Sirolimus demonstrates activity in the primary therapy of acute graft-versus-host disease without systemic glucocorticoids

Background

Advances in acute graft-versus-host disease therapy are needed.

Design and Methods

We examined the efficacy of sirolimus as primary therapy for acute graft-versus-host disease in 32 patients.

Results

Acute graft-versus-host disease involved the skin in 53% of cases, gastrointestinal tract in 66%, liver in 16%. The syndrome was overall grade 1 in 12% cases, grade 2 in 75%, and grade 3 in 13%. Sirolimus was targeted to achieve serum trough levels of 5–14 ng/mL. Sixteen (50%) patients achieved sustained, complete resolution of acute graft-versus-host disease with sirolimus alone. In contrast, 19 of 32 (59%) matched historical controls treated with standard 1 mg/kg steroids achieved complete response (P=0.47). With median follow-up time for surviving patients of 16 (range 6–26) months, one year overall survival was 56% (95% CI 38–74%). The cumulative incidence of relapse at one year was 37% (95% CI 23–60%), and mortality in remission was 20% (95% CI 10–42%). The cumulative incidence of chronic graft-versus-host disease was 55% (95% CI 39–79%). Thrombotic microangiopathy occurred in 3 cases (grade 1 n=1; grade 2 n=2), and responded to dose reduction of calcineurin inhibitor.

Conclusions

In this retrospective series, sirolimus demonstrates activity comparable to that of high-dose glucocorticoids in the primary therapy of acute graft-versus-host disease. Confirmation of this activity requires prospective clinical trials.     

The global severity of chronic graft-versus-host disease, determined by National Institutes of Health consensus criteria, is associated with overall survival and non-relapse mortality

Background

The 2005 National Institutes of Health Consensus Development Conference on chronic graft-versus-host disease proposed major changes in the classification and grading of severity of chronic graft-versus-host disease.

Design and Methods

We aimed to study the association of the proposed chronic graft-versus-host disease classification and global severity with transplantation outcomes among a consecutive series of patients who received pharmacokinetically-targeted doses of intravenous busulfan and fludarabine conditioning followed by transplantation of allogeneic peripheral blood stem cells.

Results

From a total cohort (n = 242) of patients surviving more than 100 days after hematopoietic stem cell transplantation, 181 (75% of those at risk) had some manifestations of graft-versus-host disease after day 100. Of these, at onset 13 (7%) had late acute graft-versus-host disease, 62 (34%) had classic chronic graft-versus-host disease, and 106 (59%) had the overlap subtype of chronic graft-versus-host disease. The global severity of the chronic graft-versus-host disease was mild in 25% of cases, moderate in 46%, and severe in 29%. Multivariable modeling demonstrated the independent association of global severity of chronic graft-versus-host disease with overall survival (moderate/severe versus mild; HR 2.9, 95% CI 1.8–4.7, P<0.0001) and non-relapse mortality (moderate versus mild; HR 3.86, 95% CI 1.17–12.73, P=0.03, and severe versus mild (HR 10.06, 95% CI 3.07–32.97, P<0.001). The type of onset of progressive chronic graft-versus-host disease and the platelet count at the time of diagnosis of the disease were significantly associated with overall survival. The occurrence and severity of chronic graft-versus-host disease was also significantly associated with primary disease relapse.

Conclusions

Patients with moderate to severe chronic graft-versus-host disease, as determined by National Institutes of Health Consensus criteria, have an inferior overall survival and worse non-relapse mortality. Clinical and research advances are needed to improve the outcomes of affected patients.     

Prolonged sirolimus administration after allogeneic hematopoietic cell transplantation is associated with decreased risk for moderate-severe chronic graft-versus-host disease

