Key Points• We developed an approach of T-cell-replete haploidentical HSCT with low-dose anti-T-lymphocyte globulin.• Outcomes of suitably matched URD-HSCT and HRD-HSCT are similar, and HRD-HSCT improves outcomes of patients with high-risk leukemia.We developed an approach of T-cell-replete haploidentical hematopoietic stem cell transplantation (HSCT) with low-dose anti-T-lymphocyte globulin and prospectively compared outcomes of all contemporaneous T-cell-replete HSCT performed at our center using matched sibling donors (MSDs), unrelated donors (URDs), and haploidentical related donors (HRDs). From 2008 to 2013, 90 patients underwent MSD-HSCT, 116 underwent URD-HSCT, and 99 underwent HRD-HSCT. HRDs were associated with higher incidences of grades 2 to 4 (42.4%) and severe acute graft-versus-host disease (17.2%) and nonrelapse mortality (30.5%), compared with MSDs (15.6%, 5.6%, and 4.7%, respectively; P < .05), but were similar to URDs, even fully 10/10 HLA-matched URDs. For high-risk patients, a superior graft-versus-leukemia effect was observed in HRD-HSCT, with 5-year relapse rates of 15.4% in HRD-HSCT, 28.2% in URD-HSCT (P 5 .07), and 49.9% in MSD-HSCT (P 5 .002). Furthermore, 5-year disease-free survival rates were not significantly different for patients undergoing transplantation using 3 types of donors, with 63.6%, 58.4%, and 58.3% for MSD, URD, and HRD transplantation, respectively (P 5 .574). Our data indicate that outcomes after HSCT from suitably matched URDs and HRDs with low-dose anti-Tlymphocyte globulin are similar and that HRD improves outcomes of patients with high-risk leukemia. This trial was registered at www.chictr.org (Chinese Clinical Trial Registry) as #ChiCTR

Mobilization of mesenchymal stem cells (MSCs) is a promising strategy for tissue repair and regenerative medicine. The establishment of an appropriate animal model and clarification of the underlying mechanisms are beneficial to develop the mobilization regimens for therapeutic use. In this study, we therefore established a rat MSC mobilization model and investigated the related mechanisms, using continuous hypoxia as the mobilizing stimulus. We found that MSCs could be mobilized into peripheral blood of rats exposed to short-term hypoxia (2 days) and the mobilization efficiency increased in a time-dependent manner (2-14 days). Hypoxia-inducible factor-1α (HIF-1α) was upregulated during hypoxic exposure and was expressed continuously in bone marrow. Inhibition of HIF-1α expression by YC-1 remarkably reduced the number of mobilized MSCs, suggesting that HIF-1α is essential for hypoxia-induced MSC mobilization. Further, we investigated the potential role of HIF-1α target genes, vascular endothelial growth factor (VEGF), and stromal cell-derived factor-1α (SDF-1α). VEGF expression was elevated from day 2 to day 7 of hypoxia, stimulating an increase in bone marrow sinusoidal vessels and possibly facilitating the egress of MSCs. SDF-1α protein levels were increased in the peripheral blood of rats during MSC mobilization and promoted the migration of MSCs under hypoxic conditions in vitro. These results suggest that HIF-1α plays a pivotal role in hypoxia-induced MSC mobilization, possibly acting via its downstream genes VEGF and SDF-1α. These data provide a novel insight into the mechanisms responsible for MSC mobilization and may help in the development of clinically useful therapeutic agents.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) for severe aplastic anemia (SAA) is mainly limited by the high incidence of graft failure and GvHD. Mesenchymal stem cells (MSCs) have been shown to support hematopoiesis in vivo and to display potent immunosuppressive effects to prevent or treat GvHD after HSCT. In a multicenter phase II trial, we developed an approach with co-transplantation of MSCs in patients undergoing haplo-HSCT. Forty-four patients with SAA were included. The conditioning regimen included busulfan, cyclophosphamide and thymoglobulin (ATG). The recipients received cyclosporin A (CsA), mycophenolate mofetil and short-term methotrexate for GvHD prophylaxis. Three out of 44 patients, who died early before hematopoietic engraftment, were not assessed. Evaluable patients (97.6%; 40/41) achieved hematopoietic reconstitution and sustained full donor chimerism. The median time for myeloid engraftment was 12 days (range 8-21 days) and for platelet engraftment was 19 days (range 8-154 days). The incidence was 29.3% for grade II-IV acute GvHD and 14.6% for chronic GvHD. The overall survival was 77.3% with a median 12-month (range 0.9-30.8) follow-up for surviving patients. These data suggest that co-transplantation of MSCs could reduce the risk of graft failure and severe GvHD in haplo-HSCT for SAA.

