Pretransplant Conditioning Therapy Research Articles

The practice of ontogenetically determined movements of standing up among children and adolescents at the Department of Hematopoietic Stem Cell Transplantation as a preventive measure for orthostatic hypotension

Hematopoietic stem cell transplantation (HSCT) requires a long hospitalization during which patients have to stay in transplantation wards, mainly in their single isolation rooms. One of the main goals of rehabilitation for HSCT patients is to prevent physical activity deficit by the introduction of an appropriate exercise programme and the monitoring of exercise adherence. The aim of our study was to evaluate the effectiveness of the prescribed exercise programme based on basic locomotor patterns to prevent physical activity deficit. The interim results of the prospective randomized controlled study included data from 20 patients. The study was approved by the Independent Ethics Committee and the Scientific Council of the 1Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of Ministry of Healthcare of the Russian Federation. An experimental group included 11 patients aged 8–16 years old (the median age was 12 years old); a control group included 9 patients aged 5–11 years old (the median age was 8 years old). Patients from the experimental group were preliminarily taught a set of exercises aimed at practicing ontogenically determined standing up which they should further perform during a day in their isolation rooms. The control group patients received recommendations on how to maintain physical activity in their isolation rooms. Their physical activity during a day was then monitored by the researchers. All study subjects underwent an orthoclinostatic test (i.e., transition from lying to standing and back) for the evaluation of the autonomic nervous system function on Day 1 of pre-transplant conditioning therapy and at the time of discharge from the HSCT Department. During the entire stay at the HSCT Department, the patients from both groups were daily assessed using the ADL (activities of daily living) scale. At the time of discharge from the HSCT Department, the experimental group patients showed more adaptive autonomic nervous system responses than the control group patients. Our interim analysis showed that the patients from the experimental group had higher scores of activities of daily living than the control patients. Orthoclinostatic test results confirmed that patients undergoing HSCT for acute leukemia were predisposed to develop maladaptive conditions or even orthostatic hypotension. A set of exercises including those for the training of ontogenetically determined process of standing up was shown to be an effective tool for the prevention of insufficient autonomic nervous system responses. Even a short (30 min) face-to-face session with a physical therapist significantly increased a patient's daily physical activity.

Genetically engineered pig kidney transplantation in a brain-dead human subject.

In September 2021, a kidney (with donor-specific thymic tissue) from an α1, 3-galactosyltransferase gene-knockout (GTKO) pig was transplanted into the groin (with anastomoses to the femoral vessels) of a brain-dead subject by a surgical team at New York University Langone Health (NYU). It was reported to function immediately, passing urine and excreting creatinine. The experiment was terminated after 54h and, during this period, the kidney did not show macroscopic features of rejection. Does this experiment provide information not available to us previously and does it move the field forward to clinical trials? The information provided was very limited, but the following points are worthy of note. (i) Numerous in vivo studies in nonhuman primates have predicted that the pig kidney would function immediately. (ii) Numerous in vitro studies have predicted that a GTKO pig kidney would not be rejected within the first few days after transplantation into a human subject. (iii) GTKO kidneys are not optimal for clinical transplantation, and the transplantation of a triple-knockout (TKO) pig kidney would have been more relevant. (iv) There was no purpose in transplanting a "thymokidney" without pre-transplant conditioning therapy and follow-up for several months. (v) Because the native kidneys were retained, it is difficult to determine whether the function of the graft was sufficient to support life. (vi) The experiment was announced to the media rather than published in a peer-reviewed medical journal (although hopefully this will follow), suggesting that it was primarily carried out to gain attention to the great potential of xenotransplantation (and/or possibly to NYU). In this respect the experiment was successful. Because of the very limited period of time for which a brain-dead subject can be maintained in a metabolically and hemodynamically stable state, the value of experiments in such subjects will remain very limited. It is hoped that any future similar experiments will be planned to be more relevant to the clinical situation. Nevertheless, the report has stimulated public attention towards xenotransplantation which, unless there is an adverse response to what some might consider to be a bizarre experiment, should be of significant benefit to future progress.

