Allogeneic Peripheral Stem Cell Transplantation Research Articles

Notch Signaling in Graft-Versus-Host Disease

Abstract SCI-14 Notch is a highly conserved signaling pathway with multiple functions in health and disease. In the hematopoietic system, Notch was first described for its essential role at early stages of T cell development in the thymus and for its involvement in T cell acute lymphoblastic leukemia. In addition, Notch is being increasingly recognized as a potent regulator of antigen-driven mature T cell responses with context-dependent effects. These effects are influenced by the regulated expression of Jagged and Delta-like Notch ligands in antigen-presenting cells under the control of Toll-like receptors and other innate signals. We are investigating the importance of the Notch pathway in T cell alloimmunity in the setting of allogeneic bone marrow or peripheral blood stem cell transplantation (allo-BMT). After allo-BMT, alloimmune T cell responses mediate beneficial graft-versus-tumor (GVT) activity as well as detrimental graft-versus-host disease (GVHD). Using the pan-Notch inhibitor DNMAML and other genetic models of Notch inhibition, we have discovered an essential function for Notch signaling in donor-derived alloreactive T cells. In multiple mouse models of allo-BMT, infusion of Notch-deficient T cells as compared to wild-type T cells led to dramatically reduced GVHD severity and lethality. This effect was apparent for both CD4+ and CD8+ T cells and did not involve global immunosuppression, since Notch-deficient T cells proliferated normally and expanded in host lymphohematopoietic organs. However, Notch-deficient donor T cells failed to accumulate in the gut, a key GVHD target organ, and produced markedly decreased amounts of the pro-inflammatory cytokines IFN-γ, TNF-α, and IL-2. In parallel, Notch inhibition was associated with increased accumulation of FoxP3+CD4+ T cells. Decreased cytokine production could not be explained by a classical T helper differentiation defect. In contrast to their defective induction of GVHD, Notch-deficient T cells remained capable of mediating cytotoxic and anti-tumor responses both in vitro and in vivo, leading to preserved GVT activity even against large numbers of tumor cells. We are currently investigating the molecular and cellular mechanisms of Notch action in alloreactive T cells. We are also exploring the role of individual Notch receptors and ligands at the alloimmune synapse in vivo. Altogether, our findings identify Notch inhibition in donor T cells as a novel strategy to induce beneficial immunomodulation rather than global immunosuppression after allo-BMT. Disclosures: No relevant conflicts of interest to declare.

Clostridium Difficile Infections After Allogenic Hematopoietic Stem Cell Transplantation: Comparison of Cord Blood to Other Grafts

Abstract 4573 Background:Clostridium difficile, a gram-positive sprore-forming anaerobic bacillus, associated diarrhea (CDAD) is a major cause of nosocomial antibiotic-associated diarrhea. Hematopoietic stem cell transplant (HSCT) recipients are at increased risk of developing CDAD during the early posttransplant period due to prolonged exposure to broad-spectrum antibiotics and immunosuppressive agents. The incidence of CDAD has been increasing up to as high as 20% in allogeneic bone marrow (BMT) and peripheral blood stem cell transplantation (PBSCT). Although cord blood transplant (CBT) recipients are subjected to delayed immune constitution and high incidence of infectious complications to be risk factors for CDAD, the frequency of CDAD after CBT is unclear. We therefore retrospectively investigated the incidence and clinical features of CDAD in patients receiving CBT. Objectives/Methods:During the 2-yr retrospective period (2007–2008), 201 allogeneic HSCT were performed at the Department of Hematology of Toranomon Hospital: 135 CBT, 39 BMT and 27 PBSCT. The median age of the patients was 56 yr (range, 19–82 yr). All patients with diarrhea had a minimum of one diarrheal stool sample evaluated for the presence of toxin A. A patient found to have toxigenic Clostridium difficile by ELISA was diagnosed as CDAD. The cumulative incidence of CDAD was calculated using the Gray method considering death without CDAD as a competing risk. Overall survival (OS) was estimated by the Kaplan-Meier method. The time-dependent Fine and Gray proportional hazards model was used for multivariate analysis. Results:CDAD developed within 100 days in 11 out of 135 CBT recipients at a median onset of day 18 (range, 3–56 days). The cumulative incidence of CDAD after CBT was 9% at day 100 (Fig 1), which was similar to that after BMT (6%, P= 0.55) and to that after PBSCT (16%, P=0.27). All 17 patients who developed CDAD were successfully treated with using oral metronidazole or oral vancomysin. Of the 17 patients with CDAD, 7 (41%) died within 100 days after transplant, and the direct cause of death was irrelevant to CDAD in the 7 patients. The 1-yr survival after diagnosis of CDAD was 58%, which was comparable to that in patients without developing CDAD (59%, P=0.98). The univariate analysis failed to identify any significant risk factors for CDAD as well as the multivariate analysis. Conclusions:The current study showed that CDAD developed early after CBT at the incidence similar to BMT or PBSCT, and that prompt treatment for CDAD may work in improving its prognosis. It is therefore essential to recognize CDAD as one of the differential diagnosis of diarrhea because it is treatable complication after HSCT. Disclosures:No relevant conflicts of interest to declare. [Display omitted]

