Biol Blood Marrow Transplant Research Articles

Prospective Multicenter Phase II Study of HLA-Haploidentical Peripheral Blood Stem Cell Transplantation after Reduced-Intensity Conditioning with Post-Transplant Cyclophosphamide: First Analysis of the JSCT Haplo13 Study

s / Biol Blood Marrow Transplant 21 (2015) S266eS321 S313 Mount Sinai, New York, NY; Medicine/BMT, Mount Sinai Hospital, New York NY; Mount Sinai Medical Center, New York, NY; Division of Hematology/Oncology, Mount Sinai School of Medicine, New York, NY; Bone Marrow and Stem Cell Transplantation, Icahn School of Medicine at Mount Sinai, New York, NY Background: The use of monoclonal antibodies in combination with chemotherapy for the treatment of lymphoma may improve efficacy by utilizing distinct mechanisms of tumor kill while minimizing overlapping toxicity. Bendamustine, a bifunctional alkylating agent, has been usedwith Rituximab in indolent non-Hodgkin lymphomas, chronic lymphocytic leukemia, and more recently for the treatment relapsed and refractory diffuse large B-cell lymphomas (DLBCL). Brentuximab, a monoclonal antibody targeting CD30, is approved for refractory and relapsed anaplastic large cell lymphoma, Hodgkin lymphoma relapsed after autologous stem cell transplant and may have efficacy in the treatment of DLBCL which have been shown to express CD30 in 14-25% of cases. We report the Mount Sinai Hospital experience using bendamustine and brentuximab (BB) in the treatment of relapsed-refractory lymphomas and its utility as a bridge to transplant. Methods: Retrospective analysis of the tolerability and outcomes for patients receiving BB between 2013 and 2014 as part of quality assurance review. Results: Seven patients (1 female) were identified with a median age of 39 (range 34-70). Diagnoses included Hodgkin lymphoma (4), primary mediastinal B-cell lymphoma (1), DLCBL (1), and peripheral T-cell enteropathy associated type II lymphoma (1). Median time between initial diagnosis and BB was 19 months (range 12-175 months). Patients received a mean of 4.1 prior lines of therapy (range 2-9) including allogeneic transplantation (1) and autologous transplantation (4). All patients received 90 mg/m2 of bendamustine and 1.8 mg/kg of brentuximab approximately every 3 weeks for a mean of 2.71 cycles. Two patients developed sepsis and fungal pneumonia, respectively. Of 6 patients evaluable for response, 4 had partial response, 1 hadmixed response and 1 had PR after 2 cycles, then received a stem cell boost/DLI but ultimately died from progression of disease (POD). 2 other patients received allogeneic stem transplants and 3 patients are awaiting transplantation. Conclusion: BB in our heterogeneous and heavily pretreated population has been well tolerated, has provided disease control, and has enabled patients to be bridged to transplant, both allogeneic and autologous.

Piloting a Clinical Resource Nurse Role

s / Biol Blood Marrow Transplant 21 (2015) S374eS390 S377 encouraged to voice concerns with alarm issues in daily rounds. Compliance and failures with the process are reviewed regularly with staff. Findings & Interpretation: From January 2013eJuly 2014, percent compliance with the CMCP increased from 30% to >98% compliance (median1⁄487%). Number of alarms per patient day decreased from 180 to 20 (median1⁄426) alarms/ day during this time period. The CMCP has sustained low numbers of alarms per monitored patient day. Discussion & Implications: The results of the CMCP have not only been sustained, but improved with time. The process has been integrated into the daily workflow of the BMT staff. With the number of false alarms down there is an improved work environment and re-sensitization to cardiac monitor alarms.

Defibrotide for the Treatment of Severe Hepatic Veno-Occlusive Disease: An Analysis of Clinical Benefit As Determined By Number Needed to Treat (NNT) to Achieve Complete Response and to Improve Survival

s / Biol Blood Marrow Transplant 21 (2015) S108eS113 S110 estimate) following HSCT, and 62% (by Kaplan-Meier estimate) in 45 pts following CT. Conclusions: Defibrotide in pts with VOD was generally well tolerated in this population, with manageable toxicity and promising response rates and survival. Safety results from prior studies are consistent with the favorable tolerability profile seen in this largest experience to date. Enrollment to the T-IND study continues, with updated results to be presented at the meeting. Support: Jazz Pharmaceuticals

