Miguel A. Alvarez

Autologous peripheral blood stem cell transplantation (PBSCT) mobilized with G-CSF in AML in first complete remission. Role of intensification therapy in outcome.

In order to determine if peripheral blood stem cells (PBSC) collected after priming with G-CSF in AML in first complete remission (CR) can be used for autologous transplantation and to evaluate the efficacy of early intensification therapy as in vivo purging, we studied 35 consecutive patients with AML in first CR. After standard induction and consolidation chemotherapy, 24 of them were treated with one (10 patients) or two (14 patients) cycles of high-dose cytarabine plus etoposide prior to PBSC collection. G-CSF was used as the priming agent. Of the 35 patients scheduled for peripheral blood stem cell transplantation (PBSCT), three relapsed before transplantation, and the 32 remaining underwent PBSCT. High-dose therapy consisted of either total body irradiation plus cyclophosphamide or busulphan plus cyclophosphamide. The median number of CD34+ cells infused was 3.24 × 106/kg (range 0.15–14). The median times to reach a PMN count of 0.5 × 109/l and a platelet count of 50 × 109/l were 12 (8–28) and 30 (11–345) days, respectively. There was no transplant-related mortality. Twelve patients relapsed between 2 and 21 months post-PBSCT. With a median follow-up of 28 months, actuarial disease-free survival (DFS) is 52.41 ± 9% in the intent-to-treat group and 57.4 ± 9.8% in patients who underwent PBSCT. The probability of DFS is significantly higher for patients who receive early intensification therapy prior to both PBSC collection and PBSCT as compared with patients that do not: 68.8 ± 10.27% vs 35.5 ± 12.6%, P = 0.0418. These results indicate the feasibility of PBSCT in AML using G-CSF-mobilized PBSC. The use of intensification treatment as ‘purging in vivo’ prior both to collection of PBSC and PBSCT significantly reduces the risk of relapse in this group of patients.

Prevention of graft-versus-host disease in high risk patients by depletion of CD4 + and reduction of CD8 + lymphocytes in the marrow graft

From March 1994 to September 1997, 30 patients with hematological malignancies (12 ANLL, 10 CML, four ALL and four multiple myeloma) received HLA-identical allogeneic bone marrow transplants with the marrow graft selectively depleted of CD4+ lymphocytes and the CD8+ cell content adjusted to 1 × 106/kg. Total depletion of CD4+ and partial depletion of CD8+ lymphocytes was carried out by an immunomagnetical method. All patients were considered as having high risk for developing GVHD by at least one of the following criteria: patient age >35 years; donor age >35 years; donor multiparity or marrow from an unrelated donor. Twenty-four cases received marrow from an identical sibling and six from an unrelated donor. In order to assess the role of methotrexate (MTX) in addition to cyclosporin A (CsA) after transplant, patients were randomly assigned to received either CsA alone (n = 15) or CsA plus a short course of MTX (n = 15). No case of primary graft failure was observed, but two patients developed late graft failure. Six patients presented grade II acute GVHD and no case of severe III–IV GVHD was seen. The actuarial probability of developing grade II–IV acute GVHD was 25.9 ± 9.6% for the entire population. Patients receiving post-transplant CsA + MTX had significantly less probability of acute GVHD than those receiving CsA exclusively (6.7 ± 6.4% vs 50.5 ± 17.8%, P = 0.03) and the schedule of post-transplant immunosuppression was the only factor associated with the incidence of acute GVHD in a multivariate analysis. The actuarial incidence of chronic GVHD for the entire population was 31.8 ± 12.5, and there was no significant difference between both groups with additional prophylaxis. Four patients with CML and three with ANLL relapsed: the actuarial probability of remaining in complete remission for all patients was 53.6 ± 17.3%. For patients with acute leukemia, the probability of remaining in complete remission did not differ significantly between those transplanted in first complete remission and those receiving a transplant in more advanced phases of the disease (87.5 ± 11.6% vs 72.9 ± 16.5%; P = 0.44). The incidence of mixed chimerism assessed by PCR was 34%. Nineteen patients are alive between 2 and 43 months post-transplant, the probability of overall survival being 57.8 ± 10.4%. Our data indicate that this method of selective T cell depletion is very effective in preventing acute GVHD in high risk patients, particularly when used in combination with post-transplant CsA + MTX.

Insulin response to a short stress period

In 24 healthy volunteers (seven women, 17 men; mean age 23 +/- 5 years), we studied the insulin response to a short stress period of 30 min, induced by cognitive conflict under social pressure. Insulin, growth hormone (GH), blood glucose and blood pressure (BP) determinations were performed before and after the stress period. There was a significant increase in insulin levels following the stress period (p = 0.02, paired t-test). A multiple stepwise regression analysis, with insulin difference as the dependent variable and initial GH and blood sugar levels, their increments and body mass index as predictors, showed that insulin variation was independent of any of the predictors. We discuss the influence of autonomic innervation on insulin secretion and the possible change in insulin sensitivity during stress.

