Nonmyeloablative Hematopoietic Stem Cell Transplantation Research Articles

Whole Genome Sequencing On Donor Cell Leukemia in a Patient with Multiple Myeloma Identified Gene Mutations That May Provide Insights to Leukemogenesis.

AbstractAbstract 2414Donor cell leukemia (DCL) is a rare occurrence and refers to leukemia of donor origin in patients who have received allogeneic hematopoietic stem cell transplantation (HSCT). We have previously described a male patient with IgG-κ myeloma who received non-myeloablative allogeneic HSCT from a HLA-matched brother and developed complex karyotype acute myeloid leukemia (AML) of donor origin 10 years after transplantation. He achieved complete remission (CR) with standard induction and consolidation chemotherapy but relapsed one year afterwards. We hypothesized that a comparison of the donor HSC before transplantation (pre-leukemic) and the subsequent AML at whole genome level will provide a unique dataset that may shed light on the pathogenesis of leukemia.DNA was extracted from an aliquot of donor mobilized peripheral blood mononuclear cells (mPBMNC) frozen before transplantation as well as unfractionated and CD34+ myeloblasts of the patient's bone marrow at diagnosis and subsequent relapse of AML. The complete donor origin of the AML was confirmed by PCR based on polymorphic STRs. Whole-genome sequencing (WGS) was performed to sequence paired-end reads generated by Illumina HiSeq 2000. Reads were aligned to the human referecne genome (hg19, NCBI37) by SOAP3 and analysed to detect single nucleotide variants (SNVs), small insertion and deletion (indels) and copy number variations (CNVs). Selected genes after filtering were independently validated by Sanger sequencing.There were 835M and 810M 100bp paried-end reads with insert distance of 500bp generated from donor mPBMNC and CD34+ myeloblasts of the relapsed DCL with respective mean depths of 43.2X and 42.6X after alignment. The digital karytoyping based on the read depth was consistent with that by conventional cytogenetic study. 3,979,582 and 1,020,717 SNVs and indels were detectable from both samples. Based on the Catalog of Somatic Mutations in Cancer (COSMIC) and excluding those asian specific wildtypes annotated in 1000 genome project, 11 SNVs and 15 indels within coding sequence with potential roles as tumor suppressors or oncogenes were identified. On the other hand, there were 128,752 and 56,330 SNVs and indels detected exclusively in DCL. Those putative non-pathogenic SNP and those changes locating outside the gene regions were filtered. Within the gene region, SNVs in introns and synonymous mutations were also filtered. 142 non-synonymous SNVs (139 missense and 3 nonsense mutations) were identified of which 25 were considered as statistically highly confident and 17 of them could be confirmed by Sanger Sequencing. Twelve of these were also identified from the whole BM sample of DCL at diagnosis. These candidates include transcription factor (SALL1), metabolic enzymes (UGT1A5, SPEG), membrane protein (TMC6, SCN3A), cytoskeleton protein (MYH10), ribonucleoprotein (RAVER1), secreted protein (WNT7A), protein involved in DNA damage repair (APLF) and others (PRPF8, ZNF518B and MKRN3). 26 indels were indentified in the coding region of which 5 were considered as statistically highly confident, however, only one indel could be confirmed by Sanger Sequencing in the relapse sample and was not present in the diagnostic sample.The WGS performed in paired pre-leukemic (donor HSC) and leukemic (DCL) human samples has provided us with unique opportunities to dissect the genetic changes in HSC that may contribute to the initiation of AML with complex karyotype. The potential impacts of bone marrow microenvironment in this patient with myeloma in inducing DCL are also being evaluated. Disclosures:No relevant conflicts of interest to declare.

Allogeneic hematopoietic stem cell transplantation for multiple myeloma

Despite the curative potential of allogeneic hematopoietic stem cell transplantation (allo HSCT) for patients with multiple myeloma, and reduction of transplant-related mortality with nonmyeloablative transplant approaches, rates of acute and chronic graft-versus-host disease and disease progression remain high. It is unclear if nonmyeloablative transplants are more effective than autologous (auto). Novel promising drugs and maintenance treatment strategies following auto SCT may also delay allo transplantation. In this review, we summarize the emerging data on allo HSCT and provide suggestions for its optimal role in the treatment of myeloma. Large cooperative group studies comparing allo HSCT with auto SCT as frontline therapy have been performed with reduced intensity conditioning regimens using unmanipulated peripheral blood stem cells from human leukocyte antigen (HLA)-compatible donors and standard calcineurin inhibitor graft-versus-host disease prophylaxis. Two recent reports show conflicting data. Although the Blood and Marrow Transplant Clinical Trials Network 0102 study demonstrated no progression-free or overall survival advantage at 3 years, a European study demonstrated superior 5-year outcome after auto/HLA-matched sibling allo HSCT compared with tandem auto SCT in previously untreated multiple myeloma patients. The advent of maintenance therapy could potentially improve outcomes of both transplant types. High rates of acute and chronic graft-versus-host disease currently limit the implementation of nonmyeloablative allo HSCT. Novel approaches are required so that patients with myeloma can undergo allo HSCT before resistance develops to standard drug combinations.

