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High-Dose chemotherapy with peripheral blood progenitor cell transplantation (PBPCT) is a potentially curative treatment option for patients with both hematological malignancies and solid tumors, including breast cancer. However, based on a number of clinical studies, there is strong evidence that minimal residual disease (MRD) persists after high-dose chemotherapy in a number of patients, which eventually results in disease recurrence. Therefore, several approaches to the treatment of mrd are currently being evaluated, including treatment with dendritic cell (DC)-based cancer vaccines. DCs, which play a crucial role with regard to the initiation of T-lymphocyte responses, can be generated ex vivoeither from CD34hematopoietic progenitor cells or from blood monocytes. They can be pulsed in vitrowith tumor-derived peptides or proteins, and then used as a professional antigen-presenting cell (APC) vaccine for the induction of antigen-specific T-lymphocytes in vivo. This paper summarizes our preclinical studies on the induction of primary HER-2neu specific cytotoxic T-lymphocyte (CTL) responses using peptide-pulsed DC. As HER-2neu is overexpressed on 30-40 of breast and ovarian cancer cells, this novel vaccination approach might be particularly applicable to advanced breast or ovarian cancer patients after high-dose chemotherapy and autologous PBPCT.

Macrophages (MAC) are important effector cells of the immune system but also play an essential role as regulatory cells in hematopoiesis. They originate from circulating monocytes (MO) as immature precursor cells that undergo terminal differentiation upon migration from the capillary bed into the various tissues. In the presence of serum, MAC maturation from blood MO is observed in vitro and can be followed by the expression of maturation-associated antigens (MAX.1, .3, .11, and .26; transferrin receptor, 13C2, CD16). We have tested blood MO from 22 patients with aplastic anemia (AA) for their capacity to undergo terminal maturation in vitro. After isolation, blood MO in six patients expressed CD14 molecules at low density when compared to normals. On culture for 7 days, in 15 patients various abnormalities could be shown by phenotype analysis using cell-enzyme-linked immunosorbent assay (ELISA) and an immunoperoxidase staining technique of single cells. Abnormalities ranged from the distinctive failure of mature MAC to express single surface antigens (eg, gp64-MAX.1) to complete inhibition of the development of a MAC maturation-associated phenotype. In three patients the maturational defect was found to persist in complete remission after successful therapy with antileukocyte globulin (ALG). Neither in other immunosuppressed or multiple-transfused patients nor in those with bone marrow hypoplasia secondary to cancer chemotherapy and during hematologic reconstitution following autologous bone marrow transplantation (BMT), defective MO maturation in vitro was seen. Our data provide evidence for the existence of serious disorders within the MO-MAC lineage in patients with AA. This observation may either reflect the stem-cell defect or indicate a MAC involvement in the pathogenesis of the disease.

We report on the requirements that have to be met to combine a standard- dose chemotherapy regimen with broad antitumor activity with the mobilization of peripheral blood hematopoietic progenitor cells. Thirty- two cancer patients were given a 1-day course of chemotherapy consisting of etoposide (VP16), ifosfamide, and cisplatin (VIP; n = 46 cycles), followed by the combined sequential administration of recombinant human interleukin-3 (rhIL-3) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). Control patients received GM-CSF alone or were treated without cytokines. Maximum numbers of peripheral blood progenitor cells (PBPC) were recruited on day 13 to 17 after chemotherapy, with a median of 418 CD34+ cells/microL blood (range, 106 to 1,841) in IL-3/GM-CSF-treated patients, 426 CD34+/microL (range, 191 to 1,380) in GM-CSF-treated patients, and 46 CD34+/microL (range, 15 to 148) in patients treated without cytokines. In parallel, there was an increase in myeloid (10,490 colony-forming unit-granulocyte-macrophage [CFU-GM]/mL blood; range, 1,000 to 23,400), as well as erythroid (10,660 burst-forming unit-erythroid [BFU-E]/mL blood; range, 3,870 to 24,300) and multipotential (840 CFU-granulocyte, erythrocyte, monocyte, megakaryocyte [GEMM]/mL blood; range, 160 to 2,070) progenitor cells in IL-3 plus GM-CSF-treated patients. In GM-CSF-treated patients, significantly less precursor cells of all lineages were mobilized, particularly multipotential progenitors (400 CFU-GEMM/mL blood; range, 200 to 2,150). Only small numbers of CD34+ cells and clonogenic progenitor cells could be recruited in intensively pretreated patients. Our data document that after standard-dose chemotherapy-induced bone marrow hypoplasia, IL-3 plus GM-CSF can be used to recruit PBPC, which might shorten the hematopoietic recovery after high-dose chemotherapy in chemosensitive lymphomas or solid tumors.