TAT-MYC Recombinant Fusion Protein Enhances Hematopoietic Stem Cell Graft Performance and Immune Cell Reconstitution after Transplantation

The long interval between hematopoietic stem cell transplantation (HSCT) and hematopoietic engraftment and immune reconstitution is a dangerous time for patients. Although neutrophil counts usually rise within several weeks following stem cell transplantation, recovery of functional T and B cell repertoires can often take months; during this period patients are at risk for severe infections, and graft vs. tumor effects may be attenuated. We have examined the use of a recombinant fusion protein consisting of the N-terminal 9 amino acid segment of the HIV TAT protein transduction domain (PTD) fused to the MYC protein as a means of enhancing stem cell graft performance.Methods: Coding sequences for the 9-amino acid TAT PTD and the cDNA encoding MYC were ligated in-frame and cloned into the pET101/D-Topo vector for expression in E Coli. The fusion protein, which we designated TBX-4000, was purified to homogeneity, analyzed for contaminants, and prepared to clinical grade for administration in accordance with good manufacturing practices.Results: Following a 1-hour incubation of TBX-4000 with human cord blood cells, the chimeric protein appeared in the nucleus of target cells within 2 hours, but was rapidly metabolized, vanishing by 72 hours. Whole murine marrow and purified murine LSK+ cells that were incubated with TBX-4000 significantly out-performed control cells in competitive engraftment experiments in immune deficient recipient mice, even at ratios of 1:9 treated to control cells. Significantly higher numbers of T and B cells were recovered from both peripheral blood and splenic tissue of irradiated immune deficient mice transplanted with stem cell grafts that had been cultured for 60 minutes with TBX-4000, relative to control mice. TBX-4000 cultured cells demonstrate resistance to apoptosis induced by Granzyme B, which may account for their hardiness during engraftment in marrow niches recently exposed to the stress of pre-transplant conditioning therapy. Extensive toxicologic studies demonstrated that prolonged exposure to TBX-4000 does not cause malignant transformation of hematopoietic cells in culture, and does not lead to tumor formation in mice repeatedly injected with the recombinant protein. Mice transplanted with stem cells incubated with TBX-4000 did not develop leukemia or solid tumors during 4 months of follow-up, or following three “generations” of successive, serial transplantation. Because TBX-4000 augments immune reconstitution following hematopoietic stem cell transplantation and may stimulate graft vs. host diseases reactions, T and B cell depletion of the graft will be performed in pilot studies in humans to prevent this complication.Conclusion: Brief exposure to the TAT-MYC recombinant fusion protein enhances the performance of hematopoietic stem cells in a pre-clinical transplantation model. The brief presence of the protein protects engrafting cells from apoptotic stimuli, and does not lead to transformation of the cells following transplantation. The efficacy and safety of TBX-4000 in our pre-clinical studies serve as the basis for currently planned clinical trials of this chimeric protein in patients undergoing stem cell transplantation. DisclosuresGregory:Taiga Biotechnologies, Inc.: Employment, Equity Ownership. Turner:Taiga Biotechnologies, Inc.: Employment, Equity Ownership. Refaeli:Taiga Biotechnologies, Inc.: Employment, Equity Ownership.

A Randomized Study of Pretransplant Conditioning Therapy for AML/MDS with Fludarabine ± Clofarabine and Once Daily IV Busulfan with Allogeneic Hematopoietic Transplantation for AML and MDS

A Randomized Study of Pretransplant Conditioning Therapy for AML/MDS with Fludarabine ± Clofarabine and Once Daily IV Busulfan with Allogeneic Hematopoietic Transplantation for AML and MDS

Long-Term Outcomes after Treatment with Clofarabine ± Fludarabine with Once-Daily Intravenous Busulfan as Pretransplant Conditioning Therapy for Advanced Myeloid Leukemia and Myelodysplastic Syndrome

Pretransplant conditioning regimens critically determine outcomes in the setting of allogeneic stem cell transplantation (allo-SCT). The use of nucleoside analogs such as fludarabine (Flu) in combination with i.v. busulfan (Bu) has been shown to be highly effective as a pretransplant conditioning regimen in acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS). Because leukemia relapse remains the leading cause of death after allo-SCT, we studied whether clofarabine (Clo), a nucleoside analog with potent antileukemia activity, can be used to complement Flu. In a preliminary report, we previously showed the safety and efficacy of Clo ± Flu with i.v. Bu in 51 patients with high-risk AML, CML, and MDS. The study has now been completed, and we present long-term follow-up data on the entire 70-patient population, which included 49 (70%), 8 (11%), and 13 (19%) patients with AML, MDS, and CML, respectively. Thirteen patients (19%) were in complete remission, and 41 patients (59%) received matched unrelated donor grafts. Engraftment was achieved in all patients. Sixty-three patients (90%) achieved complete remission. There were no deaths reported at day +30, and the 100-day nonrelapse mortality rate was 4% (n = 3). Thirty-one percent of patients (n = 22) developed grades II to IV acute graft-versus-host disease, and the median overall survival and progression-free survival times were 2.4 years and .9 years, respectively. Our results confirm the safety and overall and progression-free survival advantage of the arms with higher Clo doses and lower Flu doses, which was most prominent in the AML/MDS group.

