CD8+ T-cell immunity against cancer-testis antigens develops following allogeneic stem cell transplantation and reveals a potential mechanism for the graft-versus-leukemia effect

Background

Allogeneic stem cell transplantation is associated with a powerful ‘graft-versus-leukemia’ effect that is generally considered to result from an alloreactive T-cell immune response. However, disease remission can also be observed after syngeneic transplantation and we investigated whether a T-cell immune response to cancer-testis antigens can be detected in patients in the post-transplant period.

Design and Methods

The T-cell immune response against cancer-testis antigens was studied in a cohort of 41 patients who underwent allogeneic stem cell transplantation for the management of acute myeloid leukemia or multiple myeloma. The cytokine secretion assay was combined with magnetic selection to allow detection of an interferon-γ-secreting T-cell response to a panel of cancer-testis antigen peptides.

Results

A cancer-testis antigen-specific CD8⁺ T-cell immune response was observed in the peripheral blood of five patients with an average magnitude of 0.045% of the CD8⁺ T-cell repertoire. Four of these patients had undergone reduced intensity conditioning transplantation with alemtuzumab for the treatment of acute myeloid leukemia and three remain in long-term remission. T-cell immunity was focused against peptides derived from MAGE proteins and was markedly increased within the bone marrow.

Conclusions

Functional cancer-testis antigen-specific CD8⁺ T-cell immune responses develop in the early period following reduced intensity allogeneic stem cell transplantation and are preferentially localized to bone marrow. These immune responses are likely to contribute to the cellular basis of the graft-versus-leukemia effect.     

The role of matched sibling donor allogeneic stem cell transplantation in pediatric high-risk acute myeloid leukemia: results from the AML-BFM 98 study

Background

The role of allogeneic stem cell transplantation in post-remission management of children with high-risk acute myeloid leukemia remains controversial. In the multi-center AML-BFM 98 study we prospectively evaluated the impact of allogeneic stem cell transplantation in children with high-risk acute myeloid leukemia in first complete remission.

Design and Methods

HLA-typed patients with high-risk acute myeloid leukemia, who achieved first complete remission (n=247), were included in this analysis. All patients received double induction and consolidation. Based on the availability of a matched-sibling donor, patients were allocated by genetic chance to allogeneic stem cell transplantation (n=61) or chemotherapy-only (i.e. intensification and maintenance therapy; n=186). The main analysis was done on an intention-to-treat basis according to this allocation.

Results

Intention-to-treat analysis did not show a significantly different 5-year disease-free survival (49±6% versus 45±4%, P_{log rank}=0.44) or overall survival (68±6% versus 57±4%, P_{log rank}=0.17) between the matched-sibling donor and no-matched-sibling donor groups, whereas late adverse effects occurred more frequently after allogeneic stem cell transplantation (72.5% versus 31.8%, P_Fischer<0.01). These results were confirmed by as-treated analysis corrected for the time until transplantation (5-year overall survival: 72±8% versus 60±4%, P_Mantel-Byar 0.21). Subgroup analysis demonstrated improved survival rates for patients with 11q23 aberrations allocated to allogeneic stem cell transplantation (5-year overall survival: 94±6% versus 52±7%, P_log-rank=0.01; n=18 versus 49) in contrast to patients without 11q23 aberrations (5-year overall survival: 58±8% versus 55±5%, P_log-rank=0.66).

Conclusions

Our analyses defined a genetic subgroup of children with high-risk acute myeloid leukemia who benefited from allogeneic stem cell transplantation in the prospective multi-center AML-BFM 98 study. For the remainder of the pediatric high-risk acute myeloid leukemia patients the prognosis was not improved by allogeneic stem cell transplantation, which was, however, associated with a higher rate of late sequelae.     

