Adverse reactions during transfusion of thawed haematopoietic progenitor cells from apheresis are closely related to the number of granulocyte cells in the leukapheresis product.

BACKGROUND AND OBJECTIVES: The infusion of thawed haematopoietic progenitor cells from apheresis (HPC-A) is associated with minor but frequent adverse reactions (ARs), which has been mainly attributed to dimethyl sulphoxide (DMSO). Nevertheless, other factors may play a role in the pathogenesis of such toxicity. MATERIALS AND METHODS: The ARs on a cohort of 423 cryopreserved HPC-A infusions for 398 patients in HPC transplantation program were analysed. RESULTS: ARs were reported in 105 graft infusions (24·8%) and most of them were graded as mild to moderate. The most frequently reported ARs were gastrointestinal and respiratory, and three patients presented epileptic seizure. The volume of DMSO/kg (P < 0·001), volume of red-blood-cells/kg (P = 0·02), number of nuclear cells (NCs)/kg (P <0·001) and number of granulocytes/kg (P<0·001) in the infused graft were significant in the univariate analysis for the occurrence of ARs. The amount of granulocytes/kg remained significant in the multivariate analysis (P<0·001). The grade of ARs also correlated with the amount of cryopreserved granulocytes. CONCLUSION: The incidence and grade of ARs during infusion of cryopreserved HPC-A are related to the amount of granulocytes in the graft.

Prognostic factors influencing feasibility of cytogenetic and molecular analysis in leukapheresis products in chronic myelogenous leukemia patients.

Conventional cytogenetic (CC) study and molecular analysis were performed in 150 leukapheresis products from 36 patients diagnosed with chronic myelogenous leukemia who were included in an autologous stem cell transplantation program. The aims of the study were to evaluate the effectiveness of these two methods for the detection of residual disease in the harvest and to identify the factors influencing the number of cycling cells present in the apheresis products. Progenitor cell mobilization procedures performed late after diagnosis (>12 months), a short interval between interferon-alpha discontinuation and mobilization (<3.5 months), and an intensive mobilization regimen (idarubicin, cytarabine, and etoposide, ICE protocol) were associated with a low probability of obtaining 25 metaphases, which was achieved in only 41 instances (25% of the samples). In 38 samples, less than ten metaphases were obtained; a peripheral blood leukocyte count <1.0x10(9)/l at mobilization and mononuclear cell counts in the bag <0.5x10(8)/kg significantly increased the probability to obtain less than ten metaphases for CC analysis. Previous interferon-alpha treatment during > or =12 months and low mononuclear cell counts in the bag (<0.5x10(8)/kg) increased the probability of not obtaining mitosis for cytogenetic analysis. Molecular analysis by the polymerase chain reaction (PCR) technique did not give discriminate information in the samples not evaluable by cytogenetics due to the high frequency of PCR-positive results. We conclude that new techniques such as hypermetaphase fluorescence in situ hybridization (FISH), interphase FISH, or quantitative PCR need to be routinely employed in the study of leukapheresis samples of chronic myelogenous leukemia patients for a better assessment of the neoplastic contamination of the infused products.

Combined isolation of CD34+ progenitor cells and reduction of B cells from peripheral blood by use of immunomagnetic methods.

