LncRNA-NEAT1 promotes proliferation of T-ALL cells via miR-146b-5p/NOTCH1 signaling pathway.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) is a malignant tumor of the hematopoietic system, which can develop at any age, with the symptoms of weakness, fatigue, enlarged lymph nodes, or weight loss. Nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in the process of T-ALL, but the regulatory mechanism is still not known clearly. METHODS: The expression levels of NEAT1 and miR-146b-5p in T-ALL cells were performed by qRT-PCR and NOTCH1 protein level- wwWwas determined by western blot assay. Dual-luciferase reporter assay was used to detect the interaction between NEAT1 and miR-146b-5p, as well as miR-146b-5p and NOTCH1. The cell proliferation was measured by using MTT assay and colony formation assay. RESULTS: The expression levels of NEAT1 were markedly increased, but miR-146b-5p levels were reduced in T-ALL cells. Knockdown of NEAT1 or overexpression of miR-146b-5p decreased NOTCH1 expression, inhibited the proliferation of T-ALL cells. MiR-146b-5p bound both NEAT1 and NOTCH1 3'-UTR directly. Finally, inhibition of miR-146b-5p could abrogate the effects of NEAT1 knockdown on the proliferation of T-ALL cells. CONCLUSION: NEAT1 promotes the proliferation of T-ALL cells by sponging miR-146b-5p to upregulate the expression of NOTCH1. The results of this study provide new insight into the action mechanism of NEAT1 modulating T-ALL progression.

[Prognostic Value of Corrected Levels of Serum Calcium and Serum Lactate Dehydrogenase for Newly Diagnosed Multiple Myeloma Patients].

OBJECTIVE: To investigate the prognostic value of the serum calcium level corrected by serum albumin (cCA) and corrected serum lactate dehydrogenase (LDH) level for the risk stratification for newly diagnosed multiple myeloma (MM) patients. METHODS: The clinical data and survival of 186 newly diagnosed MM patients admitted to our hospital from June 1, 2015 to November 1, 2017 were collected. The patients's survival time was obtained by telephone and follow-up visits to patients and their families. The value of the prognostic system consisting of cCA levels and LDH levels in the survival time of MM patients was retrospectively analyzed. Moreover, the post-corrected hypercalcemia and high LDH as 2 factors were used for risk stratification, then according to these 2 factors, the MM patients were divided into 3 groups: group 1 (no risk factor), group 2 (1 risk factor) and group 3 (2 risk factors), and the effect of risk factors on the prognosis of MM patients was analyzed. RESULTS: The median follow-up time was 16 months. The cumulative OS rate of the post-corrected hypercalcemia group was lower than that of the non-hypercalcemia group. The 1-year cumulative OS rate in the 2 groups was 79.0%±6.7% and 88.6%±3.0%, the 3-year cumulative OS rate was 53.0%±10.5% and 74.6%±6.6% (P=0.016), respectively. The cumulative OS rate of the high LDH group [LDH ＞upper limit of normal (ULN), ULN=250 U/L] was lower than that in the normal LDH group. The 1-year cumulative OS rate in the 2 groups was 71.6%±8.6% and 90.0%±2.8%, the 2-year cumulative OS rate was 44.9%±12.1% and 83.1%±4.0%, respectively, and the median OS time was 19 months (95%CI: 15.32-23.34) and not reached (P=0.001). The risk stratification analysis showed that the median OS time of the 3 group was not reached (n=103, 57%), not reached (n=70, 39%) and 17 months (n=7, 4%, 95%CI: 5.19-28.41, P＜0.001). Patients with two risk factors had a prognosis worse than patients with 0-1 risk factor. CONCLUSION: The prognostic combination of corrected serum calcium and LDH levels may provide a basis for risk stratification and prognosis in MM patients in clinical practice.

A TET2 rs3733609 C/T genotype is associated with predisposition to the myeloproliferative neoplasms harboring JAK2(V617F) and confers a proliferative potential on erythroid lineages.

