Refractory Graft-versus-host Disease Research Articles

Umbilical cord blood-derived mesenchymal stem cells ameliorate graft-versus-host disease following allogeneic hematopoietic stem cell transplantation through multiple immunoregulations.

Although mesenchymal stem cells (MSCs) are increasingly used to treat graft-versus-host disease (GVHD), their immune regulatory mechanism in the process is elusive. The present study aimed to investigate the curative effect of third-party umbilical cord blood-derived human MSCs (UCB-hMSCs) on GVHD patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and their immune regulatory mechanism. Twenty-four refractory GVHD patients after allo-HSCT were treated with UCB-hMSCs. Immune cells including T lymphocyte subsets, NK cells, Treg cells and dendritic cells (DCs) and cytokines including interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-α) were monitored before and after MSCs transfusion. The results showed that the symptoms of GVHD were alleviated significantly without increased relapse of primary disease and transplant-related complications after MSCs transfusion. The number of CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) cells decreased significantly, and that of NK cells remained unchanged, whereas the number of CD4(+) and CD8(+) Tregs increased and reached a peak at 4 weeks; the number of mature DCs, and the levels of TNF-α and IL-17 decreased and reached a trough at 2 weeks. It was concluded that MSCs ameliorate GVHD and spare GVL effect via immunoregulations.

Linear Relationship Between Lymphocyte Subpopulations Counts in Peripheral Blood and Buffy-Coat during Extracorporeal Photopheresis in Patients with Graft-Versus-Hot-Disease Using an Off-Line System

Introduction:Extracorporeal photopheresis (ECP) is a treatment modality that entails leukapheresis followed by mixing the buffy coat with 8-methoxypsoralen (8-MOP) and exposing it to UVA light. The buffy coat is then returned to the patient. ECP may be performed employing two different techniques: an on-line and off-line procedure. The off-line system includes two steps: a processing of buffy coat before reinfused to the patient.The treatment is thought to have an immunomodulatory effect and is most commonly used to treat cutaneous T cell lymphoma, acute and chronic graft-versus-host disease (GVHD), and heart and lung allograft rejection. The exact mechanism and optimal cells dose to be treated is unknown. At the present time, a standard protocol is used generally without considering peripheral blood (PB) leukocytes counts or lymphocyte subpopulations (LP) of the patient. With the off-line system, PB leukocytes counts and LP analysis may be useful to choose the amount and distribution of cells to be infused.The first objective of this study is to examine the quantitative correlation between PB LP and the buffy-coat in order to set individualized guidelines of treatment. Once this relationship is understood, the PB LP may serve as a surrogate marker for cell dose treated and help predicting the efficiency of ECP.The second objetive of this study is to examine the mean performance of the buffy coat LP categorized according to PB leukocytes counts (<1.5 x 109/L and >1.5x 109/L).Patients and methods: Twenty two consecutive patients with refractory GVHD were prospectively studied, from november 2009 to may 2014. Apheresis procedures were perfomed with COBE Spectra system (Terumo BCT®, Lakewood, CO, USA; version 7.0) by processing 1.5-2 times the patient blood volume. The product was transferred to a UVA-permeable bag (UVA, Macopharma, France), added 5 mL (0.1 mg) of 8-methoxypsoralen (8-MOP) aqueous solution (S.A.L.F.®, Cenate Sotto, Italy), exposed to UVA irradiation (Macogenic G2, Macopharma®), and then reinfused.Peripheral blood sample was drawn before ECP. Just before reinfused, buffy coat sample was drawn. Spearman correlation analysis was performed between the LP CD3+CD4+, CD3+CD8+, CD19+, NK of the preapheresis peripheral blood patient and the buffy coat infused analyzed by multiparameter flow cytometry 5 colors (FC500-Beckman Coulter®) in the total sample and in three groups according to preapheresis leukocytes counts (<2.5, 2.5-7.5, >7.5x 109/L). The mean performance was calculated: CD3+CD4+, CD3+CD8+, CD19+, NK+ LP count in buffy coat/ CD3+CD4+, CD3+CD8+, CD19+, NK+ PB /ml x treatment volume (ml) x 100.The mean performance of the buffy coat LP categorized according to preapheresis leukocytes counts (<1.5x 109/L and >1.5 109/L) were compared by Mann-Whitney test.Results:A total of 22 patients and 136 procedures were included in the final analysis. CD3+CD4+, CD3+CD8+, CD19+, NK LP in peripheral blood significantly correlated with those in buffy coat collected by COBE Spectra system, r= 0,74, 0.78, 0,92, 0,39 respectively (table 1). The LP mean doses and mean performance in buffy coat infused are specified in Table 1. The correlations was stronger in all LP with PB leukocytes counts <2.5 x 109/L (table2). We have not found any statistical correlation between the performance of the LP CD3+CD4+, CD3+CD8+, CD19+, NK according to PB leukocytes counts (<1.5x109/L and >1.5x109/L).Conclusions:The buffy coat contains great variability in lymphocyte subpopulations with predominant levels of CD3+CD8+. There is a robust linear relationship between all PB and buffy coat LP. The mean performance LP was around 40% and it was not related to very low PB leukocyte count (<1.5x109/L). The correlation was stronger with lower leukocytes counts in PB. If we could demonstrate a relationship between cell doses infused and clinical response, we could plan the necessary dose for each patient according to the PB leukocyte count and LP preapheresis. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Alpha 1 Anti-Trypsin (AAT) Offers Potent Therapy for Steroid Resistant Gut Gvhd: Interim Results of a Phase I/II Clinical Study