The pretransplant systemic metabolic profile reflects a risk of acute graft versus host disease after allogeneic stem cell transplantation.

Allogeneic stem cell transplantation is used in the treatment of younger patients with severe hematological diseases, especially hematological malignancies, and acute graft versus host disease (GVHD) is then an important immune-mediated posttransplant complication. Several risk factors for acute GVHD have been identified, including pretransplant factors that possibly influence the posttranspant course through their effects on host immunocompetent cells. Metabolic regulation is important for immunoregulation, and we therefore investigated whether the pretransplant metabolic status of allotransplant recipients was associated with later acute GVHD. In our population-based study we investigated the systemic (serum) metabolic profile for 86 consecutive allotransplant recipients. The samples were collected before start of the pretransplant conditioning therapy. Patients who developed later acute GVHD especially showed altered pretransplant amino acid metabolism, including (1) altered metabolism of immunoregulatory branched chain amino acids (leucine, isoleucine and valine); and (2) altered levels of potentially proinflammatory tyrosine metabolites (p-cresol sulphate, 3-phenylpropionate) formed by the gastrointestinal microbial flora. However, isobutyrylcarnitine and propyonylcarnitine levels were also altered; the carnitines are important for the transport of fatty acids and may also be important for the release of immunoregulatory cytokines in allotransplant recipients. These metabolic alterations were associated with an ongoing pretransplant acute phase reaction or early hematopoietic/immune reconstitution. Thus, allotransplant recipients developing acute GVHD showed altered preconditioning/pretransplant levels of several immunoregulatory metabolites. Our hypothesis is that these metabolites alter or activate recipient immunocompetent cells and thereby enhance or initiate anti-recipient immune reactivity.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-015-0880-x) contains supplementary material, which is available to authorized users.

The histone deacetylase inhibitor SAHA sensitizes acute myeloid leukemia cells to a combination of nucleoside analogs and the DNA-alkylating agent busulfan

Fludarabine (Flu), clofarabine (Clo) and busulfan (Bu) are used in allogeneic hematopoietic stem cell transplant (allo-HSCT). We reported that combining [Flu + Clo + Bu] had a synergistic cytotoxicity in AML cells. We hypothesized that combining [Flu + Clo + Bu] with the histone deacetylase inhibitor SAHA will further enhance cytotoxicity. We exposed the acute myeloid leukemia (AML) cell lines KBM3/Bu2506 and OCI-AML3 to Flu, Clo, Bu and SAHA alone and in various combinations. [Flu + Clo + Bu + SAHA] resulted in synergistic cytotoxicity, which can be attributed to (1) activated DNA-damage response and cell cycle checkpoint activation through the ATM–CHK2–P53 (or P73) pathway or ATM–CHK2–cdc25–cdc2 pathway, (2) histone modifications and (3) activated apoptosis pathway. The [Flu + Clo + Bu + SAHA] combination causes mitochondrial outer membrane permeabilization, leakage of cytochrome c and Smac/Diablo into the cytosol with caspase activation, and release of apoptosis-inducing factor (AIF) into the nucleus resulting in nuclear fragmentation and cell death. These results provide a mechanistic basis for using SAHA in future clinical trials with double nucleoside analog-busulfan combinations in pretransplant conditioning therapy.