Sustained remission of blastic plasmacytoid dendritic cell neoplasm with ABVD chemotherapy

Blastic plasmacytoid dendritic cell neoplasm is a rare but clinically aggressive tumor known to be derived from the precursors of plasmacytoid dendritic cells with frequent cutaneous involvement. Though the majority of patients initially respond to multi-agent chemotherapy, most cases without hemopoietic stem cell transplantation relapse within a year. We describe a case of a 71-year-old man with a dark-purple subcutaneous nodule (5×3 cm) under his right auricle. Histologic examination of the excisional biopsy specimen revealed a diffuse proliferation of blast cells with irregular nuclei, fine chromatin and one to several small nucleoli in the dermis extending to the subcutaneous soft tissues. The tumor cells expressed CD123, CD56, CD4, CD7, LCA, and TdT but not CD3, CD20, CD79a, CD10, CD68, CD163, myeloperoxidase (MPO), or naphthol-ASD-chloroacetate (ASD-Ch) esterase. A diagnosis of blastic plasmacytoid dendritic cell neoplasm was made. He did not have any other lesions except for the solitary skin nodule. He had refractory cytopenia with multilineage dysplasia (RCMD) and renal dysfunction. It was difficult for him to receive hemopoietic stem cell transplantation because of his advanced age and renal dysfunction. We had previously experienced successful treatment with ABVD chemotherapy for interdigitating dendritic cell sarcoma after ineffective CHOP chemotherapy. The plasmacytoid dendritic cell is one of the precursor cells of the interdigitating dendritic cell. Therefore we tried to apply ABVD therapy to him. The first course of ABVD induced complete remission. Although the therapies were reduced and postponed because of various complications, he is now in complete remission that has lasted for 21 months. Although previously not reported, ABVD therapy is useful for patients with blastic plasmacytoid dendritic cell neoplasm who cannot receive hemopoietic stem cell transplantation.     

Endothelial cell-derived microparticles induce plasmacytoid dendritic cell maturation: potential implications in inflammatory diseases

Background

Increased circulating endothelial microparticles, resulting from vascular endothelium dysfunction, and plasmacytoid dendritic cell activation are both encountered in common inflammatory disorders. The aim of our study was to determine whether interactions between endothelial microparticles and plasmacytoid dendritic cells could contribute to such pathologies.

Design and Methods

Microparticles generated from endothelial cell lines, platelets or activated T cells were incubated with human plasmacytoid dendritic cells sorted from healthy donor blood or with monocyte-derived dendritic cells. Dendritic cell maturation was evaluated by flow cytometry, cytokine secretion as well as naive T-cell activation and polarization. Labeled microparticles were also used to study cellular interactions.

Results

Endothelial microparticles induced plasmacytoid dendritic cell maturation. In contrast, conventional dendritic cells were resistant to endothelial microparticle-induced maturation. In addition to upregulation of co-stimulatory molecules, endothelial microparticle-matured plasmacytoid dendritic cells secreted inflammatory cytokines (interleukins 6 and 8, but no interferon-α) and also induced allogeneic naive CD4⁺ T cells to proliferate and to produce type 1 cytokines such as interferon-γ and tumor necrosis factor-α. Endothelial microparticle endocytosis by plasmacytoid dendritic cells appeared to be required for plasmacytoid dendritic cell maturation. Importantly, the ability of endothelial microparticles to induce plasmacytoid dendritic cells to mature was specific as microparticles derived from activated T cells or platelets (the major source of circulating microparticules in healthy subjects) did not induce such plasmacytoid dendritic cell maturation.

Conclusions

Our data show that endothelial microparticles specifically induce plasmacytoid dendritic cell maturation and production of inflammatory cytokines. This novel activation pathway may be implicated in various inflammatory disorders and endothelial microparticles could be an important immunmodulatory therapeutic target.     

Concurrent intensive chemotherapy and imatinib before and after stem cell transplantation in newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia. Final results of the CSTIBES02 trial

Background

Imatinib, given concurrently or alternating with chemotherapy, has improved the response and survival of patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph⁺ ALL) but relapses are still frequent. The aim of this study was to evaluate the feasibility and results of giving imatinib concurrently with intensive chemotherapy, stem cell transplantation and post-transplant imatinib maintenance therapy in patients with newly diagnosed Ph⁺ ALL.

Design and Methods

This was a phase II study of patients with newly diagnosed Ph⁺ ALL given standard chemotherapy, together with imatinib (400 mg/day) until stem cell transplantation, followed by imatinib maintenance therapy for all patients regardless of the molecular status of the disease.

