Peripheral Hematopoietic Stem Cell Research Articles

Commentary on the preservation of human RBC, platelets, plasma clotting proteins and peripheral blood mononuclear hematopoietic stem cells by freezing and storage in a 80 C mechanical freezer

Commentary on the preservation of human RBC, platelets, plasma clotting proteins and peripheral blood mononuclear hematopoietic stem cells by freezing and storage in a 80 C mechanical freezer

ASXL1-Mutated Donor HSCs Evolved into FLT3-ITD Positive AML in the Unrelated Donor and FLT3-ITD Negative AML in the Recipient after Transplant

It has been shown that hematopoietic stem cells (HSCs) acquire somatic mutations over time, most commonly involving DNMT3A, ASXL1, and TET2 genes. These mutation-bearing stem cells contribute to normal blood development, harbor a selective growth advantage compared to their non-mutated normal stem/progenitor counterparts, lead to the development of clonal hematopoiesis, and can subsequently gain additional mutations that confer full malignant potential. Here we report an unusual two cases of genetically distinct AML arising from a single pre-leukemic ASXL1 L866X mutant clone.A 28-year-old female was diagnosed with acute B lymphoblastic leukemia by the classical morphology and immunophenotype. Cytogenetic evaluation showed a normal 46, XX, female karyotype. Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) from a matched unrelated donor, a 33-year-old male, was performed. Donor peripheral blood derived HSCs were mobilized and administered to the recipient. On the day +50 after Allo-HSCT, cytogenetics showed a donor derived 46, XY, male karyotype. Consistent with this, chimerism analysis showed 100% donor chimerism . Twenty months after Allo-HSCT, she relapsed with AML with monocytic differentiation. Immunophenotyping revealed the blasts were positive for CD34, CD117, HLA-DR, CD13, CD33, CD64, CD14 and MPO, and were negative for B-cell or T-cell markers. Cytogenetics showed a normal 46, XY, male donor-derived karyotype. Engraftment study continued to show complete donor chimerism in the bone marrow. Molecular study revealed the ASXL1 L866X mutation. Notably, the ASXL1 L866X mutation was not only found in DNA obtained from the recipient at the time of the original diagnosis of donor cell leukemia (AML), but also found in DNA obtained on day +50 after allo-HSCT when the recipient showed full donor engraftment as well as successful complete remission. Moreover, this ASXL1 L866X mutation was also observed in DNA from the healthy donor at the time of donation of peripheral HSCs. In contrast, this ASXL1 L866X mutation was not observed in DNA obtained from recipient specimens at diagnosis or during complete remission of acute B-lymphocytic leukemia before allo-HSCT. Therefore, these data confirmed that this ASXL1 L866X mutation originated from donor HSCs . Except for the ASXL1 L866X mutation, we did not find mutations in CEBPA, DNMT3A, ETV6 (TEL), FLT3-ITD, FLT3-TKD, IDH1, IDH2, KIT, KRAS, NPM1, NRAS, PHF6, PTPN11, RUNX1, TET2, or TP53 genes in the recipient at diagnosis, remission, or relapse of donor cell leukemia.Eighty-four months after donation peripheral HSCs, the previously healthy donor also developed AML. The blasts were positive for CD34, CD117, HLA-DR, CD13, CD33, CD123, CD38, and MPO, and were negative for B- and T-cell markers. Cytogenetic analysis revealed 46, XY, male normal karyotype. The ASXL1 L866X and FLT-3/ITD mutation were identified in leukemia cells, but were not detected in his germline fingernail DNA. After four cycles of induction chemotherapy, he received HLA-identical sibling Allo-HSCT from his brother for salvaged therapy. He acquired complete donor chimerism on day +60 and FLT-3/ITD-positive cells disappeared. Two months later, the leukemia relapsed and the patient died from the disease.The fact that 84 months after donation peripheral HSCs, the healthy donor,carrying the same ASXL1 mutation, was diagnosed AML with additional new gene mutation, FLT-3/ITD, was strongly supported the most recent theory that mutation acquisition occurs in functionally normal HSCs and clonal evolution of pre-leukemic HSCs precedes human AML. Moreover, our findings provide in vivo evidence to support a clonal expansion human model in which ASXL1 is an early initiation mutation while FLT3 is a subsequent driver mutation for abnormal HSCs clonal expansion and malignant transformation. DisclosuresNo relevant conflicts of interest to declare.

