Pedro Marin

Immune reconstitution following allogeneic peripheral blood progenitor cell transplantation: Comparison of recipients of positive CD34+ selected grafts with recipients of unmanipulated grafts

We compared the kinetic recovery of lymphocytes and their subsets in two groups of patients submitted to allogeneic peripheral blood progenitor cell transplantation (allo-PBT): those receiving lymphocyte-depleted leukaphereses by positive selection of CD34+ cells (group 1, n = 18) and those receiving unmanipulated leukaphereses (group 2, n = 15). Patients were conditioned with cyclophosphamide (120 mg/kg) and fractioned total body irradiation (13 Gy, group 1; 12 Gy, group 2). The mean number (x 10(6)/kg) of CD34+ and CD3+ cells infused was 4.0 and 0.67, respectively, in group 1 patients, and 4.7 and 274, respectively, for group 2 patients. Graft-versus-host disease prophylaxis consisted of cyclosporin A + methylprednisolone for group 1 and cyclosporin A + methotrexate for group 2. Median follow-up was 7 months (range 2-8 months) for both groups. During the first 6 months post-transplant, CD4+ cell counts were lower in group 1 as compared with group 2 (p = 0.014, 0.010, 0.011, 0.0003, and 0.052 at 0.5, 1, 2, 3, and 6 months, respectively), whereas there was no difference at 8 months. The number of CD4+CD45RA+ cells was very low throughout the study in both groups, being lower in group 1 than in group 2, especially during the first 3 months post-transplant (p = 0.007 and 0.0006 at 1 and 3 months). Normal levels of CD8+ cells were reached by 1 month post-transplant in both groups. TCR gamma delta + cell counts were lower in group 1 than in group 2 during the first 4 months post-transplant (p = 0.001, 0.004, and 0.04 at 1, 3, and 4 months). A normal number of natural killer cells (CD3-CD56+) was achieved 1 month post-transplant in both groups. B lymphocytes (CD19+) showed low or undetectable counts throughout the first 4 months in both groups, achieving the normal range at 8 months. These results show that, during the first 6 months following allo-PBT with CD34+ selected grafts, the number of CD4+, CD4+CD45RA+, and TCR gamma delta + cells is significantly lower than after unmanipulated allo-PBT; these differences disappeared at 8 months. In contrast, there are no differences between transplant groups in the recovery of CD8+, CD19+, and natural killer cells.

Effectiveness of immunosuppressive therapy in older patients with aplastic anemia

