An intron mutation in the ACVRL1 may be associated with a transcriptional regulation defect in a Chinese family with hereditary hemorrhagic telangiectasia.

PURPOSE: To identify a novel pathogenic gene mutation present in a Chinese family with hereditary hemorrhagic telangiectasia (HHT) and to determine if an intron mutation may influence the transcriptional activity of the ACVRL1 gene. METHODS: HHT family members were ascertained following the presentation of proband and involved subjects. All family members (n = 5) and 113 healthy individuals were genotyped for the variant in intron 6 c.772+27G>C of ACVRL1 gene. The genomic structure of ACVRL1 in affected HHT patients and healthy individuals was determined by long range PCR and sequencing. The expression of ACVRL1 mRNA and protein in patients with HHT was evaluated using real-time polymerase chain reaction and immunoblot analysis. Luciferase activity assay and electrophoretic mobility shift assay (EMSA) were performed to uncover the mechanism of intron-related transcriptional regulation. RESULTS: Only one novel mutation in intron 6 (c.772+27G>C) of ACVRL1 gene, no other mutation, abnormal splice, gross genomic deletion or rearrangement was found in this HHT2 family. Compared with healthy individuals, ACVRL1 mRNA and protein were significantly decreased in affected HHT2 individuals. Luciferase activity assay demonstrated that the transcriptional activity of the mutated ACVRL1 was significantly lower than that of the wild-type of intron 6; EMSA results showed that intron 6 c.772+27G>C mutation was able to inhibit the binding of transcriptional factor Sp1. CONCLUSIONS: A novel intron mutation in ACVRL1 gene is associated with familial HHT2. The mechanisms may be involved in the down-regulation of ACVRL1 gene transcription.

[Changes of HLA-DR15 and immunoglobulin, T lymphocyte subsets in patients with aplastic anemia, myelodysplastic syndrome and their significance].

The objective of this study was to detect the expression frequency of HLA-DR15 in patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS), to investigate the relation of expression frequency with diseases and to analyze the relationship between immunoglobulin, T lymphocyte subsets and HLA-DR15. HLA-DR15 expression was detected by PCR-SSP; immunoglobulin was detected by immune turbidimetry; T cell subsets were detected by flow cytometry. The results showed that the expression rates of HLA-DR15 in AA and MDS as well as normal control groups were 78.6%, 63.2% and 24.6% respectively. The difference between AA, MDS and the normal control groups was statistically significant (p < 0.01). OR (odds ratios) values of AA and MDS groups were 11.262, 4.710 respectively. Compared with normal control group, expression rate of HLA-DR15 in hematologic malignancy group was not significantly different. The immunoglobulin level and abnormal T cell subsets in AA and MDS groups were statistically different between HLA-DR15 positive and negative groups (p > 0.05). It is concluded that the frequency of HLA-DR15 antigen in AA and MDS patients is significantly higher than that in normal control and hematologic malignancy group. OR value>1 showed a positive correlation between the diseases and HLA-DR15. HLA-DR15 is a susceptible gene in AA and MDS. The abnormalities of immunoglobulin level and ratios of T cell subsets in AA and MDS are common, but are not associated significantly with the expression of HLA-DR15.

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.

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.

[Hematostatic function in myeloproliferative diseases].

