Weiyan Zheng

T-cell-replete haploidentical HSCT with low-dose anti-T-lymphocyte globulin compared with matched sibling HSCT and unrelated HSCT

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 = .07), and 49.9% in MSD-HSCT (P = .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 = .574). Our data indicate that outcomes after HSCT from suitably matched URDs and HRDs with low-dose anti-T-lymphocyte 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-OCH-12002490.

Efficient Commitment to Functional CD34+ Progenitor Cells from Human Bone Marrow Mesenchymal Stem-Cell-Derived Induced Pluripotent Stem Cells

The efficient commitment of a specialized cell type from induced pluripotent stem cells (iPSCs) without contamination from unknown substances is crucial to their use in clinical applications. Here, we propose that CD34+ progenitor cells, which retain hematopoietic and endothelial cell potential, could be efficiently obtained from iPSCs derived from human bone marrow mesenchymal stem cells (hBMMSC-iPSCs) with defined factors. By treatment with a cocktail containing mesodermal, hematopoietic, and endothelial inducers (BMP4, SCF, and VEGF, respectively) for 5 days, hBMMSC-iPSCs expressed the mesodermal transcription factors Brachyury and GATA-2 at higher levels than untreated groups (P<0.05). After culturing with another hematopoietic and endothelial inducer cocktail, including SCF, Flt3L, VEGF and IL-3, for an additional 7–9 days, CD34+ progenitor cells, which were undetectable in the initial iPSC cultures, reached nearly 20% of the total culture. This was greater than the relative number of progenitor cells produced from human-skin-fibroblast-derived iPSCs (hFib-iPSCs) or from the spontaneous differentiation groups (P<0.05), as assessed by flow cytometry analysis. These induced cells expressed hematopoietic transcription factors TAL-1 and SCL. They developed into various hematopoietic colonies when exposed to semisolid media with hematopoietic cytokines such as EPO and G-CSF. Hematopoietic cell lineages were identified by phenotype analysis with Wright-Giemsa staining. The endothelial potential of the cells was also verified by the confirmation of the formation of vascular tube-like structures and the expression of endothelial-specific markers CD31 and VE-CADHERIN. Efficient induction of CD34+ progenitor cells, which retain hematopoietic and endothelial cell potential with defined factors, provides an opportunity to obtain patient-specific cells for iPSC therapy and a useful model for the study of the mechanisms of hematopoiesis and drug screening.

Donor TLR9 gene tagSNPs influence susceptibility to aGVHD and CMV reactivation in the allo-HSCT setting without polymorphisms in the TLR4 and NOD2 genes.

Owing to ethnicity of the population, those best confirmed polymorphisms in the TLR (toll-like receptor)4 and NOD2 genes with significantly prognostic impact on allogeneic hematopoietic SCT (allo-HSCT) seem to be more applicable to Europeans and are nonpolymorphic in the Asian population. The influence of innate immunity gene polymorphisms on the outcomes of allo-HSCT in those populations has been questioned. We evaluated the influence of 10 candidate single nucleotide polymorphisms (SNPs) in the TLR1, TLR2, TLR3, TLR8 and TLR9 genes on the outcomes of allo-HSCT in a Chinese population including 138 pairs of patients and unrelated donors and a second cohort of 102 pairs of patients and HLA-identical sibling donors. We found that two tagSNPs in the TLR9 gene in the donor side, +1174 A/G (rs352139) and +1635 C/T (rs352140), influenced the risk of acute GVHD (aGVHD) and CMV reactivation. Furthermore, the presence of the susceptible haplotype (A–C) in donor may be an informative predicator of worse OS at 5 years compared with those with the G–C and G–T haplotypes (58% vs 82.9%, P=0.024). Our data suggested an unrecognized association between donor TLR9 tagSNPs and the risk of HSCT-related complications in a population without polymorphisms in the TLR4 and NOD2 genes.

