CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential.

Cellular adjuvants such as dendritic cells (DC) are in the focus of tumour immunotherapy. In DC-vaccine trials, induction of tumour antigen-specific immunity is observed frequently and well-documented clinical responses have been reported. However, the overall response rate is less than 3%, therefore alternative strategies are being investigated. CD40-activated B cells (CD40-B) have been characterized previously as an interesting alternative because they present antigen efficiently and can be expanded by several logs from small amounts of peripheral blood. To determine the central technical challenges of cell-based vaccines we performed a single-patient analysis of 502 patients from DC-based tumour vaccine trials and identified at least three factors contributing to their limited efficiency: (1) lack of cell numbers; (2) lack of documented purity thus high contamination of bystander cells; and (3) lack of quality control and thus heterogeneous or unknown expression of important surface molecules such as major histocompatibility complex (MHC) and chemokine receptors. Based on these findings we re-evaluated the CD40-B approach in cancer patients. Here, we show that proliferation of B cells from cancer patients is equivalent to that observed in healthy donors. Purity is always > 90% after 2 weeks and remains stable for several weeks. They have comparable antigen-presenting capability determined phenotypically and by allogeneic mixed lymphocyte reaction. Expression of CCR7 and CD62L was detected in all samples and B cells migrated towards the relevant homing chemokines. Taken together, CD40-B cells from cancer patients can be expanded in virtually unlimited numbers at high purity and full function concerning antigen-presentation and migratory properties.

Cell fusion is not involved in the generation of giant cells in the Hodgkin-Reed Sternberg cell line L1236.

The mechanism of multinucleated cell formation in Hodgkin's disease has not yet been elucidated. We asked whether the giant multinucleated cells of the H-RS cell line L1236 develop via fusion of the predominant smaller cells. As a positive control for the fusion assay, human B cells from the B-cell lymphoma cell line BJA-B were split into two fractions, stained with the fluorochromes CMTMR and CMFDA, respectively, and fused using the polyethylene glycol 1500 cell hybridization protocol. Double-stained cells indicating fusion of BJA-B cells were detectable for up to 5 days. In parallel, L1236 cells were split into two fractions, stained with the fluorochromes, and mixed. No double-stained L1236 cells were detected. The same result was obtained when using FACS-sorted small mononuclear L1236 cells. It is thus concluded that the large multinucleated cells of the monoclonal H-RS cell line L1236 have emerged by endomitosis rather than by spontaneous cell fusion.

Immunomagnetic enrichment of CD138 positive cells from weakly infiltrated myeloma patients samples enables the determination of the tumor clone specific IgH rearrangement.

In multiple myeloma, the polymerase chain reaction (PCR) of the Ig heavy chain with allele-specific oligonucleotide (ASO) primers is a common and well-described method of identifying the tumor clone in peripheral blood (PB), bone marrow (BM) or leukapheresis products (LA). A factor which is crucial to the detection of clonal Ig rearrangements lies in the 'purity' of the tumor tissue used for the consensus PCR. We describe the application of a method to enrich CD138 positive myeloma cells derived from weakly infiltrated PB-, BM- and LA-samples. These are subjected to immunomagnetic enrichment with the MACS system, using an CD138 antibody directly conjugated to magnetic beads to obtain an enriched tumor cell population and the subsequent amplification of tumor specific IgH rearrangements. We investigated 29 samples (ten PB, ten BM, nine LA) with a median myeloma cell content of 0.5%. The approach led to a median enrichment factor of 118. Tumor-specific rearrangements could be amplified reproducibly from samples containing less than 0.1% myeloma cells.

Atypical chronic myeloid leukaemia with trisomy 21 mosaicism as a sole chromosomal abnormality.

We present a 46-year-old patient with Ph-chromosome negative, bcr-negative chronic myeloid leukaemia (CML) in accelerated phase with a clonal trisomy 21 in the leukaemic blast cells. A rapid progress of disease with appearance of monocytosis is described, showing similar features to chronic myelomonocytic leukaemia (CMML). Heterogeneous characteristics and possible distinction of these two entities are discussed.

Suppression of the tumorigenic growth of Burkitt's lymphoma cells in immunodeficient mice by cytokine gene transfer using EBV-derived episomal expression vectors.

