Analysis of circulating tumor cells in patients with multiple myeloma during the course of high-dose therapy with peripheral blood stem cell transplantation.

In multiple myeloma (MM) circulating CD19+ cells have been considered as myeloma precursors. As these cells are also possibly a reservoir of treatment resistant disease evaluation of the CD19+ cells during the course of high-dose therapy has to be a major concern. We determined the number of tumor cells in the CD19+ as well as CD19- fractions of PB of eight patients with disease sensitive to VA[I]D chemotherapy, of 10 patients who achieved partial or complete remission post-high-dose therapy (HDT) with peripheral blood stem cell transplantation (PBSCT) and of a further seven patients with disease progression post-transplantation. CD19+ cell fractions were obtained by preparative sequential magnetic and fluorescence activated cell sorting with a median purity of 97.1%. In addition, PB samples of seven patients post-transplantation were sorted for CD20+ cells (median purity, 98.7%). The number of tumor cells in the CD19+, the CD19- and the CD20+ fractions were determined using a quantitative CDR3 PCR assay. The number of CD19+ tumor cells in patients in remission post-HDT was similar to those of the patients post-VA[I]D (median, 1.05 vs 0.92 CD19+ tumor cells/ml PB, P = 0.72) providing evidence for the persistence of this tumor cell fraction during the course of HDT. This was in contrast to the CD19- compartment, in which the number of tumor cells was significantly reduced in those patients in remission post-transplantation (median, 53 vs 0 CD19- tumor cells/ml PB; P = 0.006). In patients with progressive disease the number of tumor cells in both cell fractions was significantly higher (CD19+: median, 1.05 vs 21 tumor cells/ml PB, P = 0.05; CD19-: 0 vs 63 tumor cells/ml PB, P = 0.008). While the absolute number of CD19+ cells was reduced in the group of patients after VA[I]D treatment, a polyclonal CD19+ reconstitution had occurred in patients responding to HDT. The tumor cell content in the CD19+ fractions could be confirmed by the results obtained analyzing the CD20+ cell fractions. In conclusion, these results indicate that disease progression after PBSCT in MM is accompanied by an expansion of tumor cells in both the CD19+ and CD19- fractions. Similar numbers of CD19+ clonotypic cells post-HDT suggest that these cells persist and thus, contribute to disease dissemination and relapse.     

Immunomagnetic selection of CD34+peripheral blood stem cells for autografting in patients with breast cancer

Contamination of transplants with tumour cells may contribute to relapse after peripheral blood stem cell transplantation (PBSCT). We studied the feasibility of CD34⁺ cell selection from blood-derived autografts obtained following G-CSF-supported cytotoxic chemotherapy in a group of 25 patients with breast cancer (10 with high-risk stage II/III and 15 with stage IV without bone or bone marrow involvement).
Using immunomagnetic beads (Isolex 300 SA, Baxter) CD34⁺ cells were enriched and released by chymopapain resulting in a median purity of 95% (range 82–99%) and a median recovery of 80% (range 27–132%). The enrichment procedure did not change the proportion of CD34⁺ subsets coexpressing HLA-DR, CD38 and Thy-1, while L-selectin was removed from the cell surface following selection. Using a sensitive immunocytological technique with a cocktail of epithelial-specific antibodies (anti-cytokeratin 8, 18 and 19; HEA125; BM7 and BM8), five leukaphereses products contained epithelial cells, whereas the selected CD34⁺ cell fraction was free of tumour cells. A neutrophil count of 0.5×10⁹/l and a platelet count of 20×10⁹/l was reached after a median time of 14 and 10 d following 40 high-dose chemotherapy (HDC) cycles. Our results indicate that immunomagnetic selection of CD34⁺ cells yields highly purified autografts devoid of tumour cells whereas the engraftment ability of the progenitor and stem cells is fully retained.     

