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Ferroptosis has been demonstrated a promising way to counteract chemoresistance of multiple myeloma (MM), however, roles and mechanism of bone marrow stromal cells (BMSCs) in regulating ferroptosis of MM cells remain elusive. Here, we uncovered that MM cells were more susceptible to ferroptotic induction under the interaction of BMSCs using in vitro and in vivo models. Mechanistically, BMSCs elevated the iron level in MM cells, thereby activating the steroid biosynthesis pathway, especially the production of lanosterol, a major source of reactive oxygen species (ROS) in MM cells. We discovered that direct coupling of CD40 ligand and CD40 receptor constituted the key signaling pathway governing lanosterol biosynthesis, and disruption of CD40/CD40L interaction using an anti-CD40 neutralizing antibody or conditional depletion of Cd40l in BMSCs successfully eliminated the iron level and lanosterol production of MM cells localized in the Vk*MYC Vk12653 or NSG mouse models. Our study deciphers the mechanism of BMSCs dictating ferroptosis of MM cells and highlights the therapeutic potential of non-apoptosis strategies for managing refractory or relapsed MM patients.

Multiple myeloma (MM) is still incurable despite the successful application of proteasome inhibitors in clinic. Bortezomib represents the most common chemotherapy for MM, whereas acquired drug resistance and eventually developed relapse remain the major obstruction. In the current study, we established bortezomib-resistant myeloma cell lines and screened gene expression profiles using single cell RNA-sequencing. Resistant MM cells exhibited increased clonogenic potential, specific metabolic, and epigenetic signatures, along with the self-renewal signaling characteristic of MM stem-like cells. Aberrant activation of hedgehog (Hh) signaling was correlated with drug resistance and stem cell-like transcriptional program. The key transcriptional factor GLI2 of the Hh pathway was restricted in the high acetylation and low ubiquitination states in bortezomib-resistant myeloma cells. Further investigation revealed that SIRT1 deacetylates and stabilizes GLI2 protein at lysine 757 and consequentially activates the Hh signaling, and itself serves as a direct target of Hh signaling to format a positive regulating loop. Using combination screening with an epigenetic compound library, we identified the SIRT1 specific inhibitor S1541 and S2804 had very obvious synergetic antimyeloma effect. Sirt1 inhibition could partially impeded the Hh pathway and conferred bortezomib sensitivity in vitro and in vivo. Notably, elevated SIRT1 level was also a prominent hallmark for the resistant myeloma cells, and this expression pattern was confirmed in myeloma patients, but independent of RAS/RAF mutations. Clinically, SIRT1 expression in patients with complete response was suppressed but elevated in relapsed patients, and retrospective analysis showed patients with higher SIRT1 expression had poorer outcomes. In conclusion, the cooperation of SIRT1 and Hh is an important mechanism of drug resistance in myeloma, and therapeutics combining SIRT1 inhibitors will sensitize myeloma cells to proteasome inhibitors.

•High PPFIBP1 expression predicts poorer outcome in the clinic.•PPFPIBP1 directly binds RelA and activates NF-κB signaling pathway.