G-CSF Affects the Distribution and Clonality of TRGV and TRDV Repertoire of T Cells and the Expression Pattern of CD3 Genes

Abstract 1946The immune modulatory effect of granulocyte colony-stimulating factor (G-CSF) on T cells resulted in an unexpected low incidence of graft-versus-host disease (GVHD) in allogeneic peripheral blood stem cell transplantation (allo-PBSCT). The antigen recognition of T cells was based on a heterodimer composed either of the αβ chain or of the γδ chain of the TCR, which was associated with four transmembrane proteins, named CD3γ, CD3ε, CD3δ and CD3ζ, respectively. Recent data indicated that γδ+ T cells might participate in mediating graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation. However, whether G-CSF could influence the T cell receptors (TCR) of γδ+ T cells (TRGV and TRDV repertoire) and CD3 genes remains unclear. To further characterize this feature, we compared the distribution and clonality of TRGV and TRDV repertoire of T cells and the expression pattern of CD3 genes before and after G-CSF mobilization, and investigated the correlation between the changes of TCR repertoire and GVHD in patients undergoing G-CSF mobilized allo-PBSCT.The complementarity-determining region 3 (CDR3) sizes of three TRGV and eight TRDV subfamily genes were analyzed in peripheral blood mononuclear cells (PBMCs) from 20 donors before and after G-CSF mobilization, using RT-PCR and genescan technique. To determine the expression levels of TRGV subfamily genes and CD3 genes, we performed quantitative analysis of TRGVI∼III subfamilies and CD3γ, δ, ε and ζ chain genes by real-time PCR.All of three TRGV subfamily expression levels were decreased significantly after G-CSF mobilization. The pattern of TRGV subfamily expression levels was TRGVII>TRGV I>TRGV III before mobilization, and changed to TRGV I>TRGV II>TRGV III after G-CSF mobilization. Most TRGV and TRDV subfamilies revealed polyclonality from pre-G-CSF-mobilized and G-CSF-mobilized samples. Oligoclonality was detected in TRGV and TRDV subfamilies in 3 donors before mobilization and in another 4 donors after G-CSF mobilization. The expression patterns and clonality of TRGVII, TRDV3 and TRDV1 were found more frequently to change after G-CSF mobilization. Significant positive correlation was observed between the invariable clonality of TRDV1 gene repertoire after G-CSF mobilization and low incidence of GVHD in recipients (P=0.015, OR=0.047). In addition, the expression patterns of CD3 genes before and after G-CSF mobilization were CD3ζ> CD3ε> CD3δ > CD3γ. The expression levels of CD3 ζ and ε genes after G-CSF mobilization (2.194%, 0.867%) were significantly lower than that before mobilization (7.615%, 1.839%) (P=0.031, P=0.009); while the expression levels of CD3 δ and γ genes before mobilization (1.097% and 0.271%) were not significantly different from that after G-CSF mobilization (0.333% and 0.077%) (P=0.093, P=0.070). Significant positive correlation was observed between the relative expression levels of CD3 ζ chain genes before and after G-CSF mobilization (rs=0.576,P=0.016), but no significant correlation of gene expression levels were found in CD3γ, δ and ε genes before and after G-CSF mobilization. The expression level of CD3ζ genes before and after G-CSF mobilization and CD3δ gene after mobilization all respectively showed a negative correlation to the age of donors (rs= −0.549, P=0.023; rs= −0.679, P=0.003; rs= −0.570, P=0.017).In conclusion, G-CSF mobilization not only influences the distribution and clonality of TRGV and TRDV repertoire, but also influences the expression levels of CD3 ε and ζ genes. This alteration of TRGV and TRDV repertoire might play a role in mediating GVHD in G-CSF mobilized allo-PBSCT. Disclosures:Liu:National Natural Science Foundation of China (No.30971300), Guangdong Natural Science Foundation (No.10451051501005778), Science and Technology Planning Project of Guangdong Province of China (No.2009A030200007) and China Postdoctoral Science Foundation (: Research Funding.