Genome Editing of the Bcl11A Erythroid Specific Enhancer in Bone Marrow Derived Hematopoietic Stem and Progenitor Cells for the Treatment of Sickle Cell Disease

Ablation of Bcl11A could be a viable approach for the treatment of β-hemoglobinopathies such as β-thalassemia and sickle cell disease (SCD), since patients with Bcl11A haploinsufficiency have persistently high levels of fetal hemoglobin (HbF) (up to 30%), which are associated with development of minimal to no disease symptoms. Genome editing by engineered zinc-finger nucleases that target either the exon 2 (exon ZFN) or the GATA motif of the erythroid specific enhancer (enhancer ZFN) of Bcl11A has been shown to increase HbF level in erythroid progeny from mobilized peripheral hematopoietic stem and progenitor cells (PB-CD34+ HSPCs). However, peripheral mobilization of CD34+ cells is associated with high risk and currently is not an option for SCD patients. Therefore, we investigated the efficacy of genome editing of Bcl11A in bone marrow derived CD34+ cells (BM-CD34+ HSPCs).We first established a clinically compatible large-scale process to isolate CD34+ HSPCs from human bone marrow aspirates and to transiently express the ZFN protein by mRNA electroporation. The CD34+ isolation process resulted in ~ 95% pure CD34+ cells with greater than 90% viability. Both the exon and the enhancer ZFN drove 50-60% Bcl11A gene editing, resulting in a robust elevation of HbF in the erythroid progeny. Notably, the BM-CD34+ HSPCs were found to contain a small population (10 to 25%) of CD34+CD19+ pro-B cells that were refractory to ZFN transfection under our current electroporation condition. Since CD34+CD19+ pro-B cells are not expected to contribute to reconstituting the hematopoietic system other than B-cell lineage, the Bcl11A editing efficiency in the multipotent BM-CD34+ HSPC could be even higher.The engraftment abilities of Bcl11A edited BM-CD34+ cells were then investigated in an immunodeficient NOD/scid/gamma (NSG) mouse model. At a dose of 1 million cells per mouse, treatment with either the exon ZFN or the enhancer ZFN did not detectably impact engraftment or multi-lineage reconstitution compared with untreated cells. However, Bcl11A marking in engrafted human cells was found to be markedly higher in the mice treated by the enhancer ZFN than that by the exon ZFN. The exon ZFN resulted in a strong bias towards in-frame mutations across multi-lineages with the strongest effect observed in the B-cell lineage, suggesting that a threshold level of Bcl11A is required for efficient hematopoietic reconstitution and that cells fully lacking it due to disruption of the coding sequence are at a disadvantage. In contrast, the enhancer ZFN resulted in comparable Bcl11A marking across all lineages with no apparent selection for cells with a functional GATA sequence. Collectively, these data indicate that genome editing of the erythroid specific enhancer of Bcl11A in BM-CD34+ promotes HbF reactivation in the erythroid progeny while maintaining the engraftment and multi-lineage repopulating activities of edited BM-CD34+ HSPCs, which supports further clinical development of this approach for the treatment of SCD. DisclosuresTan:Biogen: Employment, Equity Ownership. Chang:Biogen: Employment, Equity Ownership. Smith:Biogen: Employment, Equity Ownership. Chen:Biogen: Employment, Equity Ownership. Sullivan:Biogen: Employment, Equity Ownership. Zhou:Biogen: Employment, Equity Ownership. Reik:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Urnov:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Rebar:Sangamo BioSciences: Employment. Danos:Biogen: Employment, Equity Ownership. Jiang:Biogen: Employment, Equity Ownership.

The evaluation of the efficacy and safety of original filgrastim (Neupogen®), biosimilar filgrastim (Leucostim®) and Lenograstim (Granocyte®) in CD34 + peripheral hematopoietic stem cell mobilisation for allogeneic hematopoieitic stem cell transplant donors

The evaluation of the efficacy and safety of original filgrastim (Neupogen®), biosimilar filgrastim (Leucostim®) and Lenograstim (Granocyte®) in CD34 + peripheral hematopoietic stem cell mobilisation for allogeneic hematopoieitic stem cell transplant donors

Infused autograft lymphocyte-to-monocyte ratio and survival in T-cell lymphoma post-autologous peripheral blood hematopoietic stem cell transplantation.