Objective: To improve the accurate diagnosis and treatment of blastic plasmacytoid dendritic cell neoplasm (BPDCN) without skin involvement. Methods: One case of BPDCN with skin involvement progressing to the leukemia phase was used as a positive reference for bone marrow specimens, and a comparative study was conducted on one case of leukemia phase of BPDCN without skin involvement. Results: The male control was 70 years old and presented with a mass on the right forearm skin. Pathological examination indicated BPDCN. Only one cycle of decitabine + CAG was given and 5 months later, the disease progressed to the leukemia phase. Tumor cells expressed HLA-DR, CD123, CD4, CD56, CD7, CD36, TCL1 and CD304. NGS analysis of bone marrow tumor indicated ASXL1, IDH2, and TP53 mutations. The patient survived only 6 months during follow-up. Another female patient was 73 years old and was initially diagnosed with “T lymphoblastic leukemia” based on partial immunophenotyping results for 81.5% blast cells and the morphology of blast cells showing tailing phenomena. Further immunophenotyping showed that blast cells expressed CD56, HLA-DR, and CD304, partially expressed CD4, CD33, and CD117, and did not express CD34, CD13, CD3, CD5, CD7, CD8, CD10, CD11b, CD14, CD15, CD16, CD19, CD20, CD64, CD303 or MPO. Additional immunohistochemistry of bone marrow biopsy showed that CD43 was negative, TdT was mostly positive, CD4 was slightly positive, CD56 was mostly positive, CD123 was positive, CD117 was slightly positive, and lysozyme was partially weakly positive. The above results implied that after excluding BPDCN, acute myeloid leukemia subtype M5 should be considered. According to the diagnostic criteria of BPDCN in WHO 5th edition hematologic lymphoma, two scenarios can be established for diagnosing BPDCN: (1) CD4 and/or CD56 are positive and CD123 and one of pDC markers (TCL1, CD303, CD304) are expressed. (2) If any three pDC markers are expressed (CD123, TCL1, CD303, CD304), and all expected negative markers (CD3, CD14, CD19, CD34, Lysozyme, Myeloperoxidase) are negative, So the final diagnosis of BPDCN can be established according the former creteria. The latter case had ASXL1 and TET2 mutations detected by NGS analysis, achieved complete remission after receiving venetoclax + azacitidine for one cycle, followed by intermittent venoclax +demethylation agent or low-dose chemotherapy maintenance treatment and live for more than two years now. Conclusion:BPDCN without skin lesions is clinically rare, and its diagnosis is challenging. Comprehensive immunophenotyping and cautious interpretation of immunohistochemistry results such as dim lysozyme expression are crucial. Venetoclax-containing regimens have shown promising therapeutic effects.

No relevant conflicts of interest to declare.

Objective: To increase the knowledge and experience of treating left-sided endocarditis patients complicated with hemophagocytic lymphohistiocytosis.

No relevant conflicts of interest to declare.

ObjectiveTo assess the argonaute 2 (Ago2) expression in the bone marrow of multiple myeloma (MM) and determine its association with angiogenesis.

AML cells induce changes in MSC, in short term co-cultures in vitro, or in syngenic systems in vivo, that are consistent with pro-survival, anti-apoptotic, and growth-stimulatory signals that mimic inflammatory responses. Large-scale analysis of primary AML-derived MSC confirms and extends this data. Results facilitate the development of therapeutic strategies to render the BM microenvironment inhospitable to leukemia cells but supportive of normal hematopoiesis.Lowe: Blueprint Medicines: Consultancy; Constellation Pharmaceuticals: Consultancy; Mirimus Inc.: Consultancy.

The bone marrow microenvironment (BME) in acute myeloid leukemia (AML) generates resistance signals that protect AML cells/stem cells from chemotherapy. The mechanisms how the BME might support leukemia cell survival are unclear but elucidation of this process could prove useful for therapy design. Here we report new insights specific to stroma functionality in AML. A series of novel experimental approaches were developed including : 1) nanostring micro-RNA and proteomic analysis using reverse-phase protein arrays (RPPAs) of MSC derived from AML patients and normal donors; 2) genome-wide RNA analysis of FACS-sorted MSCs using Illumina arrays of genetically-defined human and murine AML cell lines/primary AML samples after co-culture with normal MSC in vitro; 3) in vivointeraction between genetically-defined murine AML and stromal cells in syngenic C57BL/6J mice, followed by harvesting and FACS-isolation of specific MSC after leukemia engraftment; 4) use of genetically-modified human MSC in vivoin our ectopic humanized bone marrow model in NSG mice (Blood 2012 : 119,4971), followed by bioluminescence growth and homing analysis of human leukemia cells. This model allows the study of in vivoeffects of altered MSC on human AML development.