Is Lymphoid Leukemia Less Susceptible to Busulfan Than Myeloid Leukemia? Comparison of Cytotoxicities of Busulfan, Melphalan, Fludarabine, Clofarabine, Cytosine Arabinoside, and Etoposide in Human Myeloid and Lymphoid Leukemia Cell Lines

s / Biol Blood Marrow Transplant 21 (2015) S185eS205 S189 101, 529, 0.46, higher than 10 and 11.7 micro M for BU, MEL, FLU, CLO, Ara-C and VP-16) and MOLT-4 was relatively sensitive to tested agents showing lowest or lower IC50 than those of other cell lines (14.2, 0.89, 0.28, 0.021, 0.0044 and 0.017 micro M respectively). BU, MEL, FLU, CLO and Ara-C showed similar activities against myeloid leukemia cells and lymphoid leukemia cells [Fig 1 A)-E)]. On the other hand, VP-16 showed more antileukemia activities against lymphoid leukemia cells than myeloid leukemia cells [Fig 1 F)]. Conclusion: Although the number of tested cell lines is limited, BU showed similar anti-leukemic activities against myeloid and lymphoid leukemia cell lines as is shown byMEL, FLU, CLO, and Ara-C. This data supports the use of busulfan as a preparative agent in patients with lymphoid malignancies. Further studies including combination of multiple chemotherapeutic agents would show more advanced scientific rationale for strategic conditioning regimen of HSCT for leukemia.

DNA Methyltransferase-1 (DNMT1): A Flow Cytometric Pharmacodynamic Assay for Azacitidine Hypomethylating Therapy after Allogeneic Hematopoietic Stem Cell Transplantation (AlloSCT)

s / Biol Blood Marrow Transplant 21 (2015) S266eS321 S318 (higher CT value), whereas the presence of an increased number of symptoms ( 3) was associated with a higher viral load. Lower viral load at initial surveillance testing correlated with an increased likelihood of being asymptomatic initially and remaining asymptomatic throughout the viral shedding episode. These results provide initial support for the use of viral load as a clinical endpoint in interventional trials.

Treatment with Human Mesenchymal Stem Cells (Remestemcel-L) Is Effective in Pediatric Patients with Refractory Acute Graft Versus Host Disease

s / Biol Blood Marrow Transplant 21 (2015) S322eS354 S339 Severe steroid-refractory acute graft-versus-host disease (aGVHD) causes significant morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Early clinical trials of therapywith humanmesenchymal stem cells (hMSCs) in pediatric patients with severe aGVHD, resistant to multiple immunosuppressive agents, showed promising results. In this study, we evaluated the risk/benefit profile of remestemcel-L, an off-the-shelf source of culture expanded human mesenchymal stem cells, provided by Mesoblast, Inc. under an expanded access program, as a rescue agent for treatment-resistant aGVHD in pediatric patients. Children with grade B-D aGVHD failing steroids and, in most cases, other immunosuppressive agents were eligible for enrollment. Patients received 8 bi-weekly i.v. infusions of 2 106 hMSCs/kg for 4 weeks, with an additional 4 weekly infusions after day +28 for patients who achieved either a partial or mixed response. The enrolled patients compose a very challenging population with severe disease that was nonresponsive to standard of care. One hundred sixty patients (median age, 10 years; 60% male; and 61% Caucasian) were treated in this study. One hundred thirty five patients (84%) received unrelated donor transplant. Graft source was bone marrow in 65 (41%), PBSC in 34 (21%), cord blood in 54 (34%) and donor leukocyte infusion in 5 (3%). At baseline, the distribution of aGVHD grades B, C, and D was 19%, 28%, and 53%, respectively; 81% of the patients had severe aGVHD (grade C or D). Median duration of aGVHD before enrollment was 28 d (range, 1 to 237), and patients had failed a median of 3 immunosuppressive agents. Organ involvement at baseline was 89% gastrointestinal (GI), 50% skin, and 29% liver. Sixtysix patients (41%) had 2 organs involved, and 24 patients (15%) had all 3 organs involved. The rate of overall response (complete and partial response) at day +28 was 74% for aGVHD grade B, 66% for grade C, and 59% for grade D. Overall response for individual organs at day +28 was 62% for GI, 77% for skin, and 53% for liver. Collectively, overall response at day +28 for patients treated for severe refractory aGVHD was 64%, and this response was correlated with statistically significant improved survival at day +100 after hMSC infusion. Patients who responded to therapy by day +28 had a higher Kaplan-Meier estimated probability of 100-d survival compared with patients who did not respond (79% vs. 21%; P < .0001). Substantial numbers of patients benefited from additional therapy beyond the initial 4 weeks of treatment. Ninety one patients (57%) received more than 8 infusions, with 48% of those patients experiencing additional improvement by day +100. Remestemcel-L infusions were generally well tolerated, without excessive infusional toxicities or ectopic tissue formation. The results of this study support the use of remestemcel-L for pediatric patients with refractory aGVHD.