This study was purposed to investigate the change of early hemostatic function in patients with myeloproliferative diseases (MPD) and to explore its significance in combination with clinical data. The platelet aggregative function was measured by using ristomycin, adenosine diphosphate, collagen and adrenine as inductors, the plasma von Willebrand factor-associated antigen (vWF: Ag) level was measured by enzyme-linked immunosorbent assay (ELISA), the plasma von Wellebrand factor-ristomycin cofactor (vWF: Rco) activity was measured in 6 patients with obviously low ristomycin induced platelet aggregation (RIPA). The results showed that the platelet aggregative function obviously decreased in 35 patients, there were distinct differences in maximal platelet aggregative rate between patients and normal controls induced by 4 inductors respectively (P < 0.001, P < 0.001, P = 0.002, P < 0.001). There was no obvious difference between patients with MPD and healthy controls in plasma vWF: Ag level (P > 0.50). Plasma vWF: Rco activity in all 6 patients with MPD chosen was in the normal range, except one patient with essential thrombocytosis (ET) whose plasma vWF: Rco activity was much lower than normal. No correlation was found between platelet count and plasma vWF: Ag level in the patients (r = -0.180). No correlation was found between platelet count and maximal platelet aggregative rate induced by 4 inductors respectively in patients. It is concluded that the occurrence of abnormal platelet aggregative function is high in patients with MPD. The RIPA and vWF: Rco activity decrease in one patient with ET. However, the shortage of vWF polymer existed in his plasma have needs for further research. No correlation was observed between hemostasis and clinical manifestations. However, because of the high occurrence of platelet dysfunction in MPD patients, the clinical application of anti-platelet drugs should be considered carefully.

[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.

[The dynamic analysis and the clinical significance of vascular endothelial cell markers and hemolysis parameters in thrombotic thrombocytopenic purpura].

OBJECTIVE: To monitor the changes of hemolysis parameters and endothelial cell markers in thrombotic thrombocytopenic purpura (TTP) and reveal the clinical significance of these changes. METHODS: vWF-cleaving protease (vWF-CP) activity in 3 cases of TTP was detected by Western blot. The percentages of fragmented red cells (FRC) were counted throughout the entire clinical course. Levels of plasma thrombomodulin were detected by Western blot combined with density screening in TTP and healthy individuals (n = 3). Concentration of plasma VEGF was measured by enzyme-linked immunosorbent assay in TTP and healthy individuals (n = 9). Fundus fluorescein angiography was performed to search the evidence of microvascular thrombosis in one TTP patient with impaired visual acuity. RESULTS: The lower vWF-CP activity was observed in TTP patients; the percentages of FRC in 3 cases of TTP were 1.65%, 2.50%, 3.32% respectively with an average of 2.49% at the onset of and decreased with the improvement of the disease. The levels of plasma TM and VEGF were significantly elevated in TTP than those in healthy individuals, and related to the severity of TTP. Fundus photography in one TTP patient with impaired visual acuity revealed vascular occlusion in fundus arteriole and venulae. CONCLUSIONS: A decreased vWF-CP activity is in favour of TTP diagnosis. Dynamic monitoring of plasma TM and VEGF as well as percentages of FRC are useful indexes for reflecting the severity and evaluating therapeutic response of TTP. Selective fundus fluorescein angiography is useful for the judgement of microvascular thrombosis in TTP.

[The activation of JAK/STAT signal pathway in hypereosinophilic syndrome and the patients therapeutic response to imatinib].

OBJECTIVE: To determine whether JAK/STAT pathway is involved in proliferation of hypereosinophilic syndrome (HES) cells, and reveal the pathogenesis of HES; observe the dynamic change of the clinical phenotype and hematological response, the expression of janaus kinase/signal transducer and activator of transcription (JAK/STAT) protein or FIP1L1-PDGFRA mRNA in one HES patient treated with low-dose imatinib. METHODS: The granulocytes of peripheral blood of 4 HES patients, including 3 FIP1L1-PDGFRA positive cases and 1 negative case, were collected. The expression of JAK2, STAT3, and phosphorylated STAT (P-STAT5) proteins were detected by western blotting. One FIP1L1-PDGFRA fusion gene positive patient was administered with low-dose imatinib. Retrospective reverse transcription polymerase chain reaction (RT-PCR) analysis of FIP1L1-PDGFRA was performed and the expressions of JAK2, STAT3 and P-STAT5 were detected by western blotting before treatment and 10, 30, and 60 days after the beginning of treatment. RESULTS: Upregulation of JAK2, STAT3, and P-STAT5 proteins was shown in 3 FIP1L1-PDGFRA fusion gene positive HES patients, while all of these proteins were not expressed in one case of FIP1L1-PDGFRA negative HES. Continuous hematological remission was observed in one FIP1L1-PDGFRA fusion gene positive HES patient after low-dose imatinib treatment. The amount of FIP1L1-PDGFRA transcripts in peripheral blood granulocytes was significantly decreased in 30 days after therapy and turned negative 60 days after therapy. JAK2, STAT3, STAT5, and P-STAT5 expressions were all down-regulated time-dependently and were all negative 60 days after. CONCLUSION: There is excessive activation of JAK/STAT signal pathway in HES patient, which may contribute to the malignant proliferation of eosinophils. Low-dose imatinib, that induces complete hematological and molecular genetic remission, exerts significant effects on FIP1L1-PDGFRA positive HES.