Common germline single-nucleotide polymorphisms (SNPs) at JAK2 locus have been associated with Myeloproliferative neoplasms (MPN). And, the germline sequence variant rs2736100 C in TERT is related to risk of MPN, suggesting a complex association between SNPs and the pathogenesis of MPN. Our previous study (unpublished data) showed that there was a high frequency distribution in rs3733609 C/T genotype at Ten-Eleven Translocation 2 (TET2) locus in one Chinese familial primary myelofibrosis. In the present study, we evaluate the role and clinical significance of rs3733609 C/T genotype in JAK2V617F-positive sporadic MPN (n = 181). TET2 rs3733609 C/T genotype had a higher incidence (13.81%; 25/181) in JAK2V617F-positive sporadic MPN patients than that in normal controls (n = 236) (6.35%; 15/236), which was predisposing to MPN (odds ratio(OR) = 2.361; P = 0.01). MPN patients with rs3733609 C/T genotype had increased leukocyte and platelets counts, elevated hemoglobin concentration in comparison with T/T genotype. Thrombotic events were more common in MPN patients with rs3733609 C/T than those with T/T genotype (P < 0.01). We confirmed that rs3733609 C/T genotype downregulated TET2 mRNA transcription, and the mechanism may be involved in a disruption of the interaction between CCAAT/enhancer binding protein alpha (C/EBPA) and TET2 rs3733609 C/T locus.TET2 rs3733609 C/T genotype stimulated the erythroid hematopoiesis in MPN patients. Altogether, we found a novel hereditary susceptible factor-TET2 rs3733609 C/T variant for the development of MPN, suggesting the variant may be partially responsible for the pathogenesis and accumulation of MPN.

Analysis of clinical and laboratory characteristics in 42 patients with thrombotic thrombocytopenic purpura from a single center in China.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by microvascular platelet deposition and thrombus formation with resulting microangiopathic hemolytic anemia. Deficiency of the von Willebrand factor cleavage protease, also known as ADAMTS 13, has been implicated as an important etiological factor in TTP. Little studies were obtained on Chinese patients with TTP until now. Our aim was to analyze the clinical features, outcome and laboratory characteristics of Chinese TTP patients, and determine whether plasma ADAMTS 13 activity is decreased in TTP and its diagnostic value for TTP. Forty-two TTP patients (29 females; 13 males) admitted to our hospital from 1998 to 2010 were analyzed. There were 34 patients (81%) with the triad of TTP, including hemolytic anemia, thrombocytopenia and neurologic abnormalities; 7 (16.7%) had the classical pentad of TTP. Major etiologic factors were acquired autoimmunological abnormalities (31%); no familial TTP was identified in this series. The schistocytes of peripheral blood smears were present in all cases with a mean frequency of 4.6% (range from 0.3% to 13.4%). Plasma ADAMTS 13 activity was determined in 22 patients with the FRET-vWF86 assay. Only 4 idiopathic TTP patients (18.2%) had severe ADAMTS 13 deficiency (activity<10%); 9 (40.9%) had moderate decrease of ADAMTS 13 activity (activity: 10-40%); another 9 (40.91%) had normal ADAMTS 13 activity (>40%). T lymphocyte subpopulation was measured in 23 TTP patients with FACS Calibur; 14 of the 23 (60.9%) had significantly decreased CD4 cells count and CD4/CD8 ratio, suggesting cellular immune dysfunction may be involved in the pathogenesis of TTP. In the studies, plasmapheresis is the main therapeutic method. 26 of 31 patients (83.9%) accepting plasmapheresis achieved complete remission; those patients who only underwent plasma infusion had low remission rate (18.2%) and high mortality (9/11; 81.8%). Four patients with packed RBC infusion manifested transient exacerbation of neurologic or psychiatric symptoms. In conclusion, the diagnosis of TTP in China is still based on clinical features including evidence of microangiopathic hemolysis. Severe ADAMTS 13 activity deficiency might be a valuable indicator for idiopathic TTP diagnosis. Further studies are needed to determine the real value of ADAMTS 13 activity for TTP diagnosis and whether T lymphocytes subset dysregulation plays important role in TTP pathogenesis.

Icaritin shows potent anti-leukemia activity on chronic myeloid leukemia in vitro and in vivo by regulating MAPK/ERK/JNK and JAK2/STAT3 /AKT signalings.