Effective pharmacological strategies employed in allogeneic hematopoietic cell transplantation should prevent serious chronic graft-versus-host disease and facilitate donor-recipient immune tolerance. Based on demonstrated pro-tolerogenic activity, sirolimus (rapamycin) is an agent with promise to achieve these goals. In a long-term follow-up analysis of a randomized phase II trial comparing sirolimus/tacrolimus versus methotrexate/tacrolimus for graft-versus-host disease prevention in matched sibling or unrelated donor transplant, we examined the impact of prolonged sirolimus administration (≥ 1 year post-transplant). Median follow-up time for surviving patients at time of this analysis was 41 months (range 27–60) for sirolimus/tacrolimus and 49 months (range 29–63) for methotrexate/tacrolimus. Sirolimus/tacrolimus patients had significantly lower National Institutes of Health Consensus moderate-severe chronic graft-versus-host disease (34% vs. 65%; P=0.004) and late acute graft-versus-host disease (20% vs. 43%; P=0.04). While sirolimus/tacrolimus patients had lower prednisone exposure and earlier discontinuation of tacrolimus (median time to tacrolimus discontinuation 368 days vs. 821 days; P=0.002), there was no significant difference in complete immune suppression discontinuation (60-month estimate: 43% vs. 31%; P=0.78). Prolonged sirolimus administration represents a viable approach to mitigate risk for moderate-severe chronic and late acute graft-versus-host disease. Further study of determinants of successful immune suppression discontinuation is needed.     

Reduced-toxicity conditioning with treosulfan and fludarabine in allogeneic hematopoietic stem cell transplantation for myelodysplastic syndromes: final results of an international prospective phase II trial

Background

An alternative reduced-toxicity conditioning regimen for allogeneic transplantation, based on treosulfan and fludarabine, has recently been identified. The rationale for this study was to investigate the efficacy and safety of this regimen prospectively in patients with a primary myelodysplastic syndrome.

Design and Methods

A total of 45 patients with primary myelodysplastic syndromes were conditioned with 3×14 g/m² treosulfan and 5×30 mg/m² fludarabine followed by allogeneic hematopoietic stem cell transplantation. Subtypes of myelodysplastic syndromes were refractory anemia with excess blasts-2 (44%), refractory cytopenia with multilineage dysplasia (27%), refractory anemia (9%), refractory anemia with ringed sideroblasts (4%), refractory cytopenia with multilineage dysplasia and ringed sideroblasts (4%), refractory anemia with excess blasts-1 (2%), and myelodysplastic syndrome with isolated del (5q) (2%). The myelodysplastic syndrome was unclassified in 7% of the patients. Forty-seven percent of the patients had a favorable karyotype, 29% an unfavorable one, and 18% an intermediate karyotype. Patients were evaluated for engraftment, adverse events, graft-versus-host disease, non-relapse mortality, relapse incidence, overall survival and disease-free survival.

Results

All but one patient showed primary engraftment of neutrophils after a median of 17 days. Non-hematologic adverse events of grade III–IV in severity included mainly infections and gastrointestinal symptoms (80% and 22% of the patients, respectively). Acute graft-versus-host disease grade II–IV developed in 24%, and extensive chronic graft-versus-host disease in 28% of the patients. After a median follow-up of 780 days, the 2-year overall and disease-free survival estimates were 71% and 67%, respectively. The 2-year cumulative incidences of non-relapse mortality and relapse were 17% and 16%, respectively.

Conclusions

Our safety and efficacy data suggest that treosulfan-based conditioning therapy is a promising treatment option for patients with myelodysplastic syndromes. clinicaltrials.gov identifier: {"type":"clinical-trial","attrs":{"text":"NCT01062490","term_id":"NCT01062490"}}NCT01062490     

Mesenchymal stromal cells transiently alter the inflammatory milieu post-transplant to delay graft-versus-host disease

Background

Multipotent mesenchymal stromal cells suppress T-cell function in vitro, a property that has underpinned their use in treating clinical steroid-refractory graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. However the potential of mesenchymal stromal cells to resolve graft-versus-host disease is confounded by a paucity of pre-clinical data delineating their immunomodulatory effects in vivo.

Design and Methods

We examined the influence of timing and dose of donor-derived mesenchymal stromal cells on the kinetics of graft-versus-host disease in two murine models of graft-versus-host disease (major histocompatibility complex-mismatched: UBI-GFP/BL6 [H-2^b]→BALB/c [H-2^d] and the sibling transplant mimic, UBI-GFP/BL6 [H-2^b]→BALB.B [H-2^b]) using clinically relevant conditioning regimens. We also examined the effect of mesenchymal stromal cell infusion on bone marrow and spleen cellular composition and cytokine secretion in transplant recipients.