Aplastic anemia is defined as peripheral blood pancytopenia associated with unexplained hypocellularity of the bone marrow without excess of blast cells. If aplastic anemia goes untreated, patients die of bleeding or infections caused by aplasia. Bone marrow transplantation and immunosuppressive treatment have improved outcome, with remission rates of 60% to 80% (1-4). The decision between immunosuppressive therapy and bone marrow transplantation depends largely on the availability of a bone marrow donor. In many centers, the upper age limit for allogeneic bone marrow transplantation in patients with aplastic anemia has been set at 40 to 55 years (5-8). This limit is traditionally less stringent for immunosuppressive treatment (2, 9-14). However, data are scarce, and no study has specifically addressed outcomes in older patients with aplastic anemia. After immunosuppressive treatment, hematologic recovery is slow and often incomplete (15), and clonal transformations, such as myelodysplastic syndromes, paroxysmal nocturnal hemoglobinuria, or solid tumor, may occur (16-21). As a consequence, fear of life-threatening complications during prolonged aplasia as well as concerns about increased risk for clonal transformations, particularly in older patients, prevail. We sought to 1) determine the outcome of patients 50 years of age or older receiving immunosuppression therapy for aplastic anemia and 2) investigate the response and complication rate among these patients compared with that of younger patients. Methods Design This retrospective cohort study used data from 56 centers reporting to the European Group for Blood and Marrow Transplantation (EBMT) Severe Aplastic Anaemia Working Party between 1974 and 1997. Collected data included demographic information, pretreatment blood values, type of immunosuppressive therapy, date and number of courses of immunosuppressive therapy, response to therapy, date of last known vital status, cause of death, and type and date of late complications for every patient. Follow-up was completed by June 1997. Patients We included 810 patients from the EBMT Registry in whom acquired severe aplastic anemia was diagnosed according to current criteria (22), adequate immunosuppressive therapy (antilymphocyte globulin, cyclosporine, or both) was instituted, and bone marrow transplantation was not performed as second-line treatment. For the purpose of this analysis, patients were separated into three age groups: 50 to 59 years of age (n=115), 60 years of age or older (n=127), and 20 to 49 years of age (n=568). The latter patients served as the reference group. The Severe Aplastic Anaemia Working Party regularly stipulates the definitions for disease and late complications to its participants. No central slide review was performed. Table 1. Characteristics of 810 Patients with Aplastic Anemia Outcome Measures Outcome measures analyzed were overall survival, causes of death, response to immunosuppressive therapy, rate of relapse in responders, and late complications. Late complications were defined as secondary development of a myelodysplastic syndrome, leukemia, paroxysmal nocturnal hemoglobinuria, or solid tumor. Cause of death was classified as related to aplastic anemia or its treatment (bleeding or infection), secondary to late complications, unrelated to aplastic anemia, or unknown. Response to immunosuppressive therapy was defined as reaching complete independence from transfusions. Relapse was defined as dependence on transfusions after 3 months of independence from transfusions. Statistical Analysis Group differences were analyzed by using the Kruskal-Wallis test for continuous variables and the Fisher exact test for categorical variables. Survival probabilities were calculated by using the Kaplan-Meier estimator. Time at risk started at the date of first treatment and ended at the date of an event (response to immunosuppressive therapy, relapse, complication, or death) or the date of last known vital status, whichever came first. We calculated 95% CIs of survival probabilities according to the method of Rothman and Boice. Variables significantly associated with the risk for death were assessed by univariate and multivariate analysis. A two-sided log-rank test was used for comparisons. Because patients were treated at 56 centers, the possibility that center-specific differences in supportive care and patient selection would bias the results cannot be excluded. Therefore, univariate survival comparisons between age groups were made by using the log-rank test stratified on center. Sixty-eight percent of patients were treated at 6 centers (39 to 191 patients per center); 32% of patients were treated at 50 centers (1 to 24 patients per center). Grouping the latter patients led to 7 centers that were used for stratification. Proportional hazards regression analysis was used to assess the effect of known risk factors on survival. Variables considered were sex, age group, disease severity (reflected by neutrophil count at diagnosis), type of treatment (antilymphocyte globulin, cyclosporine, or both), and calendar year (1974 to 1979, 1980 to 1989, or 1990 to 1997). A backward stepwise procedure was used to eliminate nonsignificant variables (cut-off value, P>0.2). To adjust for the inherently increased risk for death with older age, the number of deaths observed after immunosuppressive treatment for aplastic anemia was compared with the expected number of deaths in a general European population matched for sex and age. The standardized mortality ratio (observed deaths/expected deaths) was calculated for each year after treatment. The 95% CI of the standardized mortality ratio was calculated by assuming a Poisson distribution of the number of observed deaths. The changes in risk for events over time were computed for the whole study sample as well as for the three groups separately. Statistical analysis was performed by using the SPSS statistical program (SPSS for Windows, release 6.1, SPSS, Inc., Chicago, Illinois). Role of the Funding Source The funding source had no role in the collection, analysis, or interpretation of the data or in the decision to submit the paper for publication. Results Patients Demographic and disease characteristics of all 810 patients are listed in Table 1. Significant differences were seen among age groups. More female patients were 60 years of age or older, and more men were in the reference group (P<0.001). Fewer cases of viral-associated aplastic anemia were seen in patients 50 to 59 years of age, and no cases were seen in those 60 years of age or older (P=0.008). More patients 60 years of age or older received cyclosporine alone, and more patients in the reference group received antilymphocyte globulin (P<0.001). The proportion of older patients increased continuously over time: Until 1979, 13% of the patients were 50 years of age or older; this proportion increased to 27% from 1980 to 1989 and to 38% since 1990. Hence, the median follow-up was 47 months for surviving patients 50 to 59 years of age and 31 months for patients 60 years of age or older compared with 44 months in reference patients (P<0.001). Severity of disease, as reflected by neutrophil counts at diagnosis and number of courses of immunosuppressive treatment, did not significantly differ among the groups. Multiple interrelations were found among calendar year, type of immunosuppressive therapy, neutrophil count at diagnosis, and age. Calendar year was significantly associated with type of treatment (Table 1). Before 1980, all patients received antilymphocyte globulin alone; since 1990, 51% patients received antilymphocyte globulin and cyclosporine, 31% received antilymphocyte globulin alone, and 18% received cyclosporine alone (P<0.001). Since 1990, however, the type of immunosuppressive therapy was not equally distributed among the age groups (Table 1). Patients 60 years of age or older more often received cyclosporine alone (32%), whereas most patients in the reference group received a combination of antilymphocyte globulin and cyclosporine (61%) (P<0.001). Finally, disease severity was associated with age and calendar year. Before 1980, neutrophil counts less than 0.2 109 cells/L were encountered in 44% of patients in the reference group, 20% of patients 50 to 59 years of age, and none of the patients 60 years of age or older. Since 1990, low neutrophil counts were observed in 19% of patients in the reference group, 15% of patients 50 to 59 years of age, and 55% of patients 60 years of age or older. Survival At the time of last follow-up, 552 of 810 patients (68%) were alive and 258 (32%) had died (survival rate at 5 years, 67% [95% CI, 65% to 69%]). Survival was influenced by age: The older patients were at diagnosis, the lower the survival rate (Table 2). Survival also improved over time; the 5-year survival rate was 52% (CI, 39% to 64%) in patients treated before 1980 compared with 65% (CI, 60% to 69%) in patients treated from 1980 to 1989 and 73% (CI, 66% to 88%) in patients treated since 1990 (P<0.001). This improvement was most pronounced in the reference group (P<0.001) and was not statistically significant in patients 60 years of age or older (Table 2). Table 2. Five-Year Survival by Univariate Analysis Causes of Death Two hundred fifty-eight patients (32%) died. The mortality rate was 39% (45 of 115) for patients 50 to 59 years of age and 43% (55 of 127) for patients 60 years of age or older compared with 28% (158 of 568) for patients in the reference group. The main causes of death were directly related to aplastic anemia: Bleeding or infection occurred in 205 of 258 patients (79%). Thirty of 258 patients (12%) died of late complications, and 23 of 258 patients (9%) died of causes unrelated to aplastic anemia or its treatment. In all three age groups, bleeding or infection was the main cause of death (Table 3). The excess of mortality in older patients was due to aplasia-related d