BackgroundThe infused autograft lymphocyte-to-monocyte ratio (A-LMR) is a prognostic factor for survival in B-cell lymphomas post-autologous peripheral hematopoietic stem cell transplantation (APHSCT). Thus, we set out to investigate if the A-LMR is also a prognostic factor for survival post-APHSCT in T-cell lymphomas.MethodsFrom 1998 to 2014, 109 T-cell lymphoma patients that underwent APHSCT were studied. Receiver operating characteristic (ROC) and area under the curve (AUC) were used to identify the optimal cut-off value of A-LMR for survival analysis and k-fold cross-validation model to validate the A-LMR cut-off value. Univariate and multivariate Cox proportional hazard models were used to assess the prognostic discriminator power of A-LMR.ResultsROC and AUC identified an A-LMR ≥ 1 as the best cut-off value and was validated by k-fold cross-validation. Multivariate analysis showed A-LMR to be an independent prognostic factor for overall survival (OS) and progression-free survival (PFS). Patients with an A-LMR ≥ 1.0 experienced a superior OS and PFS versus patients with an A-LMR < 1.0 [median OS was not reached vs 17.9 months, 5-year OS rates of 87 % (95 % confidence interval (CI), 75–94 %) vs 26 % (95 % CI, 13–42 %), p < 0.0001; median PFS was not reached vs 11.9 months, 5-year PFS rates of 72 % (95 % CI, 58–83 %) vs 16 % (95 % CI, 6–32 %), p < 0.0001].ConclusionsA-LMR is also a prognostic factor for clinical outcomes in patients with T-cell lymphomas undergoing APHSCT.

Secondary osteosarcoma arising from osteochondroma following autologous stem cell transplantation with total-body irradiation for neuroblastoma: A case report.

The present study reports the first case of malignant transformation to osteosarcoma arising from osteochondroma following childhood total-body irradiation (TBI). The association between TBI and later development of osteochondroma is well-known; however, malignant degeneration arising from radiation-induced osteochondroma is rare. The current study describes the case of a 17-year-old boy with osteosarcoma arising from osteochondroma of the left distal humerus, which developed following TBI. TBI was administered as part of a conditioning regimen received prior to autologous peripheral hematopoietic stem cell transplantation (HSCT) at the age of 6 years, following an initial diagnosis of neuroblastoma at the age of 5 years. The patient subsequently underwent preoperative chemotherapy followed by wide local excision and reconstruction with an extracorporeally irradiated autograft. Postoperative chemotherapy was administered, and the patient demonstrated no clinical or radiographic evidence of recurrence after 40 months of follow-up. To the best of our knowledge, this is only the second reported case of malignant degeneration of osteochondroma following childhood TBI, and the first reported case of transformation to osteosarcoma. The current case highlights the importance of close observation for secondary malignancies in this patient population.

Macrophage-Lineage Cells Negatively Regulate the Hematopoietic Stem Cell Pool in Response to Interferon Gamma at Steady State and During Infection.

Bone marrow (BM) resident macrophages (Mϕs) regulate hematopoietic stem cell (HSC) mobilization; however, their impact on HSC function has not been investigated. We demonstrate that depletion of BM resident Mϕs increases HSC proliferation as well as the pool of quiescent HSCs. At the same time, during bacterial infection where BM resident Mϕs are selectively increased we observe a decrease in HSC numbers. Moreover, strategies that deplete or reduce Mϕs during infection prevent HSC loss and rescue HSC function. We previously found that the transient loss of HSCs during infection is interferon-gamma (IFNγ)-dependent. We now demonstrate that IFNγ signaling specifically in Mϕs is critical for both the diminished HSC pool and maintenance of BM resident Mϕs during infection. In addition to the IFNγ-dependent loss of BM HSC and progenitor cells (HSPCs) during infection, IFNγ reduced circulating HSPC numbers. Importantly, under infection conditions AMD3100 or G-CSF-induced stem cell mobilization was impaired. Taken together, our data show that IFNγ acts on Mϕs, which are a negative regulator of the HSC pool, to drive the loss in BM and peripheral HSCs during infection. Our findings demonstrate that modulating BM resident Mϕ numbers can impact HSC function in vivo, which may be therapeutically useful for hematologic conditions and refinement of HSC transplantation protocols.