PURPOSE: To explore the effects of Icaritin on chronic myeloid leukemia (CML) cells and underlying mechanisms. METHOD: CML cells were incubated with various concentration of Icaritin for 48 hours, the cell proliferation was analyzed by MTT and the apoptosis was assessed with Annexin V and Hoechst 33258 staining. Cell hemoglobinization was determined. Western blotting was used to evaluate the expressions of MAPK/ERK/JNK signal pathway and Jak-2/Phorpho-Stat3/Phorsph-Akt network-related protein. NOD-SCID nude mice were applied to demonstrate the anti-leukemia effect of Icaritin in vivo. RESULTS: Icaritin potently inhibited proliferation of K562 cells (IC50 was 8 µM) and primary CML cells (IC50 was 13.4 µM for CML-CP and 18 µM for CML-BC), induced CML cells apoptosis and promoted the erythroid differentiation of K562 cells with time-dependent manner. Furthermore, Icaritin was able to suppress the growth of primary CD34+ leukemia cells (CML) and Imatinib-resistant cells, and to induce apoptosis. In mouse leukemia model, Icaritin could prolong lifespan of NOD-SCID nude mice inoculated with K562 cells as effective as Imatinib without suppression of bone marrow. Icaritin could up-regulate phospho-JNK or phospho-C-Jun and down-regulate phospho-ERK, phospho-P-38, Jak-2, phospho-Stat3 and phospho-Akt expression with dose- or time-dependent manner. Icaritin had no influence both on c-Abl and phospho-c-Abl protein expression and mRNA levels of Bcr/Abl. CONCLUSION: Icaritin from Chinese herb medicine may be a potential anti-CML agent with low adverse effect. The mechanism of anti-leukemia for Icaritin is involved in the regulation of Bcr/Abl downstream signaling. Icaritin may be useful for an alternative therapeutic choice of Imatinib-resistant forms of CML.

Use of all-trans retinoic acid in combination with arsenic trioxide for remission induction in patients with newly diagnosed acute promyelocytic leukemia and for consolidation/maintenance in CR patients.

In the present study, 90 patients with newly diagnosed acute promyelocytic leukemia (APL) were studied for all-trans retinoic acid (ATRA) and arsenic trioxide (As(2)O(3)) combination treatment in remission induction and postremission therapy. In addition, 20 APL patients who had achieved complete remission (CR) with an ATRA-based regimen received ATRA/As(2)O(3) combination for consolidation and maintenance were also enrolled. The results showed that ATRA/As(2)O(3) combination therapy yielded a high CR rate of 93.3% and a significantly shorter time to enter CR (median: 31 days; range: 18-59 days) compared to the ATRA-based regimen (n = 72; median: 39 days; range: 25-62 days). With the ATRA/As(2)O(3) combination for CR maintaining, regardless of the way by which CR was attained, the relapse-free survival was significantly better than with an ATRA plus cytotoxic chemotherapy regimen (92.9 +/- 3.2% vs. 72.4 +/- 7.6%, for the 3-year Kaplan-Meier estimate of relapse-free survival). The drug toxicity profile showed that with the use of As(2)O(3), the incidence of hepatotoxicity was obviously high during remission induction but decreased significantly during postremission treatment. We conclude that APL patients may benefit from the early use of the combination of ATRA and As(2)O(3), in either remission induction or consolidation/maintenance.

Fms-like tyrosine kinase (FLT) 3 and FLT3 internal tandem duplication in different types of adult leukemia: analysis of 147 patients.

AIM: To assess the expression level of fms-like tyrosine kinase 3 (FLT3), the incidence of FLT3/internal tandem duplications (ITD) mutation, and prognostic value of FLT3 changes in different types of adult leukemia. METHODS: Bone marrow mononuclear cells were isolated from 147 adult patients with leukemia. Reverse transcriptase polymerase chain reaction (PCR) was used to screen FLT3/ITD mutation and quantitative PCR was performed to evaluate the expression of the FLT3 transcript. Flow cytometry was used for detection of FLT3 receptor protein expression on bone marrow mononuclear cells. Pearson correlation analysis was performed to estimate the significance of FLT3. RESULTS: FLT3 expression was higher in acute myeloid leukemia and B-acute lymphoid leukemia than in T-acute lymphoid leukemia (P=0.006, P=0.001) and chronic myelogenous leukemia (P<0.001). In chronic myelogenous leukemia, FLT3 expression in blast transformation phase was higher than in acceleration phase (P=0.023). Surface expression of FLT3 protein was correlated with high percentage of bone marrow blasts and with FLT3 mRNA expression (r=0.366, P<0.001) in acute leukemia. FLT3/ITDs in the juxtamembrane domain were found in 25% of patients with acute myeloid leukemia and 7% of patients with acute lymphoid leukemia. FLT3/ITD positive sequences had 36, 42, and 57 nucleotides. FLT3/ITD mutation was associated with a higher white blood cell count, higher marrow blast percentage, and elevated serum lactate dehydrogenase (P=0.045, P=0.014, P<0.001, respectively) and not associated with a higher FLT3 mRNA and FLT3 protein expression, and lower complete remission (P=0.091, P=0.060, P=0.270, respectively). CONCLUSION: FLT3 expression levels differed in different types of adult leukemia. Overexpression of FLT3 and presence of a positive FLT3/ITD mutation in acute leukemia were associated with unfavorable clinical characteristics and poor prognosis.