BACKGROUND: Malignant cells may contribute to relapse after autologous hematopoietic cell transplantation The effectiveness of a double immunomagnetic purging strategy combining CD34-positive with B-negative cell selection to purge peripheral blood progenitor cells (PBPCs) from patients with chronic lymphoproliferative disorders has been analyzed. STUDY DESIGN AND METHODS: Twenty-two CD34+ cell selections from patients with follicular lymphoma (n = 14), chronic lymphocytic leukemia (n = 6), mantle cell lymphoma (n = 1), and splenic marginal zone lymphoma (n = 1) were performed by use of a magnetic cell selector followed by a negative cell selection step with anti-CD19 monoclonal antibody bound to immunomagnetic beads. RESULTS: The PBPC components contained median CD34+ cells of 1.24 percent (range, 0.38-3.92%) and CD19+ cells of 1.83 percent (range, 0.06-69.7%). After positive selection (n = 22), 49 percent (range, 16-72%) of CD34+ cells were recovered with a purity of 93 percent (range, 24-99%). The double-positive and -negative selections (n = 20) yielded 57.5 percent of CD34+ cells (range, 33.4-79.4%) with a purity of 95 percent (range, 63-99%). Logarithms of B-cell reduction in the CD34+-cell-enriched B-cell-depleted component had a median value of 3.63 (range, 2.74-4.84 log) and CD19+ and CD5+ cells for chronic lymphocytic leukemia patients with more than 4.56 log (>3.6-5.6 log). Of 13 PBPC components that had a tumor-specific clonal signal, 10 became PCR negative after the double-selection procedure. CONCLUSION: Combined positive and negative magnetic cell selection achieves a high grade of tumor cell reduction with up to 77 percent of the grafts being negative for tumor-specific clonal signal by PCR analysis. This technique preserves an adequate recovery of progenitor cells able to engraft.

Predictive factors for a successful mobilization of peripheral blood CD34+ cells in multiple myeloma.

We analyzed the prognostic factors for a successful mobilization and peripheral blood stem cell collection in a series of 57 consecutive patients with multiple myeloma (MM); a new scoring system to predict an adequate mobilization in this subset of patients was also constructed. A total of 221 aphereses were performed in 57 patients with MM. The median time from diagnosis to mobilization was 12 months (range 4-120). Only one line of chemotherapy was administered before mobilization to 36 patients and two or more to 21. The median number of alkylating chemotherapy cycles was 6 (2-33). Two patients were mobilized in complete remission, 32 in partial response, and 23 in stable/progressive disease. Significant adverse prognostic factors for collecting 2.5 x 10(6) CD34+cells/kg or more were: a period of at least 12 months from diagnosis, at least six cycles of alkylating agents, and a plasma cell infiltration of 20% or more prior to mobilization. Patients with three risk factors had a probability of only 0.38 (95% CI 0.3-0.9) for adequate mobilization. Ten patients failed to mobilize; a period from diagnosis of 12 months or more and female sex were unfavorable factors. Patients with two risk factors had a probability of 0.50 (95% CI 0.2-0.8) for failing the mobilization procedure. These findings indicate that MM patients must be mobilized early in the course of the disease, with minimal disease burden before severe hematopoietic progenitor cell injury due to cumulative therapy.

Individually adjusted prophylactic donor lymphocyte infusions after CD34-selected allogeneic peripheral blood stem cell transplantation.

T cell depletion of the graft increases graft failure and relapse rate in allogeneic PBSC transplantation. Delayed lymphocyte add-back after T cell-depleted transplants might prevent these complications. We present 22 consecutive allogeneic PBSC transplants from related histocompatible donors with positive selection of CD34+ cells. Recipients received prophylactic donor lymphocyte infusions (DLI) depending on their risk of relapse and of developing GVHD. Patients were considered at high risk of relapse with AML > first CR, ALL > second CR, and CML in accelerated or blastic phase. Patients were considered at high risk of developing GVHD if older than 35 years, or with a donor sensitized through previous pregnancy or blood transfusion. Patients at high risk of relapse and low risk of GVHD were scheduled to receive three DLI. Patients at low risk of relapse and high risk of GVHD did not receive DLI. The remaining patients were scheduled to receive two DLI. The DLI were administered on days +28 (2 x 10(5)/kg), +60 (2 x 10(5)/kg) and +90 (2 x 10(6)/kg) after transplant. G-CSF mobilized peripheral stem cells from healthy donors were positively selected by an immunomagnetic method. The mean CD34+ cells and CD3+ cells infused were 4.4 x 10(6)(range 1.9-10.6) and 0.085 x 10(5) (range 0.01-0.67). Cyclosporin A was given to prevent GVHD. All the patients engrafted. Twenty-two prophylactic DLI were performed in 12 patients: seven developed acute GVHD (one case grade III-IV) and none presented pancytopenia. At a mean follow-up of 585 days (range 89-1103), 14 patients were alive in CR, one patient was alive in relapse, four patients had died of relapse and three had died of transplant-related complication. Individually adjusted prophylactic DLI at the doses we used with an escalating schedule allowed an acceptable GVHD rate and a good engraftment of donor hematopoiesis.