[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.

[Identification of FIP1L1-PDGFRA fusion, and expression of signal transducer and activator of transcription 5 in hypereosinophilic syndrome].

OBJECTIVE: To determine whether FIP1L1-PDGFRA fusion exists in hypereosinophilic syndrome (HES) patients, explore the relationship between FIP1L1-PDGFRA fusion and clinical phenotypes, and observe and reveal the expression of signal transducer and activator of transcription 5 (STAT(5)) in granulocytes of HES and the biological significance thereof. METHODS: Specimens of peripheral blood were collected from 4 HES patients diagnosed based on the criteria of Chusid et al. Total RNA was extracted from granulocytes and cDNA was synthesized by reverse transcription. Nested-PCR was used to amplify the target fusion gene and the positive PCR fragments were sequenced directly. Total protein of the peripheral granulocytes was extracted. Western blotting was used to detect the expression of STAT(5) protein in the granulocyte lysates. RESULTS: FIP1L1-PDGFRA fusion genes were found in 3 of the 4 HES patients. The break points in PDGFRA were all located at exon 12, while in FIP1L1 the break points were highly variable, located at exon 8a, intron 8a, and exon 8 respectively. The patients with FIP1L1-PDGFRA fusion were susceptible to cardiac involvement. The expression of STAT(5) protein was upregulated in FIP1L1-PDGFRA positive HES patients, while STAT(5) protein expression was negative in HES patients without FIP1L1-PDGFRA fusion. CONCLUSION: FIP1L1-PDGFRA fusion has a universal significance for HES. The identification of FIP1L1-PDGFRA rearrangement is a useful molecular mark for HES diagnosis and works as the therapeutic target of imatinib. Furthermore, the activation of STAT(5), a downstream signal of the FIP1L1-PDGFRA fusion, indicates that HES is a malignant clonal disease of the hematopoietic tissue.

[Clinical analysis of 13 patients with hemophagocytic syndrome].

OBJECTIVE: To investigate the clinical features and prognosis of hemophagocytic syndrome (HS). METHODS: The clinical symptoms, signs, and laboratory-test data in 13 patients with HS were analysed. RESULTS: Increase in lactate dehydrogenase (LDH) and hyponatremia was found in all of the patients. Prolonged prothrombin time, hypofibrinogenemia, hyertriglyceridemia, and hyperferricemia also existed in some cases. The mature hemophagocytic histocyte and hemophagocytic phenomenon were observed with Whrigt-Geimsa and immunocytochemical staining. One (16.6%, 1/6) patient with infectious associated HS (IAHS) and 4 (80%, 4/5) with non-IAHS died of infection and primary disease. CONCLUSION: HS especially non-IAHS is an extremely dangerous state with high mortality. Obstinate hyponatremia may be a characteristic of HS in the early stage. It's important to supervise the change of hemophagocyte in peripheral blood and bone marrow of HS. Immunocytochemical studies on smear of enriched peripheral white blood cells are helpful to identify the primary pathogenesis of the benign or malignant diseases.