G-CSF-mobilized CD34+ peripheral blood stem cells are significantly less apoptotic than unstimulated peripheral blood CD34+ cells: role of G-CSF as survival factor

The mechanism of release of CD34⁺ cells into the peripheral blood (PB) after mobilization treatment with chemotherapy and/or growth factors is not clearly understood. Growth factors may induce increased proliferation and self renewal within the stem cell compartment. It is possible that they alter adhesion molecule profiles or other progenitor:stroma interactions, to allow release of these cells into the periphery. However, CD34⁺ cells are present in the PB under steady-state conditions, albeit in low number. Growth factors such as granulocyte colony-stimulating factor (G-CSF) may promote the survival of CD34⁺ cells in the PB by suppressing apoptosis. In order to test this hypothesis, we have quantitated apoptotic cells in the CD34⁺ fraction of peripheral blood stem cell (PBSC) collections, using two-colour flow cytometry, after staining with anti-CD34 antibody and the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD). 7AAD differentially stains live, apoptotic and dead cells, due to the altered accessibility of DNA in each subpopulation.
We have shown a significant reduction in the proportion of apoptotic cells in the CD34⁺ population mobilized by G-CSF compared to CD34⁺ cells in unstimulated PB, consistent with the theory that G-CSF is acting, at least in part, by suppressing apoptosis. In addition, we found that G-CSF mobilized CD34⁺ cells are less apoptotic than CD34⁺ cells of unstimulated normal bone marrow, indicating that, at the doses used, G-CSF is significantly altering the survival capacity of the mobilized cells.     

G-CSF alone mobilizes sufficient peripheral blood CD34+ cells for positive selection in newly diagnosed patients with myeloma and lymphoma

We have evaluated CD34⁺ cell positive selection from granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPC) in 26 patients with either multiple myeloma (MM, n  = 18) or follicular non-Hodgkin's lymphoma (NHL, n  = 8). 26 PBPC were collected with two leukaphereses: 16 contained sufficient numbers of CD34⁺ cells and were selected. The absolute number of CD34⁺ cells in the leukapheresis products was found to be significantly related to the duration of underlying disease and exposure to prior treatment. CD34⁺ cell positive selection allowed recovery of a median of 35% of CD34⁺ cells, the selected fraction containing a median number of 1.43 × 10⁶/kg CD34⁺ cells/kg (range 0.48–41.5). 10 patients were transplanted and received a median dose of 1.51 × 10⁶ CD34⁺ cells (range 0.48–4.2). The median time to granulocyte (>0.5 × 10⁹/l) and platelet (>20 × 10⁹/l) engraftment was 12 and 13 d respectively (ranges 10–13 and 0–95). Lymphoma cells were found by a sensitive polymerase chain reaction technique in four out of five CD34⁺ cell fractions tested.     

CD34+ cell immunoselection from G-CSF-alone-primed peripheral blood in children with low body mass

Summary. We report the data of CD34⁺ cell immunoselection from peripheral blood after G-CSF-alone mobilization (lOμg/kg/d s.c.) in nine children with neuroblastoma (median age 4 5 years (2-8), median body weight 16kg (10-20). Leukaphereses were carried out on a Cobe Spectra separator and two consecutive harvests (4 blood volumes processed) were used for immunoselection on a Ceprate(tm) column. The yield of CD34⁺ cells in the purified fraction was 50% (23-80), with a median number of 2.8xl0⁶ CD34+ cells/kg (1-9-4). All patients were reinfused with selected CD34⁺ cells after busulfan 600 mg/m²+ melphalan 180mg/m² and achieved successful haemopoietic recovery.     

Transplant dose of CD34+ and CD3+ cells predicts outcome in patients with haematological malignancies undergoing T cell-depleted peripheral blood stem cell transplants with delayed donor lymphocyte add-back