Background: Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT). Severe acute GVHD of the gastrointestinal tract can be life threatening and is associated with a loss of acute phase proteins including alpha1-antitrypsin (AAT). AAT acts as a potent anti-inflammatory agent. We and others have shown in allogeneic murine models that administration of AAT during HCT suppresses serum levels of pro-inflammatory cytokines, interferes with GVHD and reduces transplant-related mortality. We embarked upon a phase I/II clinical study to evaluate the feasibility and safety of AAT given to HCT recipients with steroid non-responsive acute GVHD. We aimed at characterizing pharmacodynamic effects of AAT and assess clinical responses as determined by changes in stool volume and overall clinical improvementMethods: Seven patients (3 female, 4 male) with hematologic malignancies were enrolled in the first and second cohort. Patients were 35-59 (median 50.4) years old and were given transplants from HLA-matched siblings (n=5) or received cord blood (n=2) following high intensity (myeloablative) or reduced intensity conditioning with cyclophosphamide +TBI (n=2), Fludarabine+TBI (n=4), or busulfan +cyclophosphamide (n=1). All patients received cyclosporine and MMF for GVHD prophylaxis. Acute GVHD of grades III-IV developed at 49 to 71 (median of 48) days, and treatment with systemic methylprednisolone, 2 mg/kg/day was instituted. Patients showing no clinically satisfactory responses after 5 days were given AAT (GlassiaTM) at 90 mg/kg iv on day 1, followed by 30 mg/kg (first cohort) or 60 mg/kg (second cohort) every other day for a total of 8 doses (15 days).Results: As AAT levels vary considerably from person to person, we determined plasma AAT concentrations before and after administration of exogenous AAT. Pre-treatment AAT levels ranged from 1.25mg/ml to 2.3mg/ml (mean 1.76 mg/ml), while post-treatment AAT levels were 2.5 mg/ml to 5 mg/ml (mean 3.18 mg/ml), i.e. in the normal range. Concurrently, levels of Angptl4, IL10 and IL-15 protein increased in all patients, while TIM-3, a marker of lymphocyte activation, was down-regulated. RQ-PCR analysis of donor-derived cells showed up-regulation of IL-10 and IL-1Ra (50 log2 and 10 log2, respectively, compared to pre-treatment samples). FACS analysis of peripheral blood cells showed a 5-fold increase in CD4+CD24+FoxP3+ Treg cells and a 3 fold increase in CD8+CD205+ dendritic cells (DC) in comparison to pre-treatment samples in all 7 patients. Concurrently, the numbers of TIM-3 expressing CD4+ T lymphocytes decreased. While endoscopic evaluation of patients before treatment revealed severe intestinal GVHD with mucosal denudation, endoscopic evaluation one week after completion of AAT administration showed evidence of re-epithelialization of the bowel wall. Concurrently, five of the seven patients developed formed stools, associated with decreased intestinal loss of AAT. One patient required additional therapy and died with biopsy proven liver GVHD. With a median follow-up of 8 months (range 3.5-12 months), five of of seven patients are alive, no longer requiring treatment for GVHD.Summary and conclusions: Continuous administration of AAT as salvage therapy for steroid resistant gut GVHD is feasible without clinically relevant toxicity. Stool sampling showed a decrease in intestinal AAT loss, as measured by AAT clearance and endoscopic evaluation confirmed healing of the bowel mucosa. These data are encouraging, and further exploration of AAT therapy in extended phase II and randomized trials as therapy of steroid refractory acute GVHD or as first line therapy are warranted. DisclosuresOff Label Use: Off label use of Alpha 1 Anti-Trypsin (AAT) as second line therapy for steroid-refractory gut GVHD..

The Number of Organ Failures, but Not Graft-Versus-Host Disease, Determines Outcome of Allogeneic Stem Cell Transplantation Recipients Requiring Intensive Care

▪Background: Although the prognosis of patients with hematological malignancies admitted to the intensive care unit (ICU) has improved over the past years, it remains poor in allogeneic stem cell transplantation (SCT) recipients and thus admission of these patients to the ICU remains controversial. Our center has flexible admission criteria, especially regarding graft-versus-host disease (GVHD). We thus performed a retrospective study to assess prognostic factors of survival in allogeneic SCT recipients who are admitted according to this policy.Methods: All allogeneic SCT recipients between 2002 and 2013 who were admitted to the medical ICU in our center were included. Details on patients, graft procedures, GVHD, ICU admission, and outcome were collected from individual medical records. Potential prognostic factors were explored by logistic regression analysis for ICU and hospital discharge. Cox regression analysis was performed for overall survival.Results: Of 344 patients who underwent allogeneic SCT during the study period, 92 patients (27%) were admitted to the ICU. Median time from transplantation to ICU admission was 53 days (range: -4 to 2273 days). Median age was 52 (range: 20-69). They received myeloablative (27%), sequential treatment with chemotherapy and reduced-intensity conditioning (RIC) (27%) or RIC (46%). Donors were mostly unrelated donors (59%) while graft source was mostly peripheral blood stem cells (78%) (bone marrow: 13%, cord blood: 9%). 66% patients could be discharged from the ICU and 46% from hospital whereas one year survival was 24%. No demographic or SCT parameter was associated with ICU or hospital discharge, or with overall survival. Grade 2-4 acute GVHD (34 patients, 37%) and refractory acute GVHD (12 patients, 13%) had a significant impact on hospital discharge (11/34 patients alive, OR: 2.4, 95% CI: 1-5.8, p=0.05; 2/12 patients alive, OR: 5, 95% CI: 1-24, p=0.05, respectively) but not with ICU discharge (20/34 patients alive, p=0.25; 7/12 patients alive, p=0.53, respectively). Chronic GVHD was present in 11 patients (12%) without impact on survival. At admission to the ICU, patients had a median SOFA (Sequential Organ Failure Assessment) of 7 (range 1-16), a median LODS (Logistic Organ Dysfunction System) of 4 (range 0-17), and a median APACHE II (Acute Physiology And Chronic Health Evaluation II) of 19 (range 8-42). The SOFA and LODS are efficient for determining ICU discharge (OR: 1.3, 95% CI: 1.1-1.5, p=0.003; OR: 1.3, 95% CI: 1.1-1.5, p=0.002, respectively). 46 patients (50%) required invasive mechanical ventilation, 28 patients (30%) required vasopressors, 16 patients (17%) required renal replacement therapy, and 26 patients (28%) had liver failure (bilirubin >34µmol/l). Mortality was greatly associated with the number of organ failures: each new organ failure worsens the prognosis (Figure). One year OS rates were 31%, 27%, 23%, and 0% among patients with 0 (26 patients, 28%), 1 (26 patients, 28%), 2 (26 patients, 28%), and 3 to 4 (14 patients, 15%) organ failures, respectively (HR: 1.7, 95% CI: 1.4-2.1, p<0.001).Conclusion: Allogeneic SCT recipients with 3 or 4 organ failures require further improvement of care. GVHD cannot be considered as a contraindication for transfer in the ICU per se, even with 2 organ failures. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Changes in the T and B- Cell Subsets Following Treatment with a CD25-Antibody in Patients with Steroid Refractory GvHD

