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Object. In this study, bioinformatics analysis of differentially expressed genes (DEGs) and signaling pathway activities in different progression stages of chronic lymphocytic leukemia (CLL) and pre- and post-chemoimmunotherapy (CIT) treatment was performed. This may provide novel ideas for molecular diagnosis and individualized treatment strategies for CLL patients. Methods. Data from single-cell RNA sequencing (RNA-seq) of CLL patients were obtained from the Gene Expression Omnibus database. The R package was utilized to analyze the data, and the relation of results was predicted via the GeneMANIA website. The information of 7 samples covered three stages: observation stage, pretreatment by CIT with rituximab, fludarabine, and cyclophosphamide (pre-CIT), and post-CIT. The differentially expressed genes (DEGs) were identified, and functional enrichment analyses were performed. B cell subpopulations and pseudotime trajectories analysis was conducted. Results. A total of 70,659 DEGs were identified. Each patient’s DEGs presented their own characteristics, with low similarity. Therefore, it is difficult to identify potential hub genes. Similarly, pathway enrichment analysis showed significant tumor heterogeneity among CLL patients. Analysis of relapsed post-CIT compared to the observation stage suggested that the TP53 pathway should be taken seriously as it is closely related to treatment strategy and patient prognosis. Conclusions. Tumor heterogeneity may be a more common manifestation of CLL. Individualized treatment should be considered for CLL. TP53 abnormality and its regulatory factors should still be the focus of CLL diagnosis and treatment.

As a relatively new paradigm of bio-inspired optimization techniques, artificial bee colony (ABC) algorithm has attracted much attention for its simplicity and effectiveness. However, the performance of ABC is not satisfactory when solving some complex optimization problems. To improve its performance, we propose a novel ABC variant by designing a dynamic multi-population scheme (DMPS). In DMPS, the population is divided into several subpopulations, and the size of subpopulation is adjusted dynamically by checking the quality of the global best solution. Moreover, we design two novel solution search equations to maximize the effectiveness of DMPS, in which the local best solution of each subpopulation and the global best solution of the whole population are utilized simultaneously. In the experiments, 32 widely used benchmark functions are used, and four well-established ABC variants are involved in the comparison. The comparative results show that our approach performs better on the majority of benchmark functions.

Background To evaluate the diagnostic and prognostic utility of monitoring the Epstein–Barr virus (EBV) load in the cerebrospinal fluid (CSF) and peripheral blood for the patients with EBV-associated central nervous system (CNS) diseases after allogeneic hematopoietic stem cell transplantation (allo-HSCT), 172 patients undergoing allo-HSCT were enrolled in the study. Methods The EBV DNA levels of blood were monitored regularly in recipients of transplants for 3 years post transplantation. The EBV DNA levels of CSF were monitored in patients with EBV-associated CNS diseases before the treatment and at different points following the treatment. Results Post-transplant EBV-associated diseases developed in 27 patients, including 12 patients with EBV-associated CNS diseases. The 3-year cumulative incidences of EBV-associated diseases and EBV-associated CNS diseases were 19.5 ± 3.5% and 8.6 ± 2.4%, respectively. Patients with EBV-associated diseases showed higher loads of EBV DNA in their blood compared with patients with EBV DNA-emia. No difference was seen between the EBV DNA levels of blood in patients with CNS involvement and patients without CNS involvement. The EBV DNA loads of blood increased 3–14 days before the clinical manifestations of EBV-associated diseases emerged. The EBV DNA loads of CSF were higher than that of blood in patients with EBV-associated CNS diseases. In 12 patients with EBV-associated CNS diseases, EBV DNA levels were declining in both blood and CSF with the control of diseases, and the EBV DNA loads of CSF decreased faster than that of blood in 5 patients who responded to treatment, and the EBV DNA levels of CSF increased in 5 patients who were unresponsive to treatment. On multivariate analysis, the use of anti-thymocyte globulin and intensified conditioning regimens were independent risk factors for EBV-associated diseases and EBV-associated CNS diseases. Conclusions EBV-associated CNS diseases are not rare after allo-HSCT. The EBV DNA loads of CSF could act as an important indicator, but the EBV DNA loads of blood could not, for the diagnosis, prognosis, and therapeutic evaluation of EBV-associated CNS diseases.

