Clinical Value of Translocator Protein Gene in Evaluating the Efficacy of FLT3-ITD/DNMT3A R882 Double-Mutated Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To observe the clinical significance of translocator proteins (TSPO) gene in the treatment of FLT3-ITD/DNMT3A R882 double-mutated acute myeloid leukemia (AML).@*METHODS@#Seventy-six patients with AML hospitalized in the Department of Hematology of the Affiliated People's Hospital of Ningbo University from June 2018 to June 2020 were selected, including 34 patients with FLT3-ITD mutation, 27 patients with DNMT3A R882 mutation, 15 patients with FLT3-ITD/DNMT3A R882 double mutation, as well as 19 patients with immune thrombocytopenia (ITP) hospitalized during the same period as control group. RNA was routinely extracted from 3 ml bone marrow retained during bone puncture, and TSPO gene expression was detected by transcriptome sequencing (using 2-deltadeltaCt calculation).@*RESULTS@#The expression of TSPO gene in FLT3-ITD group and DNMT3A R882 group at first diagnosis was 2.02±1.04 and 1.85±0.76, respectively, which were both higher than 1.00±0.06 in control group, but the differences were not statistically significant (P=0.671, P=0.821). The expression of TSPO gene in the FLT3-ITD/DNMT3A R882 group was 3.98±1.07, wich was significantly higher than that in the FLT3-ITD group and DNMT3A R882 group, the differences were statistically significant (P=0.032, P=0.021). The expression of TSPO gene in patients who achieved complete response after chemotherapy in the FLT3-ITD/DNMT3A R882 group was 1.19±0.87, which was significantly lower than that at first diagnosis, and the difference was statistically significant (P=0.011).@*CONCLUSION@#TSPO gene may be used as an indicator of efficacy in FLT3-ITD /DNMT3A R882 double-mutated AML.

Expression of BCR/ABL fusion gene in circulating endothelial cells from chronic myelogenous leukemia patients and its clinical significance / 中国实验血液学杂志

Several studies have shown that the tumor endothelial cells are different from the normal tissue endothelial cells. These tumor endothelial cells may contribute to tumor neo-vasculogenesis. This study was purposed to analyze the biologic features and determine the expression level of CD133 and BCR/ABL fusion gene in circulating endothelial cells (CEC) isolated from peripheral blood of CML patients, as well as to investigate the role of CEC in disease progression. Mononuclear cells were isolated from peripheral blood by density gradient centrifugation; CEC were sorted by MACS and harvested in the endothelial growth medium. The morphologic features of CEC were observed by microscopy, the cell growth rate was calculated by cell counting, and the cells were identified by immunofluorescence staining for the expression of CD31,CD34,VWF and CD133. The expression of BCR/ABL fusion gene was examined by FISH in 12 CML patients. The results indicated that the isolated CEC displayed the typical cobble-stone morphology. These cells could be identified by the positive immunofluorescence staining for CD31, CD34 and VWF, and showed more increased proliferative potential as compared to that of healthy donors. It was found that the positive rate of CD133 was 31.29% in CML patients, which was significantly different from that of healthy donors (P < 0.05). In 12 CML patients, CEC carried the same chromosome aberration as the leukemia cells (10.77%). Higher expression level of CD133 and BCR/ABL fusion gene positively correlated with progression of disease. It is concluded that the CEC may participate in invasion and angiogenesis in patients with CML and possibly correlate to the spreading and progression of the disease.

Effects of H3K27 methylation inhibitor EPZ005687 on apoptosis, proliferation and cell cycle of U937 cells and normal CD34 positive cells / 中国实验血液学杂志

The aim of this study was to investigate the effects of H3K27 methylation inhibitor EPZ005687 on the apoptosis, proliferation and cell cycle of U937 cells and normal CD34⁺ cells. The U937 cells and normal CD34⁺ cells were treated with different concentration of EPZ005687 at different time points. The apoptosis rate was determined by Annexin V/PI staining. The cell proliferation and cell cycle was determined using WST-1 assay and 7-AAD assay, respectively. The activity of H3K27 methylation was detected by chemiluminescent immunoassay. The results showed that the EPZ005687 induced an obvious apoptosis of U937 cells. The apoptotic rate was 3.96% ± 0.79%,5.74% ± 0.73%,13.34% ± 1.77% and 25.24% ± 2.55% in U937 cells treated with 0.5, 1, 5 and 10 µmol/L EPZ005687 for 48 hours, respectively. However, EPZ005687 had rare effect on normal bone marrow(NBM) CD34⁺ cells. The apoptotic rate was 3.64% ± 0.62%,4.28% ± 0.99%,6.18% ± 1.19% and 7.56% ± 1.34% after U937 cells were treated with 0.5, 1, 5 and 10 µmol/L EPZ005687 for 48 hours, respectively. EPZ005687 inhibited obviously the proliferation of U937 cells but had weak effect on the proliferation of NBMCD34⁺ cells. The inhibitory effect of EPZ005687 on U937 cells was time-dependent after treated with 0.5, 1, 5 and 10 µmol/L EPZ005687 from 12 to 96 hours. EPZ005687 induced G1 phase blocking (G1%, 64.18% ± 13.27% vs 49.43% ± 12.54%) and decreased the percentage of cells in S phase (9.67% ± 2.61% vs15.26% ± 5.58%) in U937 cells. However, EPZ005687 had no effect on the cell cycle of NBMCD34⁺ cells. In addition, EPZ005687 produced obviously depletion of H3K27 methylation in U937 cells (P < 0.05), but hardly had effect on the H3K27 methylation of NBMCD34⁺ cells. It is concluded that the EPZ005687 inhibites proliferation, induces apoptosis and cell cycle blocking in G1 phase in leukemia cells. This agent may have potential value in clinical application.