To identify gene mutations for one patient and his family members with protein S and antithrombin deficiency.ELISA were used to detect protein S (PS), protein C (PC) and antithrombin (AT) activities for the proband and family members, respectively. The genomic DNA was extracted from the peripheral blood of proband and family members. All exons and their flanks of protein S gene and antithrombin gene were amplified by polymerase chain reaction (PCR). The PCR products were sequenced directly. The mutation-related exons of his famliy members were amplified by PCR and sequenced directly.The proband was a 49-year-old male. He presented with sudden left lower extremity swelling and pain without casues. Regular examination revealed that his APTT, PT, and TT were all in normal levels, but D- dimmer was 5. 62mg/L, Color doppler ultrasonography showed thrombosis in his left femoral vein. The activity of PS for his family members was ‡1 0%, ‡2 0%, ‡3 0%, ‡4 130. 8%, ‡5 8. 4%, ‡1 0%, ‡2 0%, and that of AT was ‡1 129. 1%, ‡2 51. 9%, ‡3 73.2%, ‡4 119. 1%, ‡5 136. 2%, ‡1 65. 5% and ‡2 60. 1%, respectively. The sequencing analysis showed that a heterozygous missense mutation G68395T (NG_009813. 1) was detected in Exon 4 of PS gene leading to the substitution of Arg90 by Leu (NP_000304. 2) for the propositus. The heterozygous mutation (Arg90Leu) was also found in other family members. A heterozygous (nonsense) mutation G12444A (NG_012462. 1) was detected in Exon 4 of AT gene leading to Trp257Ter (NP_000479. 1) for the propositus. The mutation (Trp257Ter) was found in other family members with reduced activity of AT. These two mutations (G68395T in PS gene and G12444A in AT gene) were not reported before and were thus novel ones.The novel mutation G68395T in PS gene and G12444A in AT gene might be the causes of deficiency of PS and AT for the family.No relevant conflicts of interest to declare.

To identify F9 gene mutations in patients with hemophilia B registered in Nanjing Drum Tower Hospital Hemophilia Registeration Center. Methods: One stage method was used to detect APTT, PT, TT, Fg and the activities of endogenous coagultation factors. Correction testing was employed to exclud the existence of inhibitor with mixed normal plasma. Genomic DNA was extracted from blood samples of 19 unrelated haemophilia B patients and their traceable family members. All exons and their flanking sequences of the F9 gene were amplificated by PCR and subsequently, the products were purified and sequenced directly. Results: APTT was significantly prolonged for all 19 cases of hemophilia B patients, but could be corrected by mixed normal plasma. According to the serial number, FIX:C was 3.7%, 3.5%, 1.9%, 1.9%, 2.2%, 2.0%, 1.9%, 3.2%, 3.5%, 10.8%, 7.8%, 2.2%, 3.8%, 2.3%, 1.6%, 1.4%, 3.7%, 7.8% and 3.5%, respectively. Thirteen different mutations of F9 gene were identified, including C 20518 T, T 6427 C, C 6460 T, C 31008 G, C 17761 T, A 17759 G, G 30150 A, G 31093 C, T 30930 C, G 20565 A, G 30987 A, A 6473 G and C 9 G, respectively. The mutations were composed of 10 missense mutations, one nonsense mutation, one a donor splice site mutation and one promoter mutation. Among them, mutations sites nt6460, nt17761, nt20518, nt30150 and nt31008 were located in CpG islands, belonging to mutation hot-spots. Mutations including C 9 G, C 31008 G and G 31093 C were firstly reported. Conclusions: No inhibitors are detected in the plasma of all patients. The F9 mutations are heterogenous and the missense mutations are the most prevalent gene defects in Chinese HB patients.No relevant conflicts of interest to declare.

Abstract 5140

Abstract 4923

No relevant conflicts of interest to declare.

Abstract 4627

No relevant conflicts of interest to declare.