Results

Of the 30 patients included, 27 (90%) achieved complete remission, one was resistant to treatment and two died during induction therapy. The percentages of major and complete molecular responses were 86% and 21% after induction, and 81% and 65% after consolidation, respectively. Similar results were observed assessing minimal residual disease by flow cytometry. Of the 27 patients who achieved complete remission, 21 underwent stem cell transplantation (16 allogeneic, 5 autologous). Imatinib (400 mg/day) could be administered after transplantation for a median of 3.9 months in 12 patients, although it was interrupted in 10 patients (in 2 cases because of side effects of the drug). Nine patients relapsed, four before and five after stem cell transplantation and eight patients died of transplant-related causes. With a median follow-up of 4.1 years, the probabilities (95% CI) of disease-free and overall survival were 30% (15% to 45%) and 30% (16% to 45%), respectively.

Conclusions

These results confirm that imatinib is an effective first-line treatment for adult Ph⁺ ALL when given concurrently with chemotherapy, making stem cell transplantation feasible in a high proportion of patients. However, post-transplantation imatinib administration was limited, mainly because of transplantation-derived complications rather than drug-specific toxicity.     

Prognostic factors and outcomes of adult patients with acute myeloid leukemia after first relapse

Background

Patients with acute myeloid leukemia who are treated with conventional chemotherapy still have a substantial risk of relapse; the prognostic factors and optimal treatments after relapse have not been fully established. We, therefore, retrospectively analyzed data from patients with acute myeloid leukemia who had achieved first complete remission to assess their prognosis after first relapse.

Design and Methods

Clinical data were collected from 70 institutions across the country on adult patients who were diagnosed with acute myeloid leukemia and who had achieved a first complete remission after one or two courses of induction chemotherapy.

Results

Among the 1,535 patients who were treated with chemotherapy alone, 1,015 relapsed. Half of them subsequently achieved a second complete remission. The overall survival was 30% at 3 years after relapse. Multivariate analysis showed that achievement of second complete remission, salvage allogeneic hematopoietic cell transplantation, and a relapse-free interval of 1 year or longer were independent prognostic factors. The outcome after allogeneic transplantation in second complete remission was comparable to that after transplantation in first complete remission. Patients with acute myeloid leukemia and cytogenetic risk factors other than inv(16) or t(8;21) had a significantly worse outcome when they did not undergo salvage transplantation even when they achieved second complete remission.

Conclusions

We found that both the achievement of second complete remission and the application of salvage transplantation were crucial for improving the prognosis of patients with acute myeloid leukemia in first relapse. Our results indicate that the optimal treatment strategy after first relapse may differ according to the cytogenetic risk.     

Reduced intensity conditioning HLA identical sibling donor allogeneic stem cell transplantation for patients with follicular lymphoma: long-term follow-up from two prospective multicenter trials

Background

Allogeneic hematopoietic stem cell transplantation is an effective treatment for patients with poor risk lymphoma, at least in part because of the graft-versus-lymphoma effect. Over the past decade, reduced intensity conditioning regimens have been shown to offer results similar to those of conventional high-dose conditioning regimens but with lower toxicity early after transplantation, especially in patients with chemosensitive disease at transplant.

Design and Methods

The aim of this study was to analyze the long-term outcome of patients with follicular lymphoma who received an HLA identical sibling allogeneic stem cell transplant with a reduced intensity conditioning regimen within prospective trials. The prospective multicenter studies considered included 37 patients with follicular lymphoma who underwent allogeneic stem cell transplantation between 1998 and 2007 with a fludarabine plus melphalan-based reduced intensity conditioning regimen.

Results

The median age of the patients was 50 years (range, 34–62 years) and the median follow-up was 52 months (range, 0.6 to 113 months). Most patients (77%) had stage III-IV at diagnosis, and patients had received a median of three lines of therapy before the reduced intensity conditioning allogeneic stem cell transplantation. At the time of transplantation, 14 patients were in complete remission, 16 in partial remission and 7 had refractory or progressive disease after salvage chemotherapy. The 4-year overall survival rates for patients in complete remission, partial remission, or with refractory or progressive disease were 71%, 48% and 29%, respectively (P=0.09), whereas the 4-year cumulative incidences of non-relapse mortality were 26% (95% CI, 11–61), 33% (95% CI, 16–68) and 71% (95% CI, 44–100), respectively. The incidence of relapse for the whole group was only 8% (95% CI, 2–23).