Donor cell leukemia after allogeneic hematopoietic stem cell transplantation might provide a unique human model for our understanding of leukemogenesis in vivo. We hypothesized that the "2-genetic-hits model" may contribute to the "leukemization" of donor cells and first evaluated these genetic mutations that are implicated in the development of acute myeloid leukemia in a donor cell leukemia patient and donor. The patient and his donor-sister both harbored a germline mutation in CEBPA (584_589dup). Susceptible donor hematopoietic cells evolved to overt acute myeloid leukemia by developing 2 somatic CEBPA mutations (247dupC and 914_916dup) in the patient's microenvironment. These were identical to the acquired mutations identified in leukemic cells that originated from the patient during de novo acute myeloid leukemia. Our results provide the first report of multiple mutations of CEBPA contributing to the transformation of donor cells to the leukemic phenotype and provide clues to support the multiplegenetic-hits mechanism of donor cell leukemia. (Blood. 2011;117(19):5257-5260) IntroductionLeukemia relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) that arises in cells of donor origin in the transplant recipient, so-called donor cell leukemia (DCL), is a rare disease entity, and only 51 cases were reported since 1971. [1][2][3][4] The precise etiologic mechanisms of DCL remain unknown, and no common mechanism can be identified in most of the cases reported in the literature. Careful analysis of the mechanisms with respect to the oncogenic transformation of donor-derived cells might provide a unique human model for our understanding of leukemogenesis in vivo.According to the "2-genetic-hits model," cooperation between 2 classes of genetic mutations contributes to leukemogenesis. 5 One group (class 1) comprises mutations in the fms-related tyrosine kinase 3 gene (FLT3) or the neuroblastoma RAS viral oncogene homolog gene (NRAS), which increase the proliferation and/or survival of hematopoietic stem/progenitor cells. 6,7 The other complementation group (class 2) comprises mutations in CEBPA, the gene that encodes the CCAAT enhancer-binding protein␣ (C/EBP␣); the myeloid-lymphoid or mixed-lineage leukemia gene (MLL); or the nucleophosmin gene (NPM1), which cause impaired differentiation. [8][9][10] The most common mutations, which include internal tandem duplications restricted to exons 14 and 15 and point mutation of Asp 835 within the TK domain of FLT3, codon 12/13 in exon 1 and codon 61 in exon 2 of NRAS, the entire coding region of CEBPA, partial tandem duplications that span exons 2-6 or exons 2-8 of MLL, and mutations in exon 12 of NPM1, have been described extensively in acute myeloid leukemia (AML). [11][12][13][14] We hypothesized that the 2-genetic-hits model may contribute to the "leukemization" of donor cells in DCL. We screened these genetic mutations implicated in the development of common forms of AML in a DCL patient and donor. MethodsThe study was approved by the Zhejiang U...

