from autografts.

The efficacy of high-dose chemotherapy (HDC) and circulating progenitor cell (CPC) transplantation in metastatic breast cancer (MBC) relies mainly on giving this treatment after a response to conventional induction chemotherapy has been achieved. For this reason an optimal mobilization regimen should be therapeutically effective while minimizing the number of leucaphereses required to support the myeloablative therapy. The combination of an anthracycline and paclitaxel in chemotherapy-untreated MBC has produced impressive response rates. We evaluated the CPC-mobilizing capacity of the combination epirubicin (90 mg m(-2)) and paclitaxel (135 mg m(-2)) followed by filgrastim (5 microg kg(-1) day(-1)) starting 48 h after chemotherapy administration in ten patients with MBC who were eligible for an HDC and CPC transplantation programme. Leucaphereses were performed by processing at least two blood volumes per procedure at recovery from neutrophil nadir when CD34+ cells in the peripheral blood exceeded 20 microl(-1). In most patients (six out of 10) more than 2.5 x 10(6) CD34+ cells kg(-1), a threshold considered to be sufficient for haematopoietic reconstitution, were collected with a single apheresis. In the remaining four patients an additional procedure, performed the following day, was enough to reach the required number of progenitors. These data suggest that the epirubicin-paclitaxel combination, besides being a very active regimen in MBC, is effective in releasing large amounts of progenitor cells into circulation.