Results

Despite T-cell suppression in vitro, mesenchymal stromal cells delayed but did not prevent graft-versus-host disease in the major histocompatibility complex-mismatched model. In the sibling transplant model, however, 30% of mesenchymal stromal cell-treated mice did not develop graft-versus-host disease. The timing of administration and dose of the mesenchymal stromal cells influenced their effectiveness in attenuating graft-versus-host disease, such that a low dose of mesenchymal stromal cells administered early was more effective than a high dose of mesenchymal stromal cells given late. Compared to control-treated mice, mesenchymal stromal cell-treated mice had significant reductions in serum and splenic interferon-γ, an important mediator of graft-versus-host disease.

Conclusions

Mesenchymal stromal cells appear to delay death from graft-versus-host disease by transiently altering the inflammatory milieu and reducing levels of interferon-γ. Our data suggest that both the timing of infusion and the dose of mesenchymal stromal cells likely influence these cells’ effectiveness in attenuating graft-versus-host disease.     

Cytopenia and leukocyte recovery shape cytokine fluctuations after myeloablative allogeneic hematopoietic stem cell transplantation

Background

Allogeneic hematopoietic stem cell transplantation is associated with profound changes in levels of various cytokines. Emphasis has been placed on conditioning-associated mucosal damage and neutropenia and associated bacterial translocation as the initiating conditions predisposing to acute graft-versus-host disease. The post-transplant period is, however, also associated with increases in certain homeostatic cytokines. It is unclear how much the homeostatic drive to lymphocyte recovery and the production of cytokines from the engrafting donor immune system determine cytokine fluctuations in the peri- and immediate post-transplant period. The aim of this study was to examine the contributions of the conditioning regimen, donor engraftment, infections, and graft-versus-host disease to fluctuations in cytokines involved in homeostasis and inflammation.

Design and Methods

We examined the levels of 33 cytokines in relation to peri- and post-transplant events such as conditioning regimen, chimerism, and acute graft-versus-host disease in myeloablative, non-T cell-replete HLA-identical sibling donor stem cell transplantation for hematologic malignancies.

Results

We identified two cytokine storms. The first occurred following conditioning and reached peak levels when all the leukocytes were at their lowest concentrations. The second cytokine storm occurred concurrently with hematopoietic reconstitution and subsided with the achievement of full donor lymphocyte chimerism.

Conclusions

Our results indicate that both recipient-related and donor-related factors contribute to the changes in cytokine levels in the recipient following allogeneic hematopoietic stem cell transplantation. The study reported here was performed using plasma samples drawn from patients enrolled in the ClinicalTrials.gov-registered trials {"type":"clinical-trial","attrs":{"text":"NCT00467961","term_id":"NCT00467961"}}NCT00467961 and {"type":"clinical-trial","attrs":{"text":"NCT00378534","term_id":"NCT00378534"}}NCT00378534     

The combination of sirolimus plus tacrolimus improves outcome after reduced-intensity conditioning,unrelated donor hematopoietic stem cell transplantation compared with cyclosporine plus mycofenolate

Different types of graft-versus-host disease prophylaxis have been proposed in the setting of reduced intensity and non-myeloablative allogeneic stem cell transplantation. An alternative combination with sirolimus and tacrolimus has recently been tested although comparative studies against the classical combination of a calcineurin inhibitor and mycophenolate mofetil or methotrexate are lacking. We describe the results of a prospective, multicenter trial using sirolimus + tacrolimus as immunoprophylaxis, and compare this approach with our previous experience using cyclosporine + mycophenolate in the setting of unrelated donor transplantation setting after reduced-intensity conditioning. Forty-five patients received cyclosporine + mycophenolate between 2002 and mid-2007, while the subsequent 50 patients, who were transplanted from late 2007, were given sirolimus + tacrolimus. No significant differences were observed in terms of hematopoietic recovery or acute graft-versus-host disease overall, although gastrointestinal acute graft-versus-host disease grade ≥2 was more common in the cyclosporine + mycophenolate group (55% versus 21%, respectively, P=0.003). The 1-year cumulative incidence of chronic graft-versus-host disease was 50% versus 90% for the patients treated with the sirolimus- versus cyclosporine-based regimen, respectively (P<0.001), while the incidence of extensive chronic disease was 27% versus 49%, respectively (P=0.043). The 2-year non-relapse mortality rate was 18% versus 38% for patients receiving the sirolimus- versus the cyclosporine-based regimen, respectively (P=0.02). The event-free survival and overall survival at 2 years were 53% versus 29% (P=0.028) and 70% versus 45% (P=0.018) among patients receiving the sirolimus- versus the cyclosporine-based regimen, respectively. In conclusion, in the setting of reduced intensity transplantation from an unrelated donor, promising results can be achieved with the combination of sirolimus + tacrolimus, due to a lower risk of chronic graft-versus-host disease and non-relapse mortality, which translates into better event-free and overall survival rates, in comparison with those achieved with cyclosporine + mycophenolate. This trial was registered at www.clinicaltrials.gov as 2007-006416-32 by GEL-TAMO/GETH.     