Clinical investigation of homoharringtonine in combination with all-transretinoic acid and arsenic trioxide for acute promyelocytic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the clinical outcome, adverse effect and treatment cost of homoharringtonine (HHT) in combination with all-trans retinoic acid (ATRA) and arsenic trioxide (AS2O3) for newly diagnosed with patients acute promyelocytic leukemia (APL).</p><p><b>METHODS</b>Clinical data of treatment of newly diagnosed patients with APL in experimental group (HHT + ATRA + AS2O3, n = 14) and control group \[Idarubicin (IDA) + ATRA + AS2O3, n = 21\] were analyzed retrospectively. The therapeutic effects, side effects and costs during induction therapy were compared between the two groups.</p><p><b>RESULTS</b>(1) The complete remission (CR) rate were 92.9% (13/14) and 95.2% (20/21) in experimental group and control group, respectively. The time to achieve CR were (28.1 ± 3.8) and (31.7 ± 4.2) days, respectively (P > 0.05). The negative rate of PML-RARα fusion gene at the time of CR were 76.9% (10/13) and 75.0% (15/20), respectively, and that in CR patient at the end of the first cycle treatment were 100.0% (13/13) and 95.0% (19/20), respectively (P > 0.05). (2) 5-year overall survival (OS) rate were (92.6 ± 0.6)% and (89.9 ± 0.5)%, respectively (P > 0.05), 5-year disease free survival (DFS) rate were 100.0% and (86.8 ± 0.6)%, respectively (P > 0.05). (3) During induction therapy, the incidence of infection in experimental and control group were 23.1% (3/13), 60.0% (12/20), respectively (P < 0.05). The amount of platelet transfusion were (54.7 ± 29.6) and (76.5 ± 25.6) units, respectively (P > 0.05), and that of fresh frozen plasma were (1157.1 ± 238.4) and (1423.5 ± 324.6) ml, respectively (P > 0.05). The total medical costs (excluding HHT and IDA) in experimental and control group were (36074.9 ± 1245.6) and (50564.5 ± 3658.4)CNY, respectively (P < 0.05).</p><p><b>CONCLUSION</b>HHT in combination with ATRA and AS2O3 regimen for newly diagnosed APL has a better efficacy, a higher long-term survival rate, and a lower costs, which is one of the reasonable choice.</p>

Role of microenvironment in the process of expansion of late endothelial progenitor cell in vitro / 中国医学科学院学报

<p><b>OBJECTIVE</b>To study the role of the feeder layer cells as niche in the process of expansion of late endothelial progenitor cell in vitro.</p><p><b>METHODS</b>We cultured mononuclear cells (MNC)from human peripheral blood (PB)on the plate with the feeder layer cells which were irradiated late endothelial progenitor cells(EPC)or human umbilical vein endothelial cells (HUVEC) by EGM-2. After 21 days, the numbers of obtained late EPC colonies were counted separately, and their surface antigen of the late EPC was verified by fluorescence-activated cell sorter (FACS) analysis, and their ability of forming vessel structure with Matrigel in vitro. The differentiation of single stem cell on the feeder layer cell was traced by video-microscopy.</p><p><b>RESULTS</b>After 21 days of culture,(40.0±3.9)and(39.3±3.1)late EPC colonies that MNC of a hundred milliliter PB were cultured, respectively, on the feeder layer cells of EPC and HUVEC were much more than (2.0±1.3) colonies cultured on without the feeder layer cells (all P <0.05). These cells also expressed CD31,CD34,eNOS,FLt-1,P1H12,Sendo,VE cadherin,and CD117, as shown by FACS analysis. Furthermore, they formed vessel structure with Matrigel in vitro. The video-microscopy showed the asymmetric cell division was participated by the feeder layer cell during the expansion of single stem cell.</p><p><b>CONCLUSION</b>The massive expansion of late EPC can be achieved by the provision of the feeder layer cells, which may be involved in the stem cell asymmetric cell division.</p>