Diabetic Ketoacidosis at Diagnosis Influences Complete Remission After Treatment With Hematopoietic Stem Cell Transplantation in Adolescents With Type 1 Diabetes

OBJECTIVETo determine if autologous nonmyeloablative hematopoietic stem cell transplantation (AHSCT) was beneficial for type 1 diabetic adolescents with diabetic ketoacidosis (DKA) at diagnosis.RESEARCH DESIGN AND METHODSWe enrolled 28 patients with type 1 diabetes, aged 14–30 years, in a prospective AHSCT phase II clinical trial. HSCs were harvested from the peripheral blood after pretreatment consisting of a combination of cyclophosphamide and antithymocyte globulin. Changes in the exogenous insulin requirement were observed and serum levels of HbA1c, C-peptide, and anti-glutamic acid decarboxylase antibody were measured before and after the AHSCT.RESULTSAfter transplantation, complete remission (CR), defined as insulin independence, was observed in 15 of 28 patients (53.6%) over a mean period of 19.3 months during a follow-up ranging from 4 to 42 months. The non-DKA patients achieved a greater CR rate than the DKA patients (70.6% in non-DKA vs. 27.3% in DKA, P = 0.051). In the non-DKA group, the levels of fasting C-peptide, peak value during oral glucose tolerance test (Cmax), and area under C-peptide release curve during oral glucose tolerance test were enhanced significantly 1 month after transplantation and remained high during the 24-month follow-up (all P < 0.05). In the DKA group, significant elevation of fasting C-peptide levels and Cmax levels was observed only at 18 and 6 months, respectively. There was no mortality.CONCLUSIONSWe have performed AHSCT in 28 patients with type 1 diabetes. The data show AHSCT to be an effective long-term treatment for insulin dependence that achieved a greater efficacy in patients without DKA at diagnosis.

Differential immunoregulation in successful oocyte donation pregnancies compared with naturally conceived pregnancies

FOXP3 is critical for the development and function of CD4+CD25bright natural regulatory T cells (nTreg). Individuals harboring mutations in FOXP3 develop immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). We describe a child diagnosed with IPEX who underwent a reduced intensity, T and B cell depleted, matched unrelated donor bone marrow transplant followed by clinical resolution. Using lineage-specific donor chimerism studies, we demonstrate that non-myeloablative HSCT resolves disease in the context of low level donor hematopoietic stem cell (HSC) engraftment. Despite low-levels of donor HSC, thymically-derived nTreg and to a lesser extent CD4+ and CD8+ T cells, exhibit a selective in vivo growth advantage for populations containing a functional FOXP3 gene. Moreover, nTreg from this patient show regulatory function directly ex vivo. These results have implications for improving clinical therapy for patients with IPEX and provide mechanistic insight into the in vivo development of human nTreg and unexpectedly, non-regulatory T cells.

Acute Response of Peripheral Blood Cell to Autologous Hematopoietic Stem Cell Transplantation in Type 1 Diabetic Patient

ObjectiveAutologous nonmyeloablative hematopoietic stem cell transplantation (AHST) was the first therapeutic approach that can improve β cell function in type 1 diabetic (T1D) patients. This study was designed to investigate the potential mechanisms involved.Design and methodsWe applied AHST to nine T1D patients diagnosed within six months and analyzed the acute responses in peripheral blood for lymphocyte subpopulation as well as for genomic expression profiling at the six-month follow-up.ResultsWe found six patients obtained insulin free (IF group) and three remained insulin dependent (ID group); C-peptide production was significantly higher in IF group compared to ID group. The acute responses in lymphocytes at six-month follow-up include declined CD3+CD4+, CD3+CD8+ T cell population and recovered B cell, NK cell population in both groups but with no significant differences between the two groups; most immune-related genes and pathways were up-regulated in peripheral blood mononuclear cell (PBMC) of both groups while none of transcription factors for immune regulatory component were significantly changed; the IF group demonstrated more AHST-modified genetic events than the ID group and distinct pattern of top pathways, co-expression network as well as ‘hub’ genes (eg, TCF7 and GZMA) were associated with each group.ConclusionsAHST could improve the islet function in newly diagnosed T1D patients and elimination of the islet specific autoreactive T cells might be one of the mechanisms involved; T1D patients responded differently to AHST possibly due to the distinct transcriptional events occurring in PBMC.Trial RegistrationClinicalTrials.gov NCT00807651

Everolimus in Combination with Cyclosporin A as Pre- and Posttransplantation Immunosuppressive Therapy in Nonmyeloablative Allogeneic Hematopoietic Stem Cell Transplantation