Predictors of Survival in Multiple Myeloma Patients after Relapse from a Delayed Autologous Stem Cell Transplant

s / Biol Blood Marrow Transplant 21 (2015) S127eS146 S137 Day 0 [median count of 65.5/ul (range: 21.1e313.02) and of these 15 (60%) had achieved counts of> 20/ul on Day -1 itself [median count of 45.3/ul (range: 21.4e130.2)]. None of the patients had toxicity related to meloxicam. Subsequently 21 patients underwent auto SCT and their data was compared with 50 age and disease matched controls who had mobilization and auto SCT at our center between January 2013 and March 2014. Though mobilization rates and cell doses were not significantly different, the use of meloxicam and G-CSF was associated with faster neutrophil engraftment. Following auto SCT, there was lower Grade IIIeIV toxicity, lower transfusion requirement of red cells and reduction in the duration of hospital stay post SCT [Table 1]. Conclusion: The addition of meloxicam to G-CSF improves stem cell mobilization and is associated with faster engraftment, no additional toxicity, lower supportive care and lower duration of hospitalization. It is also potentially possible that we may be able to harvest the patients a day earlier depending upon the peripheral blood CD 34 counts achieved. This datawarrants a prospective randomized trial comparing Meloxicam + G-CSF with G-CSF alone as a mobilization strategy prior to autologous stem cell transplantation.

The Clinical Significance of Non-Tuberculosis Mycobacterium (NTM) Infection in Pediatric Hematopoietic Cell Transplant (HCT) Recipients

s / Biol Blood Marrow Transplant 21 (2015) S206eS239 S212 fared well (4/7 with DLBCL and 2/3 other B-cell lymphomas and 2/2 other T-cell lymphomas are alive and disease free). Patients in CR or PR had a better OS (18/23) compared with those patients with persistent or progressive disease at time of transplant (0/5). 11/29 patients died (9 from relapse and 2 from treatment related mortality). Conclusions: HSCT offers the possibility of cure for patients with NHL, especially for patients with ALCL and for those patients that have a good response to re-induction therapy prior to transplant. Relapsed disease post-transplant remains a major challenge for patients with LL and for patients transplanted with non-responsive disease. Post transplant therapies to target residual disease should be evaluated in these patients.

A Phase II, Safety and Efficacy Study of Fixed Dose Radioimmunotherapy (Zevalin, yttrium-90 ibritumomab tiuxetan) for Patients with Incomplete Response to Chemotherapy Prior to Autologous Stem Cell Transplant (ASCT) for Multiple Myeloma

s / Biol Blood Marrow Transplant 21 (2015) S185eS205 S199 or 4-hour pre-exposure to SAHA followed by washing then addition of [Clad+Gem+Bu] resulted in 40%, and 46% inhibition, respectively (P 1⁄4 0.005). These results underscore the importance of optimizing the sequence of drug exposure. Our results provide a basis for using [Clad+Gem+Bu] as a pre-transplant conditioning regimen for lymphoma patients undergoing HSCT, and further addition of an epigenetic modifier, such as SAHA, may play a role as a sensitizer to optimize the efficacy of the chemotherapy regimen.

Outcomes Resilience after System Stress: A Rapid-Cycle Response to Mitigate the Impact of Care Delivery System Stress on Primary Blood Stream Infections

s / Biol Blood Marrow Transplant 21 (2015) S355eS373 S373 Discussion & Implications: Other facilities should adapt a multiplatform model which addresses the growing computer literacy of patients while simultaneously assisting low literacy patients.

