ALBERT

Description: Abstract 5144 Objective Summarizing the features of bone marrow mesenchymal stem cells, the pathogenesis of aplastic anemia, and the characteristics of bone marrow mesenchymal stem cells in patient affected by aplastic anemia;investigating the feasibility of implanting bone marrow mesenchymal stem cells for the treatment of aplastic anemia, providing a new mentality for the aplastic anemia's clinical treatment. Methods Search the documents utilized by the CNKI, CBM, PubMED and Highwire Press database. According to the retrieved result, a summary about the bone marrow mesenchymal stem cells characteristic and the feasibility of implanting bone marrow mesenchymal stem cells for the treatment of aplastic anemia was made. Results 1. The bone marrow mesenchymal stem cells(bone marrow mesenchymal stem cells, BMSC)is another important ingredient in the bone marrow, besides hematopoietic stem cells. It is easy to separate, raise, multiply and purify mesenchymal stem cells. It still carrys the qualities of stem cells after many generations of rising. When raised in vitro, BMSC may secrete many kinds of cell factors and the growth factors,and has the long-term hematopoietic supporting function, which does not only participate in the production hemopoiesis micro environment support hemopoiesis directly, but also able to grow and differentiate into various hematopoiesis supporting bone marrow matrix cells in the suitable condition. 2. BMSC has the great potential in self-renewal and multi-direction differentiation. Not only can it take function as substitute cells in the tissue framework, it also present good application prospect with regard to hematopoietic reconstruction, transplant immunity, and genetic treatment. 3. BMSC can repair the damage of hematopoietic micro environment(HME), and promote the reconstruction of hematopoiesis and immunity after transplant. 4. The low immunogenic and antigen present ability of BMSC provide the molecular foundation for the wide application of BMSC in stem cells transplant. 5. The pathogenesis of aplastic anemia(aplastic anemia, AA)is very complicated. The current belief is that aplastic anemia is related to the intrinsic multiplication flaw of hematopietic stem cells, the inadequate function of the hematopoietic micro environment, and the disorder of organism immune function. Studies have proved that there's the remarkable decrease of ectogenous hematopoietic supporting function and immuno-suppression capability of BMSC in aplastic anemia. Nowadays, methods have been established in separating, raising, and multiplying BMSC outside the human body. Besides, studies have revealed that transplanting BMSC can restore the hematopoietic micro environment, and the BMSC plays an important part in modulating transplant immunity. 6. Observe the efficacy and safety of the treatment on 11 patients of aplastic anemia with BMSC and the outcome indicate that the treatment is effective and safe. Accordingly, there's a good prospect for applying BMSC transplant as the treatment for aplastic anemia. Conclusions It has a good effect and prospect to apply bone mesenchymal stem cells transplant to the treatment of aplastic anemia. It provides a new approach to the clinical treatment of aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

Description: Background Engraftment failure (EF) is a formidable complication after autologous hematopoietic stem cell transplantation (auto-HSCT). Mesenchymal stem cells (MSCs) and cord blood (CB) have been found to support hematopoiesis. Thus, we designed a multicenter randomized clinical trial to investigate the effects and safety of MSCs alone or combined with CB infusion for patients with EF. Methods Twenty-two patients were randomly assigned to receive the treatment with MSCs alone (MSCs group, n=11) or MSCs combined with CB (CB group, n=11). MSCs were administered once every 2 weeks (2 doses were a cycle) in both groups, and single-unit CB was administered at the same day with the first application of MSCs in CB group; After one cycle of treatments (within 28 days), the patients who did not response to MSCs would receive the therapeutic schedule in CB group, and those patients with partial response (PR) in MSCs group and those without complete response (CR) in CB group would continue another cycle of MSCs treatment. If patients did not obtain CR after two cycles of treatments (within 56 days), they would receive other treatments including allogeneic HSCT. Results After the first treatment cycle, the