Quality of life associated with sirolimus for prevention of graft-versus-host disease: results from a randomized trial

Several studies have examined sirolimus-based immune suppression for the prevention of graft-versus-host disease after allogeneic hematopoietic cell transplantation, but little is known regarding its effects on quality of life. The current study reports on changes in quality of life to Day 360 in a randomized phase II trial of sirolimus and tacrolimus versus methotrexate and tacrolimus. Quality of life was assessed prior to transplant and on Days 30, 90, 180, 270, and 360 with the Functional Assessment of Cancer Therapy – Bone Marrow Transplant Trial Outcome Index. Random effects models examined the effects of study arm on change in Trial Outcome Index scores from Day 30 to 360, controlling for base-line Trial Outcome Index. The sirolimus/tacrolimus arm (n=37) showed less improvement in Trial Outcome Index scores over time compared to the methotrexate/tacrolimus arm (n=34) (P=0.02). Patients receiving sirolimus and tacrolimus were more likely to endorse nausea and a lack of energy over time (PS≤0.01). These data suggest that sirolimus-based immune suppression is associated with less improvement in quality of life in the first year post-transplant compared to methotrexate/tacrolimus. Quality of life differences may be due to increased fatigue and nausea in patients treated with sirolimus. These findings should be considered in the clinical management of patients treated with sirolimus. (Clinicaltrials.gov identifier:00803010).     

Nuclear factor-�� B inducing kinase is required for graft-versus-host disease

Background

Donor T lymphocytes are directly responsible for graft-versus-host disease. Molecules important in T-cell function may, therefore, be appropriate targets for graft-versus-host disease therapy and/or prophylaxis. Here we analyzed whether nuclear factor-κ B inducing kinase might have a role in graft-versus-host disease.

Design and Methods

We studied the expression of nuclear factor-κ B inducing kinase in human samples from patients with graft-versus-host disease. We also explored the effect of nuclear factor-κ B inducing kinase in a murine model of graft-versus-host disease using donor cells from aly/aly mice (deficient in nuclear factor-κ B inducing kinase) and C57BL/6 mice (control).

Results

We detected expression of nuclear factor-κ B inducing kinase in T-lymphocytes in the pathological lesions of patients with acute graft-versus-host disease. Mice transplanted with aly/aly T lymphocytes did not develop graft-versus-host disease at all, while mice receiving C57BL/6 cells died of a lethal form of the disease. Deficiency of nuclear factor-κ B inducing kinase did not affect the engrafting ability of donor T cells, but severely impaired their expansion capacity early after transplantation, and aly/aly T cells showed a higher proportion of apoptosis than did C57BL/6 T cells. Effector T lymphocytes were the T-cell subset most affected by nuclear factor-κ B inducing kinase deficiency. We also detected lower amounts of inflammatory cytokines in the serum of mice receiving aly/aly T cells than in the serum of mice receiving C57BL/6 T cells.

Conclusions

Our results show that nuclear factor-κ B inducing kinase has a role in graft-versus-host disease by maintaining the viability of activated alloreactive T lymphocytes.     

Persistence of recipient-type endothelium after allogeneic hematopoietic stem cell transplantation

Background

The possibility that allogeneic hematopoietic stem cell transplantation performed across the ABO blood group-barrier is associated with an increase of graft-versus-host disease, in particular endothelial damage, has not been elucidated so far. For this reason, we investigated the level of endothelial cell chimerism after allogeneic hematopoietic stem cell transplantation in order to delineate the role of hematopoietic stem cells in endothelial replacement.