IL10-592 CC Genotype In the Donor Is Associated with a Significant Decrease of Relapse Rate and a Significant Increase of Event-Free Survival and Overall Survival After Allogeneic Hematopoietic Stem Cell Transplantation In Children with Hematological Malignancies

Abstract 4459IL10 is a pivotal immunomodulatory cytokine and is usually regarded as a suppressor of the immune responses. However, IL10 has been shown to have some immunostimulatory effects. The IL10-592 CC genotype is associated wit higher production of IL10. Because IL10 may promote the development of alloimmunity we hypothesized that the IL10-592 CC genotype in the donor reduces the risk of relapse after hematopoietic stem cell transplantation (HSCT). A cohort of 211 children (median age, 12 years) with acute lymphoblastic leukemia (n=100), acute myeloid leukemia (n=62), myelodysplastic syndrome (n=30) or chronic myeloid leukemia (n=19) who underwent allogeneic bone marrow (n=153) or peripheral blood stem cell transplantation (n=58; T-cell depleted: n=26) in a single center and/or their respective donors was genotyped of IL10 gene for rs1800872 using TaqMan real-time polymerase chain reaction. The donor was HLA-matched unrelated in 48% of transplants and HLA-identical related in 42% of transplants. Conditioning regimen was myeloablative in all cases. Two forms of post-transplant immunosuppression predominated, cyclosporine A and methotrexate in 69% of transplants and cyclosporine A alone in 17% of transplants. Cell samples from the donor were available in 174 cases and from the patient in 197 cases. The IL10-592 CC genotype was present in 82 of the 174 donors (47.1%) and in 104 of the 197 patients (52.8%). Interestingly, we found a significantly reduced incidence of relapse in patients who were transplanted from a donor with the IL10-592 CC genotype (15.9% versus 30.4%; p=0.016). In addition, we observed a significant increase of event-free survival (52.4% versus 33.7%; p=0.019) and a significant increase of overall survival (54.9% versus 37.0%; p=0.040) if the IL10-592 CC genotype was present in the donor. The occurrence of the IL10-592 CC genotype, in either donors or recipients, had no significant impact on treatment related mortality, acute and chronic graft-versus host disease. In conclusion, IL10-592 CC genotype in the donor is associated with a significant decrease of relapse rate and a significant increase of event-free survival and overall survival after HSCT in children with hematological malignancies. This is the first study to describe an association of IL10 gene polymorphism with relapse rate after HSCT. Selecting a donor with the IL10-592 CC genotype could be a useful therapeutic strategy for improving the final outcome after allogeneic HSCT. Disclosures:No relevant conflicts of interest to declare.

Low Incidence and severity of Graft-Versus-host disease after outpatient allogeneic Peripheral Blood Stem Cell Transplantation employing a Reduced-Intensity Conditioning

Abstract 4707 Introduction.Graft-versus-host disease (GVHD) is the most common complication of allogeneic hematopoietic cell transplantation (alloHCT) and may affect the transplant outcome. Its incidence is higher when the preparative regimen used is a non-myeloablative and the stem cell's source is peripheral blood after mobilization. Objective.Demonstrate the 10 years incidence of GVHD in 2 Mexican transplant centers using peripheral hematopoietic stem cell transplantation (PHSCT) in related donors after non-myeloablative conditioning. Patients and methods.Three hundred and four patients with hematological and non-hematological malignancies that underwent outpatient PHSCT after non-myeloablative conditioning between October 1998 and July 2008 were included. The age ranged between 1 and 71 years (median of 30.5). One hundred and eighty-five patients were men and 119 women. The median of cells CD34+ infused was 4.9 × 106/kg (0.23-17.70).They received cyclosporine 4mg/kg per day and intramuscular methotrexate 5mg/ m2 in days +1, +3, +5 and +11 for GVHD prophylaxis. Results.Two hundred thirty-nine (80%) patients were successfully engrafted. The conditioning regimen was delivered as an outpatient procedure in all individuals. One hundred and fifty-four patients (64%) developed acute and/or chronic GVHD. Sixty four patients (26.7%) developed acute GVHD, 50 (20.9%) developed chronic GVHD, and 40 (16.7%) with acute GVHD progressed to chronic. Twenty seven (26%) patients who developed acute GVHD were grade III or IV, and 30 (33.2%) of chronic GVHD patients presented in the extensive way. GVHD was the cause of dead in 40 patients; 24 of acute GVHD and 16 of chronic GVHD, even when immunosuppressive therapy (high dose steroids and rituximab) was used. Last death was 24 months ago; subsequently, alemtuzumab was included in GVHD treatment. The cumulative incidence of acute GVHD was 37.6%. Conclusion.The higher incidence of GVHD reported in other studies after PBSC transplant seems to decrease if non-myeloablative conditioning regimens are used. We suggest that the combination of a reduced-intensity conditioning transplant and the outpatient procedure is the responsible for the low incidence of GVHD in our patients. Disclosures:No relevant conflicts of interest to declare.