We sought to optimize and standardize stem cell and lymphocyte doses of T cell-depleted peripheral blood stem cell transplants (T-PBSCT), using delayed add-back of donor T cells (DLI) to prevent relapse and enhance donor immune recovery. Fifty-one patients with haematological malignancies received a T-PBSCT from an HLA-identical sibling, followed by DLI of 1 x 10(7) and 5 x 10(7) CD3(+) cells/kg on d +45 and +100 respectively. Twenty-four patients were designated as standard risk and twenty-seven patients with more advanced leukaemia were designated as high risk. Median recipient age was 38 years (range 10-56). Median (range) of CD34(+) and CD3(+) cell transplant doses were 4.6 (2.3-10.9) x 10(6)/kg and 0.83 (0.38-2) x 10(5)/kg respectively. The cumulative probability of acute GVHD was 39%. No patient died from GVHD or its consequences. The probability of developing chronic GVHD was 54% (18% extensive). The probability of relapse was 12% for the standard-risk patients and 66% for high-risk patients. In multivariate analysis, the risk factors for lower disease-free survival and overall survival were high-risk disease, CD34(+) dose < 4.6 x 10(6)/kg and CD3(+) dose < 0.83 x 10(5)/kg. Predictive factors for chronic GVHD were a T-cell dose at transplant > 0.83 x 10(5) CD3(+) cells/kg. These results further define the impact of CD34 and CD3 cell dose on transplant outcome and show that careful dosing of stem cells and lymphocytes may permit the control and optimization of transplant outcome.     

Evaluation of the kinetics of the bone marrow tumor load in the course of sequential high-dose therapy assessed by quantitative PCR as a predictive parameter in patients with multiple myeloma

The aim of this investigation was to examine the possible clinical significance of the kinetics of bone marrow (BM) tumor load during the course of sequential high-dose therapy (HDT) as assessed by quantitative PCR in patients with multiple myeloma. In 20 patients with multiple myeloma (MM) treated with two consecutive cycles of HDT followed by autologous peripheral blood stem cell transplantation (PBSCT), clonotypic cells in the peripheral blood (PB) and BM were quantitated by PCR using allele-specific oligonucleotides (ASO) prior to the first, immediately prior to the second, and after the second HDT. The median proportion of clonotypic cells in the BM was 1.27% before the first HDT (range, 0.03-70%), 0.17% after the first (range, 0.001-22%), and 0.05% after the second HDT (range, 0.00009-1.44%). The median number of circulating clonotypic cells was 65/ml (range, 0.9-10842) prior to HDT, 2.7/ml (range, 0-315) after the first, and 3.5/ml PB (range, 0.7-97) after the second HDT. While the median BM tumor load decreased during the first (P = 0.03) and second (P = 0.044) HDT cycles, only the first cycle resulted in a reduction of clonotypic cells in the PB (P = 0.00078 and P= 1.0, respectively). In seven patients, the BM tumor load did not decrease below the initial level after one or two cycles of HDT. All of these patients developed progressive disease (median, 19 months post first cycle; range, 10-21). Of the remaining 13 patients, only four relapsed (18, 19, 21 and 22 months after the first cycle of HDT), while nine remain in response (median followup, 29 months; range, 18-41) (log-rank test P = 0.0009). Our results indicate that the kinetics of the BM tumor load is a predictive parameter in patients with MM and identifies those patients who could benefit from further therapy including new treatment modalities.     

Autologous peripheral blood stem cell transplantation for Japanese multiple myeloma patients: results of a feasibility study

A feasibility study on high-dose therapy with autologous peripheral blood stem cell transplantation (HDT/PBSCT) was performed in Japanese patients with multiple myeloma (MM). Twenty evaluable patients younger than 65 years old with stage II/III MM were enrolled in this study. Three courses of VAD were used as initial chemotherapy. High-dose etoposide or cyclophosphamide followed by G-CSF was used for PBSCH, and 1.2-89.3 (median 23.4) x 106/kg of CD34+ cells were collected. Single (11 patients) or tandem (9 patients) HDT with melphalan (MEL) 200 mg/m2 or MEL 140 mg/m2 plus TBI 10 Gy were performed. The incidence of grade 4 toxicity (COG) was 10% and treatment-related mortality was 5%. Complete response and tumor reduction of more than 75% were obtained in 4 (21%) and 16 (84%) out of 19 patients, respectively. The actuarial 3-year overall survival (OS) and event-free survival (EFS) after PBSCT/HDT were 65.6% and 22.0%, respectively. The median EFS duration was 18 months. These preliminary results indicated that HDT/PBSCT is feasible for Japanese MM patients. A prospective randomized clinical trial will be required to assess the efficacy.     

