Abstract: The intestine is preferentially damaged in acute graft-versus-graft disease (aGVHD). Patients with intestinal GVHD are usually associated with drug-resistant diarrhea and microflora disturbance. Recent studies suggest that toll-like receptor (TLR) signaling can protect the intestinal epithelial barrier and confer commensal tolerance in health. But less is known about how functional versus dysfunctional TLR pathway opposes or favours the intestinal GVHD. Methods In the current study, BALB/c mice were transplanted whole spleen and T cell deleted (TCD) bone marrow cells from C57BL/6 mice as GVHD group, and transplanted TCD bone marrow cells as control group. The jejunum, ileum, colon and rectum epithelium were harvested and total RNA of the intestinal epithelium were extracted in two groups. The mRNA expression of classical TLR pathway TLRx/MYD88/IRAK4 signaling molecules (TLR2, TLR4, MYD88, IRAK4 and Tollip) and cytokines (IFN-γ, TNF-α and TGF-β) were detected by RT-PCR. Results The intestine of aGVHD recipients showed severe mucosal edema and erythema with histologic changes of apoptotic epithelial cells and crypt cell dropout, while the intestine of recipients in the control group did not show any intestinal GVHD evidence. TLR2 expression was markedly down-regulated and little TLR4 expression was observed in GVHD intestinal epithelium in comparison to control group. MYD88 and IRAK4 expression were lower in the entire intestinal epithelium of GVHD group but only significant in colon and rectum epithelium between the two groups. Tollip, a TLR signaling inhibitor by interfering IRAK, was found much higher in the GVHD group. For cytokines, both of IFN-γ and TNF-α expression were markedly up-regulated from proximal to distal intestine in GVHD group as compared to control group. There was no difference in TGF-β expression between the two groups. Conclusions We propose TLR signaling in the intestinal epithelium, especially in colon and rectum, presents disruption in intestinal graft-versus-host disease. IFN-γ and TNF-α might contribute to accelerate TLR pathway alteration. Disclosures: Liu: It was supported by 863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017).: Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174).: Research Funding. Wu:It was supported by 863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017).: Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174).: Research Funding. Zhao:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017).: Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174).: Research Funding. Wu:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Zhang:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Fan:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Fan:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Yin:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Zheng:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Yi:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Liu:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding.

Abstract: Noninfectious lung injury caused by graft-versus-graft disease (GVHD) is a life-threatening complication after allogeneic hematopoietic stem cell transplant (allo-HSCT). Epithelial injury is a central event in the pathogenesis of noninfectious lung injury. Recent studies have shown that alveolar epithelial cells are able to self-renew and reestablish a functional alveolar epithelium. In the inflammatory and fibrotic lung diseases, differentiated epithelia also can acquire a myofibroblast phenotype in the process termed epithelial to mesenchymal transition (EMT), which contributes to aberrant healing and fibrosis. Many factors such as inflammatory cytokine TGF-β have been suggested to induce EMT. However, the role of EMT in the remodeling of acute GVHD (aGVHD) induced lung injury is unclear. Methods BALB/c mice were lethally irradiated and transplanted T cell-deleted (TCD) bone marrow plus whole spleen cells from C57BL/6 mice as aGVHD group, and only transplanted TCD bone marrow cells as control group. Alveolar epithelial cells were isolated from mice of two groups and Ep-CAM expression was measured by flow cytometry. The mRNA expression of cytokines including IFN-γ, TNF-α and TGF-β was detected by RT-PCR. The mRNA and protein expressions of specific markers, including E-cadherin, vimentin, Snail and surfactant proteins (SP)-C in lung tissue, were detected by RT-PCR and western blot. Results All mice in the aGVHD group showed diffuse periluminal infiltrates and parenchymal pneumonitis by histopathology, while the mice in the control group did not show any lung injury evidence. IFN-γ, TNF-α, TGF-β mRNA expressions were markedly up-regulated in the lung injury group as compared to the control group (P=0.045, P=0.032, P=0.025). Alveolar epithelial cells of injured lung expressed higher Ep-CAM (P=0.017) and lower SP-C (P=0.023). RT-PCR and western blot analyses revealed a significant decrease in epithelial marker E-cadherin (P=0.029) and increase in mesenchymal marker vimentin (P=0.026) in the GVHD damaged lung. Snail, a key EMT related transcription factor, was significantly elevated at mRNA and protein level in comparison to control group (P=0.015). Conclusion EMT is involving in the remodeling of lung injury induced by aGVHD. TGF-β is demonstrated to induce EMT. Whether up-regulation of IFN-γ and TNF-α contributes to EMT is deserved to be further explored. Disclosures: Wu: 863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Liu:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Zhao:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Wu:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Zhang:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Fan:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Fan:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Yin:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Zheng:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Yi:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding. Liu:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding.