Immunosuppressive Cytokine Gene Polymorphisms and Outcome after Related and Unrelated Hematopoietic Cell Transplantation in a Chinese Population

Cytokine gene polymorphisms can affect the outcome of allogeneic hematopoietic stem cell transplantation. We analyzed 6 single nucleotide polymorphisms in 3 immunosuppressive cytokine genes, TGFβ1-509(C>T), +869(T>C), TGFβ1 receptor II (TGFβ1RII) +1167(C>T, codon389 AAC/AAT), and IL-10-1082(A>G), -819(T>C), -592(A>C), in a cohort of 138 pairs of recipients and their unrelated donors and a second cohort of 102 pairs of recipients and their HLA-identical sibling donors. TGFβ1-509 T/T genotype in the donors or T allele-positivity in the recipients was associated with a significant protective effect against acute graft-versus-host disease (aGVHD) and grades II-IV aGVHD in the unrelated transplantation cohort. In the combined cohort, multivariate analysis confirmed that donors with the TGFβ1-509 T/T genotype also conferred protection against the risk of aGVHD and grades II-IV aGVHD. In both the unrelated transplantation cohort and the sibling transplantation cohort, the IL-10-819 C/C and -592 C/C genotypes in either recipients or donors were significantly associated with a higher incidence of aGVHD. In the combined cohort, the IL-10 promoter haplotype polymorphisms at positions -1082, -819, and -592 influenced the occurrence of aGVHD and death in remission. Recipients without the A-T-A haplotype or those transplanted from donors without the A-T-A haplotype had a higher incidence of aGVHD than those who were A-T-A homozygotes or heterozygotes. Estimates for death in remission showed a clear advantage for recipients transplanted from donors with the A-T-A haplotype. In multivariate analysis, recipients without the A-T-A IL-10 haplotype had a higher risk of aGVHD (relative risk [RR] = 0.764; 95% confidence interval [CI]: 0.460-1.269; P = .096) and grades II-IV aGVHD (RR = 0.413; 95% CI: 0.245-0.697; P = .001). These results provide the first report of an association between TGFβ1, TGFβ1RII, and IL-10 polymorphic features and outcome of allo-HSCT in a Chinese population, and suggest an interaction between TGFβ1-509 genotypes and IL-10 promoter haplotype polymorphisms at positions -1082, -819, and -592 and the risk of aGVHD.

Genetic variations in T-cell activation and effector pathways modulate alloimmune responses after allogeneic hematopoietic stem cell transplantation in patients with hematologic malignancies.

Background Recently, several important polymorphisms have been identified in T-cell activation and effector pathway genes and have been reported to be associated with inter-patient variability in alloimmune responses. The present study was designed to assess the impact of these genetic variations on the outcomes of allogeneic hematopoietic stem cell transplantation. Design and Methods We first investigated ten single nucleotide polymorphisms in six genes, CD28, inducible co-stimulator, cytotoxic T-lymphocyte antigen 4, granzyme B, Fas and Fas ligand, in 138 pairs of patients and their unrelated donors and a second cohort of 102 pairs of patients and their HLA-identical sibling donors. Results We observed that patients receiving stem cells from a donor with the cytotoxic T-lymphocyte antigen 4 gene CT60 variant allele (AA genotype) had a reduced incidence of grades II–IV acute graft-versus-host disease; however, they experienced early cytomegalovirus infection and relapsed more frequently, which suggested an interaction between the donor cytotoxic T-lymphocyte antigen 4 gene CT60 AA genotype and reduced T-cell alloreactivity. Furthermore, an unrelated donor with the granzyme B +55 variant genotype (AA) was an independent risk factor for development of grades II–IV acute graft-versus-host disease (P=0.024, RR=1.811). Among patients with acute myelogenous leukemia, those with the Fas -670 TT genotype were at higher risk of relapse (P=0.003, RR=3.823). The presence of these susceptible alleles in the donor and/or patient resulted in worse overall survival (54.9% versus 69.5%, P=0.029). Conclusions Our data suggest that genotype analysis of T-cell activation and effector pathway genes can be used for risk assessment for patients with hematologic malignancies before hematopoietic stem cell transplantation.