Everolimus (RAD001) is an mTOR inhibitor that has been successfully used as an immunosuppressant in solid-organ transplantation. Data in allogeneic hematopoietic stem cell transplantation (HSCT) is limited. This study aimed to investigate pharmacokinetics, safety, and efficacy of RAD001 in a canine allogeneic HSCT model. First, pharmacokinetics of RAD001 were performed in healthy dogs in order to determine the appropriate dosing. Doses of 0.25 mg RAD001 twice daily in combination with 15 mg/kg cyclosporin A (CsA) twice daily were identified as appropriate starting doses to achieve the targeted range of RAD001 (3-8 μg/L) when orally administered. Subsequently, 10 dogs were transplanted using 2 Gy total body irradiation (TBI) for conditioning and 0.25 mg RAD001 twice daily plus 15 mg/kg CsA twice daily for pre- and posttransplantation immunosuppression. Seven of the 10 transplanted dogs were maintained at the starting RAD001 dose throughout the study. For the remaining 3 dogs, dose adjustments were necessary. RAD001 accumulation over time did not occur. All dogs initially engrafted. Five dogs eventually rejected the graft (weeks 10, 10, 13, 27, and 56). Two dogs died of pneumonia (weeks 8 and 72) but were chimeric until then. Total cholesterol rose from median 4.1 mmol/L (3.5-5.7 mmol/L) before HSCT to 6.0 mmol/l (5.0-8.5 mmol/l) at day 21 after HSCT, but remained always within normal range. Changes in creatinine and triglyceride values were not observed. Long-term engraftment rates were inferior to sirolimus/CsA and mycophenolate mofetil (MMF)/CsA regimen, respectively. RAD001/CsA caused a more pronounced reduction of platelet counts to median 2× 10(9)/L (range: 0-21 × 10(9)/L) and longer time to platelet recovery of 21 days (range: 14-24 days) compared with MMF/CsA. CsA c(2h) levels were significantly enhanced in the RAD001/CsA regimen, but c(0h) and area under the curve from 0 to 12 hours (AUC(0-12h)) values did not differ compared with an MMF/CsA immunosuppression. In summary, immunosuppression consisting of RAD001 and CsA is well tolerated but not as efficient as with other established immunosuppressants in a canine nonmyeloablative HSCT regimen. Hence, our study does not support the application of RAD001/CsA as standard practice in this setting.

Evidence for Quantitative and Functional Immune Deviation in Pediatric Patients with Sickle Cell Disease

Abstract 1054While hematopoietic stem cell transplantation (HSCT) is a curative therapy for individuals with sickle cell disease (SCD), these patients are at increased risk of graft rejection, especially after non-myeloablative HSCT. This suggests that SCD patients may exhibit immune activation at baseline. However, a rigorous analysis of the extent and character of this immune activation, using newly available multiplexed flow cytometric techniques, has not been previously reported. The objective of this study was to describe the extent of immune deviation in a cohort of pediatric subjects with SCD during steady state, given that this age group represents the majority of SCD patients undergoing HSCT. Methods:An IRB-approved prospective cross-sectional study design was used to compare patients, aged 10 –16y with SCD (homozygous SS or Sb0-thalassemia) during steady state (at least 21 days from an acute SCD exacerbation or other illness including infections requiring antibiotics, and, at least 8 weeks from any RBC transfusion) with an ethnic and age-matched control group of healthy individuals without SCD. Patients were recruited from the Aflac Comprehensive Sickle Cell Clinic at Children’s Healthcare of Atlanta. Controls were recruited using fliers from general pediatric clinics and other community centers in the metro Atlanta area. Inclusion criteria included a confirmed documented diagnosis of homozygous SS or sickle b0 thalassemia for cases and the absence of any sickle hemoglobinopathy for controls. Subjects could only participate once in this study, were excluded if they had renal disease, any significant illness that might be associated with an immune defect such as SLE, were on oral/parenteral corticosteroids, had liver disease (AST/ALT >3ULN), or were unable to give informed consent or complete all study procedures.We have currently enrolled 23/40 SCD patients and 18/30 controls. All patients and controls were African American. Other baseline demographic characteristics of both groups were similar with regards to gender and age (p=0.775 and 0.8314 respectively). Following informed consent, each subject had blood drawn for quantitative immune analysis: total WBC, ANC, ALC, total T cell count (CD4/CD8 T cell subsets), total B cell and NK cell count, and enumeration of CD4 and CD8 T cells for their memory subpopulations. Functional immune assessment was done on a subset of samples (18 patients, 6 controls) using multiplexed enumeration of 25 serum cytokines using the Invitrogen 25-plex human cytokine panel. Quantitative and qualitative flow cytometric and cytokine analysis was performed using FloJo software and the Prism software statistical package. Results:Our results provide evidence for quantitative and functional immune deviation in SCD patients compared to controls. In addition to the well-documented increases in total WBC and ANC (1.9-fold and 1.7-fold compared to controls, respectively, p <0.05), SCD patients also demonstrated significantly higher total lymphocytes, monocytes and both cytotoxic and cytokine-secreting NK cells (2.9, 2.4, and 2.2-fold compared to controls, p <0.01 for all). Circulating B cells were significantly higher in SCD patients (3.3 fold, p= 0.0005). Circulating T cells were also increased in SCD patients, but this effect was specific to CD4+ T cells (1.7-fold, p = 0.002). There was no difference in CD8+ T cells (p= 0.4). The increased CD4+ T cell count predominantly involved memory CD4+ T subpopulations, with both central and effector memory CD4+ T cells significantly increased compared to controls (1.9-fold and 2.5-fold, respectively, p< 0.01). SCD patients also showed evidence of functional immune activation at steady state; plasma cytokine analysis revealed higher circulating CXCL10, CCL4 and IL-15 (2.4, 2.1, and >10 fold compared to controls, p <0.05 for all). These cytokines, which are secreted by activated endothelial cells and monocytes, are implicated in promoting the proliferation (IL-15) and the activation (CXCL10 and CCL4) of both T cells and NK cells. Implications:Our results confirm that even during steady state, pediatric patients with SCD exhibit evidence of significant quantitative and functional immune deviation. This observation suggests that targeting immune pathways impacting NK cell, B cell, and CD4 T cell function may be required for successful engraftment of SCD patients during non-myeloablative HSCT. Disclosures:No relevant conflicts of interest to declare.