Low incidence and severity of graft-versus-host disease after outpatient allogeneic peripheral blood stem cell transplantation employing a reduced-intensity conditioning

The prevalence and features of graft-versus-host disease (GVHD) in patients receiving allografts using peripheral blood stem cells (PBSCs) after a reduced-intensity conditioning (RIC) regimen are not well known. Several features of GVHD in patients at two institutions using RIC were assessed. We analysed the overall survival (OS) and prevalence of GVHD in patients who underwent outpatient allogeneic PBSC transplantation after RIC between October 1998 and July 2008. We included 301 patients with a median age of 30 yrs (range, 1-71 yrs). In 37 cases, allogeneic peripheral blood stem cell transplantation was indicated for non-malignant disease, and in 264 for malignant disease. The median OS was 35 months. The estimated 3-yr OS was 48%. A total of 154 patients developed GVHD: there were 64 acute, 50 chronic and 40 cases that progressed from acute to chronic. Of the 104 patients with acute GVHD (aGVHD), 40% had grade I and 60% had grades II-IV. Of the 90 patients with chronic GVHD (cGVHD), 67% had limited and 33% had extensive forms. A total of 160 patients died, 40 as a result of GVHD (24 from aGVHD and 16 from cGVHD), 50 as a result of progressive disease and 70 from diverse causes. The incidence of GVHD was lower than in other series using conventional myeloablative preparative regimens. Most importantly, the severity of GVHD did not significantly affect the long-term survival.

CD3+/Tregs Ratio in Donor Grafts Is Linked to Acute Graft-versus-Host Disease and Immunologic Recovery after Allogeneic Peripheral Blood Stem Cell Transplantation

Graft-versus-host disease (GVHD), mediated by mature T cells present in the donor graft, remains a major complication after allogeneic peripheral blood stem cell transplantation (PBSCT). Regulatory T cells (Tregs) (CD4(+)CD25(high)Foxp3(+)) are believed to maintain tolerance and to inhibit GVHD after allogeneic PBSCT (allo-PBSCT). In this study, we analyzed the graft CD3(+)/Tregs ratio (gCD3/Tregs R) and evaluated its impact on acute GVHD (aGVHD) and immunologic recovery after myeloablative allo-PBSCT. We analyzed 65 consecutive patients who underwent transplantation with unmanipulated peripheral blood stem cells from an HLA-identical related donor (n = 45) or an HLA-identical unrelated donor (n = 20). The median CD3(+) and Tregs doses administered were 256 × 10(6)/kg of body weight (range, 67-550 × 10(6)/kg) and 12 × 10(6)/kg (range, 2-21 × 10(6)/kg), respectively; the median gCD3/Tregs R value was 18 (range, 8-250). Patients were subdivided into a high gCD3/Tregs R (≥36) group (HR; n = 26) and a low gCD3/Tregs R (<36) group (LR; n = 39). The incidence of aGVHD (grade II-IV) was lower in the LR group compared with the HR group (8/39 [20%] versus 22/26 [84%]; P < .001). Median cytomegalovirus-specific CD8(+) T lymphocytes were significantly higher in the LR group than in the HR group at 1 month (2 cells/μL versus 0 cells/μL; P < .001), 2 months (6cells/μL versus 1 cell/μL; P < .001), and 3 months (15 cells/μL versus 3 cells/μL; P < .001) months. Moreover, cytomegalovirus infection/disease was observed in 15% of patients in the LR group versus 69% of patients in the HR group (P < .001). At multivariate logistic regression, gCD3/Tregs R was correlated both with aGVHD (odds ratio, 2.50; 95% confidence interval, 1.30-4.50; P = .05) and with cytomegalovirus infection/disease (odds ratio, 2.35; 95% confidence interval, 0.9-5.00; P = .05). Taken together, our data may suggest that the balance in favor of graft Tregs content is able to mediate protective effects against aGVHD and to maintain an optimal microenviroment for the reconstitution of functional immunity.