Epigenetic modifiers enhance the synergistic cytotoxicity of combined nucleoside analog-DNA alkylating agents in lymphoma cell lines

Hematopoietic stem cell transplantation is used for treatment of lymphoma. In an attempt to design an efficacious and safe prehematopoietic stem cell transplantation 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 ~IC(10) 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 (0.6 μM) to this combination resulted in a marked (~65%) growth inhibition. The cytotoxicity of these combinations correlates with the activation of the ataxia telangiectasia mutated-CHK2 pathway, phosphorylation of KRAB-associated protein-1, 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 to (BMG+suberoylanilide hydroxamic acid) further enhances cell killing. Overall, BMG combinations are synergistically cytotoxic to lymphoma cells. Epigenetic changes induced by suberoylanilide hydroxamic acid and 5-aza-2'-deoxycytidine further enhance the cytotoxicity. This study provides a rationale for an ongoing clinical trial in our institution using (BMG+suberoylanilide hydroxamic acid) as pre-hematopoietic stem cell transplantation conditioning for lymphoma.

Autologous Hematopoietic Stem Cell Transplantation for Multiple Myeloma without Cryopreservation

High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation is considered the standard of care for multiple myeloma patients who are eligible for transplantation. The process of autografting comprises the following steps: control of the primary disease by using a certain induction therapeutic protocol, mobilization of stem cells, collection of mobilized stem cells by apheresis, cryopreservation of the apheresis product, administration of high-dose pretransplant conditioning therapy, and finally infusion of the cryopreserved stem cells after thawing. However, in cancer centers that treat patients with multiple myeloma and have transplantation capabilities but lack or are in the process of acquiring cryopreservation facilities, alternatively noncryopreserved autologous stem cell therapy has been performed with remarkable success as the pretransplant conditioning therapy is usually brief.

The Kinetics of Early T and B Cell Immune Recovery after Bone Marrow Transplantation in RAG-2-Deficient SCID Patients

The kinetics of T and B cell immune recovery after bone marrow transplantation (BMT) is affected by many pre- and post-transplant factors. Because of the profoundly depleted baseline T and B cell immunity in recombination activating gene 2 (RAG-2)-deficient severe combined immunodeficiency (SCID) patients, some of these factors are eliminated, and the immune recovery after BMT can then be clearly assessed. This process was followed in ten SCID patients in parallel to their associated transplant-related complications. Early peripheral presence of T and B cells was observed in 8 and 4 patients, respectively. The latter correlated with pre-transplant conditioning therapy. Cells from these patients carried mainly signal joint DNA episomes, indicative of newly derived B and T cells. They were present before the normalization of the T cell receptor (TCR) and the B cell receptor (BCR) repertoire. Early presentation of the ordered TCR gene rearrangements after BMT occurred simultaneously, but this pattern was heterogeneous over time, suggesting different and individual thymic recovery processes. Our findings early after transplant could suggest the long-term patients' clinical outcome. Early peripheral presence of newly produced B and T lymphocytes from their production and maturation sites after BMT suggests donor stem cell origin rather than peripheral expansion, and is indicative of successful outcome. Peripheral detection of TCR excision circles and kappa-deleting recombination excision circles in RAG-2-deficient SCID post-BMT are early markers of T and B cell reconstitution, and can be used to monitor outcome and tailor specific therapy for patients undergoing BMT.

Development and Validation of a Test Dose Strategy for Once-Daily i.v. Busulfan: Importance of Fixed Infusion Rate Dosing

Intravenous (i.v.) busulfan (Bu) administered once daily in myeloablative transplant regimens is convenient, effective, and relatively well tolerated. Therapeutic drug monitoring is recommended as nonrelapse mortality increases when daily exposure, as determined by the area under the plasma concentration versus time curve (AUC), exceeds 6000 μM·min. We describe sequential studies to achieve accurate prediction of treatment doses of Bu based on the kinetics of a smaller test dose. A total of 335 patients with hematologic malignancies were given daily i.v. Bu 3.2 mg/kg × 4 and fludarabine 50 mg/m(2) × 5. Pharmacokinetic monitoring was conducted for both the test dose and first treatment dose of Bu (day -5). Three different test dose schedules were evaluated: 12 mg Bu administered over 20 minutes, 0.8 mg/kg over 3 hours, and 0.8 mg/kg infused at 80 mg/h. The 3.2 mg/kg treatment doses were infused over a fixed time of 3 hours for the first 2 test dose trials and at a fixed rate of 80 mg/h for the final protocol. All test dose infusions were on day -7. In the first 2schedules, Bu administered over a fixed time had significantly higher clearance for the test dose compared with the treatment dose. However, when both the test and the treatment doses were administered at the same infusion rate, clearance of the drug between the 2 dosing days was equivalent. Predicted day -5 AUC (AUC(-5)) showed a high linear correlation (r(2) = 0.74) to the actual AUC(-5). The error of these predictions was <20% in 98% of patients and <10% in 80%. In 24 individuals, the test dose predicted an AUC >5500μM·min; therefore, the first Bu treatment dose was reduced to a desired target AUC. All adjusted doses fell within 20% of the targeted exposure. We conclude that a test dose strategy for therapeutic drug monitoring of daily i.v. Bu is accurate if the test and treatment doses are infused at the same rate. This approach allows targeting of therapeutic doses of Bu to desired levels and the potential for improved safety and efficacy.