Voriconazole as primary antifungal prophylaxis in children undergoing allo-SCT

Invasive fungal disease (IFD) causes significant morbidity and mortality among children undergoing allo-SCT. In this prospective pilot study, we analyze voriconazole as primary antifungal prophylaxis. From October 2004 to July 2010, 56 children <18 years of age were enrolled in this study. Patients received voriconazole doses of 5 mg/kg per 12 h (n=23) or 7 mg/kg per 12 h (n=33), with a limiting dose of 200 mg/12 h, from day −1 to day +75 or later in patients with active acute GVHD. Patients were followed up for IFD for 6 months. In this series, 37 (66.1%) patients successfully completed treatment (85.7% during neutropenic period) without empirical or preemptive antifungal therapy, adverse effects or IFD. Nine (16.1%) children needed preemptive (n=2) or empirical (n=7) antifungal therapy, and one (1.8%) of them developed a fatal probable IFD during the study period. A total of 10 (17.8%) children developed adverse effects related to voriconazole prophylaxis, leading to definitive withdrawal on median day 26.5 (in 7 patients after granulocytic recovery). The most frequent adverse effect was persistent elevation of hepatic enzymes in seven (12.5%) children. There were no differences between doses of 5 and 7 mg/kg per 12 h. Our results suggest that voriconazole can be safely used as primary antifungal prophylaxis in children undergoing allo-SCT.

Allogeneic bone marrow transplantation vs chemotherapy for the treatment of childhood acute lymphoblastic leukaemia in second complete remission (revisited 10 years on).

In 1989 we carried out a trial comparing allogeneic BMT to chemotherapy (CT) in 76 children with relapsed acute lymphoblastic leukaemia (ALL). Ten years on we have clinically revised outcome to firmly establish the role of each treatment, to analyse the importance of length of first remission and to provide long-term actuarial results for disease-free survival (DFS) and relapse rate in each group. For 21 patients within the transplantation group, probability of DFS and relapse are 42.8 ± 10.8% and 40.2 ± 11.7% (s.e.), respectively. In the chemotherapy group, probability of DFS is 10.0 ± 4.74% (P = 0.001) and probability of relapse 87.5 ± 5.2% (P = 0.0004). These results strongly reflect those at initial analysis, confirming a key role of BMT in the management of ALL in second remission. Moreover, on univariate analysis only two factors influenced DFS: treatment group and length of first complete remission (less or more than 30 months from first CR). Thus, it seems clear that the best therapeutic option in early relapse is BMT, whereas DFS in late relapse is at the limit of significance (P = 0.07), with a higher relapse rate in the CT group. Although encouraging results using intensified rotational combination chemotherapy have been published, prospective randomised studies are needed to assess with certainty the best therapeutic option in these patients.

Optimal timing of granulocyte colony-stimulating factor (G-CSF) administration after bone marrow transplantation : a prospective randomized study

The positive role of G-CSF in hastening the myeloid recovery of patients undergoing allogeneic bone marrow transplantation (ALLO-BMT) or autologous bone marrow transplantation (ABMT) has recently been established. Considerable knowledge about adequate doses and route of administration has been accumulated in the past few years. Nonetheless, the optimal time to start growth-factor administration remains undetermined. We have performed a stratified study according to the source of hematopoietic progenitors (ALLO-BMT or ABMT), underlying disease and its stage, and acute graft-versus-host disease (GVHD) prophylaxis regimen and randomized patients in two arms: group A, which started G-CSF on day 0 (36 patients), and group B, which started on day +7 post-BMT (39 patients). The same dose (5 Μg/kg/day) and route of administration were employed in both groups. We found no significant differences in the time to reach an absolute neutrophil count (ANC) of 0.1, 0.5, and 1×109/l and 50×109 platelets/l (medians: 10 and 11, 14.5 and 14, 17 and 16, 23 and 24 days, respectively, in groups A and B). We did not find differences in the days of fever or days on antibiotic treatment with less than 1×109/l ANC, rate of bacteriemia, or days of hospitalization in both groups. In contrast, a considerable saving of GCSF in B group was found (mean days of infusion in group A, 18, versus 11 in group B) (p<0.0001). This is equivalent to a saving of 1120

Early‐acting cytokine‐driven ex vivo expansion of mobilized peripheral blood CD34+ cells generates post‐mitotic offspring with preserved engraftment ability in non‐obese diabetic/severe combined immunodeficient mice

US per patient. Therefore, early use of G-CSF after BMT is useless and more expensive and provides no advantage over delayed administration.