Combined positive and negative cell selection from allogeneic peripheral blood progenitor cells (PBPC) by use of immunomagnetic methods.

Twenty-four mobilized peripheral blood products from healthy donors for allogeneic transplantation were positively selected for CD34(+) cells and depleted of CD4(+) and CD8(+) cells (+/- selection) by combining clinical grade immunomagnetic methods. A sequential, "two-step" strategy combining positive selection of CD34(+) cells by use of the Isolex 300i (versions 1 and 2) device and T cell depletion (TCD) using the MaxSep device and a simultaneous, "one-step" method of CD34(+)cell selection and TCD using the Isolex 300i (software versions 1 and 2) have been investigated. Using these magnetic bead separation systems, two groups of sequential +/- selection (Isolex 300i version 1/MaxSep and Isolex 300i version 2/MaxSep) and two groups of simultaneous +/- selection (Isolex 300i versions 1 and 2) were analysed. In the sequential +/- selection, logarithms of TCD (CD3(+) cell depletion) obtained by the positive selection step had median values of 3.7 with the version 1 (n = 5) and 4.5 with version 2 software of the Isolex 300i (n = 5) (P = 0.07). Version 2 also gave a higher CD34(+) cell purity and yield than did version 1 (92% vs77%, P < 0.05 and 55% vs 34%, P = 0.3, respectively). Additional TCD obtained in the second step with the MaxSep device for the two groups had a median value of 0.9 log and 7% CD34(+)cell losses. In the simultaneous +/- selection, the Isolex 300i version 2 (n = 10) gave a median TCD of 5.1 log and version 1 (n = 4) of 4 log (P < 0.005). Higher CD34(+)cell purity and yield were also obtained with version 2 than with version 1 (97% and 76%, P < 0.005 and 57% and 39%, P = 0.07, respectively). These data indicate that simultaneous, "one-step" +/- selection in the Isolex 300i version 2 achieves a high TCD with a high CD34(+) cell purity and an acceptable CD34(+) cell yield.

Autologous stem cell transplantation (ASCT) with immunologically purged progenitor cells in patients with advanced stage follicular lymphoma after early partial or complete remission: toxicity, follow-up of minimal residual disease and survival.

The role of autologous stem cell transplant (ASCT) in indolent lymphomas is a controversial issue. From 1994 to 1999, we performed ASCT with immunologically purged progenitor cells in 15 patients with advanced stage follicular lymphoma (FL) after early partial or complete remission. Results of the purging strategy and follow-up of minimal residual disease after transplant were analyzed with PCR amplification of bcl-2/IgH rearrangement for the t(14;18) translocation. A comparison of transplanted patients with a group of controls was carried out to evaluate differences in progression-free survival and overall survival. Eighty percent of patients received one chemotherapy regimen before ASCT and were in first remission. All the patients received cyclophosphamide plus hyperfractionated total body irradiation as the conditioning regimen. Nine patients were transplanted with bone marrow (BM) and six with peripheral blood progenitor cells (PBPC). Engraftment was delayed in one patient transplanted with BM. Two patients died during the transplant procedure. Ten of 12 evaluable patients were PCR positive in the BM for bcl-2 rearrangement at diagnosis. Six of them (60%) were still positive after chemotherapy, and one patient was transplanted with a positive hematopoietic product after purging. With a median follow-up of 27 months, six of eight evaluable patients still remain PCR negative in the BM. With a median follow-up of 4.7 years from diagnosis, progression-free survival was 83% (95% CI: 63-100). The risk of disease progression of non-transplanted patients was 19.2 times higher than that of transplanted patients (P = 0.01), but no differences were found in overall survival. Regarding patients in first remission, the risk of relapse was 12.6 times higher in non-transplanted than in transplanted patients (P = 0.04). This procedure seems to offer a good chance to achieve a PCR-negative state and prolonged freedom from recurrence. According to these results, prospective randomized trials are warranted.