Allogeneic stem cell transplantation is the therapeutic option for a variety of malignant and non-malignant haematological diseases. Graft versus Host Disease (GvHD) is a common post transplant complication. In 40% of these patients, GvHD is steroid refractory and associated with a mortality of around 60%. Basiliximab is a chimeric murine –human antibody also selective for interleukin -2 receptor (IL-2R) with a half life of 7 days. It is routinely used as part of the induction therapy in renal transplant recipients to prevent acute rejection following successful phase III studies. Phase 2 studies have demonstrated its superior efficacy in treating patients with steroid refractory GvHD (1). We administered Basiliximab in 14 patients with steroid refractory GvHD with a median age of 41 (range 20-69). M: F 7:7. All patients but one 13/14 received PBSC from unrelated donors and 6/13 had mismatched unrelated donors. Overall response was in the order of 12/14 (85%). One patient could not be assessed. 7/14 (50%) achieved a complete response to treatment. We aimed to study the in vivo T- and B-cell changes following Basiliximab administration as this would be an ideal platform to monitor the alterations in the regulatory T and B-cell compartment.PBMCs were obtained from all donors after informed consent, Immucan (Nr 11-116) approved by our local ethics committee, prior to and after weekly administration of Basiliximab 40mg for 4 weeks. Control samples were obtained from patients with steroid responsive acute GvHD.The total number of CD3+ as well as CD4+ and CD8+ T-cells remained constant during treatment and no change was observed on comparison with the controls. Gagliani et al (2) demonstrated that regulatory type 1 T-cells can be identified by the co-expression of CD49b and Lag3. No difference was observed between the % CD49d+, Lag3+ T-cells in the control cohort and the treatment cohort prior to therapy, ie day 0. The % CD49d+, Lag3+ T-cells decreased during the treatment period (statistically significant) in comparison to the control cohort. Despite the use of the CD25-antibody, a small population of CD25+, CD127+ cells could be detected and this population correlated to the % CD49d+, Lag3+ T-cells. [Display omitted] [Display omitted] The % CD19+, CD20+ B-cells were similar prior to treatment in the treatment group and control. Following the first administration a rise was observed followed by a decline over the next 3 weeks. No changes were seen in the activated (CD20+, CD86+) and anergic B-cell subsets (CD20+, CD21-) during the observation period. The % of CD24high, CD27+ regulatory B-cells were found to be twice that seen in the controls. With treatment a decrease was seen in this population. The CD24high, CD38high transitional B-cells were also found to be higher than that seen in the controls. No change was observed in this subset with treatment. [Display omitted] This is the first attempt to study the in-vivo changes induced by a CD25 antibody in patients with steroid refractory GvHD. We conclude that this antibody not only depletes the alloreactive CD25+ T and B-cell population but also alters the regulatory T and B-cell subsets in comparison to patients with steroid responsive GvHD. Our clinical data supports the efficacy of this drug in patients with steroid refractory GvHD. Contrary to the current knowledge that regulatory T-cells are required for GvHD suppression our data suggests that Basiliximab facilitates regulatory T-cell depletion. The reduction of the regulatory T-cell subset observed in patients responding to anti CD25 treatment suggests a complex regulation and potential dichotomous role of these cells in acute GvHD. 1.Schmidt-Hieber M, Fietz T, Knauf W, Uharek L, Hopfenmuller W, Thiel E, et al. Efficacy of the interleukin-2 receptor antagonist basiliximab in steroid-refractory acute graft-versus-host disease. Br J Haematol. 2005 Aug;130(4):568-74.2.Gagliani N, Magnani CF, Huber S, Gianolini ME, Pala M, Licona-Limon P, et al.Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells. Nat Med. 2013 Jun;19(6):739-46 DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow-Derived Mesenchymal Stem Cells Overexpressing Akt1 Protect Against ConA-Induced Liver Injury in Mice

Liver injury associated with veno-occlusive disease and graft-versus-host disease (GVHD) is a frequent and severe complication of hematopoietic stem cell transplantation, and remains an important cause of transplant-related mortality. Bone marrow derived mesenchymal stem cells (MSCs) have been evaluated for the prevention and treatment of refractory GVHD. However, poor cell viability has limited the therapeutic capacity of mesenchymal stromal cell therapy in vivo. In this study, we genetically engineered C57BL/6 mouse bone marrow MSCs using ex vivo retroviral transduction to overexpress Akt1, a serine threonine kinase and pro-survival signal protein, and tested the hypothesis that Akt1-expressing MSCs (Akt1-MSCs) are more resistant to apoptosis and can ameliorate acute liver injury induced by concanavalin A (ConA) in BALB/c mice. Cell proliferation and apoptosis analyses showed that, under both regular culture and high concentration IFN-γ (100 ng/mL) stimulation conditions, Akt1-GFP-MSCs had proliferation and survival (anti-apoptotic) advantages with down-regulated apoptosis pathways, compared to control GFP-MSCs. Twenty-four hours after receiving lethal dose of ConA (40 mg/kg, intravenous) (N=10 each group), no mouse survived, with or without 1x106 Akt1-MSCs or GFP-MSCs administration (intravenous); however, 3 and 1 survived in the 5×106 Akt1-MSCs group and 5×106 GFP-MSCs groups, respectively. In subsequent sub-lethal dose ConA (20 mg/kg) experiments, compared to GFP-MSCs, mice received Akt1-MSCs administration had significantly lower serum AST, ALT, TNF-α and IFN-γ levels and less histopathological abnormalities. In addition, Akt1-MSCs treated mice had significantly higher serum concentrations of IL-10, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). In vivo imaging showed that, hepatic fluorescence signal in sub-lethal ConA+Akt1-MSCs group was significantly stronger than ConA+GFP-MSCs group on day 0, and persisted up to 14 days, whereas the signal in ConA+GFP-MSCs, Akt1-MSCs and GFP-MSCs groups was negligible on both day 7 and day 14. Thus, bone marrow derived MSCs genetically enhanced with Akt1 had survival advantage in vitro and in vivo, and have the potential to be a potent therapy for prevention and amelioration of GVHD-associated liver impairment. Further translational pre-clinical studies are ongoing to further determine the efficacy, dosage and timing of administration of Akt1-MSCs in animal models. DisclosuresNo relevant conflicts of interest to declare.