Outcome of Children Who Experience Disease Relapse Following Allogeneic Hematopoietic Stem Cell Transplantation for Hematologic Malignancies,

Abstract 4101 Introduction:According to the Center for International Blood and Marrow Transplantation and Research (CIBMTR) data, relapse accounts for 41% of deaths after matched sibling donor hematopoietic stem cell transplantation (HSCT) and 34% of deaths after unrelated donor HSCT. Overall long term survival of 25–30% has been reported in patients who relapse after HSCT. Most of these data are from adult studies. In a recent study, survival following a second allogeneic HSCT ranged from 25–50%. There are limited data on the treatment outcome of children who relapse after an allogeneic HSCT for hematological malignancies. Methods:This multicenter retrospective study was done with the primary objective to examine the incidence and outcome of children who experience relapse after allogeneic HSCT. Patients with a primary diagnosis of acute lymphatic leukemia (ALL), acute or chronic myeloid leukemia (AML or CML), juvenile myelomonocytic leukemia (JMML), biphenotypic leukemia (BL), acute undifferentiated leukemia (AUL), and myelodysplastic syndrome (MDS) were included. After IRB approval, data was collected from review of the medical charts of patients who underwent HSCT at 5 institutions across North America. Results:Data on 532 patients was analyzed; these included 328 males and 204 females. These included ALL (n=254), AML (n=187), MDS (n=35), CML (n=26), JMML (n=3), sAML (n=12), Biphenotypic leukemia (n=10), AUL (n=3) and 1 each with APL and secondary ALL. The median age at HSCT was 9.7 years (range 0.44 to 24.98). Four hundred and fifty (85%) of the 532 patients were in complete remission (CR) at the time of HSCT. Five hundred and fifteen (97%) underwent a myeloablative (MA) HSCT and 16 (3%) had a non-myeloablative (NMA) HSCT. Total body irradiation (TBI) was part of the conditioning therapy in 334 (63%) while 198 (37%) had chemotherapy based regimens. Standard graft versus host disease (GvHD) prophylaxis with calcineurin inhibitors and methotrexate was used in 76% of patients. Grade 1–2 acute GvHD developed in 180 (34%) of patients and 82 (15%) had grade 3–4 acute GvHD. Chronic GvHD was seen in 118 (22%) cases with half having extensive chronic GvHD. After HSCT, 85 (16%) patients died of transplant related complications at 1 year, with infection as the leading (26%) cause of death. The 2, 5 and 10 year overall survival for the entire group was 58.5%, 46% and 44% respectively. Following HSCT, 131 (25%) patients relapsed at a median duration of 7.7 months (range 0.6 to 66.4 months).Of the 131 patients who relapsed 80 (61%) received salvage therapy, 39(30%) did not get any therapy and data were not available for another 12 (9%) patients. Salvage therapy included a combination of withdrawal of immunosuppression, ALL or AML re-induction therapy, or other remission inducing agents, clofarabine, and/or donor lymphocyte infusions (n=22). After salvage therapy 46 (57.5%) patients achieved CR, 26 (32.5%) had no response, 3 (4%) partial response, 2 unknown and 4 (5%) had marrow aplasia and no recovery of counts.Sixty-three (48%) of the relapsed patients underwent a second HSCT: 26 (41%) received a MA, 21 (33%) NMA, 3 (5%) with no conditioning and in 13 (21%) conditioning therapy was unknown. Nearly 22 different conditioning regimens were used for the second HSCT. After the second HSCT 19 (41%) patients died because of transplant related mortality (TRM) and 24 patients died because of relapse. Seventeen (27%) patients are alive and disease free. Conclusions:Relapse remains a major complication following allogeneic HSCT for hematologic malignancies. Our data shows that patients who relapse after HSCT have poor prognosis. In our study 30 different salvage therapies were used for these patients. Currently there is no standard acceptable salvage therapy and most cooperative group or industry sponsored clinical trials exclude patients who underwent HSCT. In addition there are no standard conditioning regimens designed specifically for the second HSCT, which could contribute to the high TRM seen during second HSCT. There is urgent need to develop prospective clinical trials to better understand the optimal therapy for this group of patients. Disclosures:No relevant conflicts of interest to declare.

T-Cell Depleted Allogeneic Hematopoietic Stem Cell Transplantation with Busulfan, Melphalan, and Fludarabine Conditioning Followed by Post Transplantation Donor Lymphocyte Infusions for Patients with Relapsed Multiple Myeloma and High-Risk Cytogenetics