Infused autograft lymphocyte to monocyte ratio and survival in T-cell lymphomas post-autologous peripheral blood hematopoietic stem cell transplantation.

e19500 Background: We recently reported that the infused autograft lymphocyte to monocyte ratio (A-LMR) is a prognostic factor for survival in B-cell lymphomas post-autologous peripheral hematopoietic stem cell transplantation (APHSCT). Thus, we set out to investigate if the A-LMR is also a prognostic factor for survival post-APHSCT in T-cell lymphomas. Methods: From 1998 to 2014, 109 T-cell lymphoma patients that underwent APHSCT were studied. Receiver operating characteristic and area under the curve identified an A-LMR ≥ 1 as the best cut-off value and validated by the k-fold cross-validation. Results: . Multivariate analysis showed A-LMR to be an independent prognostic factor for overall survival (OS) and progression-free survival (PFS). Patients with an A-LMR ≥ 1.0 experienced a superior OS and PFS versus patients with an A-LMR < 1.0 [median OS was not reached vs 17.9 months, 5-year OS rates of 87% (95 CI, 75%-94%) vs 26% (95 CI, 13%-42 %), p < 0.0001, respectively; median PFS was not reached vs 11.9...

The Safety of Granulocyte Colony Stimulating Factor (G-CSF) Administration in Pediatric Donors before Peripheral Hematopoietic Stem Cell Donation

The Safety of Granulocyte Colony Stimulating Factor (G-CSF) Administration in Pediatric Donors before Peripheral Hematopoietic Stem Cell Donation

Infused autograft lymphocyte to monocyte ratio predicts survival in classical Hodgkin lymphoma.

The infused autograft lymphocyte to monocyte ratio (A-LMR) as a surrogate marker of host immunity (ie, absolute lymphocyte count) and CD14+ HLA-DRlow/neg immunosuppressive monocytes (ie, absolute monocyte count) is a prognostic factor for patients with diffuse large B-cell lymphoma after autologous peripheral hematopoietic stem cell transplantation (APHSCT). Thus, we set out to investigate if A-LMR can also affect survival post-APHSCT in classical Hodgkin lymphoma. From 1994 to 2012, 183 patients with classical Hodgkin lymphoma who underwent APHSCT were studied. The patients were randomly divided into a training set (n=122) and a validation set (n=61). The receiver operating characteristic and area under the curve identified an A-LMR ≥1 as the best cut-off value and validated by the k-fold cross-validation in the training set. Multivariate analysis showed A-LMR to be an independent prognostic factor for survival in the training set. Patients with an A-LMR ≥1.0 experienced a superior overall survival (OS) versus patients with an A-LMR <1.0 (median OS not reached versus 40.4 months, 5-year OS rates of 86% [95% CI 72–93] versus 43% [95% CI 28–58], P<0.0001, respectively) in the training set. In the validation set, an A-LMR ≥1 showed a median OS of not reached versus 41.4 months for an A-LMR <1, 5-year OS rates of 90% (95% CI 73–97) versus 48% (95% CI 28–68; P<0.0001). A-LMR provides a platform to engineer an autograft versus tumor effect to improve clinical outcomes in patients with classical Hodgkin lymphoma undergoing APHSCT.

Aldehyde Dehydrogenase Activity may Replace Colony Forming Units as A Quality Marker in Hematopoietic Peripheral Stem Cells Collections

Aldehyde Dehydrogenase Activity may Replace Colony Forming Units as A Quality Marker in Hematopoietic Peripheral Stem Cells Collections

Salvage Chemotherapy Followed By Autologous Peripheral Stem Cell Transplantation for Relapsed Acute Promyelocytic Leukemia; A Single Center Experience