To investigate the feasibility of peripheral blood CD34+ cell selection and to analyze CD34+ cell-mediated engraftment after high-dose chemotherapy, we performed a phase I/II trial in 21 patients with advanced malignancies. The rationale for the selection of CD34+ cells from peripheral blood progenitor cell (PBPC) collections is based on the observation that contaminating tumor cells can be depleted approximately 3 logs using this procedure. CD34+ cells from chemotherapy+granulocyte colony-stimulating factor-mobilized PBPCs were positively selected with an avidin-biotin immunoadsorption column (CEPRATE SC system). One leukapheresis product with a median number of 2.8 x 10(6) CD34+ cells/kg was labeled with a biotinylated anti-CD34 monoclonal antibody and subsequently processed over the column. The yield of selected CD34+ cells was 73% +/- 24.6%. The purity of the CD34+ cell fraction was 61.4% +/- 19.7%. CD34+ cells were shown to represent predominantly committed progenitors coexpressing CD33, CD38, and HLA-DR molecules (lin+). They gave rise to myeloid as well as erythroid and multilineage colonies in vitro. In addition, positively selected CD34+ cells also comprised early hematopoietic progenitor cells, as shown by the presence of CD34+/lin- cells. Transfusion of positively selected CD34+ cells (2.5 x 10(6) CD34+/kg; range, 0.45 to 5.1) after high-dose VP16 (1,500 mg/m2), ifosfamide (12 g/m2), carboplatin (750 mg/m2), and epirubicin (150 mg/m2) (VIC-E) in 15 patients resulted in a rapid and stable engraftment of hematopoiesis without any adverse events. As compared with 13 historical control patients reconstituted with a comparable number of unseparated PBPCs, time to neutrophil and platelet recovery was identical in both groups (absolute neutrophil count > 500/microL, day + 12; platelet count > 50,000/microL, day + 15). These data indicate that autologous peripheral blood CD34+ cells and unseparated PBPCs mediate identical reconstitution of hematopoiesis after high-dose VIC-E chemotherapy. Because positive selection of CD34+ cells from mobilized blood results in a median 403-fold depletion of T cells, allogeneic CD34+ cells from mobilized blood should be investigated as an alternative to bone marrow cells for allotransplantation.