Clofarabine ± Fludarabine with Once Daily i.v. Busulfan as Pretransplant Conditioning Therapy for Advanced Myeloid Leukemia and MDS

Although a combination of i.v. busulfan (Bu) and fludarabine (Flu) is a safe, reduced-toxicity conditioning program for acute myelogenous leukemia/myelodysplastic syndromes (AML/MDS), recurrent leukemia posttransplantation 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 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/m(2), Arm II-20:20 mg/m(2), Arm III-30:10 mg/m(2), and Arm IV-single-agent Clo at 40 mg/m(2). The nucleoside analog(s) were/was infused over 1 hour once daily for 4 days, followed on each day by Bu, infused over 3 hours to a pharmacokinetically targeted daily area under the curve (AUC) of 6000 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 chronic myeloid leukemia (CML) (BC: 2, second AP: 3, and tyrosine-kinase inhibitor refractory first chronic phase [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 complete remission (CR), and 3 in second CR without platelet recovery (CRp), 2 were in high-risk CR1. Finally, 1 patient was in first CRp. Graft-versus-host disease (GVHD) prophylaxis was tacrolimus and mini-methorexate (MTX), and those who had an unrelated or 1 antigen-mismatched donor received low-dose rabbit-ATG (Thymoglobulin™). 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 (OS) 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, 1 patient died of pneumonia, and 1 of liver GVHD. We conclude that (1) Clo ± Flu with i.v. Bu as pretransplant 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 OS of 23 months in this high-risk patient population is encouraging. Additional studies to evaluate the antileukemic efficacy of Clo ± Flu with i.v. Bu as pretransplant conditioning therapy are warranted.

The synergistic cytotoxicity of clofarabine, fludarabine and busulfan in AML cells involves ATM pathway activation and chromatin remodeling

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/Bu250 6 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.

Interstrand crosslink inducing agents in pretransplant conditioning therapy for hematologic malignancies

Despite successful molecularly targeted, highly specific, therapies for hematologic malignancies, the DNA interstrand crosslinking agents, which are among the oldest and least specific cytotoxic drugs, still have an important role. This is particularly true in stem cell transplantation, where virtually every patient receives conditioning therapy with a DNA-alkylating agent-based program. However, due to concern about serious additive toxicities with combinations of different alkylating drugs, the last several years have seen nucleoside analogs, whose cytotoxic action follows vastly different molecular pathways, introduced in combination with alkylating agents. The mechanistic differences paired with different metabolic pathways for the respective drugs have clinically translated into increased safety without appreciable loss of antileukemic activity. In this report, we review pre-clinical evidence for synergistic antileukemic activity when nucleoside analog(s) and DNA-alkylating agent(s) are combined in the most appropriate manner(s), without a measurable decrease in clinical efficacy compared with the more established alkylating agent combinations. Data from our own laboratory using combinations of fludarabine, clofarabine, and busulfan as prototype representatives for these respective classes of cytotoxic agents are combined with information from other investigators to explain how the observed molecular events will result in greatly enhanced synergistic cytotoxicity. We further present possible mechanistic pathways for such desirable cytotoxic synergism. Finally, we propose how this information-backed hypothesis can be incorporated in the design of the next generation conditioning therapy programs in stem cell transplantation to optimize antileukemic efficacy while still safeguarding patient safety.