Background: After the addition of differentiating agents such as all-transretinoic acid (ATRA) to conventional chemotherapy, treatment outcomes of acute promyelocytic leukemia (APL) had been markedly improved. However, about 10% of patients with APL still have experienced relapse and have to receive salvage chemotherapy. Although arsenic trioxide (ATO) plays an important role in salvage chemotherapy as well as induction chemotherapy, the optimal treatment strategy remains unclear, especially for patients in whom autologous peripheral stem cell transplantation (AutoHCT) is planned. We tried to find the role of salvage chemotherapy including ATO-based regimen in relapsed APL patients, and want to share our experience in these patients.Patients and Methods: Among 77 patients diagnosed with APL from 2005 to 2014 at Yonsei University Severance Hospital, 11 (14.3 %) relapsed patients were enrolled. Clinical data of these patients was retrospectively reviewed. Except one patient refused further treatment at the time of relapse, 10 patients were included in the final analysis for treatment outcome.Results: All patients received remission induction chemotherapy with modified PETHEMA regimen, and achieved complete remission (CR) confirmed at the molecular level. The median duration of the first CR was 38.8 months (range 28.0–88.0 months). As pre-transplant salvage chemotherapy, earlier 3 patients received conventional chemotherapy regimen without ATO, but later 7 patients received ATO-based regimen after ATO was available in our country. There was no statistical difference in median duration to the second CR from salvage chemotherapy according to the regimen (68 days in conventional chemotherapy patients vs. 60 days in ATO-based chemotherapy, P>0.05). However, 3 patients receiving conventional regimen without ATO did not received AutoHCT because they died due to treatment-related complications during additional salvage chemotherapies. In contrast, 7 patients receiving ATO-based regimen received AutoHCT successfully, and all patients remains alive without further relapse. The median duration from the time of second CR to AutoHCT was 3.8 months (range 1.3–7.6 months), and median follow up duration after AutoHCT was 8.3 months (range 0.6–45.9 months). In these 7 patients receiving ATO-based regimens, duration of ATO treatment was variable (median 72 days, range 42-113 days), and a range of total dose of ATO given before AutoHCT was also variable (median 701.23 mg, range 420-1242.8 mg). Total dose and treatment duration of ATO was mainly determined by the time of molecular CR achieved again. The peripheral hematopoietic stem cells were harvested when molecular complete remission was confirmed, and median number of stem cells harvested was 5.14 x 10^6 cells/kg of recipient (range 2.90–7.76 x 10^6 cells/kg). The yield of harvest peripheral stem cells and the outcomes of followed AutoHCT were not significantly related to the total dose or treatment duration of pre-transplant ATO-based salvage therapy.Conclusion: In this study, ATO-based chemotherapy followed by AutoHCT was safe and excellent salvage treatment strategy for relapsed APL patients. The achievement of second molecular CR might be more important in this treatment strategy than total dose or treatment duration of ATO-based salvage therapy before AutoHCT. DisclosuresNo relevant conflicts of interest to declare.

Plerixafor Successfully Rescues Poor Mobilizers Resulting in Adequate CD34 Cell Numbers However Stem Cell Products Yield Lower Numbers of CFU/CD34 and Delayed Neutrophil Engraftment

Background: The CXCR4 inhibitor plerixafor was studied as a rescue agent in the setting of poor chemotherapy/filgrastim mobilization in a phase II trial of adults with lymphoma and myeloma who underwent a peripheral hematopoietic stem cell (HSC) mobilization prior to autologous transplant (ASCT) (NCT01037517). Given the wide range of expression of CD34 in myeloid precursors we were interested in the quality of the CD34 positive cells that were collected to proceed to transplant in poor mobilizers. Amongst a population of poor mobilizers would the CD34+ population contain cells of a more differentiated phenotype or express different proportions of homing receptors as CD34 cells collected from patients who mobilized easily?Methods: From 2009 to 2012 sequential adult patients with a diagnosis of lymphoma or multiple myeloma undergoing chemo-mobilization in preparation for autologous stem cell collection were enrolled on a phase II study to examine a strategy of plerixafor rescue in the setting of poor mobilization. Plerixafor (240 mcg/kg sc X 1) was administered on the evening prior to planned apheresis in subjects with a post nadir white blood cell count >1 X109/L and a peripheral blood [CD34] ≤ 10x106/L on the day prior to planned collection. Participants proceeded to stem cell apheresis the next day if their peripheral blood CD34 > 10x106/L. An aliquot from the mobilized product was analyzed for CD34+ cell subsets using markers of stemness ( CD33-, CD90, CD133 , CD166) and homing (CD26, CD49F, CD184) Differences between the groups are reported as means ±standard deviation and compared by two tailed t-tests.Results: 46/48 subjects were successfully mobilized in one day (HSC collection of >2 x 106 CD34+ cells/kg): 38 pts were not administered plerixafor (good mobilizer) and 8 pts were administered plerixafor (plerixafor rescue). The two participants not able to mobilize with plerixafor had evidence of progression of underlying disease within 2 weeks of attempted mobilization. Forty four participants (6/8 plerixafor and 38/38 good mobilizers) underwent ASCT. Median time to platelet engraftment was similar between good mobilizers (12 d, range 9-22d) and the plerixafor rescue group (13 d, range 12-13 d). The median time to neutrophil engraftment was prolonged in the plerixafor rescue group (23 d, range 17-67d) compared to good mobilizers (17 d, range 10-31d). CD34 content/kg of the apheresis product collected was similar between groups (good mobilizer 11.6±7.9 versus the plerixafor rescue group 6.4±4.6 p = 0.1). Subset analysis of CD34 positive cells in the products showed comparable expression of markers associated with more immature HSC progenitors but fewer CFU per CD34 positive cell plated. Homing receptors on the CD34 positive cells were similar despite the use of a CXCR4 inhibitor in the poor mobilizers. (See table) TableGood mobilizer N= 38 (Mean % ± SD)Plerixafor rescue N= 8 (Mean % ± SD)P valueStemmness MarkersCD90+ (Thy-1)24.4±12.730.6±19.30.3CD133+ (prominin1)59.1 ± 12.959.1 ± 16.80.9CD166+ (ALCAM)5.7±3.36.6±5.10.5CD33- (Siglec-3)80.6±3.369.8±13.10.01CFU/105 CD34+ cells1015±799276±3160.02Homing MarkersCD26 (dipeptidyl peptidase-4)5.4±410.4±13.30.05CD49F (VLA4)8.5±7.96.8±9.70.6CD184 (CXCR4)16.2±20.517.7±9.70.8Discussion: Plerixafor used to rescue patients at risk of poor mobilization resulted in collection of adequate numbers of CD34+ cells for transplantation and allowed ASCT in a majority of poor mobilizers in our series. Delayed neutrophil recovery, was seen in the poor mobilizer cohort. We did not see a skewing of the CD34+ cells in the products from poor mobilizers to a more mature phenotype but did find lower clonogenic potency with fewer CFU generated/CD34 cells plated. DisclosuresOff Label Use: Plerixafor used as initial mobilization strategy in autologous stem cell mobilization.