Cytokine-supported ex vivo expansion of peripheral blood progenitor cells (PBPCs) offers new perspectives for autografting after high-dose chemotherapy. One of the potential advantages is the possibility to reduce the volume of blood processed from the patient, thus allowing reduction of the overall tumor cell number in the final autograft. However, ex vivo expansion will only be advantageous if contaminating tumor cells are not expanded concomitantly. This question has not previously been addressed. Therefore, we analyzed unseparated PBPC preparations, CD34(+)-selected cell fractions, and ex vivo-expanded cell preparations from stage IV (n = 16) and high-risk stage II/III (n = 8) breast cancer patients for the presence of human epithelial antigen-(HEA) or cytokeratin (CK)-positive tumor cells. We found that three of 16 (18.8%) of the unseparated PBPC products from stage IV patients were HEA- and/or CK-positive, whereas none of the stage II/III patients were found to be positive after two cycles of induction chemotherapy with etoposide (VP16), ifosfamide, cisplatin, and epirubicin (VIP-E). After CD34+ cell selection (Ceprate SC; CellPro, Bothell, WA) and stem-cell factor (SCF), interleukin (IL)-1, IL-3, IL-6, and erythropoietin (EPO)-mediated ex vivo expansion of the CD34+ cells for 14 to 21 days, no tumor cells could be detected in these primary breast cancer patients at a sensitivity of 1 tumor cell per 4 x 10(5) nucleated cells. Thus, to answer the question of whether tumor cells are expanded concomitantly on ex vivo expansion of normal CD34+ cells, we cocultured defined numbers of primary renal carcinoma cells (RS-85), xenograft-derived breast cancer cells, and small-cell lung cancer cells with CD34+ cells selected from normal donors or cancer patients, either in serum or serum-free culture media. We found that none of the three epithelial tumor cell types increased significantly in number during a 14-day coculture period when compared with normal CD34+ cells alone or tumor cells alone, which increased 110- +/- 77-fold and 45- +/- 26-fold, respectively. However, during coculture, the tumor cells did not undergo cell death and were able to regrow when maintained in serum for longer time periods. We conclude that cytokine-supported expansion cultures of positively selected CD34+ PBPCs from primary high-risk stage II/III or stage IV breast cancer patients do not contain detectable tumor cells, which suggests that there is no increased risk of concomitantly expanding tumor cells. Moreover, cocultures of exogenously mixed tumor cell lines with normal CD34+ cells showed a relative disadvantage of tumor cell growth compared with the growth of hematopoietic cells, again without an apparent risk of concomitantly expanding tumor cells. However, considering the pronounced heterogeneity of tumor cell kinetics, ex vivo-expanded PBPC from cancer patients should be monitored for minimal residual disease.

Infection of hematopoietic progenitor cells with the human cytomegalovirus (HCMV) has been proposed as an explanation for the cytopenias associated with HCMV-related disease. To test this hypothesis, CD34+ cells, which include the hematopoietic progenitors, as well as mature leukocyte populations were purified on a fluorescence- activated cell sorter and analyzed for HCMV DNA by polymerase chain reaction (PCR). A total of 33 samples from 31 immunosuppressed as well as immunocompetent HCMV-seropositive individuals were studied. CD34+ cells were PCR-positive in four of seven bone marrow aspirates from allogeneic bone marrow transplant recipients, in three of eight aspirates from patients with acquired immunodeficiency syndrome, and in the first of two bone marrow samples from an immunocompetent patient with primary HCMV disease. CD34+ cells purified from peripheral blood for autologous and allogeneic transplantation were also analyzed, and 4 of 13 samples were HCMV DNA-positive. Interestingly, two of the four HCMV-positive samples were from healthy allogeneic donors. Among the mature leukocyte populations, the monocytes were most frequently found to be HCMV DNA-positive. No HCMV DNA was detected in the total bone marrow leukocytes of 13 healthy seropositive bone marrow donors or in the CD34+ cell fraction of three further seropositive donors. In conclusion, the data provide strong evidence that CD34+ hematopoietic progenitor cells can be infected with HCMV in immunosuppressed patients, while this cell population was not identified as a major viral reservoir in healthy HCMV-seropositive individuals.

Expression of tissue- and development-specific genes is coordinately regulated during maturation of hematopoietic precursor cells toward functional, end-stage peripheral blood (PB) cells. To study the expression and methylation of several myeloid-specific genes during in vitro differentiation of normal hematopoietic progenitor cells, we used a model of CD34+ selected PB progenitor cells (PBPCs). PBPCs from six patients with solid tumors were recruited by standard-dose chemotherapy and subsequent administration of recombinant granulocyte colony-stimulating factor (G-CSF). PBPCs were collected and CD34+ cells selected by immunoadsorption columns using a biotinylated anti-CD34 monoclonal antibody. Enriched cells contained between 78% and 90% (median, 84%) CD34+ cells as determined by fluorescence-activated cell sorting analysis. Cell preparations were cultured in the presence of interleukin-1 beta (IL-1 beta), IL-3, IL-6 and stem cell factor and with or without G-CSF for various time intervals up to 20 days. Genes for CD34 surface antigen, lysozyme (LZM) and myeloperoxidase (MPO) were examined by RNA and DNA analyses. A rapid and early downregulation of CD34 transcripts was observed, with concomitant, time-dependent upregulation of expression of both the LZM and MPO genes. These effects were enhanced in the presence of G-CSF. Analysis of the DNA methylation status at key sites within these genes showed a pattern of differentiation- and expression-associated demethylation of the LZM gene, which was also enhanced by G-CSF, and constitutive and unaltered demethylation at key regions of the CD34 and MPO genes. In conclusion, the genes for CD34, LZM, and MPO are regulated during in vitro culture of very immature PBPCs in the presence of stem cell factor, IL-1, IL-3, IL-6; their effects are enhanced by G-CSF.