Conclusions

We conclude that this strategy of reduced intensity conditioning allogeneic stem cell transplantation may be associated with significant non-relapse mortality in heavily pre-treated patients with follicular lymphoma, but a remarkably low relapse rate. Long-term survival is likely in patients without progressive or refractory disease at the time of transplantation.     

Favorable outcomes with alemtuzumab-conditioned unrelated donor stem cell transplantation in adults with high-risk Philadelphia chromosome-negative acute lymphoblastic leukemia in first complete remission

Background

Approximately 40% of adults with Philadelphia chromosome-negative acute lymphoblastic leukemia achieve long-term survival following unrelated donor hematopoietic stem cell transplantation in first complete remission but severe graft-versus-host disease remains a problem affecting survival. Although T-cell depletion abrogates graft-versus-host disease, the impact on disease-free survival in acute lymphoblastic leukemia is not known.

Design and Methods

We analyzed the outcome of 48 adults (median age 26 years) with high-risk, Philadelphia-chromosome-negative acute lymphoblastic leukemia undergoing T-cell depleted unrelated donor-hematopoietic stem cell transplantation (67% 10 of 10 loci matched) in first complete remission reported to the British Society of Blood and Marrow Transplantation Registry from 1993 to 2005.

Results

T-cell depletion was carried out by in vivo alemtuzumab administration. Additional, ex vivo T-cell depletion was performed in 21% of patients. Overall survival, disease-free survival and non-relapse mortality rates at 5 years were 61% (95% CI 46–75), 59% (95% CI 45–74) and 13% (95% CI 3–25), respectively. The incidences of grades II–IV and III–IV acute graft-versus-host disease were 27% (95% CI 16–44) and 10% (95% CI 4–25), respectively. The actuarial estimate of extensive chronic graft-versus-host disease at 5 years was 22% (95%CI 13–38). High-risk cytogenetics at diagnosis was associated with a lower 5-year overall survival (47% (95% CI 27–71) vs. 68% (95% CI 44–84), p=0.045).

Conclusions

T-cell depleted hematopoietic stem cell transplantation from unrelated donors can result in good overall survival and low non-relapse mortality for adults with high-risk acute lymphoblastic leukemia in first complete remission and merits prospective evaluation.     

Cytoplasmic nucleophosmin is not detected in blastic plasmacytoid dendritic cell neoplasm

Acute myeloid leukemia carrying cytoplasmic mutated nucleophosmin (NPMc⁺ AML) and blastic plasmacytoid dendritic cell neoplasm have been included as new entities in the 4^th edition (2008) WHO classification of myeloid neoplasms. These conditions may show clinical and pathological overlapping features (leukemic and skin involvement, and expression of macrophage markers). In this study, we provide evidence that aberrant cytoplasmic dislocation of nucleophosmin – the immunohistochemical surrogate for NPM1 mutations – allows the two entities to be genetically separated. In fact, nucleophosmin is consistently cytoplasmic in NPMc⁺ AML (because of the presence of NPM1 mutations), whilst it is nucleus-restricted (predictive of a germline NPM1 gene) in blastic plasmacytoid dendritic cell neoplasm. Our results clearly point cytoplasmic nucleophosmin (a full predictor of NPM1 mutations) as a new marker for distinguishing NPMc⁺ AML and blastic plasmacytoid dendritic cell neoplasm, further clarify the cell of origin of NPMc⁺ AML, and justify the inclusion of these pathological conditions as separate entities in the new WHO classification.     

Lower number of plasmacytoid dendritic cells in peripheral blood of children with bronchiolitis following respiratory syncytial virus infection

Objectives

Dendritic cells (DCs) are key mediators of allergic airway inflammation. Thus, it is important to understand the relationship between respiratory syncytial virus (RSV) infection and DCs, especially in children with RSV bronchiolitis.