[Molecular mechanism of reversing multi-drug resistance of K562/AO2 by puerarin].

OBJECTIVE: To determine the molecular mechanism of reversing multi-drug resistance of K562/AO2 by puerarin. METHODS: Effects of ADR and puerarin on NF-kappaB activity of K562,K562/AO2 were tested by immunofluorescence. The expression of survivin of K562,K562/AO2 was examined by immunocytochemistry. The p-gp expression was detected by flow cytometry. RESULTS: The NF-kappaB activity of K562 was significantly higher than that of K562/AO2. The NF-kappaB activity of K562 treated by ADR was significantly higher than untreated. The NF-kappaB activity of K562 which was pretreated by puerarin and then treated by ADR was much lower than that treated by ADR alone. The NF-kappaB activity of K562/AO2 intervened by puerarin was lower than that unintervened by puerarin.The p-gp and survivin expression of K562/AO2 was significantly higher than K562. The p-gp and survivin expression of K562 treated by ADR was higher than that untreated by ADR. But the p-gp and survivin expression of K562 which was pretreated by puerarin and then treated by ADR was much lower than that not pretreated by puerarin.The p-gp and survivin expression of K562/AO2 intervened by puerarin was lower than that unintervened by puerarin. The expression was negatively correlated to the duration of intervention. The inhibition effect demonstrated time dependence. CONCLUSION: The activation of NF-kappaB can increase the expression of p-gp and survivin, which may be part of the molecular mechanism of multi-drug resistance of K562. Puerarin can prevent and stop the multi-drug resistance in K562 and reverse the multi-drug resistance of K562/AO2 to ADR by inhibiting the activity of NF-kappaB and the expression of p-gp and survivin.

Arsenic trioxide, thalidomide and retinoid acid combination therapy in higher risk myelodysplastic syndrome patients.

OBJECTIVE: To evaluate the clinical efficacy and safety of arsenic trioxide, retinoic acid and thalidomide combination therapy in higher risk MDS. METHODS: Twenty-one patients diagnosed with higher risk MDS were administered 10mg/day arsenic trioxide intravenously for 10 days, 40mg/day retinoic acid orally for 2 weeks and 100mg/day thalidomide orally for 4 weeks per cycle. RESULTS: After at least two treatment cycles, 10 patients showed hematologic responses. One achieved CR, one achieved PR, three patients achieved major hematological improvements. The efficacy rate was 24% (5/21), and the response rate was 48% (10/21). The schedule was tolerated well by all patients and toxicities were moderate and reversible. CONCLUSION: The combination of arsenic trioxide, retinoic acid and thalidomide could have therapeutic benefit in higher risk MDS with safety.

[Clinical analysis of histocytic necrotizing lymphadenitis].

OBJECTIVE: To understand the clinical features and histopathology of histocytic necrotizing lymphadenitis (HNL) so as to better recognize the disease. METHODS: The clinical features, histopathology, and diagnosis of 10 patients admitted to our hospital were retrospectively analyzed. RESULTS: The clinical features of these 10 cases included: young females were the majority; lymphadenopathy and fever were the most common clinical manifestations; some cases were accompanied by connective tissue diseases. Histopathologic examination showed distinctive necrosis and around the necrotic foci, variable proliferations of histocytes but generally without infiltration of neutrophils. CONCLUSION: HNL has some typical histopathological alterations and relatively fine prognosis,but it tends to be misdiagnosed as lymphoma or lymphoid tuberculosis and may be accompanied by other diseases.

Myelodysplastic syndrome with transformation to acute monocytic leukemia with FLT3-ITD mutation following orthotopic liver transplantation.

A rare case of a 46-year-old man who underwent myelodysplastic syndrome, acute monocytic leukemia with FLT3-ITD mutation and splenic disruption following orthotopic liver transplantation is reported. The study of this case may be helpful to understand both the pathogenesis of acute leukemia and new complication of liver transplantation.