Infused Autograft Lymphocyte to Monocyte Ratio and Survival in Classical Hodgkin Lymphoma

Infused autograft absolute lymphocyte count (A-ALC) is a prognostic factor for survival post-autologous peripheral hematopoietic stem cell transplantation (APHSCT) for lymphoma. Myeloid-derived suppressor cells (MDSCs) affect tumor growth by suppression of host anti-tumor immunity. Thus, we set out to investigate if the infused autograft lymphocyte /monocyte ratio (A-LMR), as a biomarker of host immunity (i.e., lymphocytes) and MDSCs (i.e., monocytes), affects survival post-APHSCT in patients with classical Hodgkin lymphoma (cHL). Only patients that collected their stem cells through the peripheral blood were included in the study. From 1994 to 2012, 183 cHL patients qualified for the study. The 183 patients were randomly divided into a training set (n = 122) and a validation set (n = 61). Receiver operating characteristic and area under the curve (AUC) identified an A-LMR ≥ 1 as the best cut-off value (AUC = 0.8, p < 0.01) and validated by the k-fold cross-validation (AUC = 0.8) in the training set. Multivariate analysis showed A-LMR to be an independent prognostic factor for survival in the training set. Patients with an A-LMR ≥ 1.0 experienced a superior overall survival (OS) versus patients with an A-LMR < 1.0 (median OS was not reached vs 40.4 months, 5-year OS rates of 86% (95 CI, 72%-93%) vs 43% (95 CI, 28-58 %), p < 0.0001, respectively) in the training set. In the validation set, an A-LMR ≥ 1 showed a median OS of not reached vs 41.8 months for an A-LMR < 1, 5 year OS rates of 90% (95 CI, 73%-97%) vs 48% (95 CI, 29-68%), p < 0.0001, respectively. A-LMR provides a platform to engineer an immunocompetent autograft to improve clinical outcomes in cHL patients undergoing APHSCT. DisclosuresNo relevant conflicts of interest to declare.

Safety of Peripheral Hematopoietic Stem Cell Mobilization with Lenograstim in Allogeneic Healthy Donors ≥ 50 Years Old: A Single-Centre Experience

Background and ObjectivesThe use of allogenic hematopoietic stem cell transplantation (HSCT) is increasing in older patients because of reduced ablative regimens and improved supportive care. An old patient usually has a old sibling healthy donors (HDs). The safety of G-CSF for mobilization in elderly HDs has not been provided. In our institution a long-term active follow-up study of G-CSF-mobilized healthy volunteer donors has been implemented.Patients and MethodsAfter a median follow up of 94 months (7.8 years) we reviewed and analyzed safety data of our HDs database according to HDs age: HDs-1 (162), patients younger than 50 years old, HDs-2 (62), patients 50-59 years old and HDs-3 (23), patients 60 or more years old. Long-term follow-up included monitoring of neoplastic, cardiac or autoimmune diseases.ResultsTwo hundred and forty-seven successive donors were evaluated and their characteristics are well balanced among age-groups and no statistical differences have been detected: most of them were male (55.9% ), sibling (97.2%) and HLA matching (93.1%). Mobilization failure rate has been 4.3% (7/162), 1.6% (1/62) and 13% (3/23) in HDs-1, HDs-2 and HDs-3, respectively (p=0.075).Short- and long-term safety was not different among age-groups. Bone pain was reported as the most frequent short-term adverse event (76.5%). Other commonly observed short-term symptoms included headache (31.6%), fatigue (28.7%), insomnia (21.5%), nausea (13.0%) and fever (6.1%). For long-term safety surveillance no haematological malignancies were observed. The observed rate of solid tumors, cardiovascular and autoimmune events, was the expected incidence for these diseases in the western countries.ConclusionIn our study, mobilization with lenograstim in elderly HDs seems to be as safe as for young HDs safe. This data contribute to the growing body of evidence of the long-term safety of G-CSF for allogenic donor stem cell mobilization also for elderly HDs. DisclosuresNo relevant conflicts of interest to declare.