effect rates were not significant difference in MSCs and CB groups (7/11 vs. 9/11, P=0.635), and the median time of hematopoietic reconstruction was 22 (18-28) and 17 (13-22) days, respectively (P=0.036) in MSCs and CB group. There was statistically significant difference regarding neutrophil engraftment, with 17 (range 9-28) and 8 (range 6-14) days respectively (P=0.030), but no difference regarding platelet engraftment, with 21 (range 18-28) and 18 (range 11-21) days respectively (P=0.092) between MSCs and CB groups. After two cycles of treatments, 17 patients obtained CR, 2 PR and 3 NR. CB chimerisms were not detected by short tandem repeat (STR) at +15 and +30 days after CB infusion. None of the patients experienced any adverse events of grade 3/4 with the Common Terminology Criteria for Adverse Events v3.0 (CTCAE v3.0) and acute GVHD or chronic GVHD during the period of study treatment and follow-up. One patient with PR in MSCs group and 1 NR in CB group received allogeneic HSCT at +249 and +273 days after auto-HSCT because of EF and primary disease relapse, respectively. At a median follow-up time of 345 (range 129–784) days post-transplantation, 16 patients remained alive, 3 died of relapse of primary diseases and 1 died of CMV pneumonia following allo-HSCT. None of patients developed EBV-DNA viremia and EBV-associated diseases in two groups. The 2-year overall survival, disease-free survival and tumor relapse post-transplantation were 75.2% (95% CI, 63.2-87.2%), 79.5% (95% CI, 70.1-88.9%) and 20.5% (95% CI, 11.1-29.9%) respectively. Conclusions Our data suggest that ex-vivo-expanded MSCs derived from HLA-mismatched BM alone or combined with unrelated CB are effective to EF after auto-HSCT. CB can facilitate the effect of MSCs to EF. Both two strategies do not result in GVHD or increase the risk of primary diseases relapse in patients with EF. This trial was registered at www.clinicaltrials.govas#NCT01763099. Disclosures: Liu: It was supported by 863 Program (No. 2011AA020105) and 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.

Description: Abstract 5137 Objective (1)To explore whether MSC has inhibiting effect on the proliferation ofAA patients' Tcell;(2)To discuss whether MSC affects T cell's proliferation via adjusting the growth of DCs. Materials and Methods (1) MSC were separated and cultured in vitro. Cell morphology was observed and the cell surface antigen was determined by flow cytometry.(2) Peripheral blood mononuclear cells were extracted from 20 patients suffered AA and then T lymphocytes were separated by nylon fiber column. Flow cytometry was applied to determine the surface antigen and subpopulation of T cell.(3)mononuclear cells were separated from normal human peripheral blood. DCs were prepared under the culture condition of recombination human granulocyte-macrophage colony stimulating factor (GM-CSF) and recombinant human interleukin-4 (IL-4). After acquiring the mature DCs induced by LPS, the phenotype analysis of DCs before and after culture was examined by flow cytometry, respectively. Results (1) After co-culture of MSC and T lymphocytes from AA peripheral blood, flow cytometry showed that the ratio of D8+ in T cells reduced significantly from 38.7% to 29.7 % (p 〈 0.05), whereas the CD4+ ratio increased from 24.9% to 34.9% significantly (p 〈 0.05). Meanwhile, ELISA analysis indicated that the concentration of IL-2 and IFN-γ were significantly decreased from 38.9 and 38.5 ng/L to 6.8 and 6.6 ng/L, respectively (p 〈 0.05). However, IL-4 and IL-10 increased from 2.8 and 2.9 to 5.3 and 8.3 ng/L, respectively (p 〈 0.05).(2) After the induction of immature DCs by LPS, flow cytometry showed that the expression of CD1a increased from 2.4% to 68.4% in the treatment without MSC, while that of CD14+ decreased from 83.6% to 3.5% (p 〈 0.05).(3) After the co-culture of mature DCs and MSC, the expression of CD14+ increased from 5.8% to 62.8% when the expression of CD1a, CD83 and CD80 decreased from 48.6%, 60.8% and 50.2% to 30.7%,40.9% and 20.3%, respectively. Conclusions Our study shows that (1)MSC inhibits the proliferation of T lymphocytes from AA p-atients by regulating CD8+ and CD4+; (2)futher study indicates that MSC can inhibit the growth of DCs and reverse the status of DCs from matureness to immatureness; (3)thus suggests the possible mechanism of MSC's inhibiting effect as follows: MSC decreases the liveness by controlling the growth of DCs and further inhibits its proliferation. Disclosures: No relevant conflicts of interest to declare.

