Hematopoietic stem cell transplantation (HSCT) is used for treatment of lymphoma. In an attempt to design an efficacious and safe pre-HSCT conditioning regimen, we investigated the cytotoxicity of the combination of busulfan (B), melphalan (M) and gemcitabine (G) in lymphoma cell lines in the absence or presence of drugs that induce epigenetic changes. Cells were exposed to drugs individually or in combination and analyzed by the MTT proliferation assay, flow cytometry, and Western blotting. We used ~IC10 drug concentrations (57 μM B, 1 μM M and 0.02 μM G) which individually did not have major effects on cell proliferation. Their combination resulted in 50% inhibition of proliferation. Reduction to almost half concentration (20 μM B, 0.7 μM M and 0.01 μM G) did not have significant effects, but addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA; 0.6 μM) to this combination resulted in a marked (~65%) growth inhibition. The cytotoxicity of these combinations correlates with the activation of the ATM-CHK2 pathway, phosphorylation of KAP1, epigenetic changes such as methylation and acetylation of histone 3, and activation of apoptosis. The relevance of epigenetic changes is further shown by the induction of DNA methyltransferases in tumor cells with low constitutive levels of DNMT3A and DNMT3B. The addition of 5-aza-2′-deoxycytidine (DAC) to [BMG+SAHA] further enhances cell killing. Overall, BMG combinations are synergistically cytotoxic to lymphoma cells. Epigenetic changes induced by SAHA and DAC further enhance the cytotoxicity. This study provides a rationale for an ongoing clinical trial in our institution using [BMG+SAHA] as pre-HSCT conditioning for lymphoma.
DNA alkylator; nucleoside analog; SAHA; lymphoma; drug cytotoxicity Category; Hematological Malignancies or Stem Cell Transplantation
Delayed recovery of platelet count post allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been associated with worse transplant outcomes. Thrombopoietic agents have been successfully used in immune mediated thrombocytopenia or thrombocytopenia from bone marrow failure syndromes; however, the experience regarding their use after allo-HSCT is limited. Here we report on the safety and efficacy of romiplostim used in 3 consecutive patients with thrombocytopenia post allogeneic transplantation. Two patients had prolonged platelet recovery due to poor graft function while one had secondary failure of platelet recovery, likely immune mediated, post transplantation. Successful use of such agents post-transplant may improve platelet recovery, decrease rates of complications and potentially improve outcomes.
Post-transplant thrombocytopenia; romiplostim; allogeneic hematopoietic stem cell transplantation
Graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is mediated by the activation of recipient dendritic cells (DCs) and subsequent proliferation of donor T cells. Recently complement system has been shown to modulate adaptive immunity through the interaction between the complement system and lymphocytes. Complement proteins participate in the activation of DCs, antigen presentation to T cells, and proliferation of T cells. Our studies with a murine model of bone marrow transplantation (BMT) demonstrate that complement system regulates alloimmune responses in GVHD. Mice deficient in the central component of the complement system (C3−/−) had significantly lower GVHD-related mortality and morbidity compared to the wild type (WT) recipient mice. The number of donor-derived T cells including IFNγ+, IL17+ and IL17+IFNγ+ subsets was decreased in secondary lymphoid organs of C3−/− recipients. Furthermore, there was a reduction of recipient CD8α+CD11c+ in lymphoid organs. We conclude C3 regulates Th1/17 differentiation in BMT, and define a novel function of the complement system in GVHD.
Delayed engraftment remains a major hurdle following cord blood (CB) transplantation. It may be due, at least in part, to low fucosylation of cell surface molecules important for homing to the BM microenvironment. Since fucosylation of specific cell surface ligands is required before effective interaction with selectins expressed by the BM microvasculature can occur, a simple 30 minute ex vivo incubation of CB HPC with fucosyltransferase (FT) - VI and its substrate (GDP-fucose) was performed to increase levels of fucosylation. The physiologic impact of CB HPC hypo-fucosylation was investigated in vivo in NOD-SCID IL-2Rγnull (NSG) mice. By isolating fucosylated and non-fucosylated CD34+ cells from CB we show that only fucosylated CD34+ cells are responsible for engraftment in NSG mice. Further, since the proportion of CD34+ cells that are fucosylated in CB is significantly less than in BM and PB, we hypothesize that these combined observations might explain, at least in part, the delayed engraftment observed following CB transplantation. Since engraftment appears to be correlated with the fucosylation of CD34+ cells, we hypothesized that increasing the proportion of CD34+ cells that are fucosylated would improve CB engraftment. Ex vivo treatment with fucosyltransferase (FT)-VI significantly increases the levels of CD34+ fucosylation and, as hypothesized, this was associated with improved engraftment. Ex vivo fucosylation did not alter the biodistribution of engrafting cells, or pattern of long-term, multi-lineage, multi-tissue engraftment. We propose that ex vivo fucosylation will similarly improve the rate and magnitude of engraftment for CB transplant recipients in a clinical setting.