To evaluate the efficacy of imatinib administration before and/or after allogeneic hematopoietic stem cell transplantation (allo-HSCT) for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL).Patients with imatinib therapy time exceeding 30 days pre-/post-transplant were screened in our data. Imatinib was used in induced or consolidated chemotherapy pre-transplant, or maintenance therapy after 60 days post-transplant (therapy time was less than 180 days) regardless of the molecular status of the disease.Sixty-nine patients with Ph+ ALL were enrolled in the retrospective analysis. Forty-four patients received imatinib therapy, including 24 pre-transplant, 9 post-transplant, and 11 both pre- and post-transplant. With a median follow-up time of 395 days (range, 55-2762 days) post-transplant, 3-year estimated overall survival was 62.3 ± 16.6, 40.0 ± 21.9, 41.7 ± 22.2, and 25.9 ± 11.4%, respectively (P = 0.221), and disease-free survival (DFS) was 53.6 ± 17.9, 20.0 ± 17.9, 33.3 ± 25.5% and 23.6 ± 11.4%, respectively (P = 0.421), in patients with imatinib therapy pre-transplant, post-transplant, both pre- and post-transplant, neither pre- nor post-transplant. The incidence of relapse at 3 year for patients with imatinib therapy post-transplant (n = 20) was 63.6%, comparing with 24.2% (P = 0.018) in patients without imatinib therapy post-transplant (n = 49). The ratio of CD4+CD25+Foxp3+ cells in blood was significantly higher at 30 and 60 days after imatinib therapy than that at the time of pre-imatinib in 20 patients (P = 0.019 and 0.001, respectively).Application of imatinib pre-transplant might have benefited for patients with Ph+ ALL. Whether administration of imatinib, regardless of the molecular status of the disease post-transplant increases relapse, is a worthy goal for further study.

In this study we investigated the etiology and pathogenesis of nephrotic syndrome (NS) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in 257 patients with hematopoietic malignancies who survived more than 2 months post allo-HSCT. Associations of NS with the conditioning regimen, graft versus host disease (GVHD), and other variables were analyzed. Pathologic features of the kidney, regulatory T cells (Tregs), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were studied. NS was identified in 9 patients. The number of Tregs at day+30, 60, 90, and 180 was lower in NS patients than non-NS patients (P=0.001, 0.001, 0.007, 0.003). Serum levels of IFN-γ and TNF-α were higher in NS patients (P=0.032, 0.001, respectively). NS post allo-HSCT was associated with the occurrence of chronic GVHD (P=0.02). NS post-HSCT is an immune disorder that may involve immune complex deposition, Th1 cytokines, and Tregs.

Background Recently, a few data showed that Granulocyte-colony-stimulating factor (G-CSF) modified bone marrow (G-BM) grafts leaded a lower morbidity and mortality of graft-versus-host disease(GVHD), compared with G-CSF mobilized peripheral blood stem cells (G-PBSCs) and steady state bone marrow (SS-BM) grafts. However, the mechanism of this difference is not fully understood. In this study, we compared the immune components in the three grafts. Methods Twenty patients undergoing HLA-matched sibling donor stem cell transplantation(SCT) were enrolled in this study. Patients were randomized to receive G-BM (n=10) or G-PBSCs (n=10) transplantation. Immune components, including Human leucocyte antigen-G (HLA-G), Myeloid-derived suppressor cells (MDSCs) and T cell subsets, were detected in bone marrow (BM) and peripheral blood (PB) of donors before and at day 5 after G-CSF mobilization. Results Among 20 patients, 2 patients developed acute GVHD (aGVHD) and 5 developed chronic GVHD (cGVHD) in G-PBSCs group, and no patients developed aGVHD and only 1 developed limited cGVHD in G-BM group. For the grafts, the proportion of MDSCs in PB and BM was 1.47% ± 0.12% and 2.26% ± 0.35% (P=0.001), respectively, before mobilization. After 5 days of G-CSF mobilization, the proportion of MDSCs in PB and BM increased to be 2.73% ± 0.68% and 2.56% ± 0.12%, respectively. The increase is significant both in PB and BM (P=0.000,P=0.002, respectively), while no significant difference was found between the proportion of MDSCs in G-PB and G-BM graftes (P=0.115). The levels of HLA-G (both soluble and membrane bound) in PB and BM all increased significantly after G-CSF mobilization (all P Conclusions The differences of immune components in the grafts might play a part in the different morbidity and mortality of GVHD. The higher expression of HLA-G in the grafts might be the main reason for the lower morbidity and mortality of GVHD in G-CSF-primed bone marrow transplantation. Disclosures: Liu: It was supported by 863 Program (No. 2011AA020105), National Public Health Grand Research Foundation (Grant No. 201202017).: Research Funding; It was supported by National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174).: Research Funding.