Allogeneic peripheral blood stem cell transplantation with CD34+-cell selection and delayed T-cell add-back in adults. Results of a single center pilot study.

BACKGROUND AND OBJECTIVES: Allogeneic peripheral blood stem cell transplantation with CD34+ cell-selection (CD34+-PBSCT) allows rapid hematologic engraftment with a reduction in graft-versus-host disease (GVHD), although concerns exist regarding the increased risk of tumor relapse associated with T-cell depletion of the graft. Delayed T-cell add-back (TCAB) after such transplants may restore the graft-versus-tumor effect while achieving a reduced early transplant-related mortality due to less GVHD in a group of patients at high risk of early death (i.e., age >= 45 years). DESIGN AND METHODS: Ten patients 45 years of age or older with hematologic malignancies received a CD34+-PBSCT and cyclosporin A (CyA) to prevent acute GVHD, followed by a planned delayed donor TCAB of 107 T-cells/kg to restore the graft-versus-tumor effect. The infused graft included a median of 6.3x106 CD34+ cells/kg and 4.4x104 CD3+ cells/kg. RESULTS: Engraftment was prompt in all cases. Four patients developed acute GVHD after the CD34+-PBSCT and/or chronic GVHD after CyA withdrawal and did not proceed to TCAB, and two patients died early before the planned TCAB. Four patients proceeded to TCAB at a median of day +104 after CD34+-PBSCT (+92 to +150). Two of these patients developed acute GVHD grades I-II (IBMTR Index B) after TCAB and all four developed chronic GVHD, which was extensive in two. With a median follow-up of 611 days (range 499-847) after transplant in the seven survivors, there have been no disease progressions, and all patients show a pattern of complete donor chimerism in bone marrow and peripheral blood. INTERPRETATIONS AND CONCLUSIONS: The results of our pilot study suggest that this protocol produces an acceptable transplant-related morbidity and mortality in patients 45 years and older. However, there may be benefit in infusing CD34+-selected PBSCT with even lower T-cell contents and further delaying the TCAB.

Direct immunomagnetic method for CD34+ cell selection from cryopreserved cord blood grafts for ex vivo expansion protocols.

BACKGROUND: Cord blood is a useful source of HPCs for allogeneic transplantation. HPC ex vivo expansion of a cord blood graft has been proposed as a way to increase the speed of engraftment and thus to reduce the occurrence of transplantation-related complications. OBJECTIVE: The purpose of this study was to optimize a method for CD34+ cell selection of thawed cord blood grafts under clinical grade conditions, intended for application in a static, serum-free expansion culture. MATERIAL AND METHODS: Twelve samples were thawed and washed with dextran, albumin, and rHu-deoxyribo-nuclease I (RHu-DNase) to avoid clumping. CD34+ cells were selected by using a sensitized immunomagnetic bead and 9C5 MoAb complex. A buffer containing rHu-DNase, citrate, albumin, and immunoglobulin in PBS was used during the procedure. CD34+ cells were eluted and detached by using an immunomagnetic cell selection device. Cells from the enriched fraction were cultured for 6 days in serum-free medium supplemented with rHu-SCF, rHu-IL-3, rHu fetal liver tyrosine kinase 3 ligand, and rHu thrombopoietin (50 ng/mL each). Cells were expanded in well plates and in two semipermeable bags. RESULTS: A mean of 1.94 x 10(6) (+/- 1.55) CD34+ cells was obtained, yielding a CD34+ cell recovery of 52 +/- 12 percent. Nonspecific loss of CD34+ cells was 32 +/- 10 percent. CFU-GM and BFU-E/CFU-Mixed recoveries were 33 +/- 15 percent and 27 +/- 12 percent, respectively. CD34+ cells obtained were functionally comparable with fresh CD34+ cells selected for clonogenic potential. The capacity for expansion was not significantly different in the two types of bags studied. HPCs in wells were expanded 33 +/- 14-fold for CD34+ cells and 42 +/- 19-fold for overall colonies. The expansion rates observed in wells were significantly superior to those obtained in bags. CONCLUSION: The feasibility of a clinical-scale cord blood selection procedure based on a direct immunomagnetic method after thawing, followed by an ex vivo expansion culture using semipermeable bags, is shown. After 6 days of expansion, it was possible to generate a 9-fold increase in CD34+ cells, a 6-fold increase in CFU-GM and a 13-fold increase in BFU-E/CFU-Mixed colonies.