Design and Methods

The frequency of donor-derived endothelial cells was analyzed in 52 hematopoietic stem cell transplant recipients, in 22 normal skin biopsies, in 12 skin samples affected by graft-versus-host disease, various tissues from five autopsies and four secondary solid tumors by ABH immunohistochemistry, XY fluorescence in situ hybridization and short tandem repeat analysis of laser captured endothelial cells.

Results

Skin biopsies from two patients transplanted with minor ABO-incompatible grafts (i.e. O in A) showed 3.3% and 0.9% H antigen-positive donor-derived endothelial cells by ABH immunohistochemistry. Tumor biopsies from two recipients showed 1.2% and 2.5% donor-derived endothelial cells by combined immunohistochemistry/ fluorescence in situ hybridization. All other skin samples, heart, liver, bone-marrow, and tumor tissues failed to reveal donor-type endothelial cells up to several years after ABO-incompatible hematopoietic stem cell transplantation.

Conclusions

Endothelial cell replacement by bone marrow-derived donor cells after allogeneic hematopoietic stem cell transplantation is a rare event. It does not seem to represent a major mechanism of physiological in vivo blood vessel formation, tumor neoangiogenesis, vascular repair after graft-versus-host disease episodes or acceptance of ABO-incompatible grafts.     

Graft-versus-host disease is the major determinant of humoral responses to the AS03-adjuvanted influenza A/09/H1N1 vaccine in allogeneic hematopoietic stem cell transplant recipients

Background

Responses to influenza vaccines are poorly characterized in immunocompromised patients. The goal of this study was to assess the efficacy of the AS03-adjuvanted influenza H1N1/A/09 vaccine in allogeneic hematopoietic stem cell transplant recipients.

Design and Methods

We enrolled 65 patients and 138 controls in an open prospective study. Controls received one dose and patients 2 doses of the AS03-adjuvanted influenza H1N1/A/09 vaccine at a 3-week interval. Geometric mean titers and seroprotection/seroconversion rates were determined by hemagglutination inhibition before and four weeks after the last immunization. Clinical and biological markers, including immunoglobulins, CD3⁺, CD4⁺, CD8⁺ and naïve CD4⁺ T-cell counts were assessed in all patients.

Results

Baseline seroprotection rates were low in patients (6.6%) and controls (14.8%). After 2 doses, patients (n=57, 92.3%) achieved similar seroprotection rates (84% vs. 87%, P=0.65) and antibody titers (305 vs. 340, P=0.88) as controls (n=131, 93.9%) after one dose. In univariate analysis, transplant-to-vaccination interval less than 12 months, active graft-versus-host disease, immunosuppressive drugs, hemoglobin less than 12g/L, lymphopenia less than1G/L, IgG less than 4g/L, IgA less than 0.5g/L, IgM less than 0.5g/L and naive CD4⁺ T cells less than 150/μL were significantly associated with weaker responses. Multivariate analysis identified transplant-to-vaccination interval and active graft-versus-host disease as the most powerful negative predictors of antibody responses (P=0.04 and P=0.002, respectively). Vaccination was well tolerated in both cohorts.

Conclusions

In allogeneic hematopoietic stem cell transplant recipients, 2 doses of an adjuvanted influenza vaccine elicited comparable responses to a single dose in healthy individuals. However, vaccine responses remained poor in patients with ongoing graft-versus-host disease, supporting the need for additional strategies in this high-risk patient population. (ClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT01022905","term_id":"NCT01022905"}}NCT01022905)     

Sensitivity of changes in chronic graft-versus-host disease activity to changes in patient-reported quality of life: results from the Chronic Graft-versus-Host Disease Consortium

Background

The 2005 National Institute of Health Chronic Graft-versus-Host Disease Consensus Conference recommended collection of patient-reported outcomes in clinical trials on chronic graft-versus-host disease. We assessed whether changes in chronic graft-versus-host disease severity, determined using National Institute of Health criteria, clinicians’ assessment or patients’ self-evaluation, correlated with patient-reported quality of life as measured by the Short Form-36 and Functional Assessment of Cancer Therapy – Bone Marrow Transplant (FACT-BMT) instruments.

Design and Methods

Three-hundred and thirty-six adult patients (median age 52 years; range, 19 – 79) with chronic graft-versus-host disease from six transplant centers contributed baseline and follow-up data (from 936 visits overall).