Invasive fungal infection in allogeneic hematopoietic stem cell transplant recipients: single center experiences of 12 years

Invasive fungal infection (IFI) is a growing cause of morbidity and mortality among patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We retrospectively reviewed the records of 408 patients undergoing allo-HSCTs during the period November 1998 to December 2009, analyzed the incidence and risk factors of IFI, and examined the impact of IFI on overall survival. A total of 92 (22.5%) episodes suffered proven or probable IFI (4 patients were proven, 88 patients were probable). Candida was the most common pathogen for early IFI, and mold was the most frequent causative organism for late IFI. A prior history of IFI, human leukocyte antigen (HLA) mismatch, long-time neutropenia, and acute graft-versus-host-disease (GVHD) were risk factors for early IFI. A prior history of IFI, corticosteroid therapy, cytomegalovirus (CMV) disease, and chronic GVHD were risk factors for late IFI. IFI-related mortality was 53.26%. The 12-year overall survival (OS) rate for IFI was significantly lower than that of patients without IFI (41.9% vs. 63.6%, P<0.01).中文概要目的回顾性分析了浙江大学医学院附属第一医院骨髓移植中心1998年至2009年接受造血干细胞移植(HSCT)发生侵袭性真菌感染(IFI)的患者资料,评估HSCT后IFI发病情况、流行病学特征及危险因素,以期寻找病原菌特点及相关的危险因素,为制定更有针对性的预防治疗策略提供依据。创新点首次对本中心异基因HSCT后的IFI感染情况进行统计分析,研究年限长,样本量较大;另作为国内较成熟的移植中心之一,本中心的研究结果对国内异基因HSCT后IFI感染情况有较大的参考意义。方法对1998年11月至2009年12月在本移植中心接受异基因HSCT的408例患者进行观察分析,总结HSCT后IFI发病情况和病原菌分布,并采用多因素分析判断危险因素。结论本研究显示IFI与HSCT预后明显相关, HSCT后不同时期IFI病原菌分布及危险因素存在差异。临床应根据IFI病原学特点及HSCT后时间分布特点,采取不同的策略,更有针对性地用药及开始治疗,从而改善预后。

Silencing of Human Phosphatidylethanolamine-Binding Protein 4 Enhances Rituximab-Induced Death and Chemosensitization in B-Cell Lymphoma

Rituximab is the first line drug to treat non Hodgkin’s lymphoma (B-NHL) alone or in combination with chemotherapy. However, 30–40% of B-NHL patients are unresponsive to rituximab or resistant after therapy. Human phosphatidylethanolamine-binding protein 4 (hPEBP4) is a novel member of PEBP family and functions as an anti-apoptotic molecule. In this study, we found hPEBP4 to be expressed in up to 90% of B-cell lymphoma patients, but in only 16.7% of normal lymph nodes. Interestingly, hPEBP4 overexpression inhibited rituximab-mediated complement dependent cytotoxicity (R-CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) in B-NHL cells while downregulation of hPEBP4 augmented the therapeutic efficacy of rituximab both in vitro and in vivo. Furthermore, hPEBP4 silencing sensitized the primary B-acute lymphocytic leukemia (B-ALL) cells to R-CDC. During rituximab-mediated complement dependent cytotoxicity, hPEBP4 was recruited to the cell membrane in a PE-binding domain dependent manner and inhibited R-CDC induced calcium flux and reactive oxygen species (ROS) generation. These events contributed to the decrease of cell death induced by R-CDC in B-cell lymphomas. Meanwhile, hPEBP4 knockdown potentiated the chemosensitization of the rituximab in B-cell lymphoma cells by regulating the expression of Bcl-xl, Cycline E, p21waf/cip1 and p53 and the activation of caspase-3 and caspase-9. Considering that hPEBP4 conferred cellular resistance to rituximab treatment and was preferentially expressed in lymphoma tissue, it could be a potential valuable target for adjuvant therapy for B-cell lymphoma.

Bortezomib in combination with dexamethasone and subsequent thalidomide for newly-diagnosed multiple myeloma: A Chinese experience