Preliminary exploration of Gd-DTPA enhanced inner ear image of guinea pig using magnetic resonance scan / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To observe the distribution of Gd-DTPA in the inner ear of guinea pig by MRI at different time points after intratympanic administration, explore the optical time for observing the whole inner ear. To study the pharmacokinetic feature of Gd-DTPA in the inner ear, and find out whether discrimination of endolymph and perilymph can be obtained under current conditions.</p><p><b>METHODS</b>Sixty-five guinea pigs were randomly divided into 13 groups, after diluted Gd-DTPA intratympanic injection, each group of guinea pigs were scanned through MRI (3D-T1 FSE sequence) at different time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 48 h, 72 h, 96 h). Pixel intensity values of some locations in the inner ear were analyzed using e-Film software, then pixel intensity was converted into concentration using the results of previous in-vitro study. ABR thresholds of bilateral ears before, 1 d and 7 d after intratympanic injection were compared.</p><p><b>RESULTS</b>Six hours after transtympanic Gd-DTPA injection was the time point when contrast agent was distributed all over the inner ear, and reached a high concentration: 589.29, 552.54, 570.17, 255.08, 107.09 and 139.18 µmol/L in the vestibule, scala vestibuli and scala tympani of basal turn, the 2(nd), the 3(rd) and the apical turn individually, that was also the optimal time for observing the whole inner ear by MRI. Perilymph appeared to be preferentially enhanced relative to the endolymph, resulting in a distinction between the scales of the inner ear. There was no significant difference between the experimental ear (diluted GD-DTPA injected ear) and contrast ear (physiological saline injected ear) on 1 d and 7 d.</p><p><b>CONCLUSIONS</b>The best time getting MRI imaging after intratympanic diluted GD-DTPA injection is 6 h. After diluted agent injection perilymph can be enhanced so as to be differentiated with endolymph by MRI, diluted agent have no obvious effect on the ABR threshold. The pharmacokinetic feature of Gd-DTPA in the inner can be studied using MRI.</p>

Relationship between expression of chemokine receptor and curative effect of multiple myeloma / 中国实验血液学杂志

This study was purposed to explore the correlation of CXCR4, CCR1, CCR2 expression with curative effect of multiple myeloma (MM). Flow cytometry was used to detect the expressions of CXCR4, CCR1, CCR2 on cell surface of bone marrow from 48 newly diagnosed MM patients. These patients were divided into two groups: one group with expression of chemokine receptor (group I) and another group without expression of chemokine receptor (group II). The group I was consisted of 34 patients, but 3 out of them could not be continuously followed up. The group II was consisted of 14 patients. The MM patients of 2 groups were treated with chemotherapeutic drugs for 3 and 6 months, the curative efficacy of 2 groups were compared. The results showed that after treating for 3 and 6 months the effective rates of group I and group II were 80.6% (25/31) vs 50% (7/14) and 83.9% (26/31) vs 50% (7/14) respectively, which suggested that curative efficacy of group I was better than that of group II (p < 0.05). It is concluded that CXCR4, CCR1, CCR2 may be used as indexes for evaluating curative effect of MM patients.

Correlation of FLT3 gene expression level and internal tandem duplication mutation in acute myeloid leukemia and its clinical significance / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the prevalence of FLT3 gene expression and internal tandem duplication (ITD) mutation in acute myeloid leukemia (AML) patients and its clinical significance.</p><p><b>METHODS</b>The expression level of FLT3 mRNA was detected with real-time quantitative RT-PCR (RQ-PCR) technique in 152 bone marrow samples from 129 AML patients. The ITD of the FLT3 gene (FLT3/ITD) was detected in 75 de novo AML patients by reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>The expression levels of FLT3 mRNA was 0.0020(0.0006 - 0.0040) in normal controls, and 0.1041 (0 - 33.8736) in 80 de novo AML patients (P = 0.001). FLT3/ITDs were found in 11 (14.7%) of 75 de novo AML patients. The FLT3 expression levels in patients with FLT3/ITD were 0.0297 to 33.8736 with a median level of 0.2200, and in those without FLT3/ITD were 0 to 26.2200 with a median level of 0.0975. The FLT3 expression levels in the former patients were higher than that in the latter ones, but there was no statistical significance (P = 0.093). The complete remission (CR) rate was lower in acute promyelocytic leukemia (APL) patients with FLT3/ITD than in APL patients without FLT3/ITD (P = 0.015). In de novo AML other than APL patients without FLT3/ITD, the CR rate was lower in patients with higher levels of FLT3 expression (expression level > 0.04, CR rate 37.5%) than those with lower levels of FLT3 expression (expression level < 0.04, CR rate 100%). On follow-up of 20 patients, the FLT3 expression level was decreased at least one logarithm degree when they got CR, but it was no significant change in non-remission patients.</p><p><b>CONCLUSIONS</b>Quantification of FLT3 mRNA expression level and detection of FLT3/ITD in AML patients may serve as an index for evaluating therapeutic efficacy, predicting prognosis, and monitoring minimal residual disease