Abstract 1992Allogeneic hematopoietic stem cell transplantation (allo HSCT) is a curative therapy for patients (pts) with multiple myeloma, but conventional allo HSCT has been associated with unacceptably high rates of mortality. Non-myeloablative allo HSCT has resulted in high rates of acute and chronic graft-versus-host disease (GvHD) and progression. We report results of a pilot study of 13 pts, using T-cell depleted allo HSCT (allo TCD HSCT) from HLA compatible (matched related = 8, matched unrelated = 3, and mismatched unrelated = 2) donors. All 13 pts had relapsed myeloma within 12 mos following auto HSCT, and 12/13 pts also had high-risk cytogenetics at diagnosis [t(4;14), t(14;16), del17p by FISH and/or del13q by karyotyping]. All pts achieved at least a partial response from preceding chemotherapy or second auto HSCT. Pts underwent allo TCD HSCT with busulfan (0.8mg/kg × 10 doses), melphalan (70mg/m2 × 2 days), fludarabine (25mg/m2 × 5 days) and rabbit ATG (2.5mg/kg × 2 days). T-cell depletion was performed by positive CD34 selection (Isolex) followed by rosetting with sheep erythrocytes, achieving < 103CD3+/kg for all grafts. All pts engrafted promptly (median d+11, range d+10 to +12). Pts were eligible to receive low doses of donor lymphocyte infusions (DLI) (5×10e5 – 1×10e6 CD3+/kg) no earlier than 5mos post allo HSCT.9/13 pts are alive and now 19–45months (mos) post TCD HSCT. Four pts are in complete remission (CR) at 19, 22, 33 and 39mos following allo TCD HSCT. Two of these pts reentered CR following DLI. Two pts had stable VGPR for 24 and 26mos before progression and reentered VGPR following treatment with Rev/Vel/Dex (RVD) + DLI; they are now 36 and 45mos post TCD HSCT. Two pts with refractory myeloma, who were transplanted with residual 16% and 10% plasma cells in marrow, achieved CR for 8 and 12mos post allo TCD HSCT before they developed progression. Following salvage chemotherapy + DLI, these pts are 20 and 21mos post transplant, respectively. One pt remained in CR for 9mos post TCD HSCT before progression with soft tissues plasmacytomas. With salvage radiation + chemotherapy, the pt is now 20mos post TCD HSCT. The 1 and 2 year probability estimates for overall survival and progression free survival with their 95% confidence intervals are: 1-yr OS 0.69 (95% CI: 0.48, 1.0); 2-yr OS 0.69 (95% CI: 0.48, 1.0); 1-yr PFS 0.46 (95% CI: 0.26, 0.83), 2-yr PFS 0.35 (95% CI: 0.15, 0.78).12/13 pts were without signs of GvHD, but one pt had possible superimposed gut GvHD following fulminant C diff colitis. Four pts died early post TCD HSCT (between 2–6 mos), due to oseltamivir-resistant H1N1 infection (N=1); respiratory failure secondary to infection of unknown etiology (N=1), status epilepticus (N=1), and acute cerebral hemorrhage (N=1).In summary, these results demonstrate that long-lasting disease control can be achieved with TCD HSCT in pts with multiply relapsed and refractory myeloma including those with high-risk cytogenetics. Administration of calculated, low dose donor lymphocyte infusions can induce complete remission without inducing GvHD. Pts who failed to respond to standard chemotherapy pretransplant responded to reuse of this therapy post TCD HSCT. Based on these results, we are performing a phase II clinical trial at Memorial Sloan-Kettering Cancer Center for pts with relapsed multiple myeloma following auto SCT who had high-risk cytogenetics at diagnosis or at relapse as well as for patients with high-risk cytogenetics in the upfront setting following preceding auto SCT. Disclosures:No relevant conflicts of interest to declare.

Successful allogeneic hematopoietic stem cell transplantation for GATA2 deficiency

AbstractWe performed nonmyeloablative HSCT in 6 patients with a newly described genetic immunodeficiency syndrome caused by mutations in GATA2—a disease characterized by nontuberculous mycobacterial infection, monocytopenia, B- and NK-cell deficiency, and the propensity to transform to myelodysplastic syndrome/acute myelogenous leukemia. Two patients received peripheral blood stem cells (PBSCs) from matched-related donors, 2 received PBSCs from matched-unrelated donors, and 2 received stem cells from umbilical cord blood (UCB) donors. Recipients of matched-related and -unrelated donors received fludarabine and 200 cGy of total body irradiation (TBI); UCB recipients received cyclophosphamide in addition to fludarabine and TBI as conditioning. All patients received tacrolimus and sirolimus posttransplantation. Five patients were alive at a median follow-up of 17.4 months (range, 10-25). All patients achieved high levels of donor engraftment in the hematopoietic compartments that were deficient pretransplantation. Adverse events consisted of delayed engraftment in the recipient of a single UCB, GVHD in 4 patients, and immune-mediated pancytopenia and nephrotic syndrome in the recipient of a double UCB transplantation. Nonmyeloablative HSCT in GATA2 deficiency results in reconstitution of the severely deficient monocyte, B-cell, and NK-cell populations and reversal of the clinical phenotype. Registered at www.clinicaltrials.gov as NCT00923364.

Therapeutic in vivo selection of thymic-derived natural T regulatory cells following non-myeloablative hematopoietic stem cell transplant for IPEX

FOXP3 is critical for the development and function of CD4(+)CD25(bright) natural regulatory T cells (nTreg). Individuals harboring mutations in FOXP3 develop immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). We describe a child diagnosed with IPEX who underwent a reduced intensity, T and B cell depleted, matched unrelated donor bone marrow transplant followed by clinical resolution. Using lineage-specific donor chimerism studies, we demonstrate that non-myeloablative HSCT resolves disease in the context of low level donor hematopoietic stem cell (HSC) engraftment. Despite low-levels of donor HSC, thymically-derived nTreg and to a lesser extent CD4(+) and CD8(+) T cells, exhibit a selective in vivo growth advantage for populations containing a functional FOXP3 gene. Moreover, nTreg from this patient show regulatory function directly ex vivo. These results have implications for improving clinical therapy for patients with IPEX and provide mechanistic insight into the in vivo development of human nTreg and unexpectedly, non-regulatory T cells.