Abstract 4892

To discuss the clinical and laboratory features, diagnosis and treatment of human granulocytic anaplasmosis.We present the clinical and laboratory features, diagnosis and treatment of a patient with human granulocytic anaplasmosis.The patient with human granulocytic anaplasmosis presented with fever,cough?Adiarrhea?Amyalgia?Afacies typhosa?Arelative infrequent pulse and swelling of lymph nodes. Laboratory examination showed the patient had leukopenia?Athrombocytopenia?Aproteinuria?Aliver injury?Ablood clotting abnormal?AEBV-DNA positive. We also found the patient's ferritin?Acreatase?Aamylase and lipase increased. In the patient's bone marrow, plasma cells were increased, hemophagocyte and intragranulocytic inclusions were found. The patient did not respond to the treatment of imipenem, cefepime hydrochloride and teicoplanin. But he was treated successfully with moxifloxacin.Patient with human granulocytic anaplasmosis can present leukopenia, thrombocytopenia, blood clotting abnormal and plasma cells increased in bone marrow. It's quite necessary to make differential diagnosis with some blood diseases. The patient can be accompanied with EBV infection and hemophagocytic syndrome. The patient can be cured by antibiotics-quinolones.No relevant conflicts of interest to declare.

Autologous peripheral blood stem cells transplantation is widely used in treatment of malignant blood diseases, solid tumors and autoimmune diseases recently. Before apheresis, we filled the pipelines of Baxter CS-3000PLUS blood cell separator with the autologous blood instead of irradiated allogenetic blood.The study is aimed to explore the safety and clinic efficacy of autologous blood donation for apheresis of stem cell in low body weight pediatric patients with type 1 diabetes.There were three pediatric patients with type 1 diabetes in our hospital between August 2007 and December 2007. These patients included 2 male and 1 female with age of 5, 9, 9 respectively. Their weight was 15,18 and 23.5 kilograms respectively. Their blood test indexes were HB>120g/L,HCT>34%. On the 14th day before apheresis, 100ml autologous blood was collected and preserved in 4°C. Mobilization program was CTX 2g/m2 for 2 days and G-CSF 5∼10 μg/kg/d on the 5th day of before apheresis. Apheresis was initiated when WBC>5.0×109/L. SVSCH and SVCC were installed to blood cell separator at first. Before apheresis, we filled the pipelines of blood cell separator with the autologous blood instead of irradiated allogenetic blood.On the 9th day after the autologous blood donation, RBC ? AHB and HCT went back to the original level. In the process of apheresis.,every vital sign including blood pressure and heart rate was normal during apheresis. Hypotension or hyperthermia did not appear. The amount of CD34+ cell reached to 1.28-3.05×106/kg.It is concluded that autologous blood donation is a safe and an effective method to avoid allogenetic blood transfusion for apheresis of stem cell in low body weight pediatric patients.No relevant conflicts of interest to declare.

Abstract 4518

Abstract 4194

Objective:To increase the knowledge and understanding of acute lymphoblastic leukemia with maturation(ALLm).

Objective: To increase the knowledge and understanding of acute lymphoblastic leukemia with maturation(ALLm). Method: One ALLm case with clinical manifestation, bone marrow morphology, immunophenotype and cytogenetic results were presented and related literatures were reviewed. Result: The patient was a fifty-five year old women, the haematological feature was Pancytopenia. There were 12% lymphoblasts and 82.5% mature appearing lymphocytes in the bone marrow smear. The mature appearing leukemic cells could not be separated clearly by gating. However, the immunophenotypes of mature-appearing leukemic cells(Low FSC and Low CD45) and lymphoblasts were the same. Both results were CD33+CD34+CD19+CD22+HLA−DR+CD5−CD7−CD10−CD13−CD14−CD15−CD20−CD25−CD45−CD71−CD11b−CD103−CD117−. Bone marrow biopsy showed hypercellularity with diffuse infiltration of lymphocytes, The results were CD34++,TdT++,Pax-5+++,CD43++,CD3+(scatter),CD5+(scatter),CD20−,CD10+,CyclinD1−, lymphoblasts Ki67+, mature-appearing leukemic cells ki-67-. FISH analysis of the bone marrow revealed about 1% cells with a signal pattern suggesting loss of one copy of chromosome 8. After VDP, MA, AAG and HAG regimens Complete remission was not achieved. Conclusion: ALLm is a special morphological variant of acute lymphoblastic leukemia, most mature appearing cells are in resting G0 phase and this could be the reason why ALLm has a poor response to chemotherapy.