Abstract: 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 〈 0.001). After 5 days of rhG-CSF mobilization, the proportion in the blood and BM was 0.66% ± 0.28% and 0.72% ± 0.13%, respectively. The difference between proportion in the blood before and after mobilization was significant (P =0.017). Adhesion molecules on MDSCs surface after rhG-CSF mobilization were significant lower than that before rhG-CSF mobilization, both in blood and BM (P 〈 0.001). The expression of ARG and iNOS after mobilization were significantly higher than that before mobilization (P 〈 0.001), but not in BM (P =0.695, 0.073). There was a significant negative correlation between the number of transplanted MDSCs cells and the grading of aGVHD (correlation coefficient rs =0.445, P =0.014). The difference between the incidence of aGVHD in high MDSCs group and low MDSCs group (20.00% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(315\) \end{document} vs 66.67% \batchmode \documentclass[fleqn,10pt,legalpaper] {article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(1015\) \end{document}, P =0.011), there was no significant difference between the hematopoietic/immune reconstitution, relapse rate (26.67% \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(415\) \end{document} vs 20.00% \batchmode \documentclass[fleqn,10pt,legalpaper] {article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(315\) \end{document}, P =0.671) and disease-free survival rate (46.7% ±14.2% vs 38.9%±15.8%, P =0.833) in two groups. 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: Abstract 4139 Background Herpesvirus infections of central nervous system (CNS) are associated with encephalitis/myelitis and other neurological syndromes as well as lymphoproliferative diseases in immunocompromised individuals. Diagnosis is mainly based on the detection of virus-DNA in cerebrospinal fluid (CSF). Recently, some studies demonstrate that herpesvirus-associated diseases have increased in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT), but these mainly focus on the systematic herpesvirus infections and lack of a large-sample prospective study of CNS herpesvirus infections. Methods The eligibility criteria are as following: (1)The patients after allo-HSCT; (2)The patients who were diagnosed as Epstein-Barr virus (EBV)-associated diseases; (3)The patients with other herpesvirus-associated diseases other than EBV-associated diseases accompanying CNS manifestations; (4)The patients with unexplainable CNS manifestations. According to the criteria aforementioned, fifty-four of 250 patients undergoing allo-HSCT in our single institution between July 2008 and April 2012 were enrolled in this prospective study. Moreover, 18 patients with herpesvirus-DNA-emia who did not develop herpesvirus-associated diseases volunteered to have their CSF monitored (platelet 〉 50×109/L). Herpesvirus-DNA of CSF, blood and other body fluids was monitored by polymerase chain reaction (PCR). Once herpesvirus-associated CNS diseases were considered, immunophenotypic analysis of CSF cells and magnetic resonance imaging scanning of CNS were performed. Results Twenty-four patients were diagnosed as herpesvirus-associated CNS diseases, including 8 EBV encephalitis, 7 EBV-associated CNS post-transplant lymphoproliferative diseases (PTLD), 5 herpes simplex virus type 1(HSV-1) encephalitis, 2 cytomegalovirus (CMV) encephalitis, 1 CMV myelitis and 1 varicella zoster virus(VZV) encephalitis, respectively. The EBV-DNA levels of CSF were significantly higher than that of blood (82457 ± 6126 copies/ml vs. 18517 ± 3906 copies/ml, P=0.030). The virus of CSF was consistent with the virus of blood in all patients except one patient with EBV-associated CNS-PTLD, who was EBV-DNA positive of CSF but CMV-DNA positive of blood. The median time of herpesvirus-associated CNS diseases onset was 79 days post-transplants and 70.8% cases occurred within 100 days post-transplants. The 3-year cumulative incidence of herpesvirus-associated CNS diseases and EBV-associated CNS diseases was 12.8±2.6% and 7.5±2.0%, respectively. With a median follow-up of 198 days after the diagnosis of herpesvirus-associated CNS diseases, 13 patients survived and 11 died. The causes of death were related with herpesvirus in 7 cases and not related with herpesvirus in 4 cases. Conclusions PCR detection of CSF virus-DNA is a sensitive and specific method for diagnosing herpesvirus-associated CNS diseases. EBV-associated CNS diseases are more common than other herpesvirus-associated CNS diseases in the early times of allo-HSCT. The EBV-DNA negative in blood could not exclude EBV-associated CNS diseases. Disclosures: No relevant conflicts of interest to declare.