Description: Background : Autologus hematopoietic stem cell transplantation (auto-HSCT) has now been widely used in treatment ofhematologic malignancies, some solid tumor, and autoimmune diseases and so on. Engraftment failure (EF) is a severe complication after auto-HSCT, and is occurring in 2% to 9.5% of patients. Although this complication is becoming increasingly uncommon with the wide application of mobilized peripheral blood stem cells (PBSCs) instead of bone marrow (BM), it is still associated with considerable morbidity and mortality related to an increasing risk of hemorrhagic complications and infections. In this study, we retrospectively analyzed risk factors of primary EF after auto-HSCT. Method: For the purpose of this study, EF was defined as an absolute neutrophil count (ANC)≦0.5×109/L and a platelet (PLT) count≦20×109/L for at least 35 consecutive days post-transplantation, with hypoplastic BM and without primary disease relapse. Two hundred and five patients undergoing auto-HSCT from Nanfang Hospital of Southern Medical University, Guangzhou General Hospital of Guangzhou Military Command and Zhongshan City People’s Hospital of Guangdong Province, were included in this study between March 2004 and March 2014. They were divided into two groups according to the presence (EF group) or absence (non-EF group) of primary EF. The clinical and transplantation-related characteristics were collected and compared between groups. Then we would choose some factors to perform a risk analysis for EF. Results: The median age of 205 patients was 39 years (range 14 to 60 years). Primary diseases included acute myelogenous leukemia (n=120), non-Hodgkin lymphoma (n=49), multiple myeloma (n=36). Eighteen patients (8.78%) developed primary EF after auto-HSCT. There were significant differences regarding infection during the period from transplantation to hematopoietic reconstruction or EF, and the doses of CD34+ cells infused between groups (P values were 0.010 and 0.044). Results of univariate models of binary logistic regression analysis showed that the number of chemotherapy cycles, infection during the period from transplantation to hematopoietic reconstruction or EF and the doses of CD34+ cells infused were related to primary EF after auto-HSCT (P values were 0.017, 0.040 and 0.018). The results of multivariate analysis for the above 3 factors showed that too much chemotherapy cycles pre-transplantation (P=0.032, odds ratio [OR] = 3.140, 95% confidence interval [CI], 1.101 to 8.956) was a risk factor for primary EF after auto-HSCT and the number of CD34+ cells transfused was a protective factor for it (P=0.035, OR= 3.249, 95% CI, 1.088 to 9.704). The 5-year overall survival, disease-free survival and cumulative incidence of tumor relapse post-transplantation were 68.3% (95% CI, 60.6%-76.0%), 63.0% (95% CI, 58.0%-68.0%) and 37.0% (95% CI, 32.0%-42.0%). Conclusion: The number of chemotherapy cycles is a risk factor for primary EF after auto-HSCT, which may be due to the damaged BM microenvironment by chemotherapy drugs. However, the number of CD34+ cells transfused is a protective factor for it. Disclosures Liu: It was supported by National Natural Science Foundation of China (81270647, 81300445, 81200388); National High Technology Research and Development Program of China (863 Program) (2011AA020105); National Public Health Grand Research Foundation (201202017);: Research Funding; It was supported by Natural Science Foundation of Guangdong Province (S2012010009299); the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1);: Research Funding; It was supported by the Technology Plan of Guangdong Province of China (2012B031800403); the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027).: Research Funding.