Hematopoiesis; cord blood; transplantation; engraftment; fucosylation
Limited curative treatment options exist for patients with advanced B-lymphoid malignancies, and new therapeutic approaches are needed to augment the efficacy of hematopoietic stem-cell transplantation (HSCT). Cellular therapies, such as adoptive transfer of T cells that are being evaluated to target malignant disease, use mechanisms independent of chemo- and radiotherapy with nonoverlapping toxicities. Gene therapy is employed to generate tumor-specific T cells, as specificity can be redirected through enforced expression of a chimeric antigen receptor (CAR) to achieve antigen recognition based on the specificity of a monoclonal antibody. By combining cell and gene therapies, we have opened a new Phase I protocol at the MD Anderson Cancer Center (Houston, TX) to examine the safety and feasibility of administering autologous genetically modified T cells expressing a CD19-specific CAR (capable of signaling through chimeric CD28 and CD3-ζ) into patients with high-risk B-lymphoid malignancies undergoing autologous HSCT. The T cells are genetically modified by nonviral gene transfer of the Sleeping Beauty system and CAR+ T cells selectively propagated in a CAR-dependent manner on designer artificial antigen-presenting cells. The results of this study will lay the foundation for future protocols including CAR+ T-cell infusions derived from allogeneic sources.
Kebriaei and colleagues report on their phase 1 protocol to examine the safety and feasibility of administering genetically modified autologous T cells expressing a CD19-specific chimeric antigen receptor (CAR) to patients with high-risk B-lymphoid malignancies undergoing autologous hematopoietic stem cell transplantation. CAR was introduced to T cells by nonviral gene transfer, using the Sleeping Beauty system and signals through chimeric CD28 and CD3-ζ.
The symptom burden associated with multiple myeloma (MM) is often severe. Presently, no instrument comprehensively assesses disease-related and treatment-related symptoms in patients with MM. We sought to validate a module of the M. D. Anderson Symptom Inventory (MDASI) developed specifically for patients with MM (MDASI-MM).
The MDASI-MM was developed with clinician input, cognitive debriefing, and literature review, and administered to 132 patients undergoing induction chemotherapy or stem cell transplantation. We demonstrated the MDASI-MM’s reliability (Cronbach α values); criterion validity (item and subscale correlations between the MDASI-MM and the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) and the EORTC MM module (QLQ-MY20)), and construct validity (differences between groups by performance status). Ratings from transplant patients were examined to demonstrate the MDASI-MM’s sensitivity in detecting the acute worsening of symptoms post-transplantation.
The MDASI-MM demonstrated excellent correlations with subscales of the 2 EORTC instruments, strong ability to distinguish clinically different patient groups, high sensitivity in detecting change in patients’ performance status, and high reliability. Cognitive debriefing confirmed that the MDASI-MM encompasses the breadth of symptoms relevant to patients with MM.
The MDASI-MM is a valid, reliable, comprehensive-yet-concise tool that is recommended as a uniform symptom assessment instrument for patients with MM.
Symptoms; Assessment; Validation; Multiple myeloma; MDASI
Results of a phase II clinical trial of leuprolide administered before conditioning chemotherapy in hematopoietic stem cell transplantation patients to reduce the incidence of premature ovarian failure are presented. Leuprolide did not preserve ovarian function in patients who underwent hematopoietic stem cell transplantation using either myeloablative or nonmyeloablative regimens.
Premature ovarian failure occurs in 40%–70% of patients who receive conventional chemotherapy alone. However, the incidence is higher, 70%–100%, in patients who undergo myeloablative chemotherapy with hematopoietic stem cell transplantation (HSCT). Gonadotropin-releasing hormone (GnRH) analogs, such as leuprolide, in a continuous-release formulation, may protect the ovaries from the gonadotoxic effects of chemotherapy. In non-HSCT settings, GnRH analogs have reduced the risk for premature ovarian failure to <10%. We conducted a phase II clinical trial based on the hypothesis that giving leuprolide before conditioning chemotherapy in HSCT patients reduces premature ovarian failure incidence.
Patients and Methods.
Eligible patients were women aged ≤40 years who were HSCT candidates, were premenopausal, and had both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels ≤20 IU/L. Two 22.5-mg leuprolide doses were delivered in 3-month depot i.m. injections, the first within 2 months before HSCT. Patients were monitored for menstruation return, and ovarian function tests (FSH, LH, and estradiol) were done every 2 months starting 90 days after the last leuprolide dose.