The use of mesenchymal stem cells in therapy of steroid-resistance chronic refractory graft-versus-host disease after hematopoietic stem cell transplantation

Introduction. Chronic graft-versus-host disease (GVHD) is a life-threatening complication after allogeneic hematopoietic stem cell transplantation. Mesenchymal stem cells (MSC) are involved in tissue repair and modulating immune responses. We present report of two cases of use of third-party donor mesenchymal stem cells in the therapy of steroid-resistant chronic GVHD. Case reports. The doses of MSC in both patients were 1.08 and 1.4 x 106 cells per kg body weight respectively. Time from diagnosis of GVHD to infusion of MSC was 420 and 330 days, respectively. Both patients had single infusion of MSC. We did not observe any adverse effects after both infusions of MSC. We obtained initially a partial response and then complete response in the first patient and a partial response in the second patient. In the first patient we achieved improvement of skin involvement resulting in reduction of immunosuppression. In the second patient with scleroderma we achieved only a minor improvement. Conclusion. Administration of MSC is a safe method and might by an effective therapy for patients with chronic refractory GVHD.

The immunosuppressive properties of non-cultured dermal-derived mesenchymal stromal cells and the control of graft-versus-host disease

Mesenchymal stromal cells (MSCs) have been developed for the prevention and treatment of graft-versus-host disease (GVHD). Non-cultured natural MSCs are considered ideal, as they better maintain their biological and therapeutic properties. The skin is the largest organ in the body and constitutes an interesting alternative to bone marrow for the generation of MSCs. Large numbers of dermal-derived-MSCs (DMSCs) can be easily generated without culturing in vitro, but their therapeutic effects still remain unclear. In this study, we described for the first time the use of non-cultured DMSCs for controlling GVHD in an MHC-mismatched mouse model and investigated their immunomodulatory effects. Our results showed that non-cultured mouse DMSCs decreased the incidence and severity of acute GVHD during MHC-mismatched stem cell transplantation in mice. This effect was mediated by the inhibition of splenic cell (SPC) proliferation and the enhancement of Treg cells. Consistent with the results in vivo, the results in vitro showed that human DMSCs inhibited the proliferation of peripheral blood mononuclear cells (PBMCs) by inhibiting the proliferation of CD3+ T cells. hDMSCs prevented PBMCs from entering S phase, suppressed the activation of CD3+ T cells and increased Treg proportions. In conclusion, DMSCs should be considered as a novel MSC source for the control of refractory GVHD.

Methotrexate for the Treatment of Graft-versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation.

Glucocorticoids have been the primary treatment of graft-versus-host disease (GVHD) over the past decade. Complete responses to steroid therapy are usually expected in almost one-third of aGVHD cases and partial response is anticipated in another one-third of patients. However, for those patients not responding to corticosteroid treatment, there is no standard second-line therapy for acute or chronic GVHD. Methotrexate (MTX) for treatment of steroid refractory GVHD has been evaluated in a number of studies. Results from peer-reviewed original articles were identified and the pooled data analyzed. Despite several limitations in data collection and analysis, weekly administration of methotrexate at a median dose of 7.5 mg/m2 seems to be safe with minimal toxicities in the context of both aGVHD and cGVHD treatments. The observed overall response (OR) in patients with aGVHD to MTX treatment in the published studies was 69.9%, with complete response (CR) in 59.2% and PR in 10.6%. In cGVHD the OR was 77.6%, with CR reported in 49.6% and PR in 28% of patients. Predictors of better responses were lower grade GVHD, cutaneous involvement, and isolated organ involvement. MTX as a steroid sparing agent might reduce long-term complications and improve the quality of life of GVHD affected individuals.

Long-Term Follow-Up Of Reduced-Intensity Allogeneic Hematopoietic Stem Cell Transplantation For High Risk Follicular Lymphoma