Non-myeloablative hematopoietic stem cell transplantation in the treatment of severe idiopathic CD4+ lymphocytopenia

A 40-year-old man with severe chronic idiopathic CD4+ lymphocytopenia complicated with opportunistic infections was successfully treated with non-myeloablative allogeneic hematopoietic stem cell transplantation. After conditioning with fludarabine plus low dose of total-body irradiation, CD34+ peripheral blood stem cells obtained by leukapheresis from his HLA-identical sister were infused. T cell and myeloid complete chimerism was achieved at day +28 and remained stable during the follow-up period. The patient did not develop infectious complications during the procedure. At 35 months of follow-up, his CD4+ T cell count was 1019 cells per microliter. Non-myeloablative allogeneic hematopoietic stem cell transplantation should be considered a treatment option for patients with severe forms of idiopathic CD4+ lymphocytopenia.

Minimal residual disease testing to predict relapse following transplant for AML and high-grade myelodysplastic syndromes

Evaluation of: Lange T, Hubmann M, Burkhardt R et al. Monitoring of WT1 expression in PB and CD34+ donor chimerism of BM predicts early relapse in AML and MDS patients after hematopoietic cell transplantation with reduced-intensity conditioning. Leukemia 25, 498–505 (2011).Early detection of relapse is critical for patients who have undergone hematopoietic stem cell transplantation (HSCT) for acute myeloid leukemia (AML) or high-grade myelodysplastic syndromes (MDS), since therapy can be initiated while disease burden remains low. As these neoplasms represent a heterogeneous group of malignancies with distinct underlying mutations, no single genetic marker exists that both defines AML/MDS and can be exploited for sensitive detection of neoplastic cells prior to overt hematologic relapse. Conversely, the Wilms’ tumor gene (WT1) expression level is increased in blasts of most AML/MDS patients, and quantitative analysis of WT1 expression has been used to predict relapse following myeloablative HSCT. In this article, we review a recently published study evaluating the usefulness of multiple markers, including WT1 expression, for predicting relapse in AML/MDS patients following reduced-intensity conditioning nonmyeloablative HSCT.

Early Post-Transplant Whole Tumor Cell Vaccination Elicits Anti-Tumor T Cell Responses in Patients With Advanced Chronic Lymphocytic Leukemia (CLL)

Vaccination with whole leukemia cells early after allogeneic hematopoietic stem cell transplantation (HSCT) may facilitate expansion of anti-tumor responses. We tested this hypothesis by initiating a phase I study to vaccinate CLL patients between days 30-100 following reduced-intensity (RIC)-HSCT. One cycle of 6 vaccines consisted of 1x107 irradiated autologous CLL cells admixed with irradiated K562 bystander cells secreting the cytokine adjuvant GM-CSF. Of 20 enrolled patients, 15 initiated vaccination. Four of 20 developed GvHD before day 45, precluding vaccination. At a median of 21 months follow-up, all vaccinated patients remain disease-free--an improvement, compared with a historic relapse rate of 50% at 2 years for CLL patients undergoing RIC-HSCT at our center. We thus compared T cell immune reconstitution of study subjects with CLL patients who had undergone RIC-HSCT without vaccines. Recovery of peripheral CD3+ T cell numbers until d100 was similar between vaccinated subjects and 15 transplanted/non-vaccinated CLL controls. Specificity of the reconstituted T cells in vaccinated patients, however, was consistently directed against autologous tumor cells. Five of 5 vaccinated patients demonstrated increased circulating IFN-γ secreting CD8+ T cells by ELISPOT against autologous tumor (mean of 516 vs 184 spots/106 CD8+ T cells at d60 (post-vaccine) vs d30 (pre-vaccine)), but not against autologous hematopoietic cells (PHA blasts, 57 spots/106 CD8+ cells, d60) nor autologous non-hematopoietic cells (fibroblasts, 108 spots/106 CD8+ cells, d60). In contrast, 5 of 5 controls (+HSCT, no vaccine) did not have expanded anti-tumor responses at d60 (mean 51 spots/106 CD8+ cells). Moreover, with GvHD, T cell reactivity developed against autologous PHA blasts and fibroblasts (mean 438 and 434 spots/106 CD8+ cells at d90, respectively). After vaccination, tumor-reactive CD8+ T cells were consistently polyfunctional, secreting high levels of GM-CSF, TNF-α, and IL-2. By limiting dilution, we isolated T cell clones from PBMC of 4 vaccinated subjects, and confirmed that 30-50% of T cell clones demonstrated specific recognition of autologous CLL, but not of alloantigen-bearing cells. Our studies reveal that early post-transplant CLL/GM-K562 vaccination is associated with induction of immunity against recipient CLL cells, and suggest that this is an effective strategy for tipping the balance of immunity in favor of GvL following nonmyeloablative HSCT.

A Pilot Study of T-Cell Depleted Allogeneic Hematopoietic Stem Cell Transplantation With Busulfan, Melphalan, and Fludarabine Conditioning Followed by Post Transplantation Donor Lymphocyte Infusions for Patients With Relapsed Multiple Myeloma and High-Risk Cytogenetics

Allogeneic hematopoietic stem cell transplantation (allo HSCT) is a curative therapy for patients (pts) with multiple myeloma, but conventional allo HSCT has been associated with unacceptably high rates of mortality. Non-myeloablative allo HSCT has resulted in high rates of acute and chronic graft-versus-host disease (GvHD) and progression. We report initial results of a pilot study of 13 pts, using T-cell depleted allo HSCT (allo TCD HSCT) from HLA compatible (matched related8, matched unrelated3, and mismatched unrelated2) donors.