Allogeneic peripheral blood stem cell and bone marrow transplantation for hematologic malignancies: Meta-analysis of randomized controlled trials

Peripheral blood stem cells have emerged as an alternative to bone marrow for allogeneic transplantation. To elucidate the advantages and disadvantages of research evidence related to the effects of allogeneic peripheral blood stem cells transplantation (PBSCT) and bone marrow transplantation (BMT) for hematological malignancies, we conducted a systematic review of the literature of randomized controlled trials comparing PBSCT to BMT. We systematically searched Cochrane Library, MEDLINE, EMBASE and CNKI up to May 2011. Two reviewers independently identified the eligible studies and assessed the methodological quality of included trials. The relevant data were extracted and analysed using RevMan 5.1. Ten trials totaling 1224 patients have been assessed. Pooled comparisons of studies of PBSCT and BMT found that the overall survival in PBSCT group was non-significantly different from that in BMT group [RR 0.92, 95% CI (0.80–1.07)]. The disease-free survival and relapse rate in PBSCT group were significantly different from that in BMT group [RR 0.67, 95% CI (0.52–0.86) and RR 0.51, 95% CI (0.34–0.76), respectively]. The number of days to reach the absolute neutrophil and platelet count were shorter with PBSCT. The rates of acute and chronic graft-versus-host disease (GVHD) in PBSCT group were significantly higher than that in the BMT group. The mortality in PBSCT group was non-significantly different from that in BMT group. We concluded that PBSCT was associated with a similar overall survival and mortality, improved disease-free survival and a decrease in relapse, faster engraftment, more GVHD when compared with BMT in transplantation for hematologic malignancies.

Drug dosing and monitoring in obese patients undergoing allogenic stem cell transplantation

The effects of physiological changes in patients with obesity on pharmacokinetic parameters and the time course of drug response, especially in the field of haematology/oncology, are poorly understood. For some antimicrobial drugs, dosing considerations exist, while for cytostatic drugs, dose modifications for obese patients are not consistently recommended. Glomerular filtration rate and renal perfusion appear to be similar in obese and normal weight individuals, thus elimination of hydrophilic and extensively renally cleared drugs mainly depends upon creatinine clearance. To provide information about drug dosing in morbidly obese patients undergoing allogenic haematopoietic stem cell transplantation and to develop dosing recommendations for those patients, based on literature data, pharmacokinetic properties and own experiences. A review on the literature on drug dosing in obese patients as well as on the pharmacokinetic properties of drugs which are supposed to be used in the field of stem cell transplantation was combined with own data on drug dosing and pharmacokinetic drug monitoring in a morbidly obese patient undergoing matched-unrelated allogenic peripheral blood stem cell transplantation. For hydrophilic and extensively renally cleared drugs (e.g. piperacillin/sulbactam, cotrimoxazole, fludarabine) standard dosages for adult patients or dosing based on ideal body weight (IBW) (e.g. aciclovir, methotrexate) can be used. For ciclosporin and digitoxin we could show that high initial doses are needed to achieve sufficient plasma concentrations. After steady state distribution was completed, maintenance doses comparable to normal weight patients are sufficient. Likewise, distribution of enoxaparin and phenytoin seems to take longer in obese patients. Dosing recommendations of 25 drugs that can be used in morbidly obese patients undergoing allogenic stem cell transplantation are given. Pharmacotherapy in morbidly obese patients undergoing allogenic stem cell transplantation is possible, if pharmacokinetic properties of the drugs are considered and close monitoring of plasma concentrations is performed.

Eculizumab before and after allogeneic hematopoietic stem cell transplantation in a patient with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the triad of intravascular hemolysis, venous thrombosis, and cytopenia. Treatment of PNH is generally supportive. Bone marrow transplantation is the only curative therapy for PNH, but is associated with significant morbidity and mortality. Herein, we present a patient with PNH that received eculizumab, a humanized monoclonal antibody that blocks activation of the terminal complement at C5, before and immediately following allogeneic peripheral stem cell transplantation. Prior to hematopoietic stem cell transplantation eculizumab treatment markedly reduced hemolysis and transfusion requirement; however, 1 d post transplantation a hemolytic episode occured, which was successfully stopped with eculizumab re-treatment. Afterwards the patient did not require additional transfusions. The results of this study indicate that early administration of eculizumab may be a safe and effective therapy for hemolytic episodes associated with allogeneic peripheral stem cell transplantation in patients with PNH.