Sixty eligible patients were enrolled, 59 underwent HSCT, and 44 were evaluable (median age, 25 years; median follow-up, 355 days). Only seven of 44 patients (16%) regained ovarian function. Of the 33 who received myeloablative regimens, six (18%) regained ovarian function. However, among the 11 who received nonmyeloablative regimens, only one (9%) regained ovarian function (p = .66).
Leuprolide did not preserve ovarian function in patients who underwent HSCT using either myeloablative or nonmyeloablative regimens. Other measures that protect ovarian function need to be investigated.
Gonadotropin-releasing hormone analog; Premature ovarian failure; Ovarian function preservation; Hematopoietic stem cell transplantation
The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-γ receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of γ-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.
Immunology; Issue 72; Cellular Biology; Medicine; Molecular Biology; Cancer Biology; Biomedical Engineering; Hematology; Biochemistry; Genetics; T-Lymphocytes; Antigen-Presenting Cells; Leukemia; Lymphoid; Lymphoma; Antigens; CD19; Immunotherapy; Adoptive; Electroporation; Genetic Engineering; Gene Therapy; Sleeping Beauty; CD19; T cells; Chimeric Antigen Receptor; Artificial Antigen Presenting Cells; Clinical Trial; Peripheral Blood; Umbilical Cord Blood; Cryopreservation; Electroporation
Peripheral blood natural killer (NK) cell therapy for acute myeloid leukemia has shown promise in clinical trials after allogeneic stem cell transplantation (SCT). Cord blood (CB) is another potentially rich source of NK cells for adoptive immune therapy after SCT. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. However, despite expressing normal surface activating and inhibitory NK receptors, CB-derived NK cells have poor cytolytic activity. In this study, we investigate the cellular mechanism and demonstrate that unmanipulated CB-NK cells exhibit an impaired ability to form F-actin immunological synapses with target leukemia cells compared with peripheral blood-derived NK cells. In addition, there was reduced recruitment of the activating receptor CD2, integrin LFA-1, and the cytolytic molecule perforin to the CB-NK synapse site. Ex vivo IL-2 expansion of CB-NK cells enhanced lytic synapse formation including CD2 and LFA-1 polarization and activity. Furthermore, the acquired anti-leukemic function of IL-2-expanded CB-NK cells was validated using a NOD-SCID-IL2Rγnull mouse model. We believe our results provide important mechanistic insights for the potential use of IL-2-expanded CB-derived NK cells for adoptive immune therapy in leukemia.
We examined the effect of donor characteristics on graft failure (<5% donor chimerism within 3 months after transplantation), acute and chronic graft-versus-host disease (GVHD) and survival after unrelated donor reduced intensity conditioning (RIC)transplantation in 709 patients with hematologic malignancies. Donor-recipient pairs were HLA typed at HLA-A, -B, -C, -DRB1 (allele-level). Five hundred and one patients were >95% donor chimerism, 145 patients, 5–95% and 63 patients <5%. The only donor characteristic associated with transplant-outcome was donor-recipient HLA matching. One or two-loci mismatched transplants led to higher grade 2–4 (RR 1.27, p=0.035) and grade 3–4 (RR 1.85, p<0.001) acute GVHD and two-loci mismatched transplants, higher mortality (RR 2.22, p<0.001). Graft failure was higher after transplantation of bone marrow (RR 2.33, p=0.002). Donor age, parity and donor sex match were not associated with transplant-outcome. Donor-recipient HLA-matching is the only donor characteristic predictive for survival after RIC regimens for hematologic malignancies.
The importance of matching at the human leukocyte antigen (HLA) C locus has not been well defined for unrelated umbilical cord blood transplantation. The selection algorithm for umbilical cord blood units generally considers intermediate resolution HLA typing at A and B, and allele-level at DRB1. We aimed to determine the relative importance of matching at HLA-C in addition to current selection criteria.
We used Cox regression to retrospectively examine for the effect of donor-recipient HLA matching on outcomes of 803 single umbilical cord blood transplantations for leukemia (N=727) and myelodysplastic syndrome (N=76). The primary endpoint was transplant-related mortality. HLA typing was performed using molecular techniques with a minimum of intermediate resolution for HLA-A, -B and -C and allele-level for DRB1.