Telomeres are specific nucleoprotein structures at the ends of eukaryotic chromosomes. Telomeres and telomere-associated proteins maintain genome stability by protecting the ends of chromosomes from fusion and degradation. In normal somatic cells, the length of the telomeres gradually becomes shortened with cell division. In tumor cells, the shortening of telomeres length is accelerated under the increased proliferation pressure. However, it will be maintained at an extremely short length as the result of activation of telomerase. Significantly shortened telomeres, activation of telomerase, and altered expression of telomere-associated proteins are common features of various hematologic malignancies and are related with progression or chemotherapy resistance in these diseases. In patients who have received hematopoietic stem cell transplantation (HSCT), the telomere length and the telomerase activity of the engrafted donor cells have a significant influence on HSCT outcomes. Transplantation-related factors should be taken into consideration because of their impacts on telomere homeostasis. As activation of telomerase is widespread in tumor cells, it has been employed as a target point in the treatment of neoplastic hematologic disorders. In this review, the characteristics and roles of telomeres and telomerase both in hematologic malignancies and in HSCT will be summarized. The current status of telomerase-targeted therapies utilized in the treatment of hematologic malignancies will also be reviewed.

The standard treatment for aplastic anemia (AA) in young patients is a matched sibling hematopoietic stem cell transplant. Transfusion of a chronic AA patient with allogeneic bone marrow–derived mesenchymal stromal cells (BMMSCs) is currently being developed as a cell-based therapy, and the safety and efficacy of such transfusions are being continuously improved. Nevertheless, the mechanisms by which BMMSCs exert their therapeutic effects remain to be elucidated. In this study, mesenchymal stromal cells (MSCs) obtained from bone marrow donors were concentrated and intravenously injected into 15 chronic AA patients who had been refractory to prior immunosuppressive therapy. We showed that BMMSCs modulate the levels of Th1, Th2, Th17 and Treg cells, as well as their related cytokines in chronic AA patients. Furthermore, the percentages of Th1 and Th17 cells among the H-MSCs decreased significantly, while the percentage Treg cells increased. The Notch/RBP-J/FOXP3/RORγt pathway was involved in modulating the Treg/Th17 balance after MSCs were transfused in vitro. Additionally, the role played by transfused MSCs in regulating the Treg/Th17 balance via the Notch/RBP-J/FOXP3/RORγt pathway was further confirmed in an AA mouse model. In summary, in humans with chronic AA, BMMSCs regulate the Treg/Th17 balance by affecting the Notch/RBP-J/FOXP3/RORγt pathway.

Although steady improvements to chemotherapeutic treatments has helped cure 80% of childhood acute lymphoblastic leukemia (ALL) cases, chemotherapy has proven to be less effective in treating the majority of adult patients, leaving allogeneic hematopoietic stem cell transplantation (allo-HSCT) as the primary adult treatment option. Nevertheless relapse are the leading cause of death following allo-HSCT. The genetic pathogenesis of relapse following allo-HSCT in Philadelphia chromosome- negative ALL (Ph− ALL) remains unexplored. We performed longitudinal whole-exome sequencing analysis in three adult patients with Ph− B-cell ALL (Ph− B-ALL) on samples collected from diagnosis to relapse after allo-HSCT. Based on these data, we performed target gene sequencing on 23 selected genes in 58 adult patients undergoing allo-HSCT with Ph− B-ALL. Our results revealed a significant enrichment of mutations in epigenetic regulators from relapsed samples, with recurrent somatic mutations in SETD2, CREBBP, KDM6A and NR3C1. The relapsed samples were also enriched in signaling factor mutations, including KRAS, PTPN21, MYC and USP54. Furthermore, we are the first to reveal the clonal evolution patterns during leukemia relapse after allo-HSCT. Cells present in relapsed specimens were genetically related to the diagnosed tumor, these cells therefore arose from either an existing subclone that was not eradicated by allo-HSCT therapy, or from the same progenitor that acquired new mutations. In some cases, however, it is possible that leukemia recurrence following allo-HSCT could result from a secondary malignancy with a distinct set of mutations. We identified novel genetic causes of leukemia relapse after allo-HSCT using the largest generated data set to date from adult patients with Ph− B-ALL.

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