A comparative study of bone marrow and peripheral blood CD34+ myeloblasts in acute myeloid leukaemia

To examine the differences between primitive bone marrow (BM) and peripheral blood (PB) myeloblasts in acute myeloid leukaemia (AML), we compared CD34⁺ myeloblasts of paired BM and PB samples from 14 AML patients in terms of surface phenotype, homing and engraftment in a xenogeneic transplantation model, and gene expression, based on microarray studies and quantitative polymerase chain reaction. While there was no significant difference in surface phenotypes between these two populations, in vivo assay showed significantly better homing potential of PB CD34⁺ cells than BM CD34⁺ cells. Significant correlation between homing and engraftment in AML samples was also noted. In addition, gene expression profiling of CD34⁺ cells from five paired BM and PB leukaemic samples showed that genes involved in G-protein and prostaglandin signalling, chemotaxis and stress response, cell proliferation and apoptosis were down-regulated in PB CD34⁺ myeloblasts. These data suggested that circulating primitive myeloblasts in AML are functionally different from those residing in the marrow compartment, and such differences may be partly regulated by the BM microenvironment.     

Absolute CD4+ T-lymphocyte and CD34+ stem cell counts by single-platform flow cytometry: the way forward

To determine the potential advantage of single-platform technology in the enumeration of CD4+ T lymphocyte and CD34+ stem cells, data has been analysed from the UK NEQAS for Leucocyte Immunophenotyping schemes. The inter-laboratory CVs for CD4+ T lymphocyte counts were consistently lower for single-platform (mean 13.7%, range 10-18.3%) compared to dual-platform methodology (mean 23.4%, range 14.5-43.7%). Subgroup analysis of single-platform users demonstrated mean overall inter-laboratory CVs of 17.2%, 13% and 7.1% for the FlowCount, TruCount and volumetric approach respectively. The lowest inter-laboratory CVs obtained for a single sample by each single platform approach were 4% (TruCount), 4.4% (volumetric), 4.6% (FACSCount) and 12.7% (FlowCount). Similarly, the mean inter-laboratory CV for CD34+ stem cell enumeration using non-standardized single-platform approaches was 18.6% (range 3.1-36.9%) compared to 28.6% (range 19-44.2%) for the dual-platform technology. Our results suggest absolute cell subset enumeration should be performed by single-platform technology and that such an approach should improve the quality control of multi-centre clinical trial data for CD4+ T lymphocyte and CD34+ stem cells.     

Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of multiple myeloma patients: positive selection and transplantation of enriched CD34+ cells to remove circulating tumor cells

One advantage of the use of peripheral blood stem cells (PBSCs) over autologous bone marrow would be a reduced risk of tumor cell contamination. However, the level of neoplastic cells in the PB of multiple myeloma (MM) patients after mobilization protocols is poorly investigated. In this study, we evaluated PB samples from 27 pretreated MM patients after the administration of high dose cyclophosphamide (7 g/m2 or 4 g/m2) and granulocyte-colony stimulating factor for the detection of myeloma cells as well as hematopoietic progenitors. Plasma cells containing intracytoplasmic lg were counted by microscope immunofluorescence after incubation with appropriate antisera directed against light- and heavy-chain lg. Moreover, flow cytometry studies were performed to determine the presence of malignant B-lineage elements by using monoclonal antibodies against the CD19 antigen and the monotypic light chain. Before initiation of PBSC mobilization, circulating plasma cells were detected in all MM patients in a percentage ranging from 0.1% to 1.8% of the mononuclear cell fraction (mean value, 0.7% +/- 0.4% SD). In these patients, a higher absolute number of PB neoplastic cells was detected after chemotherapy and granulocyte colony-stimulating factor. Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors with a maximum peak falling within the optimal time period for the collection of PBSCs. The absolute number of plasma cells showed a 10 to 50-fold increase as compared with the baseline value. Apheresis products contained 0.7% +/- 0.2% SD of myeloma cells (range, 0.2% to 2.7%). Twenty-three MM patients were submitted to PBSC collection. In 10 patients, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, were cryopreserved, and used to reconstitute bone marrow function after myeloablative therapy. The median purity of the enriched CD34+ cell population was 89.5% (range, 51% to 94%), with a 75-fold increase as compared with the pretreatment samples. The median overall recovery of CD34+ cells and colony-forming unit-granulocyte-macrophage was 58% (range, 33% to 95%) and 45% (range, 7% to 100%), respectively. Positive selection of CD34+ cells resulted in 2.5- to 3-log depletion of plasma cells and CD19+ B-lineage cells as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene showed the persistence of minimal residual disease in 5 of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (1,000 cGy) and highdose melphalan (140 mg/m2). They received a median of 4 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis; the median time to 0.5 x 10(9) neutrophils and to 20 and 50 x 10(9) platelets per liter of PB was 10, 11, and 12 days, respectively. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSCs after the same pretransplant conditioning regimen. In summary, our data show the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients. Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3-log and provides a cell suspension capable of restoring a normal hematopoiesis after a total body irradiation-containing conditioning regimen.     