Prochymal Improves Response Rates In Patients With Steroid-Refractory Acute Graft Versus Host Disease (SR-GVHD) Involving The Liver And Gut: Results Of A Randomized, Placebo-Controlled, Multicenter Phase III Trial In GVHD

Steroid-refractory acute GVHD (SR-GVHD) remains a significant complication of allogeneic hematopoietic cell transplantation (HCT). Prochymal® (Mesenchymal Stem Cells, MSC, derived from unrelated volunteer adult donors) was evaluated in addition to standard of care, including institutionally selected second line treatment, in a randomized (2:1) trial in patients with SR-GVHD (Protocol 280). Patients received 8 infusions of 2 × 106 MSC/kg over 4 weeks (or volume equivalent for placebo), with an additional 4 infusions administered weekly after day 28 in patients who had a partial response, defined as improvement in at least one organ without progression in others. The primary endpoint was durable complete response (DCR) for≥28 days. Additional prospectively defined outcomes included responses in patients by organ involvement. P-values were based on Chi-square tests, unless otherwise specified. 244 patients with SR-GVHD (skin involvement n=144, GI involvement n=179, liver involvement n=61) were enrolled and treated on study: Prochymal (n=163), placebo (n=81). There were no significant differences in age, pre-transplant conditioning, graft source, HLA-matching or second-line therapy between treatment arms. For the Prochymal and placebo arms, respectively, the grades of GVHD at entry were B (22% vs. 26%), C (51% vs. 58%), and D (27% vs. 16%). The respective DCR rates were 35% vs. 30% (p=0.3) in the ITT population and 40% vs. 28% (p=0.08) in the per protocol population. Results for secondary endpoints are shown in the table. Patients with GvHD affecting all 3 organs had overall complete or partial responses rate of 63% vs. 0% (n=22, p<0.05, Fisher's exact test) at day 28. Patients treated with Prochymal had less progression of liver GVHD at weeks 2 and 4 respectively (32% vs 59%, p=0.05; and 37% vs. 65%, p=0.05). The incidence of infections was not different between arms. Incidence rates were 9% vs. 8% for recurrent malignancy, 1.8% vs. 2.5% for infusional toxicity, and 0.6% vs. 4.6% for discontinuation due to an AE in Prochymal versus Placebo, respectively. GVHD with liver or gut involvement is a life-threatening complication of HCT. Our results suggest that the addition of Prochymal produced significant improvement without additive toxicity in patients with SR-GVHD involving visceral organs.Tabled 1Overall Complete or Partial Response Rates in Patients According to Organ InvolvementDay 100 Response RateOrganProchymalPlaceboOdds Ratio95% CIP ValueOverall82%73%1.70.9-3.10.12Skin78%77%1.10.5-2.40.9Liver76%47%3.61.1-11.2<0.05Gut82%68%2.21.1-4.4<0.05 Open table in a new tab

Fludarabine-based myeloablative regimen as pretransplant conditioning therapy in adult acute leukemia/myelodysplastic syndrome: comparison with oral or intravenous busulfan with cyclophosphamide

BackgroundA combination of busulfan (Bu) and cyclophosphamide (Cy) has been used as a standard myeloablative regimen for allogeneic hematopoietic stem cell transplantation (HSCT). Recent studies postulate that fludarabine (Flu) is a less toxic substitute for Cy.MethodsForty-two patients who were diagnosed with acute leukemia or myelodysplastic syndrome and received BuFlu (n=17) or BuCy (n=25) from August, 1999 to July, 2009 at Dong-A University Medical Center were retrospectively analyzed.ResultsThe median follow-up duration was 39.75 months. The BuFlu group showed a lower incidence of mucositis (P=0.005), but there was no significant intergroup difference in the time of engraftment, nausea/vomiting, acute/chronic graft-versus-host disease, hepatic veno-occlusive disease, or hemorrhagic cystitis. Moreover, the 2 groups showed no significant difference in the cumulative risk of relapse, event-free survival, or overall survival.ConclusionBuFlu administration can be employed as a preparative regimen for allogeneic HSCT and shows efficacy and transplant-adverse effects comparable to those of BuCy. However, randomized prospective studies in more patients are warranted.

5-Aza-2′-deoxycytidine sensitizes busulfan-resistant myeloid leukemia cells by regulating expression of genes involved in cell cycle checkpoint and apoptosis

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/Bu250 6 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 p16 INK4A , 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/Bu250 6 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 pretransplant regimen in patients with ML.