Compared to transplantations matched at HLA-A, -B, -C, -DRB1 (N=69; HR 1.00), transplant-related mortality risks were higher after transplantations matched at HLA-A, -B, -DRB1 and mismatched at HLA-C (N=23;HR 3.97, 95% CI 1.27 – 12.40, p=0.018). Transplant-related mortality risk were also higher after transplantations with a single mismatch at HLA-A or -B, or -DRB1 and mismatched at HLA-C (N=234; HR 1.70 95% CI 1.06 – 2.74, p=0.029) compared to transplantations matched at HLA-C with a single mismatch at HLA-A, -B, or -DRB1 (N=127; HR 1.00). Examining for an overall effect of HLA disparity on transplant-related mortality, risks were higher with units mismatched at two (N=259; HR 3.27 95% CI 1.42 – 7.54, p=0.006), three (N=253; HR 3.34 95% CI 1.45 – 7.71, p=0.005) or four (N=75; HR 3.51 95% CI 1.44 – 8.58, p=0.006) loci compared to matched units (N=69; HR 1.00).
These data suggest that we re-evaluate the current strategy for umbilical cord blood unit selection, by considering matching at HLA-C for units that are matched at HLA-A, -B, -DRB1 or in the presence of a single locus mismatch at HLA-A, -B or DRB1 to minimize mortality risks.
National Cancer Institute, National Heart Lung and Blood Institute and National Institute for Allergy and Infectious Diseases; Scholar in Clinical Research Award, the Leukemia and Lymphoma Society; Heath Resources and Services Administration; Office of Naval Research, United States Department of Navy; Children’s Leukemia Research Association; INSERM grant TGIR.
Improving the therapeutic efficacy of T cells expressing a chimeric antigen receptor (CAR) represents an important goal in efforts to control B-cell malignancies. Recently an intrinsic strategy has been developed to modify the CAR itself to improve T-cell signaling. Here we report a second extrinsic approach based on altering the culture milieu to numerically expand CAR+ T cells with a desired phenotype. For, the addition of IL-21 to tissue culture improves CAR-dependent T-cell effector functions. We used electrotransfer of Sleeping Beauty (SB) system to introduce a CAR transposon and selectively propagate CAR+ T cells on CD19+ artificial antigen-presenting cells (aAPC). When IL-21 was present, there was preferential numeric expansion of CD19-specific T cells which lysed and produced IFN-γ in response to CD19. Populations of these numerically expanded CAR+ T cells displayed an early memory surface phenotype characterized as CD62L+CD28+ and a transcriptional profile of naïve T cells. In contrast, T cells propagated with only exogenous IL-2 tended to result in an overgrowth of CD19-specific CD4+ T cells. Furthermore, adoptive transfer of CAR+ T cells cultured with IL-21 exhibited improved control of CD19+ B-cell malignancy in mice. To provide coordinated signaling to propagate CAR+ T cells, we developed a novel mutein of IL-21 bound to the cell surface of aAPC that replaced the need for soluble IL-21. Our findings demonstrate that IL-21 can provide an extrinsic reprogramming signal to generate desired CAR+ T cells for effective immunotherapy.
IL-21; CD19; Chimeric antigen receptor; T cell; artificial antigen presenting cells
We analysed the results of haematopoietic cell transplantation (HCT) in 30 patients aged 60–78 (median 65) years, with primary myelofibrosis or myelofibrosis evolving from antecedent polycythaemia vera or essential thrombocythaemia. Donors were human leucocyte antigen (HLA)-identical siblings (N = 15) or unrelated individuals (N = 15). Various conditioning regimens were used, ranging from very low intensity (fludarabine plus 2 Gy total body irradiation) to high dose (busulfan plus cyclophosphamide). Stem cell sources were granulocyte colony-stimulating factor mobilized peripheral blood progenitor cells in 29 patients and marrow in one patient. Sustained engraftment was documented in 27 of 30 patients. Day -100 mortality was 13%. With a median follow-up of 22 (range 0.5–69) months, 3-year overall survival and progression-free survival were 45% and 40%, respectively. Currently, 13 patients are surviving. Seven patients died with disease progression at 0.5–22 months, and 10 patients died from other causes at 1.5–37.5 months after HCT. While the selection of older patients for transplantation was probably biased, the present results are encouraging. Motivated older patients with myelofibrosis without substantial comorbid conditions should be offered the option of allogeneic HCT.