Results

While the majority of the patients had stable chronic graft-versus-host disease, improvement or worsening was noted in approximately 40% of follow-up visits. Multivariable analysis demonstrated no association between change in chronic graft-versus-host disease severity evaluated by National Institute of Health criteria and change in quality of life, while clinician-reported changes in severity were associated with changes in some quality of life measures. Patient-reported changes in the severity of chronic graft-versus-host disease were associated with changes in all quality of life measures. Comparison of the Short Form-36 and the FACT-BMT suggested that the data collected in the Functional Assessment of Cancer Therapy – General (FACT-G) core survey are sufficient without the need for the Short Form-36 or the FACT–BMT subscale.

Conclusions

We conclude that serial National Institute of Health and clinician-reported chronic graft-versus-host disease severity assessments cannot substitute for patient-reported outcomes in clinical trials. Collection of just the FACT-G instead of the Short Form-36 and the full FACT-BMT will decrease respondent burden without compromising quality of life assessment.     

Mesenchymal stem cells expanded in vitro with human serum for the treatment of acute and chronic graft-versus-host disease: results of a phase I/II clinical trial

This trial evaluated the feasibility and efficacy of the infusion of mesenchymal stem cells expanded using human serum for the treatment of refractory acute or chronic graft-versus-host disease. Twenty-eight expansions were started. In 22, a minimum of more than 1x10⁶ mesenchymal stem cells/kg were obtained after a median of 26 days; this threshold was not obtained in the remaining cases. Ten patients received cells for the treatment of refractory or relapsed acute graft-versus-host disease and 8 for chronic disease. One patient treated for acute graft-versus-host disease obtained a complete response, 6 had a partial response and 3 did not respond. One of the chronic patients achieved complete remision, 3 a partial response, and 4 did not respond. The current study supports the use of this approach in less heavily treated patients for both acute and chronic graft-versus-host disease. The trial has been registered at ClinicalTrials.gov: identifier {"type":"clinical-trial","attrs":{"text":"NCT00447460","term_id":"NCT00447460"}}NCT00447460.     

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.     

Evaluation of prognostic factors among patients with chronic graft-versus-host disease

Background

Chronic graft-versus-host disease (cGVHD) is a major complication after allogeneic stem cell transplantation with an adverse effect on both mortality and morbidity. In 2005, the National Institute of Health proposed new criteria for diagnosis and classification of chronic graft-versus-host disease for clinical trials. New sub-categories were recognized such as late onset acute graft-versus-host disease and overlap syndrome.

Design and Methods

We evaluated the prognostic impact of the new sub-categories as well as the clinical scoring system proposed by the National Institute of Health in a retrospective, multicenter study of 820 patients undergoing allogeneic stem cell transplantation between 2000 and 2006 at 3 different institutions. Patients were retrospectively categorized according to the National Institute of Health criteria from patients’ medical histories.

Results

As far as the new sub-categories are concerned, in univariate analysis diagnosis of overlap syndrome adversely affected the outcome. Also, the number of organs involved for a cut-off value of 4 significantly influenced both cGVHD related mortality and survival. In multivariate analysis, in addition to NIH score, platelet count and performance score at the time of cGVHD diagnosis, plus gut involvement, significantly influenced outcome. These 3 variables allowed us to develop a simple score system which identifies 4 subgroups of patients with 84%, 64%, 43% and 0% overall survival at five years after cGVHD diagnosis (score 0: HR=15.96 (95% CI: 6.85–37.17), P<0.001; score 1: HR=5.47 (95% CI: 2.6–11.5), P<0.001; score 2: HR=2.8 (95% CI: 1.32–5.93), P=0.007).

Conclusions

In summary, we have identified a powerful and simple tool to discriminate different subgroups of patients in terms of chronic graft-versus-host disease related mortality and survival.     

Identification of 4 novel HLA-B*40:01 restricted minor histocompatibility antigens and their potential as targets for graft-versus-leukemia reactivity

Background

Patients with hematologic malignancies can be successfully treated with donor lymphocyte infusion after HLA-matched allogeneic hematopoietic stem cell transplantation. The effect of donor lymphocyte infusion is mediated by donor T cells recognizing minor histocompatibility antigens. T cells recognizing hematopoietic restricted minor histocompatibility antigens may induce selective graft-versus-leukemia reactivity, whereas broadly-expressed antigens may be targeted in graft-versus-host disease.