Abstract: We aimed to investigate outcomes of different post-remission treatment (PRT) choices based on dynamic measurable residual disease (MRD) by multiparameter flow cytometry in favorable-risk AML (FR-AML). Four hundred and three younger patients with FR-AML in first complete remission (CR1) were enrolled in this registry-based cohort study, including 173 who received chemotherapy (CMT), 92 autologous stem cell transplantation (auto-SCT), and 138 allogeneic SCT (allo-SCT). The primary endpoint was the 5-year overall survival (OS). Subgroup analyses were performed based on dynamic MRD after the 1st, 2nd, and 3rd courses of chemotherapy. In subgroups of patients with negative MRD after 1 or 2 course of chemotherapy, comparable OS was observed among the CMT, auto-SCT, and allo-SCT groups ( p  = 0.340; p  = 0.627, respectively). But CMT and auto-SCT had better graft-versus-host-disease-free, relapse-free survival (GRFS) than allo-SCT in both subgroups. For patients with negative MRD after three courses of chemotherapy, allo-SCT had better disease-free-survival than CMT ( p  = 0.009). However, OS was comparable among the three groups ( p  = 0.656). For patients with persistently positive MRD after 3 courses of chemotherapy or recurrent MRD, allo-SCT had better OS than CMT and auto-SCT ( p  = 0.011; p  = 0.029, respectively). Dynamic MRD might improve therapy stratification and optimize PRT selection for FR-AML in CR1.

Abstract: Backgroud: Although the introduction of rituximab as a first-line treatment has improved outcome of post-transplant lymphoproliferative disorder (PTLD), PTLD with central nervous system involvement (CNS-PTLD) still has a dismal prognosis because of low penetrance across the blood-brain barrier. In this prospective study, we reported intrathecal rituximab was efficacy and safety in the patients with CNS-PTLD who had failed to respond to the intravenous rituximab-based treatments. Methods: From June 2009 to November 2013, 32 cases of EBV-associated PTLD were recorded in the Southern Medical University Institute of Hematology. Fourteen patients diagnosed with CNS-PTLD were enrolled in this prospective study. For the patients who failed to response to the initial intravenous rituximab-based treatments, sequential dose-escalation schedule of intrathecal rituximab (initial dose of 20mg, increased by 10mg each week and maximum dose of 50 mg) was administrated weekly. Results: Three patients were responsive and 11 were unresponsive to the initial treatments within one week after the treatments. For the 11 patients who failed to respond to the initial treatments, 9 patients received intrathecal rituximab within 7-11 days and 2 patients refused the treatment. After two cycles of rituximab-based treatments, 10 patients achieved complete response (CR), 2 patients were partial response and 2 patients were non-response. With a median follow up of 664 (range 18 to 1545) days after the diagnosis of CNS-PTLD, 7 patients survived and 7 died. The causes of death included PTLD progressing (n=3), PTLD relapse (n=1), GVHD (n=1), CMV pneumonia (n=1) and pseudomonas aeruginosa sepsis (n=1). The 3-year probability of OS was 45.7% ±14.7% in CNS-PTLD, which was no significant difference as compared to PTLD without CNS involvement(55.6% ±11.7%, P=0.706). Conclusion: Intrathecal rituximab might be an effective and safe method for CNS-PTLD in the allo-HSCT recipients who were unresponsive to the intravenous rituximab-based treatments. Disclosures No relevant conflicts of interest to declare.