Methods

We collected peripheral blood from 71 children with RSV bronchiolitis at the time of admission and 28 children who were followed up 3 months following admission. Flow cytometry was performed to detect dendritic cell immunophenotypes.

Results

Patients with RSV bronchiolitis exhibited significantly higher number of myeloid DCs and lower number of plasmacytoid DCs at the time of admission and 3 months following discharge, compared with healthy controls. These children had a significantly higher myeloid/plasmacytoid ratio 3 months after discharge compared with healthy controls.

Conclusions

Among children with RSV bronchiolitis, there is an imbalance in peripheral blood myeloid/plasmacytoid ratio. The low number of plasmacytoid DCs in peripheral blood indicates the development of bronchiolitis due to RSV infection.     

Qualitative and quantitative polymerase chain reaction monitoring of minimal residual disease in relapsed chronic lymphocytic leukemia: early assessment can predict long-term outcome after reduced intensity allogeneic transplantation

Background

The graft-versus-leukemia effect is able to induce clinical responses in patients with chronic lymphocytic leukemia treated with a reduced intensity conditioning regimen, followed by allogeneic stem cell transplantation. We investigated whether molecular remissions could be attained after reduced intensity conditioning and allogeneic stem cell transplantation in patients with relapsed chronic lymphocytic leukemia and whether the assessment of minimal residual disease might be used to predict the clinical outcome.

Design and Methods

Minimal residual disease was monitored by polymerase chain reaction using the immunoglobulin heavy-chain gene rearrangement as a molecular marker in 29 relapsed patients who achieved complete remission following reduced intensity conditioning and allogeneic stem cell transplantation. A nested-polymerase chain reaction with patient-specific primers derived from complementarity determining regions (CDR2 and CDR3) was carried out in all the patients. Real-time polymerase chain reaction was performed in patients whose nested reaction gave positive or mixed results.

Results

Three patterns of minimal residual disease were observed: negative (31%), mixed (24%), and always positive (45%). The cumulative incidence of relapse according to the minimal residual disease status at 6 and 12 months after transplantation was significantly different between polymerase chain reaction-negative and -positive patients (p=0.031 and p=0.04, respectively). Two-year disease-free survival was 93% and 46% for polymerase chain reaction-negative and -positive patients at 6 months after transplantation, respectively (p=0.012). Similarly, 2-year disease-free survival was 100% and 57% for polymerase chain reaction-negative and -positive patients at 12 months, respectively (p=0.037). No clinical or biological factors were predictive of the achievement of polymerase chain reaction negativity after allogeneic stem cell transplantation. Graft-versus-host disease was more frequent in patients who did not relapse (p=0.04). Quantitative monitoring of minimal residual disease was able to identify polymerase chain reaction-positive patients with a higher risk of relapse.

Conclusions

These findings demonstrate that relapsed patients can achieve molecular remission after reduced intensity conditioning and allogeneic stem cell transplantation and suggest a minimal residual disease-driven intervention that might be useful to prevent overt hematologic relapse.     

Value of allogeneic versus autologous stem cell transplantation and chemotherapy in patients with myelodysplastic syndromes and secondary acute myeloid leukemia. Final results of a prospective randomized European Intergroup Trial

Background

Allogeneic stem cell transplantation is usually considered the only curative treatment option for patients with advanced or transformed myelodysplastic syndromes in complete remission, but post-remission chemotherapy and autologous stem cell transplantation are potential alternatives, especially in patients over 45 years old.

Design and Methods

We evaluated, after intensive anti-leukemic remission-induction chemotherapy, the impact of the availability of an HLA-identical sibling donor on an intention-to treat basis. Additionally, all patients without a sibling donor in complete remission after the first consolidation course were randomized to either autologous peripheral blood stem cell transplantation or a second consolidation course consisting of high-dose cytarabine.