Lymphokine-activated killer (LAK) cell generation from peripheral blood stem cells by in vitro incubation with low-dose interleukin-2 plus granulocyte-macrophage colony-stimulating factor

The ability of ex‐vivo expanded peripheral blood stem cells (PBSC) to engraft non‐obese diabetic/severe combined immunodeficient (NOD/SCID) mice has not been evaluated to date. We investigated the maintenance of primitive SCID‐repopulating cells (SRC) and long‐term culture‐initiating cells (LTCIC) in PBSC expanded with early‐acting cytokines, thrombopoietin (TPO), stem cell factor (SCF) and FlT3‐ligand (FL) with or without interleukin 3 (IL‐3) and IL‐6 in short‐term (6 d) stroma‐free serum‐free cultures. TPO + SCF + FL and TPO + SCF + FL + IL‐3 + IL‐6 produced 5·9 ± 1·97 and 18·25 ± 4·49 (mean ± SEM)‐fold increase of CD34+ cells respectively. We tracked cellular division with PKH26 and sorted post‐mitotic CD34+ PKH26low cells to assess their primitive functional properties. After culture with TPO + SCF + FL, LTCICs among post‐mitotic cells increased 12·08 ± 3·4 times, and 4·3 ± 1·6 times when IL‐3 + IL‐6 were added. CD34+ PKH26low cells cultured with TPO + SCF + FL provided human multilineage (CD34, CD33 and CD19) engraftment in NOD/SCID mice, whereas no human cells were detected in mice injected with cells cultured with TPO + SCF + FL + IL‐3 + IL‐6. Percentages of CD34+/CD38, CD34+/CD33, CD34+/DR and cells in G0/G1 phase were similar among cells cultured with both cytokine combinations, indicating that the deleterious impact of IL‐3 + IL‐6 on the ability to engraft is not translated into phenotypic or cycling features. In conclusion, TPO + SCF + FL‐expanded PBSC maintain multilineage engraftment ability in NOD/SCID mice, which is abrogated by the addition of IL‐3 + IL‐6.

Quantification of minimal residual disease levels by flow cytometry at time of transplant predicts outcome after myeloablative allogeneic transplantation in ALL

Previous reports have demonstrated granulocyte– macrophage colony-stimulating factor (GM-CSF)-mediated enhancement of lymphokine activated killer (LAK) cell function. Based on these studies we have developed a model of LAK cell generation from peripheral blood stem cells (PBSC) from cancer patients by in vitro incubation with low-dose interleukin-2 (IL-2) + GM-CSF. PBSC from seven patients were incubated for 48 h at 37°C in serum-free culture medium supplemented with IL-2 at increasing concentrations (10, 100 or 1000 IU/ml) in the presence or absence of 10 IU/ml GM-CSF. LAK activity generated in cultures with 10 IU/ml IL-2 + GM-CSF was significantly higher than that generated by 10 IU/ml IL-2 and did not differ from LAK generation at optimal concentrations of IL-2 (100 and 1000 IU/ml). PBSC from five additional patients were incubated with low-dose IL-2 + GM-CSF after sequential depletion of the CD4+ and CD8+ T cell subsets. LAK activity was significantly reduced by depletion of both CD4+ and CD8+ T cells and almost completely abolished after depletion of both subsets, suggesting that T cells and not NK cells are the main LAK precursors in this model. Six patients have received two courses of LAK cells generated in vitro by low-dose IL-2 + GM-CSF on day +1 and +8 after PBSC transplant in combination with GM-CSF and IL-2 administration in vivo. The mean LAK activity in peripheral blood of these patients dramatically increased immediately after transplant from a mean of 10% to 43.2% on day +2 and remained increased during the period studied. These results are encouraging and suggest that the administration of in vitro generated LAK cells early after transplant may have a role in the control of minimal residual disease.

BALB/C mice injected with LSTRA leukemic cell line are cured by in vivo treatment with IL-2 + GM-CSF

The potential impact on patient outcome of different Minimal residual disease (MRD) levels at time of transplant in patients with lymphoblastic leukemia undergoing allogeneic hematopoietic SCT (HSCT) remains uncertain. In this study, we quantified MRD levels at time of transplant using multiparameter flow cytometry (MFC). Mononuclear cells from marrow aspirates were obtained from 102 adult and child patients before their conditioning regimen. Quantification of MRD levels was carried out by detecting patient-specific leukemia-associated immunophenotypes using four-color MFC. Thirty patients exhibited measurable levels of MRD at the time of transplant, with low levels (0.01 to ⩽0.1%) in 12 cases, intermediate levels (>0.1 to ⩽1%) in 8 cases and high levels (>1%) in 10 cases. The leukemia-free survival (LFS) rates were 65.9±7.0%, 42.9±15.7% and 0% for negative, low levels ⩽0.1% and intermediate-high levels >0.1%, respectively (P<0.001, log-rank test). Overall survival (OS) was 52.3±7.6%, 28.6±13.8% and 0% for MRD-negative, low levels ⩽0.1% and intermediate-high levels >0.1%, respectively (P<0.001, log-rank test). Multivariate Cox analysis confirmed that detection of leukemia cells by flow cytometry at transplant was the most significantly adverse factor for OS, LFS and EFS after transplant.