Self-Renewing Human Bone Marrow Mesenspheres Promote Hematopoietic Stem Cell Expansion

Strategies for expanding hematopoietic stem cells (HSCs) include coculture with cells that recapitulate their natural microenvironment, such as bone marrow stromal stem/progenitor cells (BMSCs). Plastic-adherent BMSCs may be insufficient to preserve primitive HSCs. Here, we describe a method of isolating and culturing human BMSCs as nonadherent mesenchymal spheres. Human mesenspheres were derived from CD45- CD31- CD71- CD146+ CD105+ nestin+ cells but could also be simply grown from fetal and adult BM CD45--enriched cells. Human mesenspheres robustly differentiated into mesenchymal lineages. In culture conditions where they displayed a relatively undifferentiated phenotype, with decreased adherence to plastic and increased self-renewal, they promoted enhanced expansion of cord blood CD34+ cells through secreted soluble factors. Expanded HSCs were serially transplantable in immunodeficient mice and significantly increased long-term human hematopoietic engraftment. These results pave the way for culture techniques that preserve the self-renewal of human BMSCs and their ability to support functional HSCs.

High-dose therapy autotransplantation/intensification vs continued standard chemotherapy in multiple myeloma in first remission. Results of a non-randomized study from a single institution

The purpose of this study was to analyze the outcome of patients with multiple myeloma (MM) responding to initial chemotherapy who received intensification with high-dose therapy/autotransplantation (HDT) as compared to that of those who were continued on standard chemotherapy. From 1 January 1990 to 30 June 1998, 64 patients with MM who were younger than 65 years achieved a response to initial chemotherapy. Due to referral reasons, patients preference or inclusion in trials, 31 patients received HDT as early intensification while 33 were continued on standard chemotherapy. The presenting features were similar in both groups, except for the median age, which was lower in the HDT group (53 vs 58 years, P = 0.007). Complete response – negative immunofixation – (CR) was achieved in 12 of 31 (39%) patients intensified with HDT and in two of 33 (6%) patients who were continued on conventional chemotherapy (P = 0.002). Event-free survival (EFS) was significantly longer in the HDT group (median, 43 vs 21 months; P = 0.007). Overall survival (OS) was not significantly different between groups (median, 62 vs 38 months; P = 0.21). However, patients in the HDT group who achieved CR had an EFS (median, 51 vs 31 months; P = 0.03) as well as an OS (median, not reached vs 50 months; P = 0.0006) significantly longer than those achieving a lower degree of response. In conclusion, this non-randomized study shows that early HDT increases CR rate and prolongs EFS. In addition, these results highlight CR as a crucial step for achieving long-lasting disease control and prolonged survival in patients with MM. Bone Marrow Transplantation (2000) 26, 845–849.