Background The immune modulatory effect of granulocyte colony-stimulating factor (G-CSF) on T cells resulted in an unexpected low incidence of graft-versus-host disease (GVHD) in allogeneic peripheral blood stem cell transplantation (allo-PBSCT). Recent data indicated that gamma delta+ T cells might participate in mediating graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation. However, whether G-CSF could influence the T cell receptors (TCR) of gamma delta+ T cells (TRGV and TRDV repertoire) remains unclear. To further characterize this feature, we compared the distribution and clonality of TRGV and TRDV repertoire of T cells before and after G-CSF mobilization and investigated the association between the changes of TCR repertoire and GVHD in patients undergoing G-CSF mobilized allo-PBSCT. Methods The complementarity-determining region 3 (CDR3) sizes of three TRGV and eight TRDV subfamily genes were analyzed in peripheral blood mononuclear cells (PBMCs) from 20 donors before and after G-CSF mobilization, using RT-PCR and genescan technique. To determine the expression levels of TRGV subfamily genes, we performed quantitative analysis of TRGV I~III subfamilies by real-time PCR. Results The expression levels of three TRGV subfamilies were significantly decreased after G-CSF mobilization (P = 0.015, 0.009 and 0.006, respectively). The pattern of TRGV subfamily expression levels was TRGV II > TRGV I > TRGV III before mobilization, and changed to TRGV I > TRGV II > TRGV III after G-CSF mobilization. The expression frequencies of TRGV and TRDV subfamilies changed at different levels after G-CSF mobilization. Most TRGV and TRDV subfamilies revealed polyclonality from pre-G-CSF-mobilized and G-CSF-mobilized samples. Oligoclonality was detected in TRGV and TRDV subfamilies in 3 donors before mobilization and in another 4 donors after G-CSF mobilization, distributed in TRGV II, TRDV 1, TRDV 3 and TRDV 6, respectively. Significant positive association was observed between the invariable clonality of TRDV 1 gene repertoire after G-CSF mobilization and low incidence of GVHD in recipients (P = 0.015, OR = 0.047). Conclusions G-CSF mobilization not only influences the distribution and expression levels of TRGV and TRDV repertoire, but also changes the clonality of gamma delta+ T cells. This alteration of TRGV and TRDV repertoire might play a role in mediating GVHD in G-CSF mobilized allo-PBSCT.