Abstract: Epstein-Barr virus (EBV) infection/reactivation following allogeneic hematopoietic stem cell transplantation (allo-HSCT) can cause fatal post-transplant lymphoproliferative disorders (PTLD) and other EBV-associated diseases. The development of EBV infection/reactivation and EBV-associated diseases are closely related to the immune function. Human leukocyte antigen (HLA) molecules are responsible for antigen processing and presentation to the immune system. Thus, it is supposed that HLA polymorphisms might be associated with EBV infection/reactivation and EBV-associated diseases. In this study, HLA polymorphisms and EBV infection/reactivation or EBV-associated diseases in the recipients of allo-HSCT were analyzed. Methods Three hundred and forty-nine recipients undergoing allo-HSCT were enrolled in this study between July 2008 to June 2013. For recipients and their donors, HLA-A, -B and HLA-DR were analyzed using PCR-sequence specific oligonueleotide probing (PCR-SSOP). The EBV-DNA levels of blood were monitored regularly by quantitative real-time polymerase chain reaction (RQ-PCR). Results With a median follow-up of 389 days post-transplantation (range, 7 to 1828 days), 96 cases developed EBV infection/reactivation and 40 developed EBV-associated diseases including 27 EBV-PTLD and 13 other EBV-associated diseases (i.e. 7 fever, 1 pneumonia, 2 encephalitis, 1 hepatitis, 1 encephalitis accompanying pneumonia and 1 enteritis accompanying hepatitis). The 3-year cumulative incidence of EBV infection/reactivation and EBV-associated diseases were 29.1%±2.6% and 13.1%±2.0%, respectively. 43.8% of the recipients with HLA-A11 developed EBV infection/reactivation, compared with 56.5% of those without HLA-A11 (p=0.039). Multivariate analysis showed that HLA-A11 was a protective factor for EBV infection/reactivation (OR 0.497, 95% confidence interval [CI] 0.284-0.869, p=0.014). The recipients who had the donors with HLA-A31 had a higher incidence of EBV-associated diseases than those whose donors did not have HLA-A31 (10.3% vs. 2.9%, p=0.046); more patients carried HLA-B44 suffered EBV-associated diseases than those not carried HLA-B44 (7.7% vs. 1.3%, p=0.035). In multivariate analysis, recipient HLA-B44 were confirmed to be a risk factor for EBV-associated diseases (OR 17.749, 95% confidence interval [CI] 1.946-161.917, p=0.011). The incidence of PTLD in the recipients with HLA-A74 was 7.4%, compared with 0.6% in those without HLA-A74 (p=0.031); the incidences of PTLD in recipients whose donors had and did not have HLA-DR04 were 37.0% and 20.2%, respectively (p=0.042). Multivariate analysis showed that recipient HLA-A74 (OR 11.350, 95%CI: 1.119-115.178, p=0.040) and donor HLA-DR04 (OR 3.227, 95%CI: 1.323-7.873, p=0.010) were risk factors for development of PTLD. Conclusion Our data suggest that HLA polymorphisms might affect EBV infection/reactivation and EBV-associated diseases. Disclosures: Liu: This work was supported by the National High Technology Research and Development Program of China (863 Program) (No. 2011AA020105), the National Public Health Grand Research Foundation (Grant No. 201202017).: Research Funding; This work was also supported by National Natural Science Foundation of China (Grant No.81000231, No.30971300, No.81270647), the Science and Technology Project of Guangdong Province of China (Grant No.2009A030200007).: Research Funding; This work was also supported by the Science and Technology Program of Guangzhou of China (11A72121174).: Research Funding.