Epstein-Barr virus (EBV)-based expression vectors were tested for cytokine gene transfer-mediated induction of an immune response against human lymphoma cells. These vectors express the EBV latent gene EBNA 1 and carry the EBV latent origin of replication (ori P) for episomal replication in transfected cells. In addition, 3 human immunoglobulin light chain enhancer elements augment expression in B-cells. The suitability of these vectors for expression of cytokine genes in human lymphoma cells in vitro has been demonstrated. In order to extend these experiments in vivo, highly tumorigenic Burkitt's lymphoma (BL) cells were transfected with different cytokine genes of human and murine origin cloned into the EBNA 1/ori P vectors. Tumorigenicity of the transfectants was measured after inoculation into nude mice. No effect on tumorigenicity was observed after hIL 6 transfection and an inconsistent effect after hTNFalpha transfection. In contrast, complete suppression of tumor outgrowth occurred in hIL 10 transfectants. This tumor suppressive effect, however, was restricted to the IL 10 transfectants themselves and not directed against non-transfected cells. By comparison, mIL 4 transfected BL cells also were non-tumorigenic. However, co-inoculation of mIL 4 transfected and non transfected cells resulted in suppression of the tumorigenicity of the non-transfected cells. Thus, highly tumorigenic BL cells in nude mice are sensitive to immune effector mechanisms triggered by cytokine expression. In this experimental model, EBNA 1/ori P expression vectors are a suitable tool for cytokine gene transfer mediated induction of an anti-lymphoma immune response of the host.

[Thrombotic thrombocytopenic purpura (Moschkowitz-syndrome) caused by ticlopidine]. / Thrombotisch-thrombozytopenische Purpura (Moschcowitz-Syndrom), verursacht durch Ticlopidin.

BACKGROUND: Only in a few case reports the thrombotic thrombocytopenic purpura was related to ticlopidine with a controversial discussion about this association. CASE REPORT: In a 57-year-old female patient, who was admitted with fluctuating central neurological abnormalities and generalized purpura, was made the diagnosis of a thrombotic thrombocytopenic purpura (TTP, Moschcowitz' syndrome). On admission there were a distinct anemia and thrombocytopenia. Corresponding to the hemolysis the laboratory findings showed raised reticulocytes and elevated LDH with > 900 U/l. The peripheral blood smear showed an enrichment of fragmented red cells (15%) and the bone marrow indicated a hyperplastic erythrocytopoesis and a left shift in megakaryocytopoesis. An increase of eosinophilic granulocytes and the tissue basophilic cells directed to a possible allergic phenomenon of the underlying disease. Until 3 weeks before admission she had been on ticlopidine after a left heart catheter with stenting the left coronary artery 6 weeks earlier. Beside taking of acetylsalicylacid and thyroid hormone there was no other regular medication. An early treatment with fresh frozen plasma and plasmapheresis with plasma exchange with fresh frozen plasma led directly to an elevation of thrombocytes and a normalization of hemolytic parameters. CONCLUSION: This case demonstrates the possible relationship between thrombotic thrombocytopenic purpura and the administration of ticlopidine.

Dexamethasone, carmustine, etoposide, cytarabine, and melphalan (dexa-BEAM) followed by high-dose chemotherapy and stem cell rescue--a highly effective regimen for patients with refractory or relapsed indolent lymphoma.

We performed a phase II study to determine the efficacy of maximal cytoreductive therapy with up to five cycles of Dexa-BEAM (dexamethasone, carmustine [BCNU], etoposide, cytarabine, and melphalan) followed by high-dose chemotherapy (HDCT) and autologous stem cell transplantation (ASCT) for patients with advanced relapsed or refractory indolent lymphoma. Thirty-two patients with primary refractory or relapsed indolent lymphoma were treated with the Dexa-BEAM regimen. Thirteen patients had primary refractory disease, 4 patients partial remission, and 15 patients first or subsequent relapse. Patients achieving PR or CR received HDCT with ASCT. The conditioning regimen used was BEAM (carmustine [BCNU], etoposide, cytarabine, and melphalan). Twenty-two patients responded to Dexa-BEAM resulting in a response rate of 78%. Maximum response was observed after 3.2 (range 2-5) courses. One patient with progressive disease died in septic shock during neutropenia. Nineteen patients with partial or complete remission after Dexa-BEAM received HDCT. Hematopoietic stem cells (HSC) were collected after two cycles of Dexa-BEAM. The median number of CD34+ HSC reinfused was 3.1 x 10(6)/kg (range 1.6-8.2 x 10(6)/kg). There was no transplantation-related death. All patients receiving HDCT achieved complete remission. Overall survival (OS) and freedom from treatment failure (FFTF) for all patients are estimated to be 68% and 65% at two years, respectively. With a mean follow-up of 20 months (range 8-42 months), 16/19 patients receiving HDCT are in continuous complete remission. The Dexa-BEAM regimen is effective in overcoming drug resistance in patients with indolent lymphoma who failed to respond to conventional treatment or who relapsed. The CR rate of 100% of those patients receiving HDCT and ASCT after maximal cytoreductive treatment with Dexa-BEAM suggests the use of HDCT at the time of maximal response.