L-selectin expression is low on CD34+ cells from patients with chronic myeloid leukemia and interferon-a up-regulates this expression.

BACKGROUND AND OBJECTIVE: Altered adhesive interaction between bone marrow (BM) stroma and progenitors in chronic myeloid leukemia (CML) may be in part caused by abnormal expression of cell adhesion molecules (CAMs) on malignant progenitor cells. Treatment of CML with interferon-a (IFN-a) re-establishes normal hemopoiesis in some patients in part by restoring normal adhesive interactions between CML progenitors and BM microenvironment, which may in turn be mediated by correcting CAM expression on progenitors. DESIGN AND METHODS: We investigated the expression of CAMs (L-selectin, b((2))-integrin, LFA-3, ICAM-1, ICAM-3, NCAM) on purified BM CD34(+) cells from CML patients (n= 34) and healthy adults (n= 15) by flow cytometry. Modulation of L-selectin expression on CD34(+) cells from CML after in vitro treatment with IFN-a was also investigated. RESULTS: The mean percentage of CD34(+ )cells expressing L-selectin was significantly lower in CML patients (25.4+/-12.8%) than in normal controls (68.7+/-8.3%, n=15). CD34(+)/HLA-DR(&endash;/low) and CD34(+)/ CD38(&endash;/low) co-expressing L-selectin were also significantly lower in untreated CML (27.4+/-21.5% and 39.8+/-26.7%, respectively, n=8) than in controls (61+/-17% and 83.7+/-10%, respectively, n=7). In vitro treatment with IFN-a of purified CD34(+) BM cells from untreated CML patients (n=8) induced a significant, dose and time-dependent increase in the L-selectin expression as indicated by FACS analysis. INTERPRETATION AND CONCLUSIONS: We hypothesize that this L-selectin deficiency reflects a cell surface adhesion defect of progenitors from CML that is partially restored by in vitro IFN-a treatment. These data may help to explain the adhesive abnormalities of CML progenitors to the BM microenvironment and the in vitro restoration of adhesion capacity after IFN-a treatment.

Isolation of CD34+ progenitor cells from peripheral blood by use of an automated immunomagnetic selection system: factors affecting the results.

BACKGROUND: The isolation of CD34+ cells from mobilized peripheral blood is being increasingly used in the setting of allogeneic or autologous hematopoietic cell transplantation. Investigation of variables that may influence the effectiveness of CD34+ cell selection is of interest. STUDY DESIGN AND METHODS: Fifty-one CD34+ cell selections from peripheral blood progenitor cells (PBPCs) (39 allogeneic and 12 autologous) were performed using a magnetic cell separator (Isolex 300i, Baxter), including version 2.0 software. The results obtained were analyzed for different processing variables. The feasibility of transplanting these isolated CD34+ cells was also analyzed. RESULTS: The isolated CD34+ cell fraction had a median purity of 88.9 percent (range, 47.8-98.3). The median recovery of CD34+ cells was 45.1 percent (13.8-76.2), and the median colony-forming unit- granulocyte-macrophage (CFU-GM) content was 17. 2 percent (0.8-58.6). Logarithms of T- and B-cell depletion had median values of 3.7 and 2.8, respectively. The version 2.0 software of the Isolex 300i gave a higher CD34+ cell recovery in the enriched cell fraction (median 57.8%) than did version 1.11 (39.4%) or 1.12 (44.4%) (p = 0.01). The use of recombinant human deoxyribonuclease I during cell processing yielded more CD34+ cells (53% vs. 41%, p = 0. 01) and higher purity (92.8% vs. 87%, p = 0.03). There was a correlation between the percentage of CD34+ cells labeled with the monoclonal antibody 8G12 clone and the percentage of CD34+ cells labeled with the monoclonal antibody used during the processing technique (9C5 clone) in the initial, enriched, and depleted CD34+ cell fractions (R(2) = 0.95; 0.92; 0.78, p< 0.005, respectively). Median times for recovering >0.5 x 10(9) per L of granulocytes and >20 x 10(9) per L of platelets were 13 and 16 days in the allograft patients and 13 and 14 days in the autograft patients. CONCLUSION: CD34+ cells can be highly and effectively isolated from allogeneic and autologous grafts by use of this automated technique, with a high grade of T- and B-cell depletion. These purified CD34+ cell components can engraft normally.