Design and Methods

We analyzed in detail CD8⁺ T-cell immunity in a patient with relapsed chronic myelogenous leukemia who responded to donor lymphocyte infusion with minimal graft-versus-host disease of the skin. CD8⁺ T-cell clones specific for 4 HLA-B*40:01 restricted minor histocompatibility antigens were isolated which were identified by screening a plasmid cDNA library and whole genome association scanning. Detailed T-cell reactivity and monitoring experiments were performed to estimate the clinical and therapeutic relevance of the novel antigens.

Results

Three antigens were demonstrated to be expressed on primary leukemic cells of various origins as well as subtypes of non-malignant hematopoietic cells, whereas one antigen was selectively recognized on malignant hematopoietic cells with antigen presenting cell phenotype. Skin derived fibroblasts were only recognized after pre-treatment with IFN-γ by two T-cell clones.

Conclusions

Our data show evidence for different roles of the HLA-B*40:01 restricted minor histocompatibility antigens in the onset and execution of the anti-tumor response. All antigens may have contributed to a graft-versus-leukemia effect, and one minor histocompatibility antigen (LB-SWAP70-1Q) has specific therapeutic value based on its in vivo immunodominance and strong presentation on leukemic cells of various origins, but absence of expression on cytokine-treated fibroblasts.     

Fludarabine, cyclophosphamide, antithymocyte globulin, with or without low dose total body irradiation, for alternative donor transplants, in acquired severe aplastic anemia: a retrospective study from the EBMT-SAA working party

Background

We analyzed the outcome of 100 patients with acquired severe aplastic anemia undergoing an alternative donor transplant, after immune suppressive therapy had failed.

Design and Methods

As a conditioning regimen, patients received either a combination of fludarabine, cyclophosphamide, and antithymocyte globulin (n=52, median age 13 years) or this combination with the addition of low dose (2 Gy) total body irradiation (n=48, median age 27 years).

Results

With a median follow-up of 1665 and 765 days, the actuarial 5-year survival was 73% for the group that received fludarabine, cyclophosphamide, and antithymocyte globulin and 79% for the group given the conditioning regimen including total body irradiation. Acute graft-versus-host disease grade III–IV was seen in 18% and 7% of the groups, respectively. Graft failure was seen in 17 patients with an overall cumulative incidence of 17% in patients receiving conditioning with or without total body irradiation: 9 of these 17 patients survive in the long-term. The most significant predictor of survival was the interval between diagnosis and transplantation, with 5-year survival rates of 87% and 55% for patients grafted within 2 years of diagnosis and more than 2 years after diagnosis, respectively (P=0.0004). Major causes of death were graft failure (n=7), post-transplant-lymphoproliferative-disease (n=4) and graft-versus-host disease (n=4).

Conclusions

This study confirms positive results of alternative donor transplants in patients with severe aplastic anemia, the best outcomes being achieved in patients grafted within 2 years of diagnosis. Prevention of rejection and Epstein-Barr virus reactivation may further improve these results.     

Depletion of T regulatory cells through selection of CD127-positive cells results in a population enriched in memory T cells: implications for anti-tumor cell therapy

Background

Donor lymphocyte infusions can induce remissions in patients with relapse after allogeneic hematopoietic stem cell transplantation. Nevertheless, some grafted patients never display any signs of alloreactivity, either following allogeneic hematopoietic stem cell transplantation or after donor lymphocyte infusions. Consequently, they do not develop graft-versus-host disease and frequently do not respond to donor lymphocyte infusions. In a recently published clinical trial, we observed that elimination of CD4⁺CD25⁺Foxp3⁺ natural regulatory T cells from the donor lymphocyte product could improve alloreactivity and the associated anti-tumor effect in a small proportion of patients with relapsed hematologic malignancies. Here, we aimed to improve the effect of donor lymphocyte infusion by modifying the procedure for depletion of T regulatory cells.

Design and Methods

We directly compared depletion of regulatory T cells from human peripheral blood mononuclear cells achieved by selection of CD127-positive cells or by selection of CD25-negative cells. We tested the manipulated products (i) in vitro in mixed lymphocyte reactions and against pathogen-derived recall antigens and (ii) in vivo in experimental graft-versus-host disease.