We describe a 21-year-old woman with systemic lupus erythematosus (SLE) who first presented with an thrombotic thrombocytopenic purpura (TTP), and compare the clinical manifestations and prognosis between SLE patients with thrombotic thrombocytopenic purpura in the reported literature. At the time of admission, she was suffering from petechia, purpura and had neurological symptoms. Laboratory findings demonstrated haemolytic anaemia, thrombocytopaenia and high levels of fragmentocytes. Serological test results were highly positive for antinuclear antibodies (ANA) with a particle fluorescent pattern, and also detected antibodies against Smith antibody, RNP antibody and SSA antibody but was negative for dsDNA. Direct Coombs-test was positive and D-dimer was negtive. The activity of ADAMTS13 was significantly reduced even after 4 times plasmapheresis. TTP as presenting sign in the patient with SLE was diagnosed. After 5 times plasmapheresis treatments and immunosuppressive therapy she recovered and abnormal laboratory tests were gradually returned to normal. With a follow-up of 20 months, she had a normal life. Compared with other reports, TTP can be differentiated from other thrombotic microangiopathic syndromes by its normal levels of prothrombin time, partially activated thromboplastin time (APTT), fibrinogen and direct Coombs-test. But TTP might also be a complication of SLE and the manifestations of TTP are similar to those in SLE. The detection of the fragmentation of peripheral red blood cells helped the early diagnosis of TTP. Coomb’s test might be positive when patient with TTP had SLE. ADAMTS13 and its inhibitory antibody had an important role in the pathogenesis of TTP. Plasma exchange combined with corticosteroid and cyclophsphamide should be used as early as possible in TTP patient with SLE. The prognosis was related to the treatment time and methods including plasmapheresis.

We describe a 21-year-old woman with systemic lupus erythematosus (SLE) who first presented with an thrombotic thrombocytopenic purpura (TTP), and compare the clinical manifestations and prognosis between SLE patients with thrombotic thrombocytopenic purpura in the reported literature. At the time of admission, she was suffering from petechia, purpura and had neurological symptoms. Laboratory findings demonstrated haemolytic anaemia, thrombocytopaenia and high levels of fragmentocytes. Serological test results were highly positive for antinuclear antibodies (ANA) with a particle fluorescent pattern, and also detected antibodies against Smith antibody, RNP antibody and SSA antibody but was negative for dsDNA. Direct Coombs-test was positive and D-dimer was negtive. The activity of ADAMTS13 was significantly reduced even after 4 times plasmapheresis. TTP as presenting sign in the patient with SLE was diagnosed. After 5 times plasmapheresis treatments and immunosuppressive therapy she recovered and abnormal laboratory tests were gradually returned to normal. With a follow-up of 20 months, she had a normal life. Compared with other reports, TTP can be differentiated from other thrombotic microangiopathic syndromes by its normal levels of prothrombin time, partially activated thromboplastin time (APTT), fibrinogen and direct Coombs-test. But TTP might also be a complication of SLE and the manifestations of TTP are similar to those in SLE. The detection of the fragmentation of peripheral red blood cells helped the early diagnosis of TTP. Coomb's test might be positive when patient with TTP had SLE. ADAMTS13 and its inhibitory antibody had an important role in the pathogenesis of TTP. Plasma exchange combined with corticosteroid and cyclophsphamide should be used as early as possible in TTP patient with SLE. The prognosis was related to the treatment time and methods including plasmapheresis.