OBJECTIVE Bortezomib-dexamethasone-thalidomide has been reported to be effective in newly-diagnosed multiple myeloma (MM) with an overall response rate of 92% and a CR rate of 18% (Alexanian et al, Hematology 2007;12(3):235-9), but this regimen has not been tested in the Chinese patients. We report here our results testing with this combination in the Chinese population and to investigate the efficacy and safety of bortezomib in combination of dexamethasone plus subsequent thalidomide as primary treatment for MM. METHODS Between June 2006 and March 2008, 20 consecutive newly-diagnosed patients with symptomatic MM were treated with bortezomib at 1.3mg/m(2) IV on days 1, 4, 8 and 11 and dexamethasone at 20mg/m(2) IV daily on the day of bortezomib and the day after. All patients received daily oral thalidomide that was escalated from 100mg to 200mg. Fourteen patients were male and 6 were female. Median age was 59 years (range 43-86 years). 11 patients were stage 2 according to the International Staging System, 8 were stage 3, only 1 patient was stage 1. All patients received a median of two cycles of therapy (range 1-6). The EBMT criteria were used for response evaluation. Toxicities were evaluated according to the NCI Common Toxicity Criteria version 3. RESULTS 16 out of 20 patients (80%) achieved PR and 3 (15%) achieved CR; therefore the overall response rate was 95%. With a median follow-up duration of 7.8 months (4-22 months), no patients died. Grade 3-4 toxicities included fatigue (3/20), thrombocytopenia (10/20) diarrhea (5/20) and orthostatic hypotension (3/20) Grade 2 neuropathy occurred in four out of 20 patients and herpes zoster occurred in four out of 20 patients. Routine anticoagulation or anti-thrombosis was not used. Only 1 patient suffered from DVT/PE. CONCLUSIONS Our preliminary experience in Chinese patients indicated that bortezomib-dexamethasone-thalidomide is highly effective in newly-diagnosed MM. Grade three and 4 toxicities are rare after median 2 cycles of therapy. The relative lower rates of neuropathy and DVT/PE in the Chinese patients with MM are being cautiously observed.

The beneficial impact of missing KIR ligands and absence of donor KIR2DS3 gene on outcome following unrelated hematopoietic SCT for myeloid leukemia in the Chinese population

The effect of natural killer (NK) cell alloreactivity on the outcome of unrelated hematopoietic SCT (HSCT) remains a topic of debate. NK cell alloreactivity after allogeneic HSCT is regulated by killer-cell Ig-like receptors (KIRs). To investigate the influence of KIRs on outcome after unrelated HSCT, we retrospectively analyzed the HLA and KIR genotypes of 116 donor–recipient pairs. We found that missing KIR ligands in recipients were significantly associated with a decreased leukemic relapse risk (P=0.019, HR=0.329), mainly in myeloid disease (P=0.003, HR=0.193). This beneficial effect was seen in AML/myelodysplastic syndrome and also in chronic myeloid leukemia. In myeloid disease, missing KIR ligands also improved 5-year OS (P=0.034, HR=0.430) and disease-free survival (DFS) (P=0.024, HR=0.445). Meanwhile, the presence of donor-activating KIR2DS3 gene was associated with increased relapse risk (P=0.003, HR=5.046), decreased OS (P=0.004, HR=3.181) and DFS (P=0.003, HR=2.919) in myeloid disease. No effect was seen in patients with lymphoid disease. Our study indicated that, in unrelated HSCT for myeloid leukemia, missing KIR ligands in recipients offered a lower relapse risk and a long-term survival advantage. The presence of KIR2DS3 in the donor was an important risk factor for myeloid leukemia.

Risk and prognostic factors of transplantation-associated thrombotic microangiopathy in allogeneic haematopoietic stem cell transplantation: a nested case control study.

Transplantation‐associated thrombotic microangiopathy (TA‐TMA) is a significant complication of haematopoietic stem cell transplantation. However, it remains controversial which clinical or laboratory markers are of evident risk and prognostic value. From 2006 to 2013, a nested case control study was carried out in our centre to study the risk and prognostic factors of TA‐TMA. A total of 654 consecutive patients who underwent allogeneic haematopoietic stem cell transplantation were studied. Twenty‐six (4.0%) patients matched the established diagnostic criteria. Subjects with TA‐TMA had significantly higher 3‐year none relapse mortality compared with those without (65.4% vs 15.4%, P < 0.0001). Grades 2 to 4 aGVHD and cytomegalovirus viremia were independent risk factors, and serum LDH level >500U/L as well as hypertension were early signs of TA‐TMA occurrence. Liver dysfunction and significant gastric bleeding were independent risk factors for TA‐TMA related mortality. Subjects with either liver dysfunction or significant gastric bleeding had significantly higher 3 year TA‐TMA related mortality cumulative incidence than subjects without. These observations lead to the conclusion that allo‐HSCT recipients with grades 2 to 4 aGVHD or cytomegalovirus viremia should be monitored for TA‐TMA. Liver dysfunction and significant gastric bleeding are prognostic factors for TA‐TMA. Copyright