Reconstitution and functional analyses of neutrophils and distinct subsets of monocytes after allogeneic stem cell transplantation

The aim of the study was to investigate the recovery of the innate immune system within the first 100days after allogeneic peripheral blood stem cell transplantation (PBSCT) and to elucidate a potential correlation with such important events as severe infectious complications or graft-versus-host disease (GvHD). In 30 consecutive patients who underwent allogeneic PBSCT, absolute numbers of neutrophils and monocytes were determined and different functional analyses performed at different time points (day +30, +60 and +90, respectively). The capacity to phagocyte Escherichia coli (E. coli) as well as the induction of oxidative burst after incubation with different stimuli (Phorbol-12-myristate-13-acetate; PMA, the chemotactic peptide N-formyl-Met-Leu-Phe; f-MLP or opsonized E. coli) were analysed after engraftment. There was a rapid reconstitution concerning the capability of both neutrophils and monocytes to phagocyte E. coli without a significant increase between day +30 and +90. In contrast, a twofold increase of monocyte oxidative burst after incubation with PMA at day +90 was observed (P=0.017). Furthermore, the ability of neutrophils to induce oxidative burst after ingestion with E. coli was impaired on day +30 with a significant functional reconstitution on day +60 (P=0.01). The oxidative burst activity following incubation with f-MLP did not show significant changes after stem cell engraftment. Analysis of numeric reconstitution of CD14+CD16+ monocytes demonstrated a potential correlation with a decreased incidence of chronic GvHD. The functional recovery of neutrophils and monocytes in the early period after allogeneic PBSCT differs not only concerning phagocytosis and oxidative burst but also with respect to the stimulus and the cell population that was analysed for oxidative burst activity. The subset of CD16+CD14+ monocytes might be a predictor for a reduced risk of chronic GvHD.

Immune hemolysis after fludarabine-based reduced intensity conditioning and allogeneic PBSC transplantation for CML with minor ABO incompatibility

Dear Editor, Fludarabine is commonly used in reduced intensity conditioning (RIC) regimens and has been linked to immune hemolysis [1, 2]. We report the first case of severe immune hemolysis in the fludarabine-based RIC allogeneic peripheral blood stem cell transplantation (PBSCT) for chronic myelogenous leukemia (CML) in the setting of minor ABO incompatibility. A 47-year-old woman diagnosed with CML underwent allo-PBSCTwith an HLA-matched related donor in February 2007, while in the accelerated phase. Conditioning consisted of busulfan, 2 mg/kg/day (days −6 and −5), fludarabine, 30 mg/m2/day (days −6 to −2), and anti-thymoglobulin, 10 mg/kg/day (days −4 to −1). GVHD prophylaxis consisted of cyclosporine, 5 mg/kg/day, and mycophenolate mofetil, 45 mg/kg/day. There was minor ABO incompatibility between the donor (O Rh-positive) and the recipient (B Rhpositive). The donor's anti-B antibody titer in saline test tube at 37°C was 1:8 IgG. The conditioning regimen and stem cell infusion were both uneventful. Eight days after PBSCT, she developed mucocutaneous pallor, asthenia, and nausea. Laboratory studies indicated severe hemolysis with a drop in the hemoglobin level, an increase in the serum bilirubin and lactic dehydrogenase (LDH) levels, and complete disappearance of haptoglobin (graph). Peripheral blood contained spherocytes. No fragmentation was observed. Renal function and clotting studies were normal. Thorough microbiological studies were negative. Investigations for hepatitis A, B, and C viruses, VDRL, HTLV I and II, HIV, HSV, adenovirus, parvovirus, EBV, HHV-6, cytomegalovirus, and toxoplasmosis were negative. Computed tomography (CT) of the chest and sinuses was normal, and CT of the abdomen showed only splenomegaly. Serological screening for autoimmune diseases was negative. None of the medications she was receiving was known to cause hemolysis. The direct antiglobulin test (DAT) was positive for IgG and C3d. Recipient red blood cells (RBC) were repeatedly B positive. The serum anti-B antibody titer in saline test tube at 37°C was 1:2 IgG. Antibodies eluted from the patient's RBCs showed pan-agglutination against group O antibody panel cells. Allo-antibodies were excluded by adsorption studies. The diagnosis of immune intravascular hemolysis was established. The lowest recorded hemoglobin level was 5 g/dl, and the highest levels of bilirubin and LDH were respectively 7 mg/dl (normal, 0.2 to 1.0 mg/dl) and 791 IU/ l (normal, 120 to 200 IU/l). Methylprednisolone, 2 mg/kg/day intravenously, was administered from day +8 for five consecutive days and was then gradually withdrawn over 2 months. The hemoglobin level improved rapidly, and the hemolysis panel normalized (Fig. 1). Repeated anti-B titers in saline test tube at 37°C were respectively 1:8 and 1:16 on days +11 and +14. On day +14, the DAT was negative, and the patient's blood group was O Rh positive. She received a total of six units of type O Rhpositive packed red blood cells from days +10 to +13, and no further transfusions were necessary. VNTR analysis on R. Calixto : F. Ostronoff :M. Ostronoff :A. Sucupira : R. Florencio :A. P. S. Maior :M. C. Domingues Hematology and Bone Marrow Transplantation Unit, Real-Hospital Portugues, Recife, PE, Brazil