IntroductionFollicular lymphoma (FL) is the most frequent indolent lymphoma. Autologous hematopoietic stem cell transplantation (ASCT) allows durable progression-free survival in selected relapsed patients. Recently, the EBMT lymphoma working party has published the recommendations for ASCT and allogeneic hematopoietic transplantation (alloTPH) in FL. For those high-risk young patients relapsing after ASCT, alloTPH is a valid therapeutic option; the majority being done with reduced intensity conditioning (alloRIC). AimsOur aim is to describe the outcome of patients with FL who underwent an alloTPH in our cente. Factors potentially influencing outcome such us age, sex, FLIPI at diagnosis, disease status prior alloTPH, EBMT risk score, type of donor, graft vs host disease (GVHD) prophylaxis and development of GVHD were also analized. MethodsActuarial estimates of OS and PFS rates were calculated using the Kaplan-Meier method. OS was estimated from the date of transplantation to the date of death or last follow-up. PFS was defined as the time from transplantation until progression or death from any cause. PatientsWe retrospectively found 26 alloTPH in the period 1999 - 2013. Two alloTPH were conditioned with a myeloablative regimen. Patients who underwent an alloRIC (n=24) were all conditioned with Fludarabine 30 mg/m2/5 days and Melphalan 70 mg/m2/1 day. ResultsAlloTPH was from a matched related donor in 70.8% of cases, from a mismatched related donor in 4.2%, matched unrelated donor in 8.3% and from a mismatched unrelated donor in 16.7%.The median age was 49.5 years (range 35-61). The median time from diagnosis to alloTPH was 48 months (range 9 – 190), male: female ratio was 2:1. FLIPI at diagnosis was 0 in 16.7%, FLIPI 1 in 25%, FLIPI 2 in 33.3%, FLIPI 3 in 8.3% and FLIPI 4 in 16.7. Mean number of treatment pre alloTPH was 4.5 (range 2-6). Ten patients (41.7%) had previously received an ASTC.Response before alloTPH was complete response in 54.2%, partial response in 37.5% and stable disease in 8.4%. The majority of patients (95.8%) had an EBMT risk score higher than 3.Graft vs host disease prophylaxis consisted on cyclosporine + short course of methotrexate in 54.2%, cyclosporine + micophenolate in 41.7% and sirolimus + tacrolimus in 4.2%. In vivo T-cell depletion was done in 4 patients receiving grafts from a mismatched unrelated donor (ATG or alemtuzumab, 2 patients each).Mean CD34 cells/Kg infused was 6.45 x106/kg (range 1.92 – 15.6). The median time to granulocyte recovery (>0.5x109/L) was 15 days (range 11-19) and the median time to platelets recovery (>50x109/L) was 14 days (range 6-56).Acute GVHD was developed in 45.8% of patients; it was global grade 1-2 in 36.4% ,and grade 3-4 in 63.6%. Chronic GVHD was present in 58.3% of patients. The median time of chronic GVHD presentation was 196 days after alloTPH (range 101 – 569 days).Regarding opportunistic infections, 20.8% of patients reactivated CMV with no disease, 16.7% had intestinal disease due to CMV. Thirteen per cent of patients developed pulmonary fungal infection (i.e Aspergillus sp).With a median follow up of 48.50 months (range 0-161), the 5 years OS was 66.1% (CI 95% 43.76 - 88.44) and the 5 years PFS was 63.8% (CI 95% 43.22 - 84.4). Overall morbidity at 3, 6 and 12 months after alloTPH was 5%, 5% and 10.3% respectively. Only 1 patient relapsed at 38 months after alloTPH but is still alive. Causes of death were refractory GVHD (n=5), sepsis (n=3) and acute renal failure due to microangiopathy (n=1).Survival was influenced by GVHD prophylaxis. At the median time of follow up (48.50 months) patients receiving cyclosporine-methotrexate had an OS of 82.5% vs 30% for patients receiving cyclosporine-MMF (p<0.01).Other variables (FLIPI at diagnosis, sex, age older than 60 years, the EBMT risk score pre alloTPH and disease status) had no influence on survival. ConclusionsAlloTPH for high risk FL patients seems to overcome the negative effect of FL prognostic factors at diagnosis. This strategy shows an excellent disease control with a low transplant-related mortality. Disclosures:No relevant conflicts of interest to declare.

Single Nucleotide Polymorphisms Within The Thrombomodulin Gene (THBD) Predict Risk Of Non-Relapse Mortality In Patients With Graft-Versus-Host Disease

Purpose Acute graft-versus-host disease (GVHD) is a major complication after allogeneic stem cell transplantation (allo-SCT). With the current treatment approaches focussing on escalating immunosuppression, mortality rates of therapy-refractory disease courses are approx. 80-90%, underlining the medical need for novel therapeutic concepts. The pathophysiology of refractory GVHD is incompletely understood, however, recent evidence points towards a crucial role for endothelial damage. We have previously shown that steroid-refractory GVHD associates with increasing serum thrombomodulin (TM) levels 1, 2 and loss of endothelial TM expression 3, a constellation that indicates endothelial stress. However prior to allo-SCT, baseline TM levels did not predict steroid-refractory GVHD or survival. We hypothesized that endothelial vulnerability caused by single nucleotide polymorphisms within the TM gene (THBD) is a risk factor for refractory GVHD predicting outcome after allo-SCT. Patients and Methods Seven SNPs within the THBD gene were studied (rs1962.TC, rs1042579.TC, rs1042580.AG, rs3176123.TG, rs3176124GA, rs3176126.GA and rs3176134.CT) in a training cohort of 465 allografted patients. The relevant genotypes were then re-assessed in an independent validation cohort (n=386). Results Allele frequencies of seven THBD SNPs allowed meaningful statistical correlation with outcome after allo-SCT. An increased risk of non-relapse mortality (NRM) was associated with three SNPs: rs1962.CC, rs1042579.TT (455V) and rs1042580.GG. When patients were divided into risk cohorts (one vs. no high-risk SNP), a strong correlation with NRM was observed in both cohorts (training cohort: p=0.002, HR 2.32 CI 1.36-3.95; validation cohort: p=0.007, HR 2.13, CI 1.23-3.70) (Figure 1). NRM was predicted by THBD SNPs in particular for patients who later develop GVHD (validation cohort: p=0.00056, HR 3.03 CI 5.68-1.61, training cohort: p=0.012, HR 2.38 CI 1.21-4.69) but not in patients without GVHD. In contrast, THBD SNPs did not predict incidence of acute GVHD. Multivariate analyses adjusting for clinical variables confirmed the independent effect of THBD SNPs on NRM in both cohorts. Conclusions Our results demonstrate that THBD-SNPs predict mortality of GVHD patients without influencing GVHD incidence. This supports our hypothesis that endothelial vulnerability contributes to therapy refractory courses of the disease. Rather than focussing on the escalation of combinations of (endothelial cell toxic) immunosuppressive drugs, therapeutic approaches aiming at endothelial protection should be evaluated. 1. Dietrich S, Falk CS, Benner A, et al. Endothelial vulnerability and endothelial damage are associated with risk of graft-versus-host disease and response to steroid treatment. Biol Blood Marrow Transplant 2013; 19(1): 22-7. 2. Luft T, Dietrich S, Falk C, et al. Steroid-refractory GVHD: T-cell attack within a vulnerable endothelial system. Blood 2011; 118(6): 1685-92. 3. Andrulis M, Dietrich S, Longerich T, et al. Loss of endothelial thrombomodulin predicts response to steroid therapy and survival in acute intestinal graft-versus-host disease. Haematologica 2012; 97(11): 1674-7. Disclosures: Dreger: Riemser Pharma : Consultancy, Honoraria, Research Funding.