Description: Background: Clonal hematopoiesis of indeterminate potential (CHIP) is often observed during aging[1]. CHIP is characterized by a clonal expansion of subset blood-cell clones without other hematological abnormalities. Recent studies reported a strong connection between CHIP and cardiovascular disease (CVD). Individuals with mutations in epigenetic regulators (e.g., DNMT3A, TET2, ASXL1 and JAK2) have a higher risk for CVD[2]. In vivo studies using mouse models confirmed that Tet2-deficient HSPCs exhibited clonal expansion. Furthermore, mice transferred with Tet2-deficient HSPCs showed increased risk of atherosclerosis and worse outcome after permanent left anterior descending artery (LAD) ligation induced myocardial infarction (MI)[3,4]. Macrophages were found to be one of the most affected cell types in Tet2 deficient hematopoietic system associated with increased CVD risk[3,4]. Tet2 deficiency in macrophages is known to cause aberrant innate immune response[5]. Tet2 knockout (KO) macrophages provide a protective microenvironment to prevent melanoma progression[6], suggesting the pivotal role of Tet2 in regulating macrophage function in different context. In this study, we aim to dissect the molecular mechanism on how Tet2 deficiency disrupts normal macrophage function during heart repair. Methods: We crossed Tet2f/f mice with lysMCre mice to yield macrophage specific Tet2 deficient mice (Tet2f/f-lysMCre). We performed LAD ligation in Tet2f/f-lysMCre mice to induce MI. Age and gender matched lysMCre mice were used as controls. Cardiac function was monitored by echocardiogram every week after MI for 4 weeks. Then we sacrificed mice and performed histological analysis to evaluate heart damage and repairs in lysMCre and Tet2f/f-lysMCre mice. During the course of the experiments, we collected peripheral blood and bone marrows to examine the impact of Tet2 loss on macrophages in response to heart damage and repair. In parallel, we also collected heart tissue from lysMCre and Tet2f/f-lysMCre mice at 1 and 4 weeks after LAD ligation followed by single-cell RNA-seq analysis to further dissect the underlying molecular mechanism. Results: Prior to MI, no significant difference in heart function and macrophage numbers were observed between lysMCre and Tet2f/f-lysMCre mice at the age of 6-8 weeks old. Upon LAD-induced MI, we observed worse heart function in Tet2f/f-lysMCre mice compared to lysMCre mice. Tet2f/f-lysMCre mice showed ~40% reduction in ejection fraction and ~50% decrease in fraction shortening compared to lysMCre mice (n = 15). Further histological analysis revealed 〉2-fold increase in scar areas and infarcted areas in Tet2f/f-lysMCre compared to lysMCre mice. Single cell analysis confirmed a substantial increase of the fibroblast population in Tet2f/f-lysMCre mice upon LAD ligation. Furthermore, we also observed altered expression of a large fraction of genes related to macrophage activation and tissue repair capability, suggesting that Tet2 plays important roles in regulating macrophages function within damaged heart tissues. Further epigenomic analysis on sorted cardiac-specific macrophages is anticipated provide more valuable mechanistic information. Conclusion: Taken together, our data suggest that Tet2 deficient macrophages play an important role during heart damage and repair. Inactivation mutations of TET2 detected in CHIP patients might lead to impaired macrophage function during heart damage and therefore increase the CVD risk. References 1. Jaiswal, S., Fontanillas, P., Flannick, J. et al. (2014). N Engl J Med 371, 2488-2498. 2. Jaiswal, S., Natarajan, P., Silver, A. J. et al. (2017). N Engl J Med 377, 111-121. 3. Fuster, J. J., MacLauchlan, S., Zuriaga, M. A. et al. (2017). Science 355, 842-847. 4. Sano, S., Oshima, K., Wang, Y. et al. (2018). J Am Coll Cardiol 71, 875-886. 5. Zhang, Q., Zhao, K., Shen, Q. et al. (2015). Nature 525, 389-393. 6. Pan, W., Zhu, S., Qu, K. et al. (2017). Immunity 47, 284-297 e285. Disclosures No relevant conflicts of interest to declare.

Description: Factor VIII (FVIII) functions as a cofactor for factor IXa in the contact coagulation pathway and circulates in a protective complex with von Willebrand factor (VWF). Plasma FVIII activity is strongly influenced by environmental and genetic factors through VWF-dependent and -independent mechanisms. Single nucleotide polymorphisms (SNPs) of the coding and promoter sequence in the FVIII gene have been extensively studied for effects on FVIII synthesis, secretion, and activity, but impacts of non–disease-causing intronic SNPs remain largely unknown. We analyzed FVIII SNPs and FVIII activity in 10 434 healthy Americans of European (EA) or African (AA) descent in the Atherosclerosis Risk in Communities (ARIC) study. Among covariates, age, race, diabetes, and ABO contributed 2.2%, 3.5%, 4%, and 10.7% to FVIII intersubject variation, respectively. Four intronic FVIII SNPs associated with FVIII activity and 8 with FVIII-VWF ratio in a sex- and race-dependent manner. The FVIII haplotypes AT and GCTTTT also associated with FVIII activity. Seven VWF SNPs were associated with FVIII activity in EA subjects, but no FVIII SNPs were associated with VWF Ag. These data demonstrate that intronic SNPs could directly or indirectly influence intersubject variation of FVIII activity. Further investigation may reveal novel mechanisms of regulating FVIII expression and activity.