myelofibrosis, older patients; haematopoietic cell transplantation
DNA alkylating agents alone or with ionizing radiation have been the preferred conditioning treatment in allogeneic hematopoietic stem cell transplantation (allo-HSCT). In search of less toxic alternatives, we hypothesized that combination of busulfan (Bu), fludarabine (Flu) and clofarabine (Clo) would provide superior efficacy. At low concentrations, these drugs show synergistic cytotoxicity in Bu-resistant AML KBM3/Bu2506 cells. Similar molecular responses were observed in other AML cell lines and in primary explanted AML cells. The [Clo+Flu+Bu] combination activates an intense DNA damage response through the ATM pathway, leading to cell cycle checkpoint activation and apoptosis. Phosphorylations of SMC1 and SMC3, and methylations of histones 3 and 4, are much more pronounced in cells exposed to [Clo+Flu+Bu] than [Clo+Flu], suggesting their relevance in the efficacy of the triple-drug combination. A possible mechanism for these observed synergistic effects involves the capability of [Clo+Flu] to induce histone methylations and subsequent chromatin remodeling, which may render the genomic DNA more accessible to Bu alkylation. The Bu-mediated DNA cross-linking may provide a feedback loop which perpetuates the DNA damage response initiated by [Clo+Flu] and commits the cells to apoptosis. Our results provide a conceptual mechanistic basis for exploring this triple-drug combination in pretransplant conditioning therapy for allo-HSCT.
clofarabine; fludarabine; busulfan; synergistic cytotoxicity; chromatin remodeling
Background: Circulating tumor cells (CTCs) are an independent prognostic factor in metastatic breast cancer (MBC) patients treated by conventional dose chemotherapy. The aim of this study was to determine the role of CTCs and CTCs undergoing epithelial-mesenchymal transition (EMT) in metastatic breast cancer. We used the platform of high-dose chemotherapy (HDCT) and autologous hematopoietic stem cell transplantation (AHSCT) to study the CTCs and CTCs with EMT.
Patients and methods: CTCs were enumerated in 21 MBC patients before apheresis and 1 month after AHSCT. CD34-depleted apheresis products were analyzed for CD326+ epithelial and Aldefluor+ cancer stem cells (CSC) by flow cytometry and were depleted of CD45+ cells and assessed for EMT-inducing transcription factors (EMT-TF) by quantitative RT-PCR.
Results: Patients with ≥ 5 CTCs/7.5 mL of peripheral blood 1 month after AHSCT had shorter progression-free survival (PFS) (P=0.02) and overall survival (OS) (P=0.02). Patients with apheresis products containing high percentages of CD326+ epithelial cells or overexpressing EMT-TF had shorter PFS. In multivariate analysis, low percentage of CD326+ epithelial cells and response to HDCT with AHSCT were associated with longer PFS, whereas lower CTCs after AHSCT was associated with longer OS. High CTCs, 1 month after AHSCT correlated with shorter PFS and OS in MBC patients undergoing HDCT and AHSCT, while CTCs with EMT and CSCs phenotype in apheresis products are associated with relapse.
Conclusion: Our data suggest that CTC and CTCs with EMT are prognostic in MBC patients undergoing HDCT followed by AHSCT.
metastatic breast cancer; circulating tumor cells; epithelial-mesenchymal transition; high-dose chemotherapy; autologous hematopoietic stem cell transplantation.
Cord blood (CB) is used increasingly in transplant patients lacking sibling or unrelated donors. A major hurdle to the use of CB is low cell dose which is largely responsible for elevated risk of graft failure and significantly delayed neutrophil and platelet engraftment. Since a positive correlation has been demonstrated between the total nucleated cell (TNC) and CD34+ cell dose transplanted and time to neutrophil and platelet engraftment, strategies to increase these measures are under development. One strategy includes the ex vivo expansion of CB mononuclear cells (MNC) with mesenchymal stem cells (MSC) in a cytokine cocktail. We demonstrate that this strategy can be further improved if CD3+ and/or CD14+ cells are first depleted from the CB MNC prior to ex vivo expansion. Ready translation of this depletion strategy to improve ex vivo CB expansion in the clinic is feasible since clinical-grade devices and reagents are available. Ultimately the goal of improving TNC and CD34+ transplant doses is to further improve the rate of neutrophil and platelet engraftment in CB recipients.
Cord blood; ex vivo expansion; inhibitory “accessory” cells; mononuclear cells
Umbilical cord blood (UCB) is increasingly considered as an alternative to peripheral blood progenitor cells (PBPC) or bone marrow (BM), especially when a HLA-matched adult unrelated donor is not available.
In order to establish the appropriateness of current graft selection practices, we retrospectively compared leukemia-free survival and other outcomes for each graft source in patients aged >16 years transplanted for acute leukemia using Cox regression. Data were available on 1525 patients transplanted between 2002 and 2006 using UCB (n=165), PBPC (n=888) and BM (n=472). UCB units were matched at HLA-A and B at antigen level and DRB1 at allele level (n=10) or mismatched at one (n=40) or two antigens (n=115). PBPC and BM grafts from unrelated adult donors were matched for allele-level HLA-A, B, C and DRB1 (n=632; n=332) or mismatched at one locus (n=256; n=140).