Results

The 4-year survival of the 341 evaluable patients was 28%. After achieving complete remission, the 4-year survival rates of patients under 55 years old with or without a donor were 54% and 41%, respectively, with an adjusted hazard ratio of 0.81 (95% confidence interval [95% CI], 0.49–1.35) for survival and of 0.67 (95% CI, 0.42–1.06) for disease-free survival. In patients with intermediate/high risk cytogenetic abnormalities the hazard ratio in multivariate analysis was 0.58 (99% CI, 0.22–1.50) (P=0.14) for survival and 0.46 (99% CI, 0.22–1.50) for disease-free survival (P=0.03). In contrast, in patients with low risk cytogenetic characteristics the hazard ratio for survival was 1.17 (99% CI, 0.40–3.42) and that for disease-free survival was 1.02 (99% CI, 0.40–2.56). The 4-year survival of the 65 patients randomized to autologous peripheral blood stem cell transplantation or a second consolidation course of high-dose cytarabine was 37% and 27%, respectively. The hazard ratio in multivariate analysis was 1.22 (95% CI, 0.65–2.27) for survival and 1.02 (95% CI, 0.56–1.85) for disease-free survival.

Conclusions

Patients with a donor and candidates for allogeneic stem cell transplantation in first complete remission may have a better disease-free survival than those without a donor in case of myelodysplastic syndromes with intermediate/high-risk cytogenetics. Autologous peripheral blood stem cell transplantation does not provide longer survival than intensive chemotherapy. (Eudract number: NCT00002926; http://www.cancer.gov/clinicaltrials/EORTC-06961)     

High levels of CD34+CD38low/-CD123+ blasts are predictive of an adverse outcome in acute myeloid leukemia: a Groupe Ouest-Est des Leucemies Aigues et Maladies du Sang (GOELAMS) study

Background

Acute myeloid leukemias arise from a rare population of leukemic cells, known as leukemic stem cells, which initiate the disease and contribute to frequent relapses. Although the phenotype of these cells remains unclear in most patients, these cells are enriched within the CD34⁺CD38^low/− compartment expressing the interleukin-3 alpha chain receptor, CD123. The aim of this study was to determine the prognostic value of the percentage of blasts with the CD34⁺CD38^low/−CD123⁺ phenotype.

Design and Methods

The percentage of CD34⁺CD38^low/−CD123⁺ cells in the blast population was determined at diagnosis using flow cytometry. One hundred and eleven patients under 65 years of age with de novo acute myeloid leukemia and treated with intensive chemotherapy were retrospectively included in the study. Correlations with complete response, disease-free survival and overall survival were evaluated with univariate and multivariate analyses.

Results

A proportion of CD34⁺CD38^low/−CD123⁺ cells greater than 15% at diagnosis and an unfavorable karyotype were significantly correlated with a lack of complete response. By logistic regression analysis, a percentage of CD34⁺CD38^low/−CD123⁺ higher than 15% retained significance with an odds ratio of 0.33 (0.1–0.97; P=0.044). A greater than 1% population of CD34⁺CD38^low/−CD123⁺ cells negatively affected disease-free survival (0.9 versus 4.7 years; P<0.0001) and overall survival (1.25 years versus median not reached; P<0.0001). A greater than 1% population of CD34⁺CD38^low/−CD123⁺ cells retained prognostic significance for both parameters after multivariate analysis.

Conclusions

The percentage of CD34⁺CD38^low/−CD123⁺ leukemic cells at diagnosis was significantly correlated with response to treatment and survival. This prognostic marker might be easily adopted in clinical practice to rapidly identify patients at risk of treatment failure.     

Non-myeloablative conditioning with allogeneic hematopoietic cell transplantation for the treatment of high-risk acute lymphoblastic leukemia

Background

Allogeneic hematopoietic cell transplantation is a potentially curative treatment for patients with acute lymphoblastic leukemia. However, the majority of older adults with acute lymphoblastic leukemia are not candidates for myeloablative conditioning regimens. A non-myeloablative preparative regimen is a reasonable treatment option for this group. We sought to determine the outcome of non-myeloablative conditioning and allogeneic transplantation in patients with high-risk acute lymphoblastic leukemia.