Non-myeloablative Autologous Haematopoietic Stem Cell Transplantation with Consolidation Therapy using Mitoxantrone as a Treatment Option in Multiple Sclerosis Patients

High-dose immunosuppressive therapy with autologous haematopoietic stem cell transplantation (AHSCT) is a new and promising approach to multiple sclerosis (MS) treatment. Recently, the rationale of evolution from myeloablative to non-myeloablative (NM) transplant regimens has been discussed. We aimed to study clinical outcomes in MS patients after NM -AHSCT with consolidation therapy using Mitoxantrone. 55 MS patients were included in this study (mean age - 29.1; male/female - 23/32). Median EDSS at base-line - 4.0 (1.5-8.0), the mean follow-up duration - 26 months (range 9.0 - 50). No transplant related deaths were reported. There were no deaths in the study throughout the follow-up period. The mobilization and transplantation procedures were well tolerated. All the patients responded to the treatment. At long-term follow-up in the group with relapsingremitting MS improvement was demonstrated in 15 patients (58%) and stabilization in 11 (42%). No relapses throughout the whole follow-up period were found. In the group with progressive MS improvement was achieved in 15 patients (82%) and stabilization in 3 (18%). No active new or enlarging lesions were found according to MRI data. Thus, NM-AHSCT with consolidation therapy by Mitoxantrone appears to be a safe and effective treatment for MS. The results of our study support the feasibility of NM-AHSCT with consolidation therapy in this patient population.

Efficacy and safety of autologous hematopoietic stem cell transplantation in treating type 1 diabetes mellitus

Objective To determine the safety and the therapeutic efficacy of autolagous nonmyeloablative hematopoietic stem cell transplantation (AHST) in newly-onset type 1 diabetes mellitus patients. Methods Fifteen patients with type 1 diabetes mellitus were enrolled. Hematopoietic stem cells were mobilized with cyclophosphamide and granulocyte colony-stimulating factor and then collected from peripheral blood by leukapheresis and cryopreserved. The cells were injected intravenously after conditioning with cyclophosphamide and rabbit antithymocyte globulin. Serum levels of HbA1c, C-peptide levels, and anti-glutamic acid decarboxylase antibody (GAD-Ab)titers were measured before and after AHST. Meanwhile, adverse event was recorded.Results The average age of 18 patients (6 males and 12 females)was ( 18.8±4.4 )years, the mean follow-up was ( 414± 150 ) days. 67 % ( 12/18 ) patients became insulin free, the earliest one happened at 2 weeks after AHST, and the latest one at 6 months. 4 cases resumed insulin use because of influenza and other reasons resulting in the rise of blood glucose level. Currently, 8 patients (44.4%) were completely free of insulin therapy, and the remaining cases reduced the insulin dosage by 67.3% ±22.4%. 18 cases had lowered GAD-Ab level, the negative rate was 33.3% (6/18 ). Fasting and postprandial 2 h C-peptide levels increased significantly after A HST. Area under the curve for C-peptide ( AUCC ) increased much more markedly, and it could be maintained for 1 year. Duringtransplantation,all patients had varying degrees of gastrointestinal reactions, hair loss, fever, bone marrow suppression, and other side effects. 5 patients received blood component transfusion. No damage or other severe adverse events of heart, liver, kidney, and other organs were observed. Most side effects gradually disappeared after 2-4 weeks. The recovery of neutropenia was the slowest. Conclusion Autologous hematopoietic stem cell transplantation for treatment of newly-onset type 1 diabetes with residual islet function showed a certain effect and high safety. The widened use of this new technique should be cautious until the therapeutic mechanism has been further studied. Key words: Hematopoietic stem cell; Diabetes mellitus,type 1; Autoimmune; Autologous nonmyeloablative hematopoietic stem cell transplantation

Autologous nonmyeloablative hematopoietic stem cell transplantation in patients with severe anti-TNF refractory Crohn disease: long-term follow-up

AbstractWe evaluated the safety and clinical outcome of autologous nonmyeloablative hematopoietic stem cell transplantation (HSCT) in patients with severe Crohn disease (CD) defined as a Crohn Disease Activity Index (CDAI) greater than 250, and/or Crohn Severity Index greater than 16 despite anti–tumor necrosis factor therapy. Stem cells were mobilized from the peripheral blood using cyclophosphamide (2.0 g/m2) and G-CSF (10 μg/kg/day), enriched ex vivo by CD34+ selection, and reinfused after immune suppressive conditioning with cyclophosphamide (200 mg/kg) and either equine antithymocyte globulin (ATG, 90 mg/kg) or rabbit ATG (6 mg/kg). Eighteen of 24 patients are 5 or more years after transplantation. All patients went into remission with a CDAI less than 150. The percentage of clinical relapse-free survival defined as the percent free of restarting CD medical therapy after transplantation is 91% at 1 year, 63% at 2 years, 57% at 3 years, 39% at 4 years, and 19% at 5 years. The percentage of patients in remission (CDAI < 150), steroid-free, or medication-free at any posttransplantation evaluation interval more than 5 years after transplantation has remained at or greater than 70%, 80%, and 60%, respectively. This trial was registered at www.clinicaltrials.gov as NCT0027853.