Dexa-BEAM: an effective regimen for cytoreduction prior to high-dose chemotherapy with autologous stem cell support for patients with relapsed/refractory mantle-cell lymphoma.

Mantle-cell lymphoma (MCL) is not a curable disease using conventional chemotherapy. Patients with MCL have the shortest median time to progression and the shortest median survival of all lymphoma subtypes after first-line treatment. In the present study we determined the efficacy of maximal cytoreductive therapy with up to four cycles of Dexa-BEAM (dexamethasone, carmustine [BCNU], etoposide, cytarabine, and melphalan) followed by high-dose chemotherapy (HDCT) and autologous hematopoietic stem cell support (ASCT) for patients with advanced relapsed or refractory MCL. Nine consecutive patients with relapsed or refractory MCL were included. Three patients had partial remission (PR), three patients progressive disease (PD) upon first line tretment, and three patients first or subsequent relapse. After 2 to four cycles of Dexa-BEAM eight patients achieved complete remission (CR), resulting in a response rate of 88%. Six of 8 patients responding to Dexa-BEAM received high-dose chemotherapy HDCT (BEAM) and autologous hematopoietic stem cell transplantation (ASCT). With a median follow up of 24 months six patients are alive. Five of those six patients are still in contiuous CR (range 13-54 months).

High platelet contamination in progenitor cell concentrates results in significantly lower CD34+ yield after immunoselection.

BACKGROUND: Selection of CD34+ cells by specific immunoselection leads to a significant loss of those cells. The factors influencing the yield and purity are not well identified. The results of CD34+ selection from peripheral blood progenitor cells (PBPCs) with high and low platelet contamination that are harvested with two different cell separators are reported. STUDY DESIGN AND METHODS: A progenitor cell concentrator (Ceprate SC, CellPro) was used to select CD34+ cells from 41 PBPC concentrates from 23 consecutive patients with relapsed non-Hodgkin's lymphoma (n = 3), breast cancer (n = 17), and multiple myeloma (n = 3). PBPC collection was performed by using two cell separators (CS3000 Plus, Fenwal: Group A, n = 11; and Spectra, COBE: Group B, n = 9). To reduce platelet contamination in the Spectra PBPC concentrates, an additional low-speed centrifugation was performed before CD34+ cell selection (Group C, n = 3). Leukapheresis components were stored overnight at 4 degrees C and combined with the next day's collection before the CD34+ selection procedure in 19 patients. RESULTS: A median of 1.5 leukapheresis procedures per patient were performed. Pooled PBPC concentrates showed no statistical difference in median numbers of white cells and CD34+ cells in Groups A and B: 3.2 (0.8-9.2) versus 4.4 (1.6-8. 3) x 10(10) white cells per kg and 15.0 (4.7-24.0) versus 12.0 (5. 6-34.0) x 10(6) CD34+ cells per kg. Platelet contamination was significantly higher in Group B: 0.67 (0.15-2.4) versus 2.3 (0.5-7. 1) x 10(11) (p = 0.0273). After the selection process, there was a significantly greater loss of CD34+ cells in Group B than in Group A: 39.1 versus 63.2 percent (p = 0.0070), with a median purity of 78. 0 percent versus 81.0 percent. An additional low-speed centrifugation before CD34+ cell selection seemed to reduce CD34+ cell loss in Group C with 16.9, 31.9, and 37.5 percent, respectively. CONCLUSION: CD34+ cell selection from PBPC concentrates resulted in an increased loss of CD34+ cells in concentrates with a higher platelet content. To improve CD34+ yield, PBPC concentrates with an initially low platelet contamination should be used, or additional low-speed centrifugation should be performed.

Successful peripheral blood stem cell mobilization with etoposide (VP-16) in patients with relapsed or resistant lymphoma who failed cyclophosphamide mobilization.