CD34(+)-selected hematopoietic progenitor cells are being increasingly used for autotransplantation, and recent evidence indicates that these cells can be expanded ex vivo. Of 15 patients with solid tumors undergoing a phase I/II clinical trial using CD34(+)-selected peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy, we analyzed the frequency of long-term culture-initiating cells (LTCIC) as a measure of transplantation potential before and after ex vivo expansion of CD34+ cells. PBPCs were mobilized by combination chemotherapy and granulocyte colony-stimulating factor (G-CSF). The original unseparated leukapheresis preparations, the CD34(+)-enriched transplants, as well as nonabsorbed fractions eluting from the CD34 immunoaffinity columns (Ceprate; CellPro, Bothell, WA) were monitored for their capacity to repopulate irradiated allogeneic stroma in human long-term bone marrow cultures. We found preservation of more than three quarters of fully functional LTCIC in the CD34(+)-selected fractions. Quantitation of LTCIC by limiting dilution analysis showed a 53-fold enrichment of LTCIC from 1/9,075 in the unseparated cells to an incidence of 1/169 in the CD34+ fractions. Thus, in a single apheresis, it was possible to harvest a median of 1.65 x 10(4) LTCIC per kg body weight (range, 0.71 to 3.72). In addition, in six patients, large-scale ex vivo expansions were performed using a five-factor cytokine combination consisting of stem cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (EPO), previously shown to expand committed progenitor cells. LTCIC were preserved, but not expanded during the culture period. Optimization of ex vivo expansion growth factor requirements using limiting dilution assays for LTCIC estimation indicated that the five-factor combination using SCF, IL-1, IL-3, IL-6, and EPO together with autologous plasma was the most reliable combination securing both high progenitor yield and, at the same time, optimal preservation of LTCIC. Our data suggest that ex vivo-expanded CD34+ PBPCs might be able to allow long-term reconstitution of hematopoiesis.

Peripheral blood progenitor cells (PBPCs) are increasingly used for autografting after high-dose chemotherapy. One advantage of PBPCs over the use of autologous bone marrow would be a reduced risk of tumor-cell contamination. However, the actual level of tumor cells contaminating PBPC harvests is poorly investigated. It is currently not known whether mobilization of PBPCs might also result in mobilization of tumor cells. We evaluated 358 peripheral blood samples from 46 patients with stage IV or high-risk stage II/III breast cancer, small cell (SCLC) or non- small cell (NSCLC) lung cancer, as well as other advanced malignancies for the detection of epithelial tumor cells. Monoclonal antibodies against acidic and basic cytokeratin components and epithelial antigens (HEA) were used in an alkaline phosphatase-anti-alkaline phosphatase assay with a sensitivity of 1 tumor cell within 4 x 10(5) total cells. Before initiation of PBPC mobilization, circulating tumor cells were detected in 2/7 (29%) patients with stage IV breast cancer and in 2/10 (20%) patients with extensive-disease SCLC, respectively. In these patients, an even higher number of circulating tumor cells was detected after chemotherapy with VP16, ifosfamide, and cisplatin (VIP) followed by granulocyte colony-stimulating factor (G-CSF). This approach has previously been shown to be highly effective in mobilizing PBPCs. In the 42 patients without circulating tumor cells during steady state, tumor cells were mobilized in 9/42 (21%) patients after VIP+G-CSF induced recruitment of PBPCs. The overall incidence of tumor cells varied between 4 and 5,600 per 1.6 x 10(6) mononuclear cells analyzed. All stage IV breast cancer patients and 50% of SCLC patients were found to concomitantly mobilize tumor cells and PBPCs. Kinetic analyses showed two patterns of tumor cell recruitment depending on the presence or absence of bone marrow disease: (1) early after chemotherapy (between days 1 and 7) in patients without marrow infiltration, and (2) between days 9 and 16 in patients with marrow infiltration, ie, within the optimal time period for the collection of PBPCs. We show that there is a high proportion of patients with circulating tumor cells under steady-state conditions, and in addition a substantial risk of concomitant tumor cell recruitment upon mobilization of PBPCs, particularly in stage IV breast cancer patients with bone marrow infiltration. The biologic and clinical significance of this finding is unknown at present.