Description: Background Refractory acute graft versus host disease (aGVHD) is a major cause of death after allogenetic haematopoietic stem cell transplantation (allo-HSCT). This study evaluated the efficacy and safety of mesenchymal stem cells (MSCs) from bone marrow of a third-party donor to refractory aGVHD. Methods Twenty-five patients with refractory aGVHD were enrolled in this prospective study. MSCs were given at a median dose of 1×106 cells/kg once weekly until aGVHD got complete response (CR) or MSCs were infused for a total of 8 doses. In order to evaluate infections, tumor relapse and cGVHD, 25 refractory aGVHD patients with MSCs treatment was matched with grade II-IV aGVHD patient without MSCs in the corresponding period Results After a total of 112 doses of MSCs were administered, with a median of 4 (range:2-12) doses per patient, the overall response (OR) rate for aGVHD was 72%,including CR in 56% and partial response (PR) in 16%. The efficacy of MSCs to aGVHD was significantly related with the severity and the number of involved organs of aGVHD, but unrelated with the onset timing of MSC. The incidence of CMV and EBV infections, including viremia and associated disease, was not different between MSCs and non-MSCs groups during aGVHD treatment and follow-up. At a median follow-up time of 126 (range: 49–1067) days post-transplantation, only two patients relapsed in MSCs group, compared with four relapsed in the control group. Tumor relapse was not different between the two groups (P=0.771). No toxic side effects and other secondary tumors were observed after MSCs treatment except for one EBV-associated post-transplant lymphoproliferative disorders (PTLD). The incidence of cGVHD in MSCs group, especially extensive cGVHD, was lower than that in non-MSCs group (25.0% Vs 68%, P=0.001; 20.0% Vs 64%, P=0.043, respectively). The ratio of CD3+CD4+/CD3+CD8+ T cells and the proportion of CD4+CD25+ regulatory T cells (Tregs) at 8 weeks after MSCs were higher than that before treatment (P=0.037 and P=0.020, respectively), and there were not different from the healthy people at 36 weeks after treatment. Conclusions MSCs derived from bone marrow of third-party donors are effective to refractory aGVHD. It might not increase the risks of infections, tumor relapse, but reduce the incidence and severity of cGVHD. Disclosures: Liu: Science and Technology Program of Guangzhou of China (11A72121174): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647): Research Funding; National Public Health Grand Research Foundation (Grant No. 201202017): Research Funding; 863 Program (No. 2011AA020105): Research Funding.

Description: Abstract 1930 Background: Invasive fungal infection (IFI) is a major cause of death in recipients of allogeneic hematopoietic stem cell transplant (allo-HSCT). Although antifungal prophylaxis has been demonstrated effective for prevention of IFI after allo-HSCT, the optimal agents and the term of prophylaxis remain a matter of discussion. To investigate the effect of prophylaxis term for IFI after allo-HSCT, this multicenter, randomized, open-label study was conducted to compare the efficacy and safety between long-term and short-term administration of itraconazole for primary antifungal prophylaxis in recipients of allo-HSCT. Methods: A total of 128 patients without a history of IFI were enrolled in this study. The primary antifungal prophylaxis was initiated when WBC count 〈 1.0*10⋀9/L or on day 0. Itraconazole was administered intravenously with a loading dose of 400 mg·d−1for 2 days followed by 200 mg·d−1and switched to itraconazole orally on day +14 or when WBC count 〉 1.0*10⋀9/L until 30 days post-HSCT in short-term arm or 90 days in long-term arm. The primary endpoint was the incidence of proven or probable IFI at 30 days and 90 days post-HSCT. Results: Of the 129 enrolled patients, date of 121 cases were used to determine the primary endpoint in the intent-to-treat population (59 for long-term and 62 for short-term) and 7 patients died of transplant complication or other causes before the use of itraconazole. The baseline demographic and transplant characteristic were similar in the two arms. The incidence of proven and probable IFI within 90 days post-transplants was 1.69%. Intent-to-treat analysis showed that there was not a significant difference in the rate of breakthrough IFI (proven or probable IFI) between