High-dose chemotherapy (HDCT) followed by autologous blood stem cell transplantation is considered the treatment of choice for patients with relapsed or resistant aggressive non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD). However, several authors report failure of standard mobilization regimens in 29% to 56% of these patients making the completion of HDCT impossible and as a result, negatively influencing long-term outcome. Thus, effective new regimens for patients failing initial mobilization are needed. Here we report the results of using etoposide as a mobilizing agent in 16 patients with primary resistant or relapsed malignant lymphoma who had failed prior mobilization of peripheral blood stem cells (PBSC) with cyclophosphamide (4 g/m2) followed by G-CSF. The use of etoposide 500 mg/m2 (days 1-4) + G-CSF resulted in the successful collection of adequate numbers of PBSC with a median harvest of 3.6 x 10(6)/kg (range 2.2-12.6) CD34+ cells in all 16 patients. In 7/16 (44%) patients, the target yield of at least 2.0 x 10(6) CD34+ cells was harvested by a single apheresis and the maximum number of separations for all patients was two. No excessive toxicities appeared, allowing all patients to proceed to myeloablative chemotherapy. In addition, median peak values of circulating CD34+ cells were significantly higher after etoposide as compared to cyclophosphamide (49.2/microl vs 4.7/microl; P = 0.0004). These results indicate that etoposide + G-CSF is a highly effective mobilization regimen in patients who have failed cyclophosphamide mobilization.

Using at least 5x10(6)/kg CD34+ cells for autologous stem cell transplantation significantly reduces febrile complications and use of antibiotics after transplantation.

For autologous stem cell transplantation, it is common practice to infuse at least 2 x 10(6)/kg CD34+ cells to ensure rapid engraftment. However it was recently claimed that increasing the threshold to 5 x 10(6)/kg leads to a faster platelet engraftment. To evaluate these threshold values in our patient population we undertook a retrospective analysis of 127 autologous transplants performed at our institution between 1992 and 1998. Diagnoses included Hodgkin's and non-Hodgkin's lymphoma, myeloma, acute leukaemias and solid tumours. The transplant was peripheral blood stem cells in 107 cases and CD34-selected peripheral blood stem cells in 20 cases. The median number of transplanted CD34+ cells was 3.2 x 10(6)/kg (range 0.64-25.9 x 10(6)/kg). Haematopoietic recovery to a neutrophil count >0.5 x 10(9)/l took a median of 10 (range 5-16) days from transplant. When comparing patients receiving at least 5 x 10(6)/kg and 2-5 x 10(6)/kg CD34+ cells we found a significant reduction in the median number of days with fever (1 vs 3.5 days, P = 0.0025), incidence of fever (78.8 vs 92.1%, P = 0.032) as well as duration of antibiotic treatment (7 vs 10 days, P = 0.038). This was paralleled by a faster neutrophil recovery to 0.5 x 10(9)/l (9 vs 10 days, P = 0.047). There was no significant difference in the number of platelet or red cell transfusions between the two groups. We conclude that transplantation with a stem cell dose of at least 5 x 10(6)/kg CD34+ cells reduces infectious complications and should thereby increase the safety of this type of therapy while reducing duration (and cost) of antibiotic therapy. The transplantation threshold should thus not remain at 2 x 10(6)/kg particularly in patients with a good stem cell mobilisation capacity.

Dexa-BEAM is not effective in patients with relapsed or resistant aggressive high-grade non-Hodgkin's lymphoma.

The aim of the present study was to evaluate the feasibility and response of the Dexa-BEAM regimen as a salvage therapy followed by high-dose chemotherapy (HDCT) with peripheral blood stem cell transplantation (PBCST) in responding patients with high-grade relapsed or resistant aggressive non-Hodgkin's lymphoma (NHL). Sixteen pretreated patients (mean age 44, range 26-59) with relapsed (8) or resistant (8) NHL were treated with 1-4 cycles of Dexa-BEAM (dexamethasone, BCNU, etoposide, cytarabine, melphalan) in order to attain maximal response. Patients achieving complete response (CR) or partial response (PR) received HDCT with PBSCT. The conditioning regimen used was BEAM. Three patients achieved CR and one patient PR, resulting in an overall response rate of 25%. Three of four responding patients underwent high-dose chemotherapy and were successfully transplanted with autologous blood stem cells. Progressive disease developed in one patient after transplantation. Myelosuppression (WHO grade III- grade IV), the major side effect, was observed in all courses of Dexa-BEAM. Myelosuppression-related infection WHO grade IV occurred in four patients. The protocol was not well tolerated in this heavily pretreated group of patients with four severe myelosuppression-related infections WHO grade IV and one treatment-related death. The overall response rate in this study is not comparable to other salvage regimens published and led to the discontinuation of the trial. In conclusion Dexa-BEAM was only effective in a minority of patients with refractory or relapsed aggressive NHL and was not useful as a cytoreductive regimen prior to HDCT.