Results

In vitro, we found that depletion of regulatory T cells through CD127 positive selection improved both alloreactive and pathogen-specific immune responses. In vivo, we observed accelerated donor T-cell division and enhanced graft-versus-host disease due to efficient regulatory T-cell depletion accompanied by enrichment in memory T cells.

Conclusions

Our results show that the strategy of CD127 positive selection is an efficient way of eliminating regulatory T cells from donor lymphocyte infusions and improves alloreactivity. This supports the investigation of CD127 positive selection in place of elimination of CD25-positive cells for clinical applications.Key words: CD127 positive selection, Treg, alloreactivity, donor lymphocyte infusion     

Long-term follow up after allogeneic stem cell transplantation in patients with severe aplastic anemia after cyclophosphamide plus antithymocyte globulin conditioning

Background

Due to increased rates of secondary solid organ cancer in patients with severe aplastic anemia who received an irradiation-based conditioning regimen, we decided some years ago to use the combination of cyclophosphamide and antithymocyte globulin. We report the long-term follow up of patients who underwent hematopoietic stem cell transplantation from an HLA-matched sibling donor after this conditioning regimen.

Design and Methods

We analyzed 61 consecutive patients transplanted from June 1991 to February 2010, following conditioning with cyclophosphamide (200 mg/kg) and antithymocyte globulin (2.5 mg/kg/day × 5 days).

Results

Median age was 21 years (range 4–43); 41 of the 61 patients were adults. Median duration of the disease before hematopoietic stem cell transplantation was 93 days. All but 2 patients received bone marrow as the source of stem cells and all but 2 engrafted. Cumulative incidence of acute grade II–IV graft-versus-host disease was 23% (95%CI 13–34) and 18 developed chronic graft-versus-host disease (cumulative incidence 32% at 72 months, 95% CI 20–46). In multivariate analysis, a higher number of infused CD3 cells was associated with an increased risk of developing chronic graft-versus-host disease (P=0.017). With a median follow up of 73 months (range 8–233), the estimated 6-year overall survival was 87% (95% CI 78–97). At 72 months, the cumulative incidence of avascular necrosis was 21% and 12 patients presented with endocrine dysfunction (cumulative incidence of 19%). Only one patient developed a secondary malignancy (Hodgkin’s lymphoma) during follow up.

Conclusions

Cyclophosphamide and antithymocyte globulin is an effective conditioning regimen for patients with severe aplastic anemia and is associated with low treatment-related mortality. Long-term complications include avascular necrosis and endocrine dysfunction.     

Bone marrow versus peripheral blood as the stem cell source for sibling transplants in acquired aplastic anemia: survival advantage for bone marrow in all age groups

Background

Bone marrow has been shown to be superior to peripheral blood, as a stem cell source, in young patients (<20 years of age) with acquired aplastic anemia undergoing a matched sibling transplant. The aim of this study was to test whether this currently also holds true for older patients with acquired aplastic anemia.

Design and Methods

We analyzed 1886 patients with acquired aplastic anemia who received a first transplant from a human leukocyte antigen identical sibling between 1999 and 2009, with either bone marrow (n=1163) or peripheral blood (n=723) as the source of stem cells.

Results

In multivariate Cox analysis negative predictors for survival were: patient’s age over 20 years (RR 2.0, P<0.0001), an interval between diagnosis and transplantation of more than 114 days (RR 1.3, P=0.006), no anti-thymocyte globulin in the conditioning (RR 1.6, P=0.0001), a conditioning regimen other than cyclophosphamide (RR=1.3, P=0.008) and the use of peripheral blood as the source of stem cells (RR 1.6, P<0.00001). The survival advantage for recipients of bone marrow rather than peripheral blood was statistically significant in patients aged 1–19 years (90% versus 76% P<0.00001) as well as in patients aged over 20 years (74% versus 64%, P=0.001). The advantage for recipients of bone marrow over peripheral blood was maintained above the age of 50 years (69% versus 39%, P=0.01). Acute and chronic graft-versus-host disease were more frequent in peripheral blood transplants. Major causes of death were graft-versus-host disease (2% versus 6% in bone marrow and peripheral blood recipients, respectively), infections (6% versus 13%), and graft rejection (1.5% versus 2.5%).

Conclusions

This study shows that bone marrow should be the preferred stem cell source for matched sibling transplants in acquired aplastic anemia, in patients of all age groups.