Dynamic cell cycle kinetics of normal CD34+ cells and CD38+/− subsets of haemopoietic progenitor cells in G-CSF-mobilized peripheral blood

Using a recently developed flow cytometric assay for the simultaneous measurement of cell surface antigens, DNA content and bromodeoxyuridine incorporation, we have for the first time determined the labelling index (LI), the duration of the S-phase (Ts) and the potential doubling time (Tpot) of purified CD34+ cells mobilized by G-CSF from 10 normal donors. Although CD34+ cells were not actively cycling immediately following purification, up to 5% could nevertheless traverse cell cycle without exogenous stimulation during the first 24 h of culture. In addition, it was possible to induce CD34+ cells to enter cycling by stimulation with haemopoietic growth factors (IL-3, IL-6 and SCF), resulting in median Tpot values of 18.2 d at 21 h, 7.7 d at 29 h, and 4.5 d at 37 h. Importantly, stimulation of CD34+ cells was seen almost exclusively within the CD38+ subset (mean Tpot value 2.8 d), whereas CD38- cells were not recruited into cycle (mean Tpot value 35.9 d). In conclusion, although cell cycle entry and progression can easily be induced in differentiated CD34+/CD38+ cells, immature CD34+/CD38- cells will remain dormant in most of the clinical and laboratory stimulation protocols hitherto employed. This assay can be used to obtain detailed cell cycle kinetics in leucocyte subsets in health and disease.     

Persistence of residual tumour cells after cytokine-mediated ex vivo expansion of mobilized CD34+ blood cells in multiple myeloma

Mobilized CD34⁺ blood cells were immunomagnetically enriched from leukapheresis products in five multiple myeloma (MM) patients. Thawed samples of selected CD34⁺ cells were cultured for up to 21 d in a liquid and stroma-free culture system with different combinations of recombinant cytokines. The most successful cell expansion was obtained when a combination of rh-IL-1β, rh-IL-3, rh-IL-6, rh-SCF, rh-G-CSF and rh-GM-CSF was used. After 14 d this mixture gave a 120–187-fold overall increase of total nuclear cells and a 4–8-fold overall increase of early CFU-GM numbers. In four patients a very sensitive patient-specific PCR analysis showed the presence of monoclonal cells in the initial leukapheresis products. After immunomagnetic separation a tumour cell depletion of 2–4 logs was observed, although all samples still contained malignant cells. Cell suspensions that were cultured with the most potent cytokine combination showed tumour contamination in two-thirds of evaluable cases at the moment of maximal CFU-GM output. Serial cDNA dilution experiments indicated that the positive PCR results at day 14 reflected the persistence of pre-culture tumour cells rather than in vitro expansion of tumour cells in two cases. This study demonstrates that ex vivo expansion of myeloid precursor cells from mobilized CD34⁺ cells in MM patients does not always result in an effective purging of residual tumour cells. On the other hand, our culture conditions do not seem to favour in vitro expansion of malignant cells, despite the use of a cytokine cocktail that includes potential myeloma growth factors.     

Molecular monitoring of the tumor load predicts progressive disease in patients with multiple myeloma after high-dose therapy with autologous peripheral blood stem cell transplantation.