Successful allogeneic peripheral blood stem cell transplantation with reduced intensity conditioning in a patient with severe aplastic anemia and active infection

Infection is the major cause of mortality in patients with severe aplastic anemia (SAA) and often results in postponement of immunotherapy or transplantation treatment. We report on a 23-year-old man with very SAA with almost no neutrophils who was primarily treated with peripheral blood stem cell transplantation from an HLA-identical sibling. He received reduced dose conditioning with cyclophosphamide and antithymocyte globulin during active infection with typhlitis and pneumonia. Cyclophosphamide 50 mg/kg/d was given on Day 4 and Day 3 before transplantation and antithymocyte globulin Fresenius 20 mg/kg/d was given on Day 3 and Day 2. Neutrophils and platelets were engrafted on Day +18 and Day +20 after transplantation. Symptoms and signs of acute or chronic graft versus host disease were not observed as of Day +545 after transplantation. Allogeneic hematopoietic stem cell transplantation with reduced intensity conditioning may be considered as the primary therapy for SAA complicated by severe infection.

Development of immunity against hepatitis B virus after donor lymphocyte infusion in a peripheral blood stem cell transplantation recipient with chronic hepatitis B

Hepatitis B virus (HBV) infection is one of the main concerns in blood and marrow transplantation (BMT) patients for possible breakthrough hepatitis. Active recipient immunization against HBV was found to be ineffective and many studies had showed that the adoptive transfer of immunity against hepatitis B virus would be possible by BMT with unknown duration and mechanism. A 46-year-old female patient with chronic hepatitis B had persistent detectable HBV DNA and positive serum hepatitis B e antigen (HBeAg), even while on long-term lamivudine and adefovir therapy. She received allogeneic matched unrelated donor peripheral blood stem cell transplantation (allo-MUD-PBSCT) for her refractory acute myeloid leukemia (AML). The HBV DNA became undetectable and she developed HBeAg seroconversion after PBSCT. Her hepatitis B surface antigen (HBsAg) remained positive, which disappeared later, along with the development of antibody to HBsAg after one shot of donor lymphocyte infusion (DLI) as a boost against her AML. In summary, BMT from an immunized donor would probably bring adoptive immunity against HBV. This adoptive immunity might be further enhanced by the subsequent DLI.

High-dose chemotherapy followed by autologous and allogeneic peripheral blood stem cell transplantation for recurrent disseminated trilateral retinoblastoma

Trilateral retinoblastoma (TRb) is an intracranial neurogenic tumor associated with unilateral or bilateral retinoblastoma and has very poor prognosis. Patients typically die from leptomeningeal tumor dissemination. A 3-year-old girl who had been diagnosed with TRb had a disseminated relapse after a tumorectomy, cerebrospinal irradiation, and conventional chemotherapy. The disseminated tumor disappeared after the first autologous peripheral blood stem cell transplantation (PBSCT) with high-dose melphalan and thiotepa. During the second complete remission, a second autologous PBSCT with high-dose busulfan and melphalan was performed. Seven months after the first PBSCT, the second relapse occurred, and we subsequently performed an allogeneic PBSCT with myeloablative chemotherapy consisting of melphalan, thiotepa, and cyclophosphamide. The patient showed clinical improvement after the allogeneic PBSCT. Although high-dose chemotherapies have a curative effect for some patients with TRb, the prognoses of disseminated tumors are still poor. Further examination of the high-dose chemotherapy is necessary for the time, the conditioning drugs, and the hematopoietic stem cell sources.