Discrimination of HLA null and low expression alleles by cytokine-induced secretion of recombinant soluble HLA

The disruption of disulfide bridges can decrease or abolish the cell surface expression of HLA class I molecules. Such disulfide bridges are formed by cysteine residues between amino acid (aa) positions 101/164 (α2 domain) and 203/259 (α3 domain). Sequence alterations in codons 101, 164, 203 and 259 have been observed in eleven HLA-A molecules. All of these variants except of A*3014L and A*3211Q have been reported to result in null expression alleles. In the case of HLA-A*3014L, a transversion at nucleotide position 563 replaces cysteine by serine at position 164 of the mature polypeptide. HLA-A*3014L is not detectable by standard microlymphocytotoxicity assay.To verify low or non-expression of this allele, we cloned soluble HLA-A*3014L and the reference allele HLA-A*3001 into a eukaryotic expression vector and transfected K562, C1R and HEK293 cells. Expression of soluble HLA-A*3014L and HLA-A*3001 was measured in the supernatants of transfected and untransfected cells incubated with or without IFN-γ and/or TNF-α using a W6/32 and anti-β2-microglobulin-based sandwich ELISA. Expression of mRNA transcripts of both alleles was determined by real-time RT-PCR.HLA-A*3014L was not detected in the supernatant of unstimulated transfectants. Stimulation with IFN-γ and/or TNF-α led to an increase of HLA-A*3014L secretion to a detectable level and increased HLA-A*3001 expression up to 8-fold, but did not show any difference in the increase of mRNA levels between HLA-A*3014L and A*3001. Because of this lack of any difference in the mRNA transcription, the protein expression defect is most likely caused by the missing disulfide bond formation in the α2 domain.Thus, exposing the cells to cytokine stress allows to distinguish between low- and non-expressed alleles and to classify alleles with a questionable expression pattern (Q alleles). Classifying HLA alleles in expressed and non-expressed variants is essential for matching assessments. Additionally, this discrimination between cytokine inducible and non-inducible defect alleles may be important in allotransplant settings in which a cytokine storm usually occurs following pre-transplant myeloablative conditioning or post-transplant immunosuppressive therapy.

Epigenetic Regulations Influence CML Cell Sensitivity to Busulfan: Relevance to Pretransplant Conditioning Therapy

Epigenetic Regulations Influence CML Cell Sensitivity to Busulfan: Relevance to Pretransplant Conditioning Therapy

Upregulation of XAF1 by 5-Aza-2′-Deoxycytidine Sensitizes Busulfan-Resistant Human CML Cells; Implications for Pretransplant Conditioning Therapy in Myeloid Leukemia

The DNA-alkylating drug busulfan (Bu) is commonly used in myeloablative pretransplant conditioning therapy in patients with chronic myelogenous leukemia (CML). A major obstacle to successful treatment is inherent or acquired cellular Bu-resistance. We hypothesized that cellular Bu-resistance can be reversed by epigenetic upregulation of pro-apoptotic genes. We established a Bu-resistant CML cell line B5/Bu2506 which is 4-fold more resistant to Bu than the parental B5 cells but 4-fold collaterally more sensitive to DNA demethylating agent 5-aza-2′-deoxycytidine (DAC). Exposure to DAC synergistically increased Bu-mediated cytotoxicity in B5/Bu2506 cells as evaluated by the MTT assay and analyzed by the median-effect method (combination index CI < 0.5). The DAC-induced sensitivity to Bu of B5/Bu2506 cells was associated with PARP1 cleavage, phosphorylation of histone 2AX and activation of caspase 8. Real-time PCR and immunostaining analyses of the expressions of various pro-apoptotic genes, which are known to be epigenetically regulated, showed a highly significant increase (approximately 40-fold) in the expression of XAF1 (X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1) in B5/Bu2506 cells that were exposed to 0.5 microM DAC for 48 hrs. Analysis of the XAF1 promoter by methylation-specific PCR showed a significant decrease in its methylation status which correlates with the alteration in its gene expression in the presence of DAC. These findings will be discussed relative to our analysis of XAF1 gene methylation status in leukemia cell samples obtained from patients who underwent allogeneic stem cell transplantation after high dose Bu-based conditioning therapy and either remained in remission for more than one year or who experienced rapidly progressive leukemia. Our results suggest that DNA methylation status (eg. XAF1 gene) may be used to identify leukemia patients who could benefit from Bu-DAC combinations to achieve improved leukemic cytoreduction with the conditioning program.