Kurzfassung: 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.

Kurzfassung: Mixed phenotype acute leukemia (MPAL) is a complex entity expressing both lymphoid and myeloid immunophenotyping. In the present study, 47 MPAL, 60 lymphoid antigen-positive acute myeloid leukemia (Ly + AML), and 90 acute myeloid leukemia with common myeloid immunophenotype (Ly − AML) patients were investigated. We found that, in MPAL patients, there were high proportions of blast cells in bone marrow and incidence of hepatosplenomegaly, lymphadenopathy, and Philadelphia chromosome. The overall survival (OS) and relapse-free survival (RFS) in MPAL patients were significantly shorter than those in Ly + AML and Ly − AML. With regard to the patients with normal karyotype only, the OS and RFS of MPAL were significantly lower than those of the Ly + AML and Ly − AML; but there were no significant differences in OS and RFS among the patients with complex karyotype. The OS rates of 3 groups with complex karyotype were lower than those of patients with normal karyotype. In Cox multivariate analysis, complex karyotype was an independent pejorative factor for both OS and RFS. Therefore, MPAL is confirmed to be a poor-risk disease while Ly + AML does not impact prognosis. Complex karyotype is an unfavorable prognosis factor in AML patients with different immunophenotype. Mixed immunophenotype and complex karyotype increase the adverse risk when they coexist.

Kurzfassung: Gene mutations play a critical role in leukemogenesis of AML1-ETO-positive acute myeloid leukemia (AE-AML). Nevertheless, gene mutation profile in this subtype leukemia remains unclear, and their clinical effect might be underestimated. In this study, we detested gene mutations at diagnosis and relapse with next-generation sequencing in 64 newly diagnosed AE-AML patients, and verified the results with Sanger sequencing at the same time. Our results showed that 68.8% patients presented recurrent mutations at diagnosis and 6/11 cases underwent genetic alterations at relapse. C-KIT mutation was the most common event at diagnosis, with an incidence of 42.2%, followed by ASXL1 (15.6%), MET (12.5%), MLH1 (9.4%) , TET2 (7.8%), and FBXW7, TP53 and DNMT3A (7.8%), etc. Also, C-KIT mutation was the most common molecular event associated with relapse (7/11, 63.6%). No significant difference in the clinical characteristic between the gene mutation and wild type (WT) groups was observed, except of higher incidence of additional cytogenetic abnormalities (ACAs) (P=0.025) in TP53 mutation patients. C-KIT (exon 8, 17) mutation but not exon 10 adversely affected on survival. Also ASXL1 and TP53 mutation were poor for the RFS (P 〈 0.05), and ASXL1, MET, FBXW7 and TP53 mutation negatively impacted on the OS (P 〈 0.05). Multivariate analysis showed C-KIT (exon 8, 17) and ASXL1 mutations were the independent adverse factors for survival. Further, co-mutation of these two genes showed even worse effect on survival. Taking together, additional gene mutations are critical in the development and progression of AE-AML. C-KIT and ASXL1 mutations are the two most common molecular events in this subtype leukemia. Single mutation of C-KIT (exon 8, 17) or ASXL1 poorly affects on survival, even worse in the co-mutation. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Kurzfassung: 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.