Abstract 4068 Human leucocyte antigen G (HLA-G) is a nonclassical HLA class I molecule, the tolerogenic role of HLA-G is highly supported in pregnancy immunization, tumor immune escape and organ transplant. HLA-G molecules also act as an inhibitor of the T-lymphocytes, NK cells and antigen presenting cells. HLA-G can be expressed as 7 different isoforms including 4 membrane-bound (HLA-G1 to -G4) and 3 soluble (HLA-G5 to-G7) proteins. The soluble HLA-G5 isoform encoded by intron-4 retaining spliced transcript has been previously detected in vivo in sera and grafts from solid transplanted patients who had significantly better graft acceptance. In the present study, we analyzed the expression levels of HLA-G in patients after hematopoietic stem cell transplantation (HSCT) and investigated the relationship between HLA-G and GVHD. The levels of soluble HLA-G (sHLA-G) in the plasma of peripheral blood from 90 patients underwent HSCT were determined by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to detect the expression of membrane bound HLA-G (mHLA-G). The levels of mHLA-G of patients at pre-HSCT, +15d post-HSCT and +30d post-HSCT were similar with the normal individuals. High levels of sHLA-G in the plasma of recipients were found after allogeneic HSCT (allo-HSCT). The soluble HLA-G5 and HLA-G6 levels in the plasma of recipients at +15d post-HSCT and +30d post-HSCT were significantly higher than that at pre-HSCT, but the soluble HLA-G7 levels in the plasma of recipients at +15d post-HSCT or +30d post-HSCT were similar with that at pre-HSCT. In contrast, the levels of sHLA-G in the plasma of recipients had no significant change after autologous HSCT (auto-HSCT). High level secretion of HLA-G5 after allo-HSCT (the levels of sHLA-G increased more than 1.0 fold compared to the pre-HSCT group) significantly reduced the incidence of acute GVHD and chronic GVHD; High level secretion of HLA-G6 after allo-HSCT also significantly reduced the incidence of chronic GVHD, but had no influence to the incidence of acute GVHD; High level secretion of HLA-G7 after allo-HSCT had no influence to the incidence of acute GVHD or chronic GVHD. The increased levels of HLA-G5 after allo-HSCT which compared with the pre-HSCT group had a significant negative correlation with the severity of GVHD. In conclusion, the high level secretion of HLA-G5 might inhibit the incidence of acute and chronic GVHD. Detected the secretion level of HLA-G5 after HSCT might benefit to predict and evaluate the GVHD. Disclosures: Liu: National Natural Science Foundation of China (No.30971300), Science and Technology Planning Project of Guangdong Province of China (No.2009A030200007): Research Funding.

Abstract 4069 Myeloid derived suppressor cells (MDSCs) are a group of heterogeneous cells, derived from bone marrow progenitor cells and immature myeloid cells. In tumor development and progression, MDSCs play a role of immunosuppressive effect. Recently, researchers found that granulocyte colony-stimulating factor (G-CSF) could mobilize MDSCs from bone marrow (BM) to peripheral blood (PB), but there is unknown about the effect of G-CSF to MDSCs in the human body and the relationship between MDSCs and graft-verse-host disease (GVHD). So we investigated the association between MDSCs and acute graft-verse-host disease (aGVHD). The expression of MDSCs and adhesion molecules in the BM, PB and peripheral blood stem cell grafts in 30 related donors, were detected by flow cytometry before and after recombined human G-CSF (rhG-CSF) mobilization. The expression of arginase (ARG) and inducible nitric oxide synthase (iNOS) were detected by enzyme-linked immunosorbent assay. According to the median number of MDSCs in peripheral blood stem cell harvests (9.60×106/kg), 30 leukemia patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) were divided into two groups, high MDSCs group and low MDSCs group. The hematopoietic/immune reconstitution, incidence of aGVHD, relapse, and survival rate were compared between two groups. MDSCs could be detected in the blood and BM in healthy donors, the proportion in nuclear cells in the blood and BM was 0.30% ± 0.09% and 0.53% ± 0.16%, respectively. MDSCs in BM was higher than that in PB (P In conclusion, in the human body, rhG-CSF could transfer more MDSCs from BM into the blood. RhG-CSF mobilization could increase the number of MDSCs in graft, which may be related to low incidence of aGVHD after allo-HSCT. The mechanism of rhG-CSF mobilized MDSCs to blood may be related to adhesion factor on MDSCs surface. The incidence of aGVHD in patients who accepted high MDSCs cells was lower; there was a significantly negative correlation between MDSCs transplant value and the grading of aGVHD. MDSCs cell infusion did not affect the function of immune reconstitution and the effect of graft versus leukemia. Disclosures: Liu: National Natural Science Foundation of China (No.30971300), Science and Technology Planning Project of Guangdong Province of China (No.2009A030200007): Research Funding.