CD7 expression on CD34+ cells from chronic myeloid leukaemia in chronic phase.

Thirty-seven patients with chronic phase chronic myeloid leukaemia and fourteen healthy controls have been evaluated for lineage differentiation with immunological markers on purified bone marrow CD34 positive cells by multiparameter flow cytometry. The myeloid-associated antigen CD33 and the stem cell factor receptor (CD117, c-kit) was expressed by 82.3% and 73.5% on CP-CML patients and by 57% and 57.5% on healthy donors, respectively (P < 0.005). CD34+/CD19+ or CD34+/CD10+ B-lymphoid cell population represented 9. 1% and 10.7% of the CD34+ cells in CML whereas in normal controls this subpopulation was expressed by 27.9% and 30.4% of the CD34+ cells, respectively (P< 0.005). The T-lineage associated markers (CD7 and CD2) were detected on a minor population of CD34+ BM cells of healthy controls (mean, 3.6% and 4.6%, respectively). The CD2 positive cells represented 1.5% of the CD34+ cells in CML patients. CP-CML patients co-expressed the CD7 antigen on a mean of 32.6% of the CD34+ BM cells. Moreover, 93% of this CD34/CD7 double positive subpopulation co-expressed CD33 antigen in CML patients. Co-expression of CD7 on CD34+ cells was induced to decrease significantly after short-term in vitro culture with the differentiation-inducing agent phorbol ester (PMA) and with a combination of cytokines (stem-cell factor, interleukin-3 and granulocyte colony-stimulating factor). In conclusion, a high co-expression of CD7 antigen is demonstrated on CD34+ cells of chronic phase-chronic myeloid leukaemia patients. The loss of CD7 marker following incubation with PMA and cytokines suggests that this antigen is expressed transiently in early myeloid leukaemic CML haemopoiesis.

Immunomagnetic bone marrow (BM) and peripheral blood progenitor cell (PBPC) purging in follicular lymphoma (FL).

Twenty-nine B cell follicular lymphoma (FL) patients had their BM (n = 12) or PBPC (n = 17) purged using a panel of monoclonal antibodies and immunomagnetic beads (IMB). The median recovery of nucleated cells (NC) and CD34+ cells was 59.3% (40.5-74) and 56.1% (30.8-82.9) in BM and 77.2% (64.7-88.3) and 73.5% (61.5-98.6) in PBPC (P<0.0005). A median of >1.62 and >1.02 log of target cell depletion was achieved as judged by flow cytometry analysis in BM and PBPC, respectively. Of 29% of initial harvests that had a bcl2 PCR-amplified signal, 37.5% became PCR negative in the final purged products. Absorbed cells containing IMB-target cell complexes gave bcl2 rearrangement signal in 20% of samples in which the start and final purged components were negative. Twenty-three of 26 patients receiving an autologous purged product are evaluable for engraftment. Median time to reach an ANC >0.5x10(9)/l and platelet count >20x10(9)/l was 21 (11-43) and 41 days (13-70) for BM (n = 9) and 14 (10-31) and 14 (8-37) for PBPC (n = 14) autografted patients (P = 0.01 and 0.001). One patient did not engraft and was rescued with a back-up BM. These data demonstrate that this indirect immunomagnetic technique is able to achieve a high grade of lymphoma cell depletion in BM and PBPC and that these purged products are capable of rapid engraftment after autologous transplantation.