Leukemia-free survival after UCB transplantation was comparable to that observed after 8/8 and 7/8 allele-matched PBPC or BM transplantation. Transplant-related mortality, however, was higher after UCB transplantation compared to 8/8 allele-matched PBPC (HR 1.62, p<0.01) or BM (HR 1.69, p<0.01). Grades 2–4 acute and chronic graft-versus-host disease were lower in UCB recipients compared to allele-matched PBPC (HR 0.57, p<0.01 and HR 0.38, p<0.01, respectively), while chronic and not acute graft-versus-host disease was lower after UCB compared to allele-matched BM transplantation (HR 0.63, p=0.01).
Together, these data support the use of UCB for adults with acute leukemia when an HLA-matched unrelated adult donor is lacking and when transplant is urgently needed.
We analyzed the outcomes of 283 patients receiving unrelated donor allogeneic hematopoietic cell transplantation for non-Hodgkin lymphoma (NHL) facilitated by the Center for International Blood & Marrow Transplant Research /National Marrow Donor Program (CIBMTR/NMDP) between 1991 and 2004. All patients received myeloablative conditioning regimens. The median follow-up of survivors is 5 years. Seventy-three (26%) patients are alive. The day 100 probability of death from all causes is estimated at 39%. The cumulative incidence of developing grade III-IV acute graft-versus-host disease (GVHD) at day 100 is 25%. The estimated five-year survival and failure free survival are 24% (95% CI; 19–30) and 22% (95% CI; 17–28) respectively. Factors adversely associated with overall survival included increasing age, decreased performance status, and refractory disease. Follicular lymphoma and Peripheral T-cell lymphoma had improved survival compared to aggressive B-cell lymphomas. Factors adversely associated with progression free-survival included performance status, histology and disease status at transplant. Long-term failure-free survival is possible following unrelated donor transplantation for NHL, although early mortality was high in this large cohort.
Lymphoma; unrelated donor; myeloablative
Infections due to post transplant immune deficiency is a major problem following allogeneic stem cell transplantation, particularly in patients receiving cord blood or T-cell depleted haploidentical transplants. We evaluated the incidence and type of infectious complications that occurred in these two types of transplant for 65 patients, 37 cord blood and 28 haploidentical, who received the same conditioning regimen: fludarabine, melphalan and thiotepa. While incidence of infections appeared similar in both types of transplant, viral infections were more frequent than bacterial or fungal infections and were the most common cause of death in both groups. Patients in the haploidentical group were 1.7 times (95% CI: 1.1 to 2.5) more likely to have a viral infection (p=0.013). Bacterial, fungal and CMV infections still quite frequent but contributed less to mortality. Pneumonia was the most common clinical syndrome and the number one cause of death in both groups. Both pneumonia and bacteremia occurred within the first 100 days in the majority of HSCT patients while CBT patients had a bimodal distribution, with more than one third of episodes after 6 months post transplant.
Infectious complications; cord blood transplantation; T-cell depleted haploidentical stem cell transplantation
Allogeneic hematopoietic stem-cell transplantation can cure some patients with high-risk B-cell malignancies, but disease relapse following transplantation remains a significant problem. One approach that could be used to augment the donor T cell-mediated anti-tumor effect is the infusion of allogeneic donor-derived T cells expressing a chimeric antibody receptor (CAR) specific to the B-cell antigen CD19. However, the use of such cells might result in toxicity in the form of graft-versus-host disease mediated by CD19-specific (CD19-CAR) T cells possessing alloreactive endogenous T cell receptors. We therefore investigated whether non-alloreactive tumor-specific human T cells could be generated from peripheral blood mononuclear cells of healthy donors by the combination of CD19-redirection via CAR expression and subsequent alloanergization by allostimulation and concomitant blockade of CD28-mediated costimulation. Alloanergization of CD19-CAR T cells resulted in efficient and selective reduction of alloresponses in both CD4+ and CD8+ T cells including allospecific proliferation and cytokine secretion. Importantly, T-cell effector functions including CAR-dependent proliferation and specific target cytolysis and cytokine production were retained after alloanergization. Our data supports the application of CD19-redirection and subsequent alloanergization to generate allogeneic donor T cells for clinical use possessing increased anti-tumor activity, but limited capacity to mediate graft-versus-host disease. Therapy with such cells could potentially reduce disease relapse after allogeneic transplantation without increasing toxicity, thereby improving the outcome of patients undergoing allogeneic transplantation for high-risk B-cell malignancies.