Abstract 1946 The immune modulatory effect of granulocyte colony-stimulating factor (G-CSF) on T cells resulted in an unexpected low incidence of graft-versus-host disease (GVHD) in allogeneic peripheral blood stem cell transplantation (allo-PBSCT). The antigen recognition of T cells was based on a heterodimer composed either of the αβ chain or of the γδ chain of the TCR, which was associated with four transmembrane proteins, named CD3γ, CD3ε, CD3δ and CD3ζ, respectively. Recent data indicated that γδ+ T cells might participate in mediating graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect after allogeneic hematopoietic stem cell transplantation. However, whether G-CSF could influence the T cell receptors (TCR) of γδ+ T cells (TRGV and TRDV repertoire) and CD3 genes remains unclear. To further characterize this feature, we compared the distribution and clonality of TRGV and TRDV repertoire of T cells and the expression pattern of CD3 genes before and after G-CSF mobilization, and investigated the correlation between the changes of TCR repertoire and GVHD in patients undergoing G-CSF mobilized allo-PBSCT. The complementarity-determining region 3 (CDR3) sizes of three TRGV and eight TRDV subfamily genes were analyzed in peripheral blood mononuclear cells (PBMCs) from 20 donors before and after G-CSF mobilization, using RT-PCR and genescan technique. To determine the expression levels of TRGV subfamily genes and CD3 genes, we performed quantitative analysis of TRGVI∼III subfamilies and CD3γ, δ, ε and ζ chain genes by real-time PCR. All of three TRGV subfamily expression levels were decreased significantly after G-CSF mobilization. The pattern of TRGV subfamily expression levels was TRGVII>TRGV I>TRGV III before mobilization, and changed to TRGV I>TRGV II>TRGV III after G-CSF mobilization. Most TRGV and TRDV subfamilies revealed polyclonality from pre-G-CSF-mobilized and G-CSF-mobilized samples. Oligoclonality was detected in TRGV and TRDV subfamilies in 3 donors before mobilization and in another 4 donors after G-CSF mobilization. The expression patterns and clonality of TRGVII, TRDV3 and TRDV1 were found more frequently to change after G-CSF mobilization. Significant positive correlation was observed between the invariable clonality of TRDV1 gene repertoire after G-CSF mobilization and low incidence of GVHD in recipients (P=0.015, OR=0.047). In addition, the expression patterns of CD3 genes before and after G-CSF mobilization were CD3ζ> CD3ε> CD3δ > CD3γ. The expression levels of CD3 ζ and ε genes after G-CSF mobilization (2.194%, 0.867%) were significantly lower than that before mobilization (7.615%, 1.839%) (P=0.031, P=0.009); while the expression levels of CD3 δ and γ genes before mobilization (1.097% and 0.271%) were not significantly different from that after G-CSF mobilization (0.333% and 0.077%) (P=0.093, P=0.070). Significant positive correlation was observed between the relative expression levels of CD3 ζ chain genes before and after G-CSF mobilization (rs=0.576,P=0.016), but no significant correlation of gene expression levels were found in CD3γ, δ and ε genes before and after G-CSF mobilization. The expression level of CD3ζ genes before and after G-CSF mobilization and CD3δ gene after mobilization all respectively showed a negative correlation to the age of donors (rs= −0.549, P=0.023; rs= −0.679, P=0.003; rs= −0.570, P=0.017). In conclusion, G-CSF mobilization not only influences the distribution and clonality of TRGV and TRDV repertoire, but also influences the expression levels of CD3 ε and ζ genes. This alteration of TRGV and TRDV repertoire might play a role in mediating GVHD in G-CSF mobilized allo-PBSCT. Disclosures: Liu: National Natural Science Foundation of China (No.30971300), Guangdong Natural Science Foundation (No.10451051501005778), Science and Technology Planning Project of Guangdong Province of China (No.2009A030200007) and China Postdoctoral Science Foundation (: Research Funding.