The clinical relevance of the assessment of minimal residual disease (MRD) in patients with multiple myeloma (MM) to predict disease recurrence has not been proven. In the present study, we retrospectively analyzed the tumor load in peripheral blood (PB) and bone marrow (BM) samples of 13 patients with MM both in remission after high-dose therapy (HDT) with autologous PBSC transplantation (PBSCT) and at the time of progressive disease (PD). For six patients, subsequent samples obtained in remission could be included in the study. Tumor cells were assessed by means of quantitative PCR with allele-specific oligonucleotides (ASO-qPCR) based on the method of limiting dilutions. PD was documented with ASO-qPCR in BM samples (median concentration of tumor cells in remission vs at PD: 0.18% vs 4.6%) representing a significant increase by a median factor of 8.7. In PB, the median tumor load was 799 cells/ml in remission and 23 400 cells/ml at PD. With a median factor of 45, the increase was much more pronounced. Comparing the results of the molecular monitoring in PB with those of the determination of the monoclonal protein, routinely applied as parameter for the course of the disease, revealed a superiority of the molecular monitoring because of the significantly higher increase in the tumor load. Analyzing the subsequent remission samples showed an increase of the malignant cells in four out of six PB samples and in all four BM samples available, indicating the potential of ASO-qPCR for an early PD recognition.     

Outcome of transplantation of highly purified peripheral blood CD34+ cells with T-cell add-back compared with unmanipulated bone marrow or peripheral blood stem cells from HLA-identical sibling donors in patients with first chronic phase chronic myeloid leukemia

Outcomes of highly purified CD34(+) peripheral blood stem cell transplantation (PBSCT) for chronic phase chronic myeloid leukemia (CML) (n = 32) were compared with those of PBSCT (n = 19) and of bone marrow transplantation (BMT) (n = 22) in the HLA-compatible sibling donor setting. Median follow-up was 18 months after CD34(+)-PBSCT and unmanipulated PBSCT and 20 months after BMT. CD34(+)-PBSCT was associated with delayed T-cell immune reconstitution at 3 months and 12 months after transplantation compared with PBSCT (P <.001) or BMT (not significant [NS]). The estimated probability of grades II to IV acute graft-versus-host disease (GVHD) was 60% +/- 13% for the PBSCT group, 37% +/- 13% for the BMT group, and only 14% +/- 8% for the CD34(+)-PBSCT group (CD34-PBSCT versus BMT, P <.01; and CD34-PBSCT versus PBSCT, P <.001). The probabilities for molecular relapse were 88% for CD34(+)-PBSCT, 55% after BMT, and 37% after PBSCT (CD34(+)-PBSCT versus PBSCT, P <.03). Cytogenetic relapse probability was 58% after CD34(+)-PBSCT, 42% after BMT, and 28% after PBSCT (NS). After CD34(+)-PBSCT, 26 of 32 patients received a T-cell add-back. Hematologic relapse occurred in 4 of 22 patients after BMT, in 3 of 19 patients after PBSCT, and in only 1 of 32 patients after CD34(+)-PBSCT. The occurrence of a hematologic relapse in patients receiving CD34(+)-PBSC transplants was prevented by donor leukocyte infusions, which were applied at a median of 4 times (range, 1-7 times) with a median T-cell dose of 3.3 x 10(6) x kg/body weight [at a median] beginning at day 120 (range, 60-690 days). The estimated probability of 3-year survival after transplantation was 90% in the CD34(+)-PBSCT group, 68% in the PBSCT group, and 63% in the BMT group (CD34-PBSCT versus BMT, P <.01; and CD34-PBSCT versus PBSCT, P <.03). Transplantation of CD34(+)-PBSCs with T-cell add-back for patients with CML in first chronic phase seems to be safe and is an encouraging alternative transplant procedure to BMT or PBSCT.     