Phase 1 Trial Of Bone Marrow Stromal Cells (Bone Marrow-derived MSCS) To Treat Tissue Damage In Allogeneic Stem Cell Transplant Recipients: Biological Markers Correlate With Clinical Responses and Survival

Bone marrow stromal cells (BMSC, also known as bone marrow-derived “mesenchymal stem cells”) have been used to treat acute graft-versus-host disease (GVHD) and other complications following allogeneic hematopoietic stem cell transplantation (SCT). We conducted a phase I trial using third party, early passage, BMSC for patients with steroid-refractory liver or gastrointestinal GVHD, tissue injury or marrow failure following SCT to investigate safety and clinical responses following BMSC infusion. To identify mechanisms of BMSC immunomodulation and tissue repair, patients were monitored for plasma GVHD biomarkers, cytokines, growth factors, and lymphocyte phenotype before and after BMSC infusion. BMSCs were prepared from marrow aspirates from healthy volunteers with the expansion of 3 passages. Ten subjects were infused a fixed dose of 2 x 106 BMSCs /kg weekly for 3 doses. There was no treatment related toxicity (primary endpoint). Eight subjects were evaluable for response assessment at 4 weeks after the last infusion. Five of the seven patients with steroid-refractory acute GVHD achieved complete remission (CR), two of two patients with tissue injury (pneumomediastinum/ pneumothorax) achieved resolution but there was no response in two subjects with delayed marrow failure. Rapid reductions in inflammatory cytokines occurred after the first BMSC infusion (fig1). Clinical responses correlated with a fall in biomarkers (Reg 3α, CK18, and Elafin) relevant for the site of GVHD, or CK18 for tissue injury. The GVHD complete responders survived significantly longer (>300 days vs a median of 33 days), had higher baseline absolute lymphocyte and central memory CD4 and CD8 counts but there was no clear difference in natural or induced Tregs. Cytokine changes also segregated with survival. These results confirm that BMSC are associated with rapid clinical responses and biomarker normalization in steroid-refractory GVHD and PM. However BMSC were ineffective in patients with more aggressive GVHD with lower lymphocyte counts, which suggest that effective GVHD control by BMSC, requires a relatively intact immune system. Early detection and BMSC treatment appear important in patients with refractory GVHD.▪ Disclosures:No relevant conflicts of interest to declare.

Recent Progress in Managing Graft-Versus-Host Disease and Viral Infections Following Allogeneic Stem Cell Transplantation

Despite recent reductions in transplant-related mortality, post-transplant complications such as graft-versus-host disease (GvHD) remain major obstacles to the successful application of allogeneic hematopoietic transplantation. Steroid-refractory GvHD has a poor outcome. Although there are a variety of new approaches to the treatment of refractory GvHD, many have limited evidence of efficacy. Other approaches appear to be unacceptably toxic. It would be preferable to improve GvHD prophylaxis. There is good evidence that rates of GvHD can be reduced without unacceptable reduction of the graft-versus-leukemia effect or compromising overall survival. However, prophylactic measures aimed at reducing T-cell numbers or functions are associated with high rates of reactivation of latent viruses. New technologies that allow rapid generation of virus-specific T-cells show promise to reduce the frequency and severity of such reactivations and have the potential to revolutionize the approach to post-transplant infectious complications.

A Phase II Study Using Post-Transplant Cyclophosphamide (PTC) As Graft Versus Host Disease (GVHD) Prophylaxis in Patients Undergoing HLA Matched Sibling Donor Stem Cell Transplant (SCT) for Severe Aplastic Anemia (SAA)

Abstract 4199Between September 2010 and June 2012, 15 patients with SAA underwent HLA identical sibling donor SCT using post transplant cyclophosphamide for GVHD prophylaxis. The conditioning regimen consisted of Inj Fludarabine 30 mg/m2/day from day -7 to -2 and Inj Cyclophosphamide 50 mg/kg IV on days -3 and -2. Total Body irradiation (TBI) 200 cGy as single fraction was added on day -1 from December 2011 because of concern regarding graft failure. GVHD prophylaxis consisted of Inj Cyclophosphamide administered at 50 mg/kg/day IV on day +3 and +4 post SCT. G-CSF mobilized peripheral blood stem cells (PBSC) was the graft source.There were 10 males and 5 females with a median age of 25.9 years (range: 8 – 42). The median time from diagnosis to SCT was 7.5 months (range: 2 – 36). All had 6/6 HLA matched sibling donors except 1 who had a 9/10 matched family donor. The median PBSC cell dose infused was 5.4 × 108 MNC/Kg (range: 2.4 – 8.5) and 9.58 × 106CD34/Kg (range: 5.4 – 17.2). Thirteen patients engrafted (86.6%) with a median time to ANC >0.5 × 109/L of 15.4 days (range: 15–17) and Platelet count >20 × 109 of 16.6 days (range: 12–32). Grade II –IV acute GVHD occurred in 3 patients (23%) at 42, 49 and 68 days post SCT. GVHD was grade II in 2 patients and grade IV in 1 patient. Two responded to a combination of cyclosporine and prednisolone while one patient with grade IV GVHD expired with refractory GVHD at median time of 64 days post SCT. Of the 11 evaluable patients, 4 (36.3%) developed chronic GVHD which was limited in all. Two patients who developed de novo chronic GVHD were managed with prednisolone alone. Overall 7 patients (46.6%) have not required any immunosuppression after SCT while 3 have required immunosuppressive therapy for 114, 127 and 225 days respectively At a median follow up of 11 months (range: 1 – 22), 11 (73.3%) are alive and well including 7 patients who did not require any immunosuppressive therapy following SCT.In conclusion, the use of post transplant cyclophosphamide as GVHD prophylaxis following sibling donor transplant for SAA is associated with low rates of GVHD. What makes it more attractive is the fact that 46% did not require any immunosuppression post SCT. Larger studies are required to understand the utility of this prophylaxis in sibling donor transplants for aplastic anemia.This trial is registered in the Clinical Trials Registry India - CTRI/2010/091/001480. Disclosures:No relevant conflicts of interest to declare.