Clinical features and risk factors analysis of acute graft-versus-host disease in patients with related HLA-haploidentical non T cell-depleted in vitro peripheral hematopoietic stem cell transplantation

To study the clinical features of acute graft-versus-host disease (aGVHD) and its risk factors for the related HLA-haploidentical non T cell-depleted in vitro peripheral hematopoietic stem cell transplantation (RHNT-PBSCT). From July 2002 to December 2012, 104 patients who underwent the RHNT-PBSCT were enrolled to analyze the incidences, location and its risk factors of aGVHD, compared with those of the 103 patients who received the HLA-matched sibling non T cell-depleted in vitro PBSCT (MSNT-PBSCT) in the same period. (1)The cumulative incidence of aGVHD in the RHNT-PBSCT group was significantly higher than the MSNT-PBSCT group [(56.2±4.7)% vs (34±3.6)%, P<0.05], but the cumulative incidences of II-IV and III-IVgrade aGVHD had no significant difference between the two groups[(39.5±2.9)% vs (21.2±5.4)%, P>0.05; (12.6±4.1)% vs (10.8±2.4)%, P>0.05]. (2)The cumulative incidence of cutaneous aGVHD was significantly higher in RHNT-PBSCT group than that in MSNT-PBSCT group [(42.3±3.2)% vs (17.5±2.3)%, P<0.05]. The cumulative incidences of liver and gastrointestinal aGVHD between the two groups had no significant difference [(7.7±2.1)% vs (12.6±3.4)%, P>0.05; (16.3±4.5)% vs (10.3±2.5)%, P>0.05]. (3)The 3-year disease free survival (DFS) and overall survival(OS) of RHNT-PBSCT group and MSNT-PBSCT group were (63±5.5)%, (65.2±4.7)% and (74.2±5.4)%, (77.4±5)% respectively, without significance (P=0.078, P=0.052). (4)aGVHD occurrence with HLA haplotype (P=0.003) and matched loci (P=0.002) were significantly correlated by univariate analysis. Multivariate analysis showed that only the HLA typing is a risk factor for aGVHD (HR=1.891, P=0.03). Although the incidence of total aGVHD in RHNT-PBSCT protocol is higher than that in MSNT-PBSCT, but there was no significance in severe aGVHD and cutaneous aGVHD was the common type, which indicates that RHNT-PBSCT protocol is feasible.

The loss of CD34+ cells in peripheral hematopoietic stem cell products cryopreserved by non-controlled rate freezing and stored at −80 °C after overnight storage

Although peripheral blood stem cell (PBSC) products cryopreserved by non-controlled rate freezing and stored at −80 °C after overnight storage are used frequently, data regarding the rate of loss of CD34+ cells in these products are limited. In this prospective study, CD34+ cells were counted at three (fresh, post-overnight and post-thaw) points in 83 PBSC products from 41 patients by flow cytometry. Compared to fresh products, the mean losses of post-overnight and post-thaw total CD34+ cells are 16.3% and 38.4% (p= 0.02), and the mean losses of post-overnight and post-thaw viable CD34+ cells are 16.5% and 48.5%, respectively (p< 0.001). The numbers of fresh viable, post-thaw total and post-thaw viable CD34+ cells were inversely correlated with the durations of neutrophil and platelet engraftment. Our results indicate that the mean loss of post-thaw total and viable CD34+ cells is approximately 20% higher than that observed in standard cryopreservation methods. In addition, fresh viable, post-thaw total and especially post-thaw viable CD34+ cell levels are valuable predictors of both neutrophil and platelet engraftments.