CD8+/CD57+ cells and apoptosis suppress T-cell functions in multiple myeloma

The aim of this study was to evaluate the role of CD8⁺/CD57⁺ lymphocytes in the immune dysregulation of multiple myeloma (MM). Cytofluorimetry of peripheral blood lymphocytes (PBL) purified from 39 MM patients showed an inverse relationship between the percentage of CD8⁺/CD57⁺ cells and CD4/CD8 ratio. Analysis of their activation antigens revealed that they were prevalently HLA-DR⁺ and Fas⁺. Removal of CD8⁺/CD57⁺ cells from MM PBL significantly improved cell proliferation and pokeweed mitogen (PWM)-induced polyclonal Ig production in vitro, whereas the addition of supernatants from patients' CD8⁺/CD57⁺ cell cultures to normal PBL suppressed both the PWM-driven Ig synthesis and the proliferative rate of stimulated PBL, supporting the contention that CD8⁺/CD57⁺ cells release in vitro an inhibitory factor that is directly involved in T-cell regulatory function. However, since the proliferative recovery of PWM- and phytohaemagglutinin (PHA)-stimulated MM PBL in the absence of CD8⁺/CD57⁺ lymphocytes was only partial, a dysregulated activation-induced apoptosis was anticipated. In fact, patients' PBL displayed an increased susceptibility to apoptosis and this was significantly enhanced after PWM and, even more, after PHA stimulation. Analysis of CD57 antigen expression on apoptotic or viable cells demonstrated a substantial defect of apoptosis in the CD8⁺/CD57⁺ population. Our results indicate that both the immunosuppressive effect of CD8⁺/CD57⁺ cells and the enhanced susceptibility to apoptosis of PBL could be involved in the pathogenesis of the immunodeficiency observed in this disease.     

CD34 selections from myeloma peripheral blood cell autografts contain residual tumour cells due to impurity, not to CD34+ myeloma cells

Malignant cells in haemopoietic autografts can contribute to post-transplant relapse. Engraftment of myeloma patients with CD34⁺ peripheral blood progenitors selected from total autografts reduces the number of tumour cells infused by 2.7–4.5 logs. Residual tumour cells detected in CD34⁺ selected cells may be due to selection impurity or the existence of malignant CD34⁺ progenitors. In three patients we evaluated the CD34 purity and tumour load of total autografts, CD34⁺ progenitors selected with immunomagnetic beads and highly purified CD34⁺ progenitors obtained in two rounds of selection (combining magnetic with flow cytometry activated cell sorting) to determine the cause of residual tumour cells in CD34 selections. Using allele-specific oligonucleotides (ASO) complementary to the unique Ig heavy chain sequence (CDRIII region) of the malignant clone, semi-quantitative ASO-PCR was capable of detecting one malignant cell in 10⁴–10⁵ normal white blood cells. Selection of CD34⁺ cells from bone marrow (BM) with approximately 20% malignant plasma cells resulted in a 1.4 log reduction of tumour burden. Using two-colour flow-cytometry we observed CD34^?, BB4⁺ malignant plasma cells contaminating this CD34 selection. Prior to sorting, peripheral blood cell autografts (PBCA) contained approximately 0.1% malignant cells. Selection of >99% pure CD34⁺ cells using immunomagnetic beads (Dynal) resulted in an approximate 2 log reduction of malignant cells, but residual tumour cells were still detectable. ASO-PCR detected no malignant cells in >99.9% pure CD34⁺ peripheral blood progenitors obtained with two rounds of selection (combining magnetic with flow cytometry activated cell sorting). We conclude that CD34⁺ malignant cells are not detectable in myeloma PBCA and that residual tumour cells in CD34 selections are due to contaminating CD34-negative cells.     

Interferon-γ+ and interleukin-2+ T cells in peripheral blood from multiple myeloma patients in relation to disease status and maintenance therapy with interferon-α2b (intron A)

Peripheral blood T cells from 83 patients with multiple myeloma (MM) were examined for the production of interferon-γ (INFγ) and interleukin-2 (IL-2) using three-colour flow cytometry. Comparisons were made between the percentage of cytokine-positive lymphocytes in normal donors and in patients during remission or relapse. Patients were divided into those who were on maintenance therapy with interferon-α2b (intron A) and those who had no further treatment after high-dose melphalan (HDM) with or without autologous bone marrow (ABMR) or peripheral blood stem cell rescue (PBSCR). The percentage of INFγ⁺/CD3⁺, INFγ⁺/CD45R0⁺/CD3⁺ and IL-2⁺/CD8⁺ was higher in patients on INFα2b during remission and relapse compared with normal donors (P < 0.005). During remission INFγ⁺/CD45R0⁺/CD3⁺ and IL-2⁺/CD8⁺ lymphocytes were higher in patients not on INFα2b (P < 0.05 and P < 0.005, respectively). In relapsed patients INFγ⁺/CD3⁺ and INFγ⁺/CD45R0⁺/CD3⁺ were increased in patients not taking INFα2b (P < 0.005). There was no significant difference between the percentages of cytokine-positive lymphocytes in patients taking or not taking INFα2b either during remission or relapse. Plasma IL-6 levels were similar in both groups of patients during remission. The data suggest that if maintenance therapy with INFα2b induces the synthesis of INFγ and IL-2 in vivo, the magnitude of the effect is small and may be unimportant in providing an anti-tumour effect in the majority of patients.     