Patterns of Acute and Chronic Graft-Versus-Host Disease Following ATG-Based Conditioning for Allogeneic Hematopoietic Cell Transplantation

AbstractAbstract 4479 Background:In vivo T-cell depletion with antithymocyte globulin (ATG) is known to decrease the incidence of acute and chronic graft vs. host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). However, the detailed patterns of GVHD (incidence, severity, timing, and quality) after ATG-based conditioning have not been examined in large patient cohorts, and it is unknown whether they differ from those seen in patients who do not receive ATG during conditioning therapy. Patients and Methods:We analyzed the incidence and characteristics of acute and chronic GVHD, requirements for immunosuppressive therapy (IST) and survival in a cohort of 174 patients who underwent a first HCT for hematologic malignancy with ATG as a part of their conditioning regimen. The median age was 54 years (range 19–76); all but 5 pts received PBSC, and median follow-up of survivors was 16.9 months (range 3–70 months). Donors were matched related in 18% (n=32), matched unrelated in 44% (n=77), and mismatched unrelated in 37% (n=65). Conditioning regimens were myeloablative in 33% (n=57) and reduced intensity in 67% (n=117). Additional GVHD prophylaxis included tacrolimus in all patients combined with either methotrexate (42%) or MMF (58%). Results:The cumulative incidence of grade II-IV and III-IV acute GVHD at 100 days was 34% and 7%, respectively, with the median time of onset at 43 days (range 11–98 days) after transplant. Eleven patients (6.3%) required additional immunosuppressive treatment due to steroid refractory GVHD. Late/persistent acute GVHD without any evidence of chronic occurred in 25% of patients. NIH chronic GVHD developed in 25 patients, with a cumulative incidence of 24.4% at 2 years. Forty four percent of these patients were classified as classic chronic, and 56% as overlap. The onset of chronic GVHD was quiescent in 20 (80%), progressive in 3 (12%), and de-novo in 2 (8%) patients. The global severity was mild in 9 (36%), moderate in 11 (44%) and severe in 5 (20%) cases. The median time of onset for chronic GVHD was 185 days (range 99–763). In a multivariate analysis of factors predictive for development of chronic GVHD, the only factor associated with development of chronic GVHD was prior grade II-IV acute GVHD (HR 2.5, p =.03). The most common diagnostic organ was mouth (n=16), followed by skin (n=8) and eye (n=1). The median number of sites involved during the course of chronic GVHD was 4 (range 1–7), and the median number of systemic immunosuppressive agents for treatment was 2 (range 0–4). Among the 25 chronic GVHD patients, 5 have discontinued immunosuppression at a median time of 13.1 months (range 6–26) since the diagnosis of chronic GVHD. The cumulative incidence of discontinuation of IST was 23% at one year and 50% at two years. Three deaths in the overall cohort were attributed to complications related to acute (n=2) or chronic GVHD (n=1). At 2 years, the overall survival among all 174 pts was 62.4%, cumulative incidence of relapse was 23.1%, and non-relapse mortality was 22.7%. Conclusion:These data from a large, uniformly treated and graded, predominantly peripheral blood stem cell transplant recipient population, confirm that ATG decreases both the incidence and severity of acute and chronic GVHD. In particular, the rate of moderate to severe chronic GVHD is extremely low, resulting in minimal need for tertiary treatment and decreased duration of immunosupression. Disclosures:No relevant conflicts of interest to declare.

Steroid refractory psoriasiform cutaneous graft versus host disease successfully treated by extracorporeal photopheresis: A case report

Graft-versus-host disease (GVHD) is a frequent complication occurring after allogeneic hematopoietic stem cell transplantation. It can be classified as acute and chronic GVHD based on the time of onset following transplantation and clinical presentation. Cutaneous involvement is the most common feature of acute GVHD, with maculopapular exanthema and perifollicular papular lesions. Steroid refractory GVHD is associated with a significant morbidity and mortality. We present a very rare case with acute cutaneous GVHD mimicking psoriasis vulgaris occurring after allogeneic peripheral blood stem cell transplantation for chronic lymphocytic leukemia. The patient’s rash resembled psoriasis vulgaris and showed histologic features of both psoriasis and acute GVHD. Despite concomitant cyclosporine A and methylprednisolone therapy no response was obtained. Therefore, we administered extracorporeal photopheresis and achieved the desired therapeutic effect.

Audit of Peripheral Stem Cell Transplantation for Aplastic Anemia in Multitransfused Infected Patients

IntroductionAllogeneic hematopoietic stem cell transplantation is a curative modality for aplastic anemia; the preferred stem cell source is bone marrow. However, allogeneic peripheral blood stem cell transplantation (PBSCT) used in high-risk patients is associated with higher risk of chronic graft-versus-host disease (GVHD). Our center receives multitransfused, alloimmunized, infected, late referrals for transplant. MethodsForty-one patients of median age 22 years (range 8–37) received allogeneic-PBSCT from human leukocyte antigen (HLA)-matched sibling donors. The median time since diagnosis was 12 months (range 4–65) and median pretransplant transfusions were 37 (range 6–160). Six patients were platelet refractory and one alloimmunized for pan-red blood cell (RBC) antigens. Several patients had pretransplant icterus or renal dysfunction and 26 (63.4%) had unresponsive bacterial/fungal infections. Our conditioning regimen included fludarabine 30 mg/m2 for 6 days (days –10 to –5), cyclophosphamide 60 mg/kg/d for 2 days (days –6 to –5), and antithymocyte globulin (ATGAM) 30 mg/kg/d for 4 days (day –4 to –1), which was reduced to 2 days in 2 patients. We used standard GVHD prophylaxis with cyclosporine and methotrexate on days 1, 3, 6, 11. ResultsThe median follow-up period was 29 months (range 6–78) and median engraftment time 10 days (range 8–17). Thirty-one patients (75.6%) were treated for infections, with 20 of these on antifungals for preexisting infections. There were two graft rejections and 10 (24.4%) deaths, with three intracranial hemorrhages, two rejections with infection, three cases of refractory GVHD (acute/overlap syndrome) with cytomegalovirus reactivation, and two invasive fungal infections. Overall incidence of acute GVHD was 39% with 2 grade IV cases. Ten (25%) cases developed chronic GVHD, with extensive GVHD in four. ConclusionWith more experience using shortened course of ATGAM, HLA-matched donor transfusions, and availability of newer antifungals, we have been able to decrease PBSCT-related mortality. Further improvement will be possible with early referrals.