Cellular Immunotherapy; Anergy; Chimeric antigen receptor; CD19; Gene therapy; allogeneic stem cell transplantation
While a combination of IV busulfan (Bu) and fludarabine (Flu) is a safe, reduced-toxicity conditioning program for AML/MDS, recurrent leukemia post transplantation remains a problem. To enhance the conditioning regimen’s antileukemic effect we decided to supplant Flu with clofarabine (Clo), and assayed the interactions of these nucleoside analogs alone and in combination with Busulfan (Bu) in Bu-resistant human cell lines in vitro. We found pronounced synergy between each nucleoside and the alkylator but even more enhanced cytotoxic synergy when the nucleoside analogs were combined prior to exposing the cells to Bu. We then designed a 4-arm clinical trial in patients with myeloid leukemia undergoing allogeneic stem cell transplantation (allo-SCT); Patients were adaptively randomized as follows: Arm I - Clo:Flu 10:30 mg/m2, Arm II - 20:20 mg/m2, Arm III - 30:10 mg/m2, and Arm IV - single-agent Clo at 40 mg/m2. The nucleoside analog(s) were/was infused over one hour once daily for 4 days, followed on each day by Bu, infused over 3 hours to a pharmacokinetically targeted daily AUC of 6,000 μMol-min +/− 10%. Fifty-one patients have been enrolled with a minimum follow-up exceeding 100 days. There were 32 males and 19 females with a median age of 45 years (range: 6-59). Nine patients had CML (BC: 2, second AP: 3, and tyrosine-kinase inhibitor refractory first CP: 4). Forty two patients had AML: 14 were induction failures, 8 in first chemotherapy-refractory relapse, 7 in untreated relapse, 3 in second or subsequent relapse, 4 were in second CR and 3 in second CR without platelet recovery (CRp), 2 were in high-risk CR1. Finally, 1 patient was in first CRp. Graft vs host disease- (GVHD) prophylaxis was tacrolimus and mini-MTX, and those who had an unrelated or one Ag-mismmatched donor received low-dose rabbit-ATG (Thymoglobulin™). RESULTS: All patients engrafted. Forty-one patients had active leukemia at the time of transplant, and 35 achieved CR (85%). Twenty of the 42 AML patients and 5 of 9 CML patients are alive with a projected median overall survival of 23 months. Marrow and blood (T-cell) chimerism studies at day +100 revealed that both in the lower dose Clo groups (groups 1+2) and the higher dose Clo groups (groups 3+4) the patients had a median of 100% donor (T-cell)-derived DNA. There has been no secondary graft failure. In the first 100 days one patient died of pneumonia, and one of liver GVHD. We conclude that 1) Clo±Flu with IV Bu as pretranslant conditioning is safe in high-risk myeloid leukemia patients, 2) Clofarabine is sufficiently immunosuppressive to support allo-SCT in myeloid leukemia, and 3) the median overall survival (OS) of 23 months in this high-risk patient population is encouraging. Additional studies to evaluate the antileukemic efficacy of Clo±Flu with IV Bu as pretransplant conditioning therapy are warranted.
Clofarabine; Fludarabine; IV Busulfan; CML; AML; MDS; Allogeneic Stem Cell Transplantation
Busulfan (Bu) is a DNA-alkylating drug used in myeloablative pretransplant conditioning therapy for patients with myeloid leukemia (ML). A major obstacle to successful treatment is cellular Bu-resistance. To investigate the possible contribution of DNA hypermethylation to Bu-resistance, we examined the cytotoxic activity of combined 5-aza-2′-deoxycytidine (DAC) and Bu. Exposure of Bu-resistant B5/Bu2506 ML cells to 0.5 μM DAC resulted in G2-arrest and apoptosis. The observed G2-arrest was associated with hypomethylation and subsequent expression of epigenetically controlled genes including p16INK4A, activation of the p53 pathway, and phosphorylation of CDC2. The DAC-mediated apoptosis was partly due to hypomethylation and up-regulation of XAF1, which resulted in down-regulation of the anti-apoptotic proteins XIAP, cIAP1 and cIAP2. The pro-apoptotic PUMA and BNIP3 proteins were up-regulated while pro-survival STAT3 and c-MYC were suppressed. Combination of 0.05 μM DAC and 5 μg/ml Bu resulted in synergistic cytotoxicity, which was associated with PARP1 cleavage and activation of caspases 3 and 8, suggesting induction of an apoptotic response. P53 inhibition in B5/Bu2506 cells using pifithrin-α alleviated these effects, suggesting a role for p53 therein; this observation was supported by the relative resistance of p53-null K562 cells to [DAC+Bu] combinations and by the effects of an anti-p53 shRNA on the OCI-AML3 cell line. We conclude that the synergistic effects of [DAC+Bu] are p53-dependent and involve cell-cycle arrest, apoptosis induction and down-regulation of pro-survival genes. Our results suggest that, depending on tumor p53 status, incorporation of DAC might synergistically improve the cytoreductive efficacy of Bu-based pre-transplant regimen in patients with ML.