Persistent Fatigue in STEM CELL Transplant Survivors: A Biobehavioral Perspective

s / Biol Blood Marrow Transplant 21 (2015) S114eS126 S126 Discussion & Implications: Giving students the opportunity to experience blood and marrow transplant nursing in a controlled setting has allowed for smoother transitions into the role of RN, allowing for shorter new nurse orientation, low turnover rates and high retention rates. The opportunity of working with experienced blood and marrow transplant nurses provides new nurses with the tools and support needed to be successful in a challenging specialty. TRANSPLANT NURSING RESEARCH

Safety and Efficacy of Non-Myeloablative (NMA) Melphalan-Based Conditioning for Haploidentical Allogenic Stem Cell Transplantation (HaploSCT) in Patients with Advanced Lymphoma

s / Biol Blood Marrow Transplant 21 (2015) S185eS205 S196 and low TRM and should be further investigated in prospective clinical trials.

Alemtuzumab, Fludarabine, and Melphalan Reduced Intensity Conditioning Hematopoietic Cell Transplantation: Experience in over 200 Pediatric and Young Adult Patients with Primary Immune Deficiency, Metabolic, and Marrow Failure Disorders

s / Biol Blood Marrow Transplant 21 (2015) S206eS239 S232 use. Prospective collaboration among pediatric AlloHCT centers may help to provide conclusive evidence.

Patient and Provider Preferences for Survivorship Care Plans for Allogeneic Hematopoietic Cell Transplantation (HCT) Survivors: A Qualitative Study

s / Biol Blood Marrow Transplant 21 (2015) S171eS184 S174 malignancy in 34 patients (64%), lymphoid malignancy in 16 (30%) and plasma cell dyscrasia in 3 (6%). Sixteen (30%) had myeloablative conditioning. Median time from onset of symptoms to diagnosis was 2 days (0-15) and median time from transplant to diagnosis was 11 months (0-48). Twentyeight patients (53%) had URTI symptoms at presentation, 11 (21%) had LRTI and 14 (26%) had progression from URTI to LRTI. Stratifying per the ISI criteria, 16 patients (30%) were low risk, 31 (59%) were moderate and 6 (11%) were high risk. Median survival post RSV diagnosis was 17 months (0-78). Forty-six patients received oral ribavirin, 4 received aerosolized ribavirin and 3 were not treated. Five patients (3 high and 2 moderate risk) had RSV associated mortality with a median time to death of 8 days (7-18) due to respiratory failure and/or acute respiratory distress syndrome. Case fatality among high, moderate and low risk patients were 50%, 6% and 0%, respectively. Overall survival was statistically significant among the risk groups (P 0.0112). Predicting LRTI at diagnosis and risk of URTI to LRTI progression was however not significant (P 0.1375 and 0.4505, respectively). Conclusion: Use of oral ribavirin in low and moderate risk patients was associated with an excellent outcome. The ISI predicted survival but not risk of LRTI post RSV infection in an independent cohort of allogeneic stem cell transplant recipients treated with oral ribavirin. Elevated case fatality in high risk cases highlights the need for better therapies in this group of patients.

Targeting BCL-2 and BCR-Abl Activity in Ph+ALL

s / Biol Blood Marrow Transplant 21 (2015) S185eS205 S190 time to relapse from day 30 post-transplant was 69 days (range 41-230 days). The cumulative incidence of acute GVHD grade II-IV was 44% with 10% grade III-IV. The cumulative incidence of chronic GVHD at 2 years was 48%, with 22% extensive chronic GVHD. Conclusion: In this cohort of patients with AML/ MDS, MCP-1 levels at day 30 post allogeneic HCT in patients who had achieved complete remission and full chimerism were predictive of relapse two months on average prior to overt hematological relapse. Larger studies may find potential role of MCP-1 in predicting relapse post-transplant and development of early strategies for prevention.

Autologous Transplant/Gene Therapy for Adenosine Deaminase-Deficient Severe Combined Immune Deficiency

