[Effect of BAFF/APRIL mRNA expression induced by glucocorticoid and bortezomib in multiple myeloma cells in vitro].

The study was purposed to detect BAFF/APRIL gene expression changes in bone marrow mononuclear cells (BMMNC) and myeloma cell line U266 after interference with glucocorticoid and bortezomib. After separation of BMMNC from 7 patients with multiple myeloma, BAFF/APRIL mRNA expression in BMMNC and U266 cell line was detected by real-time PCR after treated with dexamethasone 100, 200 µg/ml, methylprednisolone 100, 200 µg/ml, bortezomib 0.1 µg/ml alone and dexamethasone or methylprednisolone combined with bortezomib respectively for 48 hours. The results showed that U266 cells and BMMNC of untreated MM patients highly expressed BAFF/APRIL genes. When dexamethasone, methylprednisolone or bortezomib was added to U266 cells or BMMNC alone, BAFF/APRIL gene expression decreased as compared with the blank control (p < 0.01). The inhibiting effect of bortezomib to BAFF/APRIL expression was obviously strong(p < 0.05). When dexamethasone or methylprednisolone combined with bortezomib, the BAFF/APRIL gene expression further decreased compared with dexamethasone or methylprednisolone alone (p < 0.01). As compared with the group of methylprednisolone combined with bortezomib, BAFF/APRIL gene expression decreased in dexamethasone combined with bortezomib with a statistically significant difference (p < 0.05). It is concluded that the expression of BAFF/APRIL gene is down-regulated after bing treated with glucocorticoids and bortezomib, which suggests that besides the glucocorticoid receptor and proteasomes targets, BAFF/APRIL and their receptor sites may be new targets of glucocorticoids and bortezomib.

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.

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.

[Specific cell immune response mediated by dendritic cells in multiple myeloma patients in vitro].

OBJECTIVE: To explore KM3 multiple myeloma (MM) cell line specific cytotoxic T lymphocyte (CTL) response in vitro mediated by autologous dendritic cells (DCs) aroused by KM3 cells' lysates and acid-eluted peptides. METHODS: Monocytes were isolated from the peripheral blood of healthy individuals and MM patients, and were cultured in serum medium with IL-4, GM-CSF, and TNF-alpha to generate DCs. These DCs were pulsed by KM3 cells' lysates or the acid-eluted peptides, then incubated with autologous T lymphocytes for 3 days to induce KM3 cell antigen specific CTL. MTT assay was performed to examine the specific KM3 cells' lysing ability of CTL. RESULTS: DCs were generated in peripheral blood monocytes cultured in the serum medium containing GM-CSF, IL-4, and TNF-alpha. Autologous T lymphocytes induced by IL-2 and DCs pulsed with KM3 cells' lysates or the acid-eluted peptides showed strong killing effect on KM3 cells. CONCLUSION: The DCs pulsed by KM3 cells' lysates or the acid-eluted peptides incubated with autologous T lymphocytes can induce KM3 cell antigen specific CTL.

[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.

[Clinical significance of serum vascular endothelial growth factor and interleukin-6 in multiple myeloma].

OBJECTIVE: To investigate the clinical significance of the changes of vascular endothelial growth factor ( VEGF ) and interleukin-6 ( IL-6 ) level in multiple myeloma (MM), solid tumor following bone metastasis. METHODS: Thirty- seven MM patients, including 7 in Stage I , 8 in Stage II , 22 in Stage III, 8 solid tumor with bone metastasis patientsly, and 17 healthy controls were enrolled in this study. The serum VEGF and IL-6 levels were determined by ELISA. RESULTS: Serum VEGF and IL-6 concentrations in patients with MM and solid tumor were significantly higher than those of the healthy controls (P <0.01 ), and the VEGF level was higher in MM than in solid tumor with bone metastasis. There was significant difference in VEGF and IL-6 levels in various clinical stages of MM. VEGF levels in Stage II were significantly higher than in Stage I (P < 0.05 ) and IL-6 levels in Stage II were significantly higher than in Stage I (P < 0.05). The levels of IL-6 showed great difference according to bone lesion scores (P < 0.05). There was a positive correlation between IL-6 and serum calcium or C-reactive protein( P <0.01) , and there was a positive correlation between VEGF and serum Cr or urinary Bene-Jones protein lambda (P < 0.01 ). The IL-6 levels had significant differences between patients with the normal serum CRP, serum calcium, and beta2-MG and patients with abnormal ones (P < 0.05). VEGF levels showed significant differences between the patients with normal serum Cr, serum calcium Bene-Jones protein lambda, and urinary Bene-Jones protein lambda and patients with abnormal ones (P < 0.05). CONCLUSION: Serum VEGF and IL-6 levels are helpful to diagnose the clinical stages, and understand bone lesion and serevity of MM.

[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.