[Identification of IgG subclass and FVIII binding epitope of an acquired FVIII inhibitor in a bullous pemphigoid patient].

OBJECTIVE: To identify the clinical and laboratory diagnosis of a bullous pemphigoid patient with acquired hemophilia A (AH-A). To identify FVIII binding epitope and IgG subclass of the FVIII inhibitor, and explore the molecular mechanism for AH-A pathogenesis. METHODS: Plasma FVIII activity( FVIII: C) was determined by one-stage assay, the titre of FYIII inhibitor by Bethesda Unit (BU). IgG purification of patient plasma or normal pooled plasma was finished by protein A-agarose column chromatography. Activated partial thromboplastin time (APTT) was assayed for uncovering FVIII inhibitor effect on FVIII in vivo. Combined Western blot analysis by anti-IgG1, IgG2, IgG3 and IgG4 antibodies was used to determine the relative concentration of patient' s IgG subclass. IgG subclass concentrations were quantified by nephelometric method. Solid-phase binding assay of FVIII and FVIII inhibitor, combined with Western blot was used to recognize the binding epitope at which the FVIII inhibitor bound to FVIII. RESULTS: (1) Plasma APTT value of patient was prolonged evidently and could not be corrected by normal pooled plasma. Patient's FVIII: C was < 1.5%. The titre of FVIII inhibitor in patient plasma was 147.8 BU. (2) The purified patient IgG was able to inhibit FVIII: C of normal pooled plasma significantly with a dose dependent manner, and the patient plasma could prolong rabbit plasma APTT markedly with a time dependent manner. (3) The FVIII inhibitor was predominantly then of IgG4 subtype with a minority IgG1, and the concentration of IgG4 and IgG1 in the patient was higher than that in normal. The FVIII inhibitor reacted with FVIII 44 x 10(3) fragment epitope. CONCLUSIONS: The inhibiting effect of FVIII inhibitors on FVIII: C in the bullous pemphigoid patient with AH-A is determined and the IgG subclass of the FVIII inhibitor is identified. A binding epitope for the FVIII inhibitor is a FVIII 44 x 10(3) fragment. The results provides evidence for understanding the pathogenesis of AH-A.

[Acute monocytic leukemia after orthotopic liver transplantation: clinical features, molecular genetics, and significance thereof].

OBJECTIVE: To study the clinical features and molecular genetics of acute monocytic leukemia (AML) after orthotopic liver transplantation and significance thereof. METHODS: The clinical manifestations, laboratory findings, development, diagnosis, treatment, and prognosis of the first case of AML after orthotopic liver transplantation in the world, a Chinese, male, aged 46, were observed. RT-PCR was used to analyze the mRNA expression of FLT3, Pim-1, and Hsp-70 in the bone marrow mononuclear cells (BMMCs) of the patient. Nucleotide sequence analysis was used to detect the mutation of FLT3-ITD. Flow cytometry was used to examine the protein expression of C-kit, a stem cell factor receptor, and platelet derived growth factor receptor-alpha (PDGFRalpha). RESULTS: (1) Three months after the orthotopic liver transplantation the patient manifested symptoms and signs of anemia and thrombocytopenia. Laboratory tests found increase of white blood cells and myeloblasts with Auer's rods. Cytogenetic analysis showed a genotype of 46XY/47XY with an extra chromosome 8. Bone marrow examination revealed increased promonocytes. The diagnosis of chronic myelomonocytic leukemia was made. Five months after the liver transplantation the disease developed to AML. The patient underwent combined chemotherapy (HA or DA regimens) for 5 courses and showed a partial remission both hematologically and in bone marrow examination at first, however, became resistant to all chemotherapeutic agents. RT-PCR showed absence of wild type FLT3 allele. At last the patient died of infection. (2) A FLT3-ITD mutation of "insertion" type was identified in the BMMCs. The Pim-1 mRNA was weakly expressed and the expression of Hsp-70 mRNA was upregulated in the BMMCs. (3) The protein expression of C-kit and that of PDGFRalpha were both upregulated in the BMMCs as showed by flow cytometry. CONCLUSION: Orthotopic liver transplantation may be complicated with acute leukemia heterogeneous in clinical features and hematology. Certain defects in cytogenetics/molecular genetics may attribute to unfavorable prognosis.