To provide sufficient numbers of peripheral blood progenitor cells (PBPCs) for repetitive use after high-dose chemotherapy, we investigated the ability of hematopoietic growth factor combinations to expand the number of clonogenic PBPCs ex vivo. Chemotherapy plus granulocyte colony-stimulating factor (G-CSF) mobilized CD34+ cells from 18 patients with metastatic solid tumors or refractory lymphomas were cultured for up to 28 days in a liquid culture system. The effects of interleukin-1 beta (IL-1), IL-3, IL-6, granulocyte-macrophage-CSF (GM-CSF), G-CSF, macrophage-CSF (M-CSF), stem cell factor (SCF), erythropoietin (EPO), leukemia inhibitory factor (LIF), and interferon- gamma, as well as 36 combinations of these factors were tested. A combination of five hematopoietic growth factors, including SCF, EPO, IL-1, IL-3, and IL-6, was identified as the optimal combination of growth factors for both the expansion of total nucleated cells as well as the expansion of clonogenic progenitor cells. Proliferation peaked at days 12 to 14, with a median 190-fold increase (range, 46- to 930- fold) of total clonogenic progenitor cells. Expanded progenitor cells generated myeloid (colony-forming unit-granulocyte-macrophage), erythroid (burst-forming unit-erythroid), as well as multilineage (colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte) colony-forming units. The number of multilineage colonies increased 250- fold (range, 33- to 589-fold) as compared with pre-expansion values. Moreover, the absolute number of early hematopoietic progenitor cells (CD34+/HLA-DR-; CD34+/CD38-), as well as the number of 4-HC-resistant progenitors within expanded cells increased significantly. Interferon- gamma was shown to synergize with the 5-factor combination, whereas the addition of GM-CSF significantly decreased the number of total clonogenic progenitor cells. Large-scale expansion of PB CD34+ cells (starting cell number, 1.5 x 10(6) CD34+ cells) in autologous plasma supplemented with the same 5-factor combination resulted in an equivalent expansion of progenitor cells as compared with the microculture system. In summary, our data indicate that chemotherapy plus G-CSF-mobilized PBPCs from cancer patients can be effectively expanded ex vivo. Moreover, our data suggest the feasibility of large- scale expansion of PBPCs, starting from small numbers of PB CD34+ cells. The number of cells expanded ex vivo might be sufficient for repetitive use after high-dose chemotherapy and might be candidate cells for therapeutic gene transfer.

Whether or not peripheral stem cells have an unlimited capacity for self renewal is debated. However, everyday haematopoietic requirements are met by progenitors; and it seems that few ‘real’ stem cells are needed. Although we may not yet have identified these ‘true’ stem cells, for practical purposes the long term culture-initiating cells (LTC-ICs) are a close approximation. To date, experience in peripheral blood progenitor cell (PBPC) transplantation is largely confined to non-ablative regimens. It is therefore difficult to determine the number of PBPCs needed to effect long-term reconstitution. The number of tumour cells present among mobilised PBPCs can be reduced using the CD34 affinity colunm and by positive purging methods. The ex vivoexpansion of CD34 cells also has the effect of diluting tumour cell concentration.