s / Biol Blood Marrow Transplant 21 (2015) S79eS107 S102 success using HLA-matched related donors; however, use of alternative donors has been associated with increased graft failure, graft versus host disease (GVHD), and transplantrelated mortality (TRM). HSCT using alternative donors with post-transplantation cyclophosphamide (PT/Cy) has been performed for hematologic malignancies with engraftment, GVHD, and TRM comparable to that seen with HLA-matched related donors. There are limited reports of HSCT in nonmalignant disorders using alternative donors and PT/Cy. Design: We transplanted 9 patients with non-malignant conditions (CGD1⁄43, DKC1⁄42, DBA1⁄41, HyperIgM1⁄41, XIAP1⁄41, IPEX1⁄41) using an alemtuzamab/fludarabine based reduced intensity conditioning (RIC). All patients received GVHD prophylaxis with PT/Cy, with the addition of mycophenolate mofetil and tacrolimus for HSCT with haploidentical donors and for alkylator-sensitive diagnoses (DKC). Six patients had 10/10 HLA-matched unrelated donors, and 3 had HLA-haploidentical related donors. Results: All 9 patients successfully engrafted by day 30. Ultimately, all patients have had sustained donor engraftment sufficient to eliminate manifestations of their underlying diseases. 6 of 9 patients are full donor chimeras off immunosuppression,1 patient is a stablemixed donor chimera (76% CD3+ T cells) off immunosuppression, 1 patient is a stable mixed donor chimera on a calcineurin inhibitor (CNI), and 1 patient had secondary graft failure but was ultimately retransplanted with myeloablative conditioning using the same donor, resulting in full donor chimerism and elimination of disease. One patient developed Grade 1 and one patient Grade 2 acute GVHD, both treated successfully with steroids and a CNI. Mild skin chronic GVHD developed in 1 patient, treated successfully with phototherapy. No serious infections occurred and there was no TRM. 1 patient developed veno-occlusive disease, treated successfully with defibrotide. Disease-free survival is 89% with a median follow-up of 14 months (6-30 months). The patient with secondary graft failure is now disease-free after myeloablative transplant, for an overall disease-free survival of 100% at a median of 14 months follow-up (6-60 months). Overall survival is 100%. Conclusion: We have observed successful engraftment sufficient to eliminate manifestations of disease, limited GVHD, and no TRM in 9 patients with nonmalignant disorders using alternative donors, RIC, and PT/Cy. RIC HSCT with PT/Cy shows promise for curing nonmalignant pediatric disorders, and potentially eliminates the need for CNI use after MUD BMT. Development of prospective clinical trials to confirm these observations is warranted.

Haploidentical Hematopoietic Stem Cell Transplantation with Treosulfan-Based Conditioning Regimen for Acute Leukemia Relapsing after Initial Allogeneic Transplantation

▪Background. Patients who experienced leukemia relapse after allogeneic hematopoietic stem cell transplantation (HSCT) have a dismal prognosis. Strategies to prevent or overcome post-transplant disease recurrence are limited and their efficacy still remains unclear. Recently, HSCT using haploidentical T-replete source has found increasing feasibility and acceptance thanks to new transplant platforms (Luznik L. et al., Biol Blood Marrow Transplant, 2008; Peccatori J. et al., Leukemia, 2014), allowing lower rates of graft-versus-host disease (GvHD) and non-relapse mortality (NRM). We evaluated the outcome of haploidentical transplant approach performed as second transplant (HSCT2) for patients who suffered of acute leukemia (AL) relapse after a first allogeneic HSCT (HSCT1).Methods. We analyzed 42 consecutive patients (median age 48 years) who underwent HSCT2 from a haploidentical donor, different from the previous one, between 2007 and 2013 in our Institute. Patients suffered of acute myeloid leukemia (n=31), acute lymphoblastic leukemia (n=9) or biphenotypic AL (n=2) relapsing after HSCT1 (matched related: 21; unrelated: 17; mismatched related: 4; umbilical cord blood: 4). Thirty-seven patients (88%) received re-induction therapy before HSCT2: 13 of them achieved complete remission (CR), 16 did not respond and 8 patients were transplanted directly in aplasia. In 5 cases HSCT2 was performed without re-induction therapy, in active disease (AD). Median Hematopoietic Cell Transplant-Co-morbidity Index was 2 (range 0-6) at time of HSCT2.All patients received treosulfan (14 g/m2 for 3 days) and fludarabine (30 mg/m2 for 5 days) as part of preparative regimen for HSCT2; conditioning was intensified with total body irradiation (4 Gray) in 18 (43%) or with melphalan (70 mg/m2 for 2 days) in 6 patients (14%). GvHD prophylaxis consisted of mycophenolate mofetile and sirolimus combined with pre-transplant anty-thymocyte globuline (n=36) or with post-transplant cyclophosphamide (n=6). Unmanipulated peripheral blood stem cells were used as graft source in all cases.Results. Three out of 42 patients (7%) experienced early death in aplasia, while all the others achieved neutrophil engraftment (n=39, 93%), with no primary graft rejection. Complete remission of disease at day +30 was reached in 37 out of 39 evaluated cases (95%). Estimated overall survival (OS) and leukemia-free survival (LFS) (± SE) rates at 1 year from HSCT2 were 37% (± 8%) and 35% (± 7%), respectively (Figure 1A). One-year relapse incidence (± SE) amounted to 32% (± 7%) and NRM (± SE) to 31% (± 7%) (Figure 1A) (7 patients died for infectious, 5 for GvHD and 1 for central nervous system toxicity). Sixteen patients (38%) experienced of acute GvHD grade II-IV and 20 (47%) of chronic GvHD. Disease status at HSCT2 (AD vs CR) had no impact on OS (HR:1.26, 95% CI 0.58-2.76, p=0.564) and relapse (HR:1.35, CI 95% 0.46-3.76, p=0.61). Conversely, time of remission from HSCT1>6 months provided the better outcome in terms of estimated OS (±SE) at 1 year (50% ± 10%) compared to patients with shorter duration of remission (18% ± 9%) (HR:0.49, 95% CI 0.24-1.02, p=0.05) and reduced risk of relapse (HR:0.28, 95% CI 0.11-0.75, p=0.011) (Figure 1B). After a median follow-up time of 22 months (range 6-68), 11 patients (26%) were alive with sustained leukemia remission.Conclusions. These results demonstrate the feasibility of HSCT2 from a haploidentical donor as treatment for AL relapse after initial allogeneic transplant, showing rates of OS, LFS and NRM comparable to the reported in literature for HSCT2 from matched related or unrelated graft, using the same or different donor (Christopeit M. et al., J Clin Oncol, 2013). Interestingly, disease status at HSCT2 did not influence the outcome of transplant. While, in accordance with previous reports, duration of remission after HSCT1 is the major factor involved in predicting outcome of a second transplant due to an increased relapse incidence after HSCT2 in patients with shorter HSCT1 remission, reflecting diseases biologically different. If confirmed in a larger cohort, these data could be useful in decision making after HSCT1 failure for disease recurrence. [Display omitted] DisclosuresBonini:MolMed S.p.A.: Consultancy.