Autologous stem cell transplantation in advanced follicular lymphoma. A single center experience.

BACKGROUND AND OBJECTIVE: The use of intensive therapy supported by autologous stem cell transplantation (ASCT) is being investigated as treatment for poor-prognosis follicular lymphomas (FL). A single-center experience is herein reported. DESIGN AND METHODS: From September 1990 to October 1997, 30 consecutive patients (pts) with advanced FL received transplants, 8 of bone marrow and 22 of peripheral blood. Thirteen harvests were purged by an immunomagnetic method using anti-B antibodies. Twenty-seven patients received salvage chemotherapy (CT) before ASCT with the objective of reaching this procedure in the best possible response. The disease status at ASCT was: 1(st) CR in 7 pts, > or =2(nd) CR in 6 pts, PR in 10 pts, untreated relapse in 2 pts and chemoresistant disease in 5 pts. RESULTS: There was only one transplant-related death (one month after ASTC). With a median follow-up of 19 (1-89) months, 27 pts are alive, 8 pts have relapsed/progressed at a median time of 11 (6-22) months after ASCT. The estimated 2-year PFS and OS are 57% (95% CI, 34-81%) and 83% (95% CI, 64-100%). When comparing the progression-free interval (PFI) before salvage CT and ASCT and the PFI after ASCT, of 17 evaluable pts, 10 had a PFI after ASCT longer than the previous interval, and 5 additional pts remain in CR/PR with a follow-up that has not yet reached the duration of pre-transplant response. By contrast, 2 pts had a short post-transplant response. INTERPRETATION AND CONCLUSIONS: High-dose therapy followed by ASCT obtains a high rate of responses, frequently longer than any previous PFI. Additional follow-up is necessary to determine whether there is any "plateau" in response duration and to define what proportion of pts may be cured with ASCT in this setting.

Mini-ICE regimen as mobilization therapy for chronic myelogenous leukaemia patients at diagnosis.

Between April 1996 and May 1998, 20 consecutive patients with Ph chromosome-positive CML in first chronic phase without an HLA-identical sibling received the mini-ICE regimen shortly after diagnosis to mobilize progenitor cells into the peripheral blood (PBPCs). The sex distribution was 12 males and eight females and the median (range) age 48.5 (22-62) years. The time interval between diagnosis and mobilization was a median (range) of 2 (0-5) months. Leukaphereses were initiated during recovery from chemotherapy-induced aplasia. A median number of 3 (1-7) aphereses per patient were performed to collect >/=2.0 x 106 CD34+cells/kg. Cytogenetic analysis was performed on the aphereses products of 18 patients. Complete cytogenetic Ph chromosome negativity was observed in four patients, nine had a partial negativity, three a minimal negativity and two no negative cells. Southern blot for bcr-abl was negative in the remaining two patients but the polymerase chain reaction analysis was positive. Following reinfusion, severe neutropenia was present for a median of 8.5 (3-19) days and severe thrombocytopenia lasted a median of 8 (3-18) days. Ten patients did not develop febrile neutropenia with four of them being treated on an outpatient basis. Treatment-related mortality was not observed. In conclusion, our experience demonstrates the feasibility of mobilizing PBPCs shortly after the diagnosis of CML with a safe regimen. Of note, mini-ICE allowed the collection of apheresis products with at least a major component of Ph-negative cells in almost 75% of the patients.

Low-dose donor CD8+ cells in the CD4-depleted graft prevent allogeneic marrow graft rejection and severe graft-versus-host disease for chronic myeloid leukemia patients in first chronic phase.