High-dose therapy/autologous stem cell transplantation in patients with chemosensitive multiple myeloma: predictors of complete remission

Summary:High-dose therapy (HDT) followed by autologous stem cell support is widely used as intensification treatment in patients with multiple myeloma (MM) responsive to the initial chemotherapy. However, there is growing evidence that only the subset of patients who achieve complete remission (CR) actually benefit from this approach. The aim of this study was to identify pretransplant predictors of CR in responding myeloma patients intensified with HDT. A total of 59 patients with chemosensitive disease received myeloablative therapy. The intensification regimen consisted of MEL-200 (23), MEL-140/TBI 12 Gy (21) or busulfan-based regimens (15). Serum and urine negative immunofixation were required for CR. After HDT, the CR rate increased from 8 to 37%. For the overall series, the median event-free survival (EFS) and overall survival (OS) from the initiation of therapy were 41 and 68 months, respectively. Patients who achieved CR had an EFS (median 47 vs 36 months; P=0.023) as well as an OS (median not reached vs 60 months; P=0.006) significantly longer than those attaining a lower degree of response. Finally, the pretransplant features significantly associated to CR were a low M-protein size (serum ⩽10 g/l and urine <0.5 g/24 h) (P=0.0003) and a proportion of bone marrow plasma cells of ⩽5% (P=0.02).

Serial chimerism analyses indicate that mixed haemopoietic chimerism influences the probability of graft rejection and disease recurrence following allogeneic stem cell transplantation (SCT) for severe aplastic anaemia (SAA): indication for routine assessment of chimerism post SCT for SAA

Ninety‐one patients were studied serially for chimeric status following allogeneic stem cell transplantation (SCT) for severe aplastic anaemia (SAA) or Fanconi Anaemia (FA). Short tandem repeat polymerase chain reaction (STR‐PCR) was used to stratify patients into five groups: (A) complete donor chimeras (n = 39), (B) transient mixed chimeras (n = 15) (C) stable mixed chimeras (n = 18), (D) progressive mixed chimeras (n = 14) (E) recipient chimeras with early graft rejection (n = 5). As serial sampling was not possible in Group E, serial chimerism results for 86 patients were available for analysis. The following factors were analysed for association with chimeric status: age, sex match, donor type, aetiology of aplasia, source of stem cells, number of cells engrafted, conditioning regimen, graft‐versus‐host disease (GvHD) prophylaxis, occurrence of acute and chronic GvHD and survival. Progressive mixed chimeras (PMCs) were at high risk of late graft rejection (n = 10, P < 0·0001). Seven of these patients lost their graft during withdrawal of immunosuppressive therapy. STR‐PCR indicated an inverse correlation between detection of recipient cells post‐SCT and occurrence of acute GvHD (P = 0·008). PMC was a bad prognostic indicator of survival (P = 0·003). Monitoring of chimeric status during cyclosporin withdrawal may facilitate therapeutic intervention to prevent late graft rejection in patients transplanted for SAA.

Efficacy and toxicity of a high-dose G-CSF schedule for peripheral blood progenitor cell mobilization in healthy donors.