Infused Autograft Lymphocyte to Monocyte Ratio and Survival in Diffuse Large B Cell Lymphoma

Infused autograft absolute lymphocyte count is a prognostic factor for survival after autologous peripheral hematopoietic stem cell transplantation (APHSCT) for diffuse large B cell lymphoma (DLBCL). CD14+ HLA-DRlow/neg immunosuppressive monocytes affect tumor growth by suppressing host antitumor immunity. Thus, we set out to investigate if the infused autograft lymphocyte to monocyte ratio (A-LMR), as a biomarker of host immunity (ie, lymphocytes) and immunosuppression (ie, monocytes), affects survival after APHSCT. From 1994 to 2012, 379 DLBCL patients who underwent APHSCT were studied. The 379 patients were randomly divided into a training set (n = 253) and a validation set (n = 126). Receiver operating characteristic and area under the curve identified an A-LMR ≥1 as the best cut-off value, which was validated by the k-fold cross-validation in the training set. Multivariate analysis showed A-LMR to be an independent prognostic factor for survival in the training set. Patients with an A-LMR ≥ 1.0 experienced superior overall survival (OS) compared with patients with an A-LMR <1.0 (median OS: 167.2 versus 17.6 months; 5-year OS: 73% [95% confidence interval (CI), 63% to 80%] versus 30% [95% CI, 2% to 38%], P < .0001, respectively) in the training set. In the validation set, an A-LMR ≥ 1 showed a median OS of 181.2 months versus 19.5 months for an A-LMR <1, and 5-year OS rates of 67% (95% CI, 52% to 79%) versus 35% (95% CI, 25% to 47%), P < .0001, respectively. The A-LMR provides a platform to engineer immunocompetent autograft to improve clinical outcomes in DLBCL patients undergoing APHSCT.

Clinical observation of haploidentical-hematopoietic stem cell transplantation combined with human umbilical cord-derived mesenchymal stem cells for severe aplastic anemia-II

This study was purposed to investigate the efficacy and safety of haploidentical hematopoietic stem cells (allo-HSCT) transplantation combined with human umbilical cord-derived mesenchymal stem cell infusion (hUC-MSC) for severe aplastic anemia-II (SAA-II). Eight SAA-II patients received haploidentical allo-HSCT, the G-CSF mobilized peripheral hematopoietic stem cells and bone marrow haploidentical hematopoietic stem cells were selected as graft, the human umbilical cord-derived mesenchymal stem cells (hUC-MSC) were infused as the third party. Conditioning regimen consisted of rabbit anti-thymic lymphocytes protein(ATG), cyclophosphamide(CTX) and fludarabine(Flu). For two patients out of 8 SAA-II patients the conditioning regimen was combined with busulfan(BU). The graft versus host disease(GVHD) was prevented with CSA, MTX, ATG, CD25 and mycophenolate mofetil. The results showed that the average number of nucleated cells were 9.13×10(8)/kg, and number of CD34(+)cells were 3.76×10(6)/ kg. All the 8 SAA-II patients achieved hematopoietic reconstitution. The average time of neutrophils count>0.5×10(9)/L was 11.9 days, and average time of Plt level >20×10(9)/L was 14.6 days. The incidence of acute GVHD of I-II grade was 25%, and that of III-IVgrade was 12.5%, the transplantation-related mortality was 25%. It is concluded that haploidentical allo-HSCT combined with umbilical cord MSC infusion is an effective approach to cure SAA.

Clinical analysis of 81 cases of malignant lymphoma treated with autologous hematopoietic stem cell transplantation

To investigated the curative effect of autologous hematopoietic stem cell transplantation (ASCT) for malignant lymphoma. The clinical data of 81 patients with malignant lymphoma received ASCT from April 1999 to October 2013 were retrospectively analyzed. Of 81 patients, 70 were non-Hodgkin's lymphoma (NHL) and 11 Hodgkin's lymphoma (HL). High dose of etoposide combined with G-CSF was used to mobilize peripheral hematopoietic stem cell. Preconditioning regimen was BEAM (carmustine + cytarabine + etoposide + melphalan). Enough peripheral blood stem cells were collected from all patients. All of the patients after transplantation achieved hematopoietic reconstitution, the median time of the absolute neutrophil count (ANC) recovery to >0.5×10⁹/L time was 10(7-16) d, and the median time of platelet count recovery to >20×10⁹/L was 10(6-17) d. With the follow-up of 23(2-139) months, progression free survival (PFS) was 72.7%, and overall survival (OS) was 88.6%. The median PFS and OS were not reached. Complete remission (CR) before ASCT was an independent prognostic factor of PFS. No transplant related death happened. ASCT was a safe and effective method for treatment of malignant lymphoma.