Based on previous experiences in animals and humans, low doses of CD8+ lymphocytes infused together with the marrow graft seem to enhance engraftment after allogeneic T cell-depleted marrow transplantation. From April 1994 to February 1997, 12 patients with chronic myelogenous leukemia in first chronic phase receiving a bone marrow transplant (BMT) from an HLA-identical sibling were included in a pilot study of T cell subset depletion. Total depletion of CD4+ cells of the marrow graft and partial depletion of CD8+ cells was performed by immunomagnetic separation. In order to improve the engraftment rate, we infused a low fixed number of CD8+ lymphocytes (0.25 x 10(6)/kg). All the patients were at high risk of developing acute graft-versus-host disease (GVHD), with a recipient age of >30 years, and/or donor sensitized by previous pregnancies or transfusions. All of them received cyclosporin A and methotrexate post-BMT. No graft failure was observed. The grade III-IV GVHD rate was 16.6%, and the actuarial survival at 3 years is 81.8%. Immunological recovery showed persistent CD8+ HLA-DR+ lymphocytosis 8 months after transplant. Relapses were not observed. This experience shows the importance of CD8+ cells to ensure correct engraftment, decreasing the GVHD rate.

Isolation of CD34+ hematopoietic progenitor cells in chronic myeloid leukemia by magnetic activated cell sorting (MACS).

We have evaluated an easy and fast immunomagnetic method for positive selection of cells expressing the CD34 antigen from BM, peripheral blood (PB) and apheresis products (AP) of CML patients and healthy adults (HA) in order to further characterize them by immunophenotypic analysis. From an initial frequency of CD34+ cells in the original sample of 1.8 +/- 1.7%, CD34+ cells were rapidly and efficiently enriched up to 91.5 +/- 6.4% by high-gradient magnetic cell sorting (MACS) (yield 53 +/- 21%). A five-dimensional flow cytometric analysis of the immunomagnetic isolated CD34+ cells demonstrated little overlap between CD34+HLA-DRlo and CD34+CD38lo subpopulations in both BM-HA and in BM-CML. Only 16 and 6% of the CD34+HLA-DRlo and CD34+CD38lo cells respectively, showed lack of expression of both Ag (CD34+HLA-DRloCD38lo) in BM-CML samples. Between 60 and 70% of the CD34+ cells expressed the stem cell factor (SCF) receptor (c-KIT, CD117) and there were no differences between BM-HA and BM-CML patients. Moreover, more than 60% of the CD34+HLA-DRlo cells, co-expressed c-KIT. MACS-enriched BM-CD34+ cells showed normal hematopoietic colony formation in vitro in all the sources analyzed with a higher colony-forming efficiency than the unfractionated sample (MNC).

Translocation (1;14)(p34;q11) and trisomy 8 in a T-cell acute lymphoblastic leukemia patient.

A case of T-cell acute lymphoblastic leukemia (T-ALL) in a 25-year-old man cytogenetically characterized by a t(1;14)(p34;q11) and trisomy 8 is reported. These chromosome abnormalities were observed in 82% of the analyzed metaphases. Fluorescence in situ hybridization (FISH) with a probe for the centromere of chromosome 8 was used to correlate cellular morphology in bone marrow (BM) cells with May-Grünwald-Giemsa smears and the cytogenetic alterations. By FISH analysis, trisomy 8 (and presumably the associated t[1;14]) was demonstrated only in lymphoid blast cells but not in the rest of the cells belonging to other hematopoietic lineages.

[Identification of marker chromosomes and complex chromosomal translocations, using non radioactive in situ hybridization, in malignant hemopathies]. / Identificación de cromosomas marcadores y translocaciones cromosómicas complejas, mediante hibridación in situ no radioactiva, en hemopatías malignas.

We describe five patients with hematological malignancies in which fluorescence in situ hybridization technique (FISH) has been combined with classical cytogenetic study for characterizing chromosomal abnormalities. Three of them were chromosome Philadelphia positive; using a 22 chromosome painting probe it was confirmed the presence or absence of a variant complex translocation. In patients 4 and 5, the karyotype showed a marker chromosome which was definitively identified by FISH as a deletion of X chromosome and a 18 ring chromosome, respectively. The value of this methodology in the cytogenetic diagnosis of hematological malignancies is commented.