Design and Methods

Fifty-one patients (median age 56 years) underwent allogeneic hematopoietic cell transplantation from sibling or unrelated donors after fludarabine and 2 Gray total body irradiation. Twenty-five patients had Philadelphia chromosome-positive acute lymphoblastic leukemia. Eighteen of these patients received post-grafting imatinib.

Results

With a median follow-up of 43 months, the 3-year overall survival was 34%. The 3-year relapse/progression and non-relapse mortality rates were 40% and 28%, respectively. The cumulative incidences of grades II and III-IV acute graft-versus-host disease were 53% and 6%, respectively. The cumulative incidence of chronic graft-versus-host disease was 44%. Hematopoietic cell transplantation in first complete remission and post-grafting imatinib were associated with improved survival (P=0.005 and P=0.03, respectively). Three-year overall survival rates for patients with Philadelphia-negative acute lymphoblastic leukemia in first remission and beyond first remission were 52% and 8%, respectively. For patients with Philadelphia chromosome-positive acute lymphoblastic leukemia in first remission who received post-grafting imatinib, the 3-year overall survival rate was 62%; for the subgroup without evidence of minimal residual disease at transplantation, the overall survival was 73%.

Conclusions

For patients with high-risk acute lymphoblastic leukemia in first complete remission, non-myeloablative conditioning and allogeneic hematopoietic cell transplantation, with post-grafting imatinib for Philadelphia chromosome-positive disease, can result in favorable long-term survival. (Clinicaltrials.gov identifier: NCT0036738)     

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.     

Mammalian target of rapamycin activity is required for expansion of CD34+ hematopoietic progenitor cells

Background

The mammalian target of rapamycin is a conserved protein kinase known to regulate protein synthesis, cell size and proliferation. Aberrant regulation of mammalian target of rapamycin activity has been observed in hematopoietic malignancies, including acute leukemias and myelodysplastic syndromes, suggesting that correct regulation of mammalian target of rapamycin is critical for normal hematopoiesis.

Design and Methods

An ex vivo granulocyte differentiation system was utilized to investigate the role of mammalian target of rapamycin in the regulation of myelopoiesis.

Results

Inhibition of mammalian target of rapamycin activity, with the pharmacological inhibitor rapamycin, dramatically reduced hematopoietic progenitor expansion, without altering levels of apoptosis or maturation. Moreover, analysis of distinct hematopoietic progenitor populations revealed that rapamycin treatment inhibited the expansion potential of committed CD34⁺ lineage-positive progenitors, but did not affect early hematopoietic progenitors. Further examinations showed that these effects of rapamycin on progenitor expansion might involve differential regulation of protein kinase B and mammalian target of rapamycin signaling.

Conclusions

Together, these results indicate that mammalian target of rapamycin activity is essential for expansion of CD34⁺ hematopoietic progenitor cells during myelopoiesis. Modulation of the mammalian target of rapamycin pathway may be of benefit in the design of new therapies to control hematologic malignancies.     

Effectiveness of subcutaneous low-dose alemtuzumab and rituximab combination therapy for steroid-resistant chronic graft-versus-host disease

Background

Chronic graft-versus-host disease is a common late complication of allogeneic hematopoietic stem cell transplantation. Corticosteroids are the standard initial treatment. Second-line treatment has not been well defined. We evaluated the effectiveness and safety of low doses of alemtuzumab plus low doses of rituximab in the treatment of steroid-refractory chronic graft-versus-host disease.

Design and Methods

Ten men and 5 women were prospectively included in the study. All patients received one cycle of subcutaneous alemtuzumab 10 mg/day/3 days and intravenous rituximab 100 mg on Days +4, +11, +18 and +25. The therapeutic response was measured on Days +30, +90 and +365 of the protocol.

Results

Median age was 41 years. The main site involved was the oral mucosa (86.7%) followed by the eyes (66.7%), liver (60%), skin (53%), lungs (13.3%) and intestinal tract (6.7%). The overall response was 100% at Day +30 evaluation: 10 patients (67%) had partial remission, 5 (33%) had complete remission. At Day +90 evaluation, 7 (50%) patients had partial remission, 4 (28%) had complete remission; 3 (21%) had relapsed chronic graft-versus-host disease and one patient did not reach the evaluation time point. So far, 5 patients have reached the Day +365 follow-up evaluation; 2 (40%) had partial remission, 2 had complete remission and one experienced chronic graft-versus-host disease progression. Adverse effects were mainly infections in 67% of patients; these were all quickly solved, except for one patient who died from pneumonia.