busulfan; 5-aza-2′-deoxycytidine; myeloid leukemia; drug resistance; DNA methylation; cell cycle signaling; apoptosis
To test the hypothesis that the outcome of hematopoietic stem cell grafts is at least partially determined by the cellular composition of the graft, the National Marrow Donor Program analyzed the correlation of cellular phenotypes of unrelated grafts with graft outcome. Samples from 94 bone marrow (BM) and 181 peripheral blood progenitor cell (PBPC) grafts for transplantations at 40 U.S. transplant centers between 2003 and 2005 were analyzed at a single immunophenotyping reference laboratory. Samples were shipped from transplant centers upon receipt of graft. Graft cellular composition included analysis of leukocyte total cell numbers, and subsets of myeloid [CD34+, CD34+ CD38−], lymphoid [CD3+, CD3+ CD4+, CD3+ CD8+], and activated lymphoid cells [CD3+ CD25+, CD3+ CD69+, CD3+ HLA-DR+] coexpressing CD3+. There was substantial variability in the cellular composition of BM and PBPC grafts before and after graft processing by red blood cell (RBC) removal or plasma depletion in preparation for transplant. With BM grafts, cellular composition was not associated with hematopoietic recovery, graft-versus-host disease (GVHD), or survival. With PBPC grafts, survival rates were higher with CD34 + >5 × 106/kg, 59% compared to 34% with CD34+ ≤5 × 106/kg at 1-year. Platelet recovery was higher with PBPC containing CD3+ CD8+ >8 × 107/kg. Neutrophil recovery or GVHD could not be predicted by any cellular subsets of PBPC grafts. Though survival was superior with PBPC grafts containing >5 × 106 CD34+/ kg an optimal graft mix of myeloid, lymphoid and activated lymphoid subsets was not identified.
Graft composition; unrelated donor transplant; hematopoietic recovery; overall survival
Incorporation of the anti-CD20 monoclonal antibody rituximab into front-line regimens for diffuse large B-cell lymphoma (DLBCL) has resulted in improved survival. Despite this progress, many patients develop refractory or recurrent DLBCL and then receive autologous hematopoietic stem cell transplantation (AuHCT). It is unclear to what extent pre-transplant exposure to rituximab affects outcomes following AuHCT. Outcomes of 994 patients receiving AuHCT for DLBCL between 1996 and 2003 were analyzed according to whether rituximab was (n=176, “+R” group) or was not (n=818, “ −R” group) administered with front-line or salvage therapy prior to AuHCT. The +R group had superior progression-free survival (50% versus 38%, p=0.008) and overall survival (57% versus 45%, p=0.006) at 3 years. Platelet and neutrophil engraftment were not affected by exposure to rituximab. Non-relapse mortality (NRM) did not differ significantly between the +R and −R groups. In multivariate analysis, the +R group had improved progression-free survival (relative risk of relapse/progression or death 0.64, p<0.001) and improved overall survival (relative risk of death of 0.74, p=0.039). We conclude that pre-transplant rituximab is associated with a lower rate of progression and improved survival following AuHCT for DLBCL, with no evidence of impaired engraftment or increased NRM.
autologous hematopoietic stem cell transplantation; lymphoma; rituximab
We evaluated effects of graft transport time on outcomes after transplantation of 938 unrelated donor bone marrow (BM) or 507 peripheral blood progenitor cells (PBPC) in patients with acute or chronic leukemia and myelodysplasia. BM grafts were collected at 107 centers and PBPC, 89 centers. Median time from end of collection to infusion was 14 hours for BM and 15 hours for PBPC. Platelet recovery was less likely in BM recipients when the interval from end of collection to receipt at transplant center was ≥ 20 hours (odds ratio 0.47, p=0.010) and when the interval from receipt to infusion was ≥ 6 hours (odds ratio 0.57, p=0.001). Mortality rates were higher in recipients of HLA-matched BM when the interval from end of collection to receipt at transplant center was ≥ 20 hours (relative risk 2.67, p<0.001) after adjustment for other significant prognostic factors. Mortality after HLA-mismatched BM transplants was not associated with transport time. Transport times had no demonstrable effect on outcomes after PBPC transplants. These data support a general review of current transport procedures, especially for BM grafts requiring longer transport time and every effort made to minimize time from collection to infusion.
transport of unrelated donor grafts; transport times; transplant-outcomes