Feasibility of Low-Dose Interleukin-2 Therapy Following T-Cell-Depleted Nonmyeloablative Allogeneic Hematopoietic Stem Cell Transplantation From HLA-Matched or -Mismatched Family Member Donors

Introduction: High relapse rates and infections remain primary causes of failure in nonmyeloablative transplantation. Interleukin-2 (IL-2) may stimulate the immune system and improve outcomes. The primary objective of this pilot study was to evaluate the feasibility of administering IL-2 following a T-cell-depleted nonmyeloablative hematopoietic stem cell transplant. Methods: Patients received T-cell-depleted nonmyeloablative transplant from a matched or mismatched related donor. Those with allogeneic engraftment, <grade 2 acute GVHD at time of study entry, and no severe end organ damage were eligible and received IL-2 starting 6 weeks after the first day of stem cell infusion. Patients received 1 mu/m2 daily for 5 days each week for 4 weeks followed by a 2-week rest period for a 6-week cycle to continue for up to 1 year. Results: Eight patients aged 28–69 years were treated. Significant toxicities were limited to GVHD of the skin ≤grade 2 in 3 patients and severe fatigue in 4 patients, limiting the duration of therapy. Two of the 8 patients died of relapsed disease and 1 from CMV. With a median overall duration of follow-up of survivors of 48 months, 5 patients (63%) remain alive and in continuous complete remission.

Interactions Between Donor CD4 Cells and Residual Host Cells After Nonmyeloablative Hematopoietic Stem Cell Transplantation Suppress Hematopoiesis and Lead to Graft Rejection

AbstractAbstract 75While non-myeloablative conditioning significantly reduces morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT), the risk of graft rejection increases, because persistent host cells mediate host-vs-graft reactivity. Here we studied the dynamics of engraftment and hematopoietic reconstitution after non-myeloablative radiation (XRT) in a minor antigen-mismatched model with high barriers to engraftment. BALB.K mice received purified hematopoietic stem cells (HSC: cKit+Thy1.1loSca1+Lin−) +/− TC from AKR/J donors. Recipients of pure HSC or HSC+CD8 TC regularly achieved stable mixed chimerism; however, if grafts contained CD4+CD25− TC (CD4con), recipients failed to engraft. This lack of engraftment was associated with BM hypocellularity (median 3.8 vs 13.3 x10^6 cells/2 legs in HSC+CD4con vs HSC recipients, respectively; p=0.0003), and lymphopenia (<5% vs >40%) at 2 weeks (w) post-HCT. In addition, 2w post-HCT the BM of mice given HSC+CD4con contained increased proportions of CD4 TC which expressed high levels of IFNγ (median 45% of donor vs. 13% of host CD4 TC; p<.001) that exceeded the levels of IFNγ present their spleens or in the BM of control groups (HSC only, wild type [WT], or XRT <10%). A primary determinant of naïve TC immunoreactivity is antigen presentation by dendritic cells (DC). Plasmacytoid DC (CD11c+B220+), which have tolerogenic activity, dominated the BM DC pool in mice given pure HSC at 1–2w post-HCT, but were lacking in recipients of HSC+CD4con. In this latter group, primarily myeloid DC (CD11c+Mac1+) were present that strongly expressed MHCII, CD40, and CD80. This inflammatory profile of myeloid DC was more pronounced in the BM than in the spleens of HSC+CD4con recipients, and IL-12 secretion was measurable in the myeloid DC of these recipients, even without the external endotoxin stimulation which is usually required to detect cytokine production. The IL-12/IFNγ axis is reported to be important in autoimmune pathologies and acute graft rejection of solid organs. To elucidate the mechanism by which IFNγ producing donor CD4 TC suppress engraftment and lymphopoiesis in our system, the progenitor pool in the BM of HSC+/−CD4con recipients was analyzed at 1–2w post-HCT. As expected, the BM recipients of purified HSC had all maturation stages of stem and progenitor cells (HSC: cKit+Sca1+Lin−Flt3−CD34−CD150+ [LT=long-term]/CD150− [ST=short term]; multipotent progenitors, MPP: cKit+Sca1+Lin−Flt3+/−CD34+/−). In contrast, mice given HSC+CD4con lacked MPP, while their population of ST (>LT) HSC was enlarged. These HSC were not apoptotic (as assessed by annexin V staining), but were cell cycle arrested (assessed by DNA content analysis). To study the functionality of these HSC outside the inflammatory environment, LT+ST HSC from HSC+CD4con recipients were FACS purified at 2w post-HCT and infused into secondary Rag2γc-/- recipients. These adoptively transferred HSC and promptly sustained multilineage hematopoiesis derived from the primary BALB.k host. Our results suggest that interactions between naïve donor CD4 TC and immunocompetent residual host cells, such as DC, create a pro-inflammatory environment involving the IL-12/IFNγ axis. We evaluated several other MHC-identical strain combinations which demonstrated lower levels of cytokine production with improved hematopoiesis compared to the AKR into BALK.K combination. These differences imply that impaired hematopoiesis is due to activation of donor CD4 TC in response to disparate minor alloantigens, and not due to non-specific inflammation. Such immune reactions appear to prevent engraftment of donor HSC and also inhibit the HSC expansion required to restore hematopoiesis early post-HCT. We conclude that HCT with pure HSC can result in superior immune reconstitution and donor chimerism after non-myeloablative conditioning. Pure HSC are immunologically anergic, and do not trigger inflammation in the BM microenvironment. As it is generally believed that TC augment donor HSC engraftment, our results are of broad significance because they reveal critical interactions between donor and host populations after non-myeloablative conditioning. Disclosures:No relevant conflicts of interest to declare.