Conclusions

This combination therapy appears to be an efficacious and safe treatment for steroid-refractory chronic graft-versus-host disease. Longer follow up to determine the durability of response and survival is required.     

Allogeneic hematopoietic stem cell transplantation allows long-term complete remission and curability in high-risk Waldenstr?m’s macroglobulinemia. Results of a retrospective analysis of the Société Fran?aise de Greffe de Moelle et de Thérapie Cellulaire

Background

Patients with poor-risk Waldenström’s macroglobulinemia have suboptimal response and early post-treatment relapse with conventional therapies. Hence, new therapeutic approaches such as allogeneic stem cell transplantation should be evaluated in these patients.

Design and Methods

We examined the long-term outcome of allogeneic stem cell transplantation in Waldenström’s macroglobulinemia by studying the records of 24 patients reported in the SFGM-TC database and one transplanted in the bone marrow unit in Hamburg.

Results

Median age at the time of transplant was 48 years (range, 24–64). The patients had previously received a median of 3 lines of therapy (range, 1–6) and 44% of them had refractory disease at time of transplant. Allogeneic stem cell transplantation after myeloablative (n=12) or reduced-intensity (n=13) conditioning yielded an overall response rate of 92% and immunofixation-negative complete remission in 50% of evaluable patients. With a median follow-up of 64 months among survivors (range, 11–149 months), 5-year overall survival and progression-free survival rates were respectively, 67% (95% CI: 46–81) and 58% (95% CI: 38–75). The 5-year estimated risk of progression was 25% (95% CI: 10–36%), with only one relapse among the 12 patients who entered complete remission, versus 5 in the 12 patients who did not. Only one of the 6 relapses occurred more than three years post-transplant.

Conclusions

Allogeneic stem cell transplantation yields a high rate of complete remissions and is potentially curative in poor-risk Waldenström’s macroglobulinemia.     

Oxygen tension plays a critical role in the hematopoietic microenvironment in vitro

Background

In the bone marrow mesenchymal stromal cells and osteoblasts form functional niches for hematopoietic stem and progenitor cells. This microenvironment can be partially mimicked using in vitro co-culture systems. In this study, we examined the oxygen tension in three distinct compartments in a co-culture system of purified CD34⁺ cells and mesenchymal stromal cells with regard to different spatial localizations.

Design and Methods

Hypoxic cells in the co-culture were visualized by pimonidazole staining. Hematopoietic cell distribution, and functional and phenotypic characteristics were analyzed by flow cytometry. The secretion of vascular endothelial growth factor and stromal-derived factor-1 by mesenchymal stromal cells in low oxygen co-cultures was determined by an enzyme-linked immunosorbent assay. The effect of co-culture medium on the hematopoietic cell migration potential was tested in a transwell assay.

Results

In co-cultures under atmospheric oxygen tension, regions of low oxygen tension could be detected beneath the feeder layer in which a reservoir of phenotypically more primitive hematopoietic cells is located in vitro. In low oxygen co-culture, the adhesion of hematopoietic cells to the feeder layer was decreased, whereas hematopoietic cell transmigration beneath mesenchymal stromal cells was favored. Increased vascular endothelial growth factor-A secretion by mesenchymal stromal cells under low oxygen conditions, which increased the permeability of the monolayer, was responsible for this effect. Furthermore, vascular endothelial growth factor-A expression in low oxygen mesenchymal stromal cells was induced via hypoxia-inducible factor signaling. However, stromal cell-derived factor-1 secretion by mesenchymal stromal cells was down-regulated under low oxygen conditions in a hypoxia-inducible factor-independent manner.

Conclusions

We demonstrate for the first time that differences in oxygen tension cause selective modification of hematopoietic cell and mesenchymal stromal cell interactions in a co-culture system, thus confirming that oxygen tension plays a critical role in the interaction between hematopoietic cells and the niche environment.