CD34+ Cells in Bone Marrow Are Quantitatively and Functionally Impaired in Patients with Poor Graft Function Following Allogeneic Hematopoietic Stem Cell Transplantation

▪Background: Poor graft function (PGF), defined as the presence of 2 or 3 cytopenic counts (ANC ≤0.5×109/L and PLT ≤20×109/L or Hb ≤70 g/L) for at least 3 consecutive days beyond day +28 post-transplantation with transfusion requirement, in the presence of complete donor chimerism, and in the absence of severe graft-versus-host disease (GVHD) or hematological relapse, remains a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Emerging evidence from mouse studies suggested that effective hematopoiesis depends on a particular bone marrow (BM) microenvironment in which hematopoietic stem cells (HSCs) reside. We recently found that the impaired BM microenvironment may contribute to the occurrence of PGF post-HSCT using a prospective nested case-control study (Kong Y, et al. Biol Blood Marrow Transplant.2013;19:1465-1473). However, it remains unclear whether the quantitative and functional abnormalities of HSCs are involved in the pathogenesis of PGF.Aims: To evaluate whether the quantitative and functional abnormalities of CD34+cells in BM are involved in the pathogenesis of PGF after allo-HSCT.Methods: The normal multilineage differentiation capacity of the CD34+ cells sorted from the donors BM of PGF and good graft function (GGF) patients underwent allo-HSCT were investigated in vivo using sublethally irradiated NOD-PrkdcscidIL2rgnull mice by intra-BM injection. Subsequently, a prospective nested case-control study was performed enrolling 15 patients with PGF, 30 matched patients with GGF after allo-HSCT and their healthy donors (HDs). To minimize the potential influence of the length of time after allo-HSCT, the PGF and GGF patients had their HSCs tested at a matched median time point after allo-HSCT. Quantification of CD34+ cells was performed by flow cytometry. Owning to the limited numbers of CD34+cells in PGF patients, colony-forming cell (CFC) assay, instead of xenograft assay, was performed to investigate the multi-pluripotency of post-HSCT HSCs in vitro. Additionally, the quiescent cell cycle status and reactive oxygen species (ROS) production of HSCs in the post-HSCT BM were further analyzed by flow cytometry in the aforementioned three groups of subjects. The study was approved by the Ethics Committee of Peking University People’s Hospital and written informed consent was obtained from all subjects.Results: CD34+ BM cells from donors of PGF and GGF patients demonstrated no significant differences of normal multilineage differentiation capacity in NOD-PrkdcscidIL2rgnull mice. In the prospective nested case-control study, the demographic and clinical characteristics showed no significant difference between allo-HSCT patients with PGF and those with GGF. The percentage of CD34+ cells significantly decreased in the BM of PGF patients post-HSCT, although similar amounts of CD34+ cells were transplanted. We observed a remarkable decrease in stem cell/progenitor frequency in post-HSCT CD34+ BM cells of PGF patients as determined by CFC assay. Additionally, post-HSCT BM of PGF patients contained a significantly lower number of quiescent cells in the CD34+CD38low fraction compared to the BM of GGF patients and HDs. Moreover, significantly higher levels of ROS were observed in BM CD34+, CD34+CD38low, CD34+CD38high fractions of PGF patients following allo-HSCT compared to that in GGF patients and HDs.Summary/Conclusion: Although the frequency and function of CD34+ BM cells were revealed normal pre-HSCT, CD34+ BM cells were quantitatively and functionally impaired in patients with PGF following allo-HSCT. Our data indicate that the diminished regenerative capacity of BM of PGF patients following allo-HSCT is possibly related to a loss of quiescence and a reduced tolerability of CD34+ cells to oxidative stress.Acknowledgment: Supported by the National Natural Science Foundation of China (grant nos. 81370638&81230013), the Beijing Municipal Science and Technology Program (grant no. Z141100000214011), and Peking University People’s Hospital Research and Development Funds (grant no. RDB2012-23). DisclosuresNo relevant conflicts of interest to declare.