CD3-induced T-cell activation in the bone marrow of myeloma patients: major role of CD4+ cells

Summary. A large expansion of activated T cells (CD3⁺CD25⁺) with the potential to act as anti-tumour effector cells is inducible in multiple myeloma (MM) patients by culturing bone marrow mononuclear cells (BMMCs) with the anti-CD3 monoclonal antibody (mAb) OKT3. The aim of this study was to provide a greater characterization of CD3-activated T cells. On day 6, most T cells coexpressed the CD1 la, CD18, CD54, CD45R0 antigens and consisted of activated (CD25⁺) CD4⁺ and CD8⁺ cells in nearly equal proportions. Kinetics studies showed that CD4⁺CD25⁺ cells proliferated more rapidly and peaked earlier than CD8⁺CD25⁺ cells. When experiments were performed with purified subpopulations by removing CD4⁺ cells (resulting in CD8⁺ BMMCs) or by removing CD8⁺ cells (resulting in CD4⁺ BMMCs), T-cell activation and autologous plasma cell decrease were observed in CD4⁺ BMMCs only. Transwell cultures showed that CD4 help was necessary to make CD8⁺ BMMCs susceptible to CD3 stimulation. Relevant amounts of IL-2 were found in the supernatants of CD4⁺ BMMCs cultures, whereas no secretion of IL-4 was detected, indicating a Thl-like profile of CD3-activated CD4⁺ cells.
These data indicate that CD4⁺ cells proliferate earlier and provide optimal help to induce the subsequent expansion of CD8+ cells after CD3 stimulation of MM BMMCs. Adequate stimulation of CD4⁺ cells is therefore essential in any strategy aiming to recover T-cell-mediated immunity in MM.     

High-dose therapy in patients with Hodgkin's disease: the use of selected CD34(+) cells is as safe as unmanipulated peripheral blood progenitor cells.

Register data suggest that patients with Hodgkin's disease (HD) given high-dose therapy (HDT) with peripheral blood progenitor cells (PBPC) have a less favourable prognosis as compared to those given bone marrow as stem cell support. Since this can be due to infusion of tumour cells contaminating the PBPC grafts, we initiated a feasibility study in which PBPC grafts from HD patients were purged by CD34(+) cell enrichment. Controversy exists about whether the use of CD34(+) enriched stem cells leads to a delayed haematological and immune reconstitution. We compared these parameters, including risk of infections and clinical outcome after HDT, in patients with HD given either selected CD34(+) cells or unmanipulated PBPC as stem cell support. From October 1994 to May 2000, 40 HD patients with primary refractory disease or relapse were treated with HDT and supported with either selected CD34(+) cells (n = 21) or unmanipulated PBPC (n = 19) as stem cell support. All patients had chemosensitive disease at the time of transplantation. A median of 5.8 (range 2.7-20.0) vs 4.5 (range 2.3-17.6) x 10(6) CD34(+) cells per kilo were reinfused in the CD34(+) group and PBPC group, respectively. No difference was observed between the two groups with regard to time to haematological engraftment, reconstitution of B cells, CD56(+) cells and T cells at 3 and 12 months and infectious episodes after HDT. Two (5%) treatment-related deaths, one in each group, were observed. The overall survival at 4 years was 86% for the CD34(+)group and 74% for the PBPC group with a median follow-up of 37 months (range 1-61) and 46 months (range 4-82), respectively (P = 0.9). The results of this study demonstrate that the use of CD34(+) cells is safe and has no adverse effects either with respect to haematological, immune reconstitution or to infections after HDT.