Expression of tissue- and development-specific genes is co-ordinately regulated during maturation of hematopoietic precursor cells toward functional, end-stage peripheral blood (PB) cells. To study the expression and methylation of several myeloid-specific genes during in vitro differentiation of normal hematopoietic progenitor cells, we used a model of CD34+selected PB progenitor cells (PBPCs). PBPCs from six patients with solid tumors were recruited by standard-dose chemotherapy and subsequent administration of recombinant granulocyte colony-stimulating factor (G-CSF). PBPCs were collected and CD34+cells selected by immu-noadsorption columns using a biotinylated anti-CD34 monoclonal antibody. Enriched cells contained between 78% and 90% (median, 84%) CD34+cells as determined by fluorescence-activated cell sorting analysis. Cell preparations were cultured in the presence of interleukin-1β (IL-1β), IL-3, IL-6 and stem cell factor and with or without G-CSF for various time intervals up to 20 days. Genes for CD34 surface antigen, lysozyme (LZM) and myeloperoxidase (MPO) were examined by RNA and DNA analyses. A rapid and early downregulation of CD34 transcripts was observed, with concomitant, time-dependent upregulation of expression of both the LZM and MPO genes. These effects were enhanced in the presence of G-CSF. Analysis of the DNA methylation status at key sites within these genes showed a pattern of differentiation-and expression-associated demethylation of the LZM gene, which was also enhanced by G-CSF, and constitutive and unaltered demethylation at key regions of the CD34 and MPO genes. In conclusion, the genes for CD34, LZM, and MPO are regulated during in vitro culture of very immature PBPCs in the presence of stem cell factor, IL-1, IL-3, IL-6; their effects are enhanced by G-CSF.

CD34+-selected hematopoietic progenitor cells are being increasingly used for autotransplantation, and recent evidence indicates that these cells can be expanded ex vivo. Of 15 patients with solid tumors undergoing a phase l/ll clinical trial using CD34+-selected peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy, we analyzed the frequency of long-term culture-initiating cells (LTCIC) as a measure of transplantation potential before and after ex vivo expansion of CD34+cells. PBPCs were mobilized by combination chemotherapy and granulocyte colony-stimulating factor (G-CSF). The original unseparated leukapheresis preparations, the CD34+-enriched transplants, as well as nonabsorbed fractions eluting from the CD34 immunoaffinity columns (Ceprate; CellPro, Bothell, WA) were monitored for their capacity to repopulate irradiated allogeneic stroma in human long-term bone marrow cultures. We found preservation of more than three quarters of fully functional LTCIC in the CD34+-selected fractions. Quantitation of LTCIC by limiting dilution analysis showed a 53-fold enrichment of LTCIC from 1/9,075 in the unseparated cells to an incidence of 1/169 in the CD34+fractions. Thus, in a single apheresis, it was possible to harvest a median of 1.65 × 104LTCIC per kg body weight (range, 0.71 to 3.72). In addition, in six patients, large-scale ex vivo expansions were performed using a five-factor cytokine combination consisting of stem cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (EPO), previously shown to expand committed progenitor cells. LTCIC were preserved, but not expanded during the culture period. Optimization of ex vivo expansion growth factor requirements using limiting dilution assays for LTCIC estimation indicated that the five-factor combination using SCF, IL-1, IL-3, IL-6, and EPO together with autologous plasma was the most reliable combination securing both high progenitor yield and, at the same time, optimal preservation of LTCIC. Our data suggest that ex vivo–expanded CD34+PBPCs might be able to allow long-term reconstitution of hematopoiesis.

A process for direct chlorination of rare earth ores without the necessary preparation of carbides or other intermediate compounds is described. Complete chlorination of all non-volatile components of the ores is obtained in a special, electrically heated furnace at temperatures between 1000 and 1200°C. The construction of the furnace permits quick replacement of some important components while the furnace is still hot. This reduces the unavoidable corrosion problem. The flowsheet of the process is presented. The process permits a high productive capacity with a small space, low investment, and low labor input. It is possible to process the most varied ores, such as Bastnasite, Monazite, Allanite, Cerite, Xenotime, Euxenite, Fergusonite, Gadolinite, etc., in the same installation. Reference is made to the treatment of off-gases. Fused anhydrous rare earth chlorides, without contaminants, are obtained as the final product, which is very suitable for the production of rare earth metals. This process can also be used to convert pure rare earth oxides to anhydrous chlorides.