Desialylation Induces Apoptosis and Phagocytosis of Platelets in Patients with Prolonged Isolated Thrombocytopenia after Allogeneic Hematopoietic Stem Cell Transplantation

Introduction:Prolonged isolated thrombocytopenia (PIT) represents a significant complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and is associated with an adverse patient prognosis and higher transplant-related mortality owing to a higher risk of infection events, severe (grades 3 to 4) acute graft-versus-host disease (GVHD) and chronic GVHD. PIT is defined as a peripheral platelet count less than 100×109/L without sustained anemia or leukopenia for more than 3 months after allo-HSCT (Zhang X, et al. Biol Blood Marrow Transplant, 2011). However, the underlying mechanisms remain unclear. Different kinds of functional glycoproteins are expressed on the platelet surface, with sialic acid residues at the end of their glycan. Desialylation of platelet glycoproteins has been found to be associated with accelerated platelet clearance in refrigerated platelets (Gerard Jansen AJ, et al. Blood 2012). Platelet-specific glycoprotein GPIbα,the functional subunit of the von Willebrand factor receptor, was the majorly desialylated glycoprotein; NEU1, one of the four human sialidases, was the enzyme that catalyzed GPIbα desialylation. However, few studies have focused on this mechanism in patients suffering PIT after allo-HSCT. In this study, we hypothesized that desialylation on platelet surfaces is associated with PIT after allo-HSCT. The mechanisms participating in this process may include GPIbα clustering, platelet apoptosis and phagocytosis by macrophages.Patients and methods:Blood samples were collected 90 days after allo-HSCT from 70 patients with PIT. Samples from post-transplantation patients who have normal platelet counts were taken as controls. Sialylation and desialylation were measured by detecting specific lectins via flow cytometry. Human sialidase expression was determined by immunofluorescence, flow cytometry and reverse transcription PCR. Platelet apoptosis markers were measured by flow cytometry, and macrophages stimulated from THP-1 cells were used in the phagocytosis assay.Results:We tested sialic acid residues and the desialylation markers, including β-galactose and β-N-Acetyl glucosamine, on the platelet surface, and found that platelets from PIT patients had significantly higher desialylation levels. Serum sialic acid levels were measured, and the results showed higher levels in PIT patients. Further, NEU1 was found to be over-expressed on the surface of platelets from PIT patients, and was found to be the enzyme that catalyzed the platelet surface desialylation. To further reveal the mechanism that lead to PIT, we proved that GPIbα was the desialylated glycoprotein on platelets from PIT patients. We found that GPIbα desialylation and clustering in PIT patients induced changes in the expression of Bcl-2 family protein, as a 2-fold increase in active Bax expression and a similar decrease in Bcl-XL expression were observed. Depolarization of the inner mitochondria membrane was augmented in desialylated platelets from PIT patients, indicating increased platelet apoptosis. Moreover, macrophages stimulated from the THP-1 cell line preferred to phagocytose desialylated platelets from PIT patients in vitro; this process could be blocked by the sialidase inhibitor, DANA. In the in vitrostudy, we found that dexamethasone led to a 32% decrease in phagocytosis, whereas oseltamivir, an antiviral medicine that can block sialidase from influenza virus, could also partially function on human sialidase and protect 43% of platelets from phagocytosis.In conclusion, our results demonstrate that desialylation played a role in the mechanism of prolonged isolated thrombocytopenia after allo-HSCT, most likely through platelet apoptosis induction and increased phagocytosis by macrophages in the peripheral circulation. Dexamethasone and oseltamivir could decrease platelet apoptosis and inhibit platelet phagocytosis in vitro, implying a novel potential method for treating PIT after allo-HSCT. DisclosuresNo relevant conflicts of interest to declare.