In Vivo T Cell Depletion With Dexamethasone Reduces Graft Versus Host Disease (GVHD) With Minimal Side Effects and No Additional Risk of Early Relapse After Allogeneic Peripheral Blood Stem Cell (PBSC) Transplantation

Cytokine mobilized stem cells collected from peripheral blood are routinely used for allogeneic hematopoietic stem cell transplantation (alloHSCT). Advantages to PBSC use include better donor tolerance and more rapid engraftment than marrow source stem cells. Disadvantages of PBSC include the increased incidence and severity of chronic GVHD due to higher numbers of alloreactive T cells.1.2

Comparative Analysis of Outcomes of Allogeneic Peripheral Blood Stem Cell Transplantation From Related and Unrelated Donors for Acute Myeloid Leukemia

Comparative Analysis of Outcomes of Allogeneic Peripheral Blood Stem Cell Transplantation From Related and Unrelated Donors for Acute Myeloid Leukemia

Comprehensive healthcare for individuals with sickle cell disease

Comprehensive healthcare for individuals with sickle cell disease

Prognostic Implications of the NIH Consensus Criteria in Children with Chronic Graft-versus-Host Disease

PurposeIn this study, we analyzed a cohort of children with chronic graft-versus-host disease (GvHD) according to the NIH consensus classification (NCC) in order to observe whether global assessment at diagnosis correlates with GvHD-specific endpoints. We then studied the clinical course of these patients, specifically with regards to episodes of GvHD exacerbation requiring treatment escalation.Materials and MethodsRecipients of either allogeneic bone marrow transplantation (BMT) or peripheral blood stem cell transplantation (PBSCT) from January 2006 to August 2008 at the Department of Pediatrics, The Catholic University of Korea were evaluated for chronic GvHD, which was diagnosed according to the NCC. The course of chronic GvHD in these patients was then followed.ResultsOf 59 evaluable patients, 23 developed chronic GvHD for a cumulative incidence of 39.3%. Upon multivariate analysis, previous acute GvHD (≥grade II) had a significant impact on chronic GvHD incidence. With a median duration of systemic treatment for chronic GvHD of 501 days, no significant relationship was found between initial global severity of chronic GvHD and either duration of immunosuppressive treatment or final clinical response to treatment. Fifteen patients (65%) experienced at least one episode of chronic GvHD exacerbation during the period of follow-up, with a median of four exacerbations in the subgroup of patients who experienced such events. Lung GvHD resulted in the highest number of exacerbations per diagnosed patient, followed by oral GvHD.ConclusionAnalysis of this small cohort indicates that global assessment as proposed by the NCC may have limited correlations with GvHD-specific endpoints, possibly due to the favorable response of children to treatment.

Adoptive transfer and selective reconstitution of streptamer‐selected cytomegalovirus‐specific CD8+ T cells leads to virus clearance in patients after allogeneic peripheral blood stem cell transplantation

Cytomegalovirus (CMV) disease constitutes a serious complication after allogeneic stem cell transplantation. For the clearance of CMV, CD8+ T cells are pivotal. Here, the novel streptamer technology was used at good manufacturing practice (GMP) level for adoptive transfer of CMV-specific T cells into acute leukemia patients with recurrent high CMV antigenemia after allogeneic stem cell transplantation. After a single transfusion, the frequency of CMV-specific CD8+CD45RA+CCR7- effector T cells increased dramatically from 0.0% to a maximum of 27.1% of all T cells. These T cells were clearly donor derived and did not stem from intrinsic reconstitution, as demonstrated by analysis of 1) donor chimerism through single-tandem repeats, 2) T-cell receptor excision circles, and 3) Vβ-chain typing by polymerase chain reaction. Clinically, the specific T-cell transfer resulted in a persistent clearance of the CMV antigenemia, which allowed the patients to discontinue toxic antiviral drug therapy without further high-level reactivation of CMV, demonstrating the power of the streptamer technology. Taken together, the streptamer technology offers the advantage of selecting virus-specific CD8+ T cells at GMP level for adoptive T-cell transfer, thus inducing long-lasting specific CD8+ T-cell responses without increasing the risk for graft-versus-host disease.