Background: Promising results have been observed in our previous phase-II study evaluating the combination of Bendamustine plus Rituximab (B-R) in patients with relapsed/refractory indolent or mantle cell lymphomas. An overall response rate (ORR) of 90%, including a 60% rate of complete remissions (CR) was documented. Objective: In October 2003 we initiated a multicenter randomized phase-III study to compare efficacy and safety of the combination B-R versus CHOP plus Rituximab (CHOP-R) as first-line therapy for follicular, indolent and mantle cell lymphomas. Methods: Patients (pts) were randomized to receive Rituximab 375 mg/qm (day 1) plus either Bendamustine 90 mg/qm (days 1+2) every 28 days or the standard CHOP regimen every 21 days for a maximum of 6 cycles. The primary endpoint was event-free survival (EFS). A sample size of 474 pts were calculated to power the study sufficiently to demonstrate a non-inferior EFS associated with B-R treatment, as defined by a difference in EFS between the two regimes of less than 10% after 3 years. An event was defined by a response less than a partial response, disease progression, relapse, or death from any cause. Results: So far 439 patients have been randomized. 273 patients are evaluable for response for this first interim analysis (B-R: n=139; CHOP-R: n=134). Median patient age is 63 years. Histologies are equally distributed between arms: follicular 52%, mantle cell 20%, and other indolent lymphomas 28% in both treatment groups, each. The ORR for pts treated with B-R was similar to that associated with CHOP-R (94% vs 93%, respectively). CR was also similar at 51% for B-R compared to 40% for CHOP-R. The median observation time for both groups is 17 months. Thus far, 15 deaths have been observed (B-R: 7; CHOP-R: 8). Progressive or relapsed disease has been documented during the follow-up period: 27 in pts treated with B-R and 32 in the CHOP-R group. The B-R regimen appears to have a better toxicity profile, as evidenced by a lower rate of total alopecia (40% CHOP-R vs. 0% with B-R) and a lower number of infectious complications (41 in CHOP-R pts vs. 19 in the B-R group). Correlating, the CHOP-R regimen was more hematotoxic: WHO grade 3/4 leukocytopenia was reported in 41% CHOP-R treated pts compared with 12% in pts treated with B-R. Conclusions: In this first interim analysis the combination of Bendamustine plus Rituximab appears to be non-inferior to the standard CHOP-R while showing a better tolerability profile. The study will be closed in December 2007 according to the planned patient recruitement schedule. Updated results will be presented at this time.

Background: Promising results have been observed in our previous phase-II study evaluating the combination of Bendamustine plus Rituximab (B-R) in patients with relapsed/refractory indolent or mantle cell lymphomas. An overall response rate (ORR) of 90%, including a 60% rate of complete remissions (CR) was documented.

Vaccination of cancer patients using dendritic cells (DC) was shown to be effective for B cell lymphoma and malignant melanoma. Here we provide evidence that patients with advanced breast and ovarian cancer can be efficiently vaccinated with autologous DC pulsed with HER-2/neu or MUC1 derived peptides. Ten patients were included in this pilot study. The DC vaccinations were well tolerated with no side effects. One patient developed a complete regression of her multiple subcutaneous lesions. In 5 out of 10 patients, antigen specific cytotoxic T-cells (CTL) could be detected in the peripheral blood using both intracellular IFN-γ staining and 51Cr-release assays. The major CTL response in vivo was induced with the HER-2/neu derived E75 and the MUC1 derived M1.2 peptide which lasted for more than 6 months, suggesting that these peptides might be immunodominant. In addition, in one patient vaccinated with the MUC1 peptides, CEA- and MAGE-3 peptide-specific T-cell responses were detected after several vaccinations. In a second patient immunized with the HER-2/neu peptides, MUC1 specific T-cells were induced after 7 immunizations, suggesting that antigen spreading in vivo might occur after successful immunization with a single tumor antigen. Our results show that vaccination of DC pulsed with a single tumor antigen may induce immunological and clinical responses in breast and ovarian cancer. This study may be relevant to the design of future clinical trials of other peptide-based cancer vaccines.