Extracorporeal photopheresis in refractory chronic graft-versus-host disease: The influence on peripheral blood T cell subpopulations. A study by the Hellenic Association of Hematology

Extracorporeal photopheresis (ECP) has been established as an effective treatment modality for patients with chronic extensive graft-versus host disease (GVHD). In the present study, we evaluated the influence of ECP on the numbers of CD4+, CD8+, CD20+, CD56+ cells, and on T-regulatory (Tregs), as well as on the numbers of naïve, central memory (CM), and effector memory (EM) T-cells in patients treated for refractory chronic GVHD. Flow cytometric analysis of peripheral blood lymphocytes was performed for the calculation of the different T-cell subsets. Patients with GVHD had a higher percentage of EM-CD4+ cells in comparison with healthy donors (p=0.046). The percentages of naïve-CD8+, naïve-CD4+, CM-CD8+, CM-CD4+, EM-CD8+, and Tregs were not different between patients with GVHD and healthy donors. Similarly there was no statistical difference in the percentages of naïve, CM, and EM CD4+ and CD8+ cells before and after 3months of treatment with ECP. However, in the subset of Tregs a statistically significant increase was observed after 3months of treatment with ECP (p=0.015). Responders to ECP had statistically significantly higher absolute numbers of CD4+, and CD8+ cells, in comparison with non-responders. These data further support the concept that ECP does not cause immune-suppression, but should be better considered as an immune-modulating treatment.

Infliximab Treatment for Steroid-Refractory Acute Graft-Versus-Host Disease After Reduced-Intensity Cord Blood Transplantation in Adults

Abstract 4553 IntroductionAcute graft versus host disease (GVHD) remains the most frequent complication after allogeneic hematopoietic stem cell transplantation (SCT). In reduced intensity cord blood transplantation (CBT) previous studies have reported a lower incidence of severe acute GVHD compared with conventional allo-SCT. However, in these studies the incidences of grade II-IV acute GVHD varied widely from 26% to 51% (H.Narimatsu Stem cell int. 2011: 607569). In particular, post transplant immune disorders, including early immune reactions (PIR) and acute GVHD, are potential complications following CBT in adult patients. Such reactions might increase the risk of organ dysfunction, leading to high rates of transplantation-related mortality, particularly in patients who do not respond to primary therapy, which usually consists of steroids. Infliximab, a chimeric monoclonal antibody against tumor necrosis factor alpha, has shown activity against steroid refractory acute GVHD. Patients and MethodsWe retrospectively reviewed 275 patients who underwent single-unit reduced intensity cord blood transplantation consecutively from March 2007 to December 2011 at our institute. Patients in whom PIR or acute GVHD developed received methylprednisolone 1–2 mg/kg per day. If no partial or complete resolution of symptoms occurred, they were considered steroid-refractory and proceeded to infliximab treatment. The dose of infliximab was 5 mg/kg/day once weekly for at least 1 course. An antifungal drug, itraconazole or micafungin or voriconazole was used until all immunosuppressive drus were withdrawn. ResultsIn this study we retrospectively evaluated 54 patients who had steroid refractory acute GVHD. Of these, 21 who received infliximab were analyzed. GVHD prophylaxis was with only tacrolimus(n=15) and mycophenolate mofetil+tacrolimus(n=6). All of them developed grade III to IV GVHD. Median follow-up time of survivors was 548 days (range, 222–1152). The overall response rate was 62% (n=13), and 9 patients (43%) experienced complete response (CR). 5 patients (24%) did not respond and 3 (14%) had progressive GVHD. The Kaplan-Meier estimate of overall survival was 31%, with no signs of chronic GVHD (n=2). Four patients who responded subsequently died, one of exacerbation of GVHD, three of infections. All the 8 unresponsive patients died of GVHD or infections. Five patients (21%) had non-Candida invasive fungal infections. Sixteen patients(79%) had bacterial infections. ConclusionInfliximab was well tolerated and active for the treatment of steroid-resistant acute GVHD even following reduced intensity cord blood transplantation. However, it is associated with a high rate of infections. Controlled studies to assess the pharmacokinetics and most effective dosing regimen of infliximab for the treatment of steroid refractory acute GVHD are warranted. Disclosures:No relevant conflicts of interest to declare.

Allogeneic Hematopoietic Stem Cell Transplantation for Blastic Plasmacytoid Dendritic Cell Neoplasm

Abstract 4938 BackgroundBlastic plasmacytoid dendritic cell neoplasm is a rare malignancy that typically follows a highly aggressive clinical course,formerly known as blastic natural killer (NK)-cell lymphoma and CD4+/CD56+ hematodermic neoplasm. Here we report 2 cases of blastic plasmacytoid dendritic cell neoplasm that underwent allogeneic stem cell transplantation. Patients and MethodsCase one was recognized as acute lymphocyte leukemia and then was revised to BPDC at recurrence, and another initially presented with typical cutaneous involvement and was promptly diagnosed by biopsy. After induction/re-induction and subsequent consolidation chemotherapy, 2 cases of BPDC received HLA identical-sibling donor allogeneic stem cell transplantation (Allo-HSCT) after BU/Cy, TBI/Cy+VP-16 conditioning regimen, respectively. Both achieved rapid hematopietic recovery in 2 weeks. Multi-color labeling flow cytometry was performed to monitor minimal residual disease (MRD) and the patient received donor lymphocyte infusion (DLI) to control primary malignancy as MRD-triggered or preventive strategy. ResultsCase 1 survived refractory acyclovir-resistant Epstein-Barr virus viremia after rituximab treatment, but failed in DLI-related grade IV graft versus host disease (GVHD) mixed with thrombotic microangiopathy (TMA) on 1 year post-transplantation. Case 2 died from relapse. Conclusion:Allo-HSCT seems to be a promising treatment for aggressive BPDC aided with MRD monitoring and MRD-triggered DLI. GVHD mixed with TMA deserves more attention for treatment of refractory GVHD. Disclosures:No relevant conflicts of interest to declare.