Prolonged Isolated Thrombocytopenia (PIT) Is Associated with Platelet Aggregation, Which Is Affected By Endothelial Progenitor Cells (EPCs) Following Allo-HSCT

IntroductionAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for malignant hematological diseases, but prolonged isolated thrombocytopenia (PIT) following allo-HSCT seriously decreases patient survival after transplantation. PIT was defined as a platelet count <100×109/L for more than 3 months after HSCT, recovery of all other cell counts, and no apparent cause for thrombocytopenia(Zhang XH et al, Biol Blood Marrow Transplant, 2011). Endothelial progenitor cells (EPCs) are a minor subpopulation of peripheral blood mononuclear cells with surface expression of both endothelial and stem cell (CD34) lineage antigens. These cells were able to promote vasculogenesis at sites of ischemia in vitro. Recent research has shown that EPCs inhibit platelet aggregation by secreting prostacyclin (PGI2) (Abou-Saleh et al., Circulation, 2009). However, to our knowledge, there is no research regarding the relationship between EPCs and PIT after allo-HSCT. We hypothesize that PIT after allo-HSCT is associated with an abnormal increase of EPCs, which might secret high levels of PGI2 and inhibit platelet activation and aggregation.Methods and resultsWe enrolled 78 patients who underwent allo-HSCT in our department due of various types of hematological malignancies, of which 38 patients were suffering from PIT and 40 were matched controls.In the patients suffering from PIT after allo-HSCT, a significantly higher percentage of BM EPCs percent were found compared with the control group (0.041%±0.022% v 0.021%±0.012%, p <0.05) at +3 months after allo-HSCT. However, when the PIT patients were classified into subgroups according to the PLT count at +3 month after allo-HSCT, the difference only existed in patients with lower platelet counts (PLT<50 x 109/L) and controls (0.052%±0.025 v 0.021%±0.012%, p<0.05), but there was no significant difference between the subgroups of PIT patients.Then, we tested whether PGI2 levels had the same tendency as the EPCs. We found that in PIT patients, the serum PGI2 levels seemed to be higher than in the control group, but the difference did not reach statistical significance (379.23±43.41 pg/ml v 234.75±38.49 pg/ml, p=0.08). However, when classified into subgroups, serum PGI2 levels of patients with lower platelet counts were significantly higher than those of the controls (433.32±76.43 pg/ml v 234.75±38.49 pg/ml, p<0.05).We also measured platelet aggregation and activation in the two groups. We found that platelet aggregation and activation (measured by CD62P and PAC-1, respectively) in PIT patients were significantly lower than in the control group (PLT aggregation: 21.2%± 5.5% v 29.2% ±8.7%, p <0.05; PLT activation-PAC-1: 21.6%± 7.3% v 28.7% ±8.3%, p <0.05; PLT activation-CD62P: 43.7%± 8.3% v 38.9% ±9.4%, p =0.22). However, when further classified into subgroups, none of the groups showed significant differences.ConclusionPatients suffering PIT after allo-HSCT have a higher percentage of BM EPCs, higher serum PGI2 levels and lower platelet activation and aggregation. These data might lead to new insight into the mechanism of PIT following allo-HSCT, and provide a potential avenue for treatment for PIT after allo-HSCT.Disclosures: No relevant conflicts of interest to declare.