he long-term and short-term arm (1.69% vs. 0%; P=0.311). The incidence of IFI (proven, probable, and possible) in long-term arm was higher than short-term arm within 90 days post-transplants (6.78% vs. 0%, P=0.03). Acute GVHD was the risk factor affected the occurrence of IFI after HSCT (OR= 2.750, 95%CI 1.939–3.901, P=0.023). Prophylaxis was interrupted in 11 patients due to event of intolerance, including 1 in the short-term arm and 10 in the long-term arm (P=0.004). The incidences of drug-related adverse events in the two arms were similar (6.78% vs. 3.23%, P=0.43). The incidence of overall survival at 6 months was 75.74% in long-term arm and 84.56% in short-term arm (P=0.459). Conclusions: This study indicates that itraconazole was effective for prevention of IFI within 30 days post-HSCT. Whether the prolonged term administration of antifungal agent for prevention fungal infection is benefit to the recipients in allo-HSCT is worthy of further exploration. This trial was registered at www.clinicaltrials.gov as NCT01160952. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1904 Background Poor graft function (PGF) is a refractory complication that occurs in 5–27% patients, and is associated with considerable morbidity and mortality related to infections or hemorrhagic complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Mesenchymal stem cells (MSCs) possess the capacity to differentiate into several types of mesenchymal tissues, to suppress immunological responses, and to support hematopoiesis. Clinical applications of MSCs are evolving rapidly with goals of improving hematopoietic engraftment, preventing and treating graft-versus-host disease (GVHD) after allo-HSCT and so on. In the present study, we prospectively evaluated the efficacy and safety of MSCs expanded from the bone marrow of a third-party donor to patients with PGF after allo-HSCT. Methods Twenty patients, including primary PGF in 7 and secondary in 13, were enrolled in this prospective multicenter trial. Bone marrow-derived MSCs were obtained from HLA-unrelated third-party donors. MSCs were cultured and expanded with human MSCs growth medium (low-glucose Dulbecco's modified Eagle's medium [L-DMEM], Hyclone, Logan and UT) with 10% (v/v) fetal bovine serum. MSCs were harvested after 4–5 passages for clinical administration. MSCs were given at 4 weeks intervals at the dose of 1×106 cells/kg. If ANC counts did not achieve 〉1.5×109/L and PLT counts not 〉50×109/L within 28 d after MSCs treatment, a second course of MSCs treatment was given. Flow cytometry was used to examine the lymphocyte subsets in peripheral blood pre- and post-MSCs treatment. Results Seventeen patients were responsive and 3 (primary PGF in 2 and secondary in 1) were not to 1–3 cycles of MSCs treatment. Within the first 100 d after MSCs treatment, 13 patients developed 20 episodes of infections. Moreover, 5 patients experienced cytomegalovirus-DNA viremia and 7 experienced Epstein-Barr virus (EBV)-DNA viremia within the first 100 d after MSCs treatment. Within a median follow-up of 125 d (range 11–579 d) after MSCs treatment, 6 patients (primary PGF in 3 and secondary in 3) experienced CMV-DNA viremia, and 8 (primary PGF in 2 and secondary in 6) developed EBV-DNA viremia, including 3 with EBV-associated post-transplant lymphoproliferative disorders (PTLD). One patient developed gradeII acute GVHD and 2 developed local chronic GVHD after MSCs treatment; 2 developed acute GVHD and chronic GVHD, respectively, after donor lymphocyte infusion because of PTLD. The proportions of CD3+ T cells and CD3+CD4+ T cells at 56 d after MSCs treatment were higher than those prior to MSCs treatment (88.05 ± 1.26% vs 79.05 ± 3.23%, P=0.015 and 24.09 ± 1.95% vs 17.40 ± 1.57%, P=0.012, respectively). The proportion of CD3+CD8+ T cells at 56 d after MSCs treatment was lower than that prior to MSCs treatment (51.91 ± 2.86% vs 61.39 ± 3.43%, P=0.043), and the ratio of CD3+CD4+ to CD3+CD8+ T cells was higher after MSCs treatment (0.31 ± 0.04 vs 0.51 ± 0.06, P=0.01). The proportions of CD4+CD25+ FoxP3 regulatory T cells, CD19+ B cells, and NK cells did not change significantly after MSCs treatment. With a median follow-up time of 508 d (range, 166–904 d) post-transplantation, 9 patients were alive and 11 died. No short-term toxic side effects were observed after MSCs treatment. Two patients experienced leukemic relapse. With the exception of 3 patients with PTLD, no secondary tumors occurred. Conclusions MSCs derived from the bone marrow of a third-party donor are effective to both primary and secondary PGF after allo-HSCT. However, whether such treatment might increase the risks of EBV infection and reactivation or the development of EBV-associated PTLD should be studied further. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4166 Background High resolution typing of HLA-A, B, C, DQB1 and DRB1 at the allelic level allows transplantation of hematopoietic progenitor cells (HPC) from matched allogeneic donors. The extent of matching has been recognized as one of the most important predictors of survival. High resolution typing relies on Sanger sequence-based typing (SBT) and PCR with sequence-specific primers (SSP), both of which detect nucleotide changes in exons that alter the HLA antigens. Next generation exome sequencing (NGES) offers the potential for rapid and less expensive HLA typing for transplantation of matched allogeneic HPC, but the role of NGES for high resolution HLA typing has not been examined. In this study, we designed a bioinformatic pipeline to account for the hundreds to thousands of alleles at each HLA locus in the population, and used it to explore the possibility of high resolution HLA typing using NGES data. Methods Indexed sequencing libraries were constructed from 3 HapMap samples (NA19129, NA18507, and NA19240), target enriched with SureSelect Human All Exon V3 kit (Agilent Technologies), and sequenced in multiplex as paired-end 2×101 bp reads on an Illumina HiSeq2000. The above process was repeated for NA19240 to assure reproducibility. The NGES-based HLA typing consisted of 3 major steps: 1) Filtering reads: cDNA and genomic sequences of all alleles of each HLA locus [ImMunoGeneTics project (IMGT)/HLA database] were concatenated to form five references, to which input reads were aligned using NovoalignMPI V.2.07.07 with standard options and no more than 1 mismatch per read. 2) Eliminating unsupported alleles: Curated alignment of all alleles of each HLA locus was obtained from IMGT/HLA database. Each polymorphic position was termed a D position. K-mers (a DNA sequence with length k), each containing a D position, was searched against the reads from step 1. Alleles were credited or penalized based on presence or absence of their k-mers in the reads. Alleles with negative overall scores were eliminated and this step was repeated until no elimination occurred. 3) Ranking allele pairs: all possible pairs of candidate alleles from step 2 were evaluated; a pair was credited at each D position if k-mers of both alleles were supported by the reads or penalized if otherwise. The allele pair (or pairs if a tie) ranked at the top was the presumed type of the locus examined. The NGES-based high resolution typing was run on a computing node (IBM x3650-m2). The results were compared with the results of conventional typing (SBT and SSP), followed by examination of the disagreement between the methods and adjustment of k. Results The conventional high resolution typing results comprised 15 pairs of alleles that were uniquely different (one pair of alleles at each locus of HLA-A, B, C, DQB1 and DRB1 for 3 samples). The method was established using the information on HLA-A alleles of NA19129, leaving 14 pairs of alleles for validation. Initial NGES-based typing indicated that the specificity of the method was dependent on k-mer length, with increased length required to eliminate interference from mimicking reads (e.g. mimics of an HLA-C allele). Thus, with a k-mer length of 50 bp, NGES-based typing correctly determined 12 of 14 pairs of alleles, while with k-mer lengths of 60 or 65 bp, it correctly determined 14 of 14 pairs of alleles. All 5 pairs of alleles of NA19240 were also correctly determined with the longer k-mers using reads from the replicate sequencing. After read filtering, the typing took approximately 5 minutes per sample. Conclusion In this first proof-of-concept study, high resolution HLA typing was successfully performed using NGES data. With k-mer lengths of 60 or 65 bp, NGES-based typing correctly identified 100% of tested allele pairs, and demonstrated a similar level of resolution and accuracy as conventional high resolution typing. Note: C.L. and X.Y. contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.