An important issue in allogeneic peripheral blood progenitor cell transplantation is the optimization of the regimen of mobilization of progenitor cells from normal donors. It has been shown that for G-CSF doses up to 10 μg/kg/day, a dose–response relationship exists for the degree of progenitor cell mobilization. Formal comparisons with doses higher than 10 μg/kg/day, however, have not been reported. The aim of this study was to compare the mobilization and collection results of two different G-CSF (Filgrastim) schedules: 10 μg/kg/12 h (n = 20; group A) vs 10 μg/kg/24 h (n = 20; group B). Apheresis sessions were started on day 5 (after 4 days of G-CSF). Adverse events consisted of bone pain, headache, and fatigue which required treatment with acetaminophen ± codeine in both donor groups. Discontinuation of G-CSF administration for intolerable side-effects was not necessary in any case. The increase in peripheral leukocyte and lymphocyte counts × 109/l on day 5 was higher in group A (56.2 (37.1–75.2) and 4.4 (2.1–14.6), respectively) than in group B (27.5 (13.2–53.9) and 2.6 (1.9–5.1), respectively) (P < 0.0001 and P = 0.008). Platelets × 109/l decreased in group A from 228 (161–286) before G-CSF administration to 207 (155–328) on day 5 (P = 0.03), whereas no change was observed in group B. Following the first apheresis, a significant decrease in platelet count was observed with both G-CSF schedules without any differences between groups. The number × 106/kg of both nucleated and CD34+ cells collected after the first apheresis session was higher in group A (672 (462–992) and 5.9 (3.4–10.4), respectively) than in group B (427 (319–608) and 3.1 (1.1–6.8), respectively) (P = 0.0003 in both cases). The median number of CD3+cells × 106/kg collected after one apheresis session was similar with both G-CSF schedules (212 (91–430) in group A and 170 (110–291) in group B) (P = NS). In conclusion, the schedule of 10 μg/kg/12 h was well tolerated and resulted in the collection of a higher number of progenitor cells than 10 μg/kg/24 h without increasing the T cell content. This approach could avoid the donor having to undergo a second apheresis, and facilitate further graft manipulation.

CRITERIA FOR SEVERE APLASTIC ANAEMIA

To study the validity of currently accepted international criteria for severe aplastic anaemia, 213 consecutive cases of bone marrow aplasia from a single institution were analysed. The distribution percentiles of peripheral blood values and multivariate analysis showed that the current reticulocyte count limit of 1% (corrected for haematocrit) is inadequate an an indicator of severe disease and should be substantially lowered. Since the choice of treatment in aplastic anaemia may depend on the prognosis current criteria for severe aplastic anaemia should be modified.

Impact of constitutional polymorphisms in VCAM1 and CD44 on CD34+ cell collection yield after administration of granulocyte colony-stimulating factor to healthy donors

Background The number of CD34+ cells mobilized from bone marrow to peripheral blood after administration of granulocyte colony-stimulating factor varies greatly among healthy donors. This fact might be explained, at least in part, by constitutional differences in genes involved in the interactions tethering CD34+ cells to the bone marrow. Design and Methods We analyzed genetic characteristics associated with CD34+ cell mobilization in 112 healthy individuals receiving granulocyte colony-stimulating factor (filgrastim; 10 μg/kg; 5 days). Results Genetic variants in VCAM1 and in CD44 were associated with the number of CD34+ cells in peripheral blood after granulocyte colony-stimulating factor administration (P=0.02 and P=0.04, respectively), with the quantity of CD34+ cells ×106/kg of donor (4.6 versus 6.3; P<0.001 and 7 versus 5.6; P=0.025, respectively), and with total CD34+ cells ×106 (355 versus 495; P=0.002 and 522 versus 422; P=0.012, respectively) in the first apheresis. Of note, granulocyte colony-stimulating factor administration was associated with complete disappearance of VCAM1 mRNA expression in peripheral blood. Moreover, genetic variants in granulocyte colony-stimulating factor receptor (CSF3R) and in CXCL12 were associated with a lower and higher number of granulocyte colony-stimulating factor-mobilized CD34+ cells/μL in peripheral blood (81 versus 106; P=0.002 and 165 versus 98; P=0.02, respectively) and a genetic variant in CXCR4 was associated with a lower quantity of CD34+ cells ×106/kg of donor and total CD34+ cells ×106 (5.3 versus 6.7; P=0.02 and 399 versus 533; P=0.01, respectively). Conclusions In conclusion, genetic variability in molecules involved in migration and homing of CD34+ cells influences the degree of mobilization of these cells.

Flow Cytometry-Based Approach to ABCG2 Function Suggests That the Transporter Differentially Handles the Influx and Efflux of Drugs

To better characterize the function of the ABCG2 transporter in vitro, we generated three cell lines (MXRA, MXRG, and MXRT) stably expressing ABCG2 after transfection of wild‐type ABCG2 and two mutants (R482G and R482T), respectively.