Stable nontumorigenic phenotype of somatic cell hybrids between malignant Burkitt's lymphoma cells and autologous EBV-immortalized B cells despite induction of chromosomal breakage and loss.

Fusion of the highly tumorigenic Burkitt's lymphoma (BL) cell line BL60-P7 with the nontumorigenic autologous EBV-immortalized lymphoblastoid cell line (LCL) IARC 277 results in suppression of the tumorigenic phenotype of the parental cell line BL60-P7 after s.c. inoculation into T cell-deficient nude mice. We analyzed whether, after long-term cultivation of these lymphoma hybrid cells, expression of tumorigenicity could be observed and correlated to the loss of particular chromosomes or chromosomal fragments, akin to numerous nonlymphoid hybrid cell models described previously. Two years after fusion, in vitro proliferation of some BL x LCL hybrid cells accelerated, and they partially lost LCL-typical aggregation. However, no major changes in the expression pattern of B cell-associated surface antigens and the EBV latent membrane protein LMP 1 were observed. Cytogenetic evaluation of these cells revealed spontaneous loss of chromosomes. Karyotyping of long-term cultivated hybrid cells demonstrated the occurrence of disomy for each chromosome in at least one metaphase analyzed. Therefore, if suppression of tumorigenicity in these hybrid cells would have been the result of the presence of a single LCL-derived chromosome, there should have been a high probability of its loss, leading to tumorigenic segregants. Surprisingly, the tumorigenic phenotype remained suppressed in nude mice. To induce chromosomal breakage and maldistribution, in addition to spontaneous chromosomal loss, the hybrid cell lines were irradiated at various doses. Again, none of the hybrid cell clones treated in this manner became tumorigenic in nude mice. Immunohistological analysis of the regressing hybrid cell tumors revealed that the hybrid cells had retained their LCL-like differentiation phenotype in vivo. In addition, infiltration with mononuclear cells of murine origin was observed in these regressing hybrid grafts. We conclude that suppression of the tumorigenic Burkitt's lymphoma phenotype in these hybrid cells cannot be attributed to a function encoded by a distinct chromosome or chromosomal fragment. Rather, the unexpected stable nontumorigenic phenotype reflects a LCL-specific activated B-cell phenotype of these hybrids, most probably induced by the expression of numerous copies of episomal latent EBV proteins.

Suppression of Burkitt's lymphoma tumorigenicity in nude mice by co-inoculation of EBV-immortalized lymphoblastoid cells.

EBV-immortalized B-lymphoblastoid cell lines (LCL) inoculated s.c. into T-cell-deficient nude mice regress completely after a short initial growth period. We tested whether the putative host response underlying this phenomenon might also be directed against progressively growing Burkitt's lymphoma (BL) tumors in nude mice. Outgrowth of BL tumors was suppressed when cells of the highly tumorigenic BL cell line BL 60 were mixed with cells of the autologous LCL IARC 277 before s.c. inoculation into nude mice. Even when the cells were inoculated separately and simultaneously into contralateral flanks of the mice, regression of initially growing BL tumors could be observed, albeit with reduced frequency and dependent on the dose of LCL cells. Tumor growth of BL 60 cells could also be suppressed by co-inoculation with the non-autologous LCL IARC 174 and IARC 277 cells could suppress growth of the non-autologous BL cell line Eli. Pronounced infiltration with murine (m)CD-11b-positive mouse macrophages and mCD-8a-positive mouse lymphoid cells, most probably natural killer cells, was seen in histological tissue sections of regressing BL 60 tumors when LCL cells were inoculated contralaterally. In regressing BL tumors, these mouse cells were present not only in necrotic areas but also in vital BL tissue, indicating that infiltration of mouse cells had taken place before the development of necrosis. Since tumor-infiltrating mouse cells can be activated at least by some human cytokines, we measured cytokine production of BL 60 and IARC 277. High amounts of IL 6 and IL 10 were produced by the LCL cells, whereas IL-6 and IL-10 production by the BL 60 cells was beyond or close to the detection threshold. In addition, IL 8 was secreted up to 5-fold more by the LCL than by the BL cells. The results presented here thus suggest a host response of the nude mouse, which is triggered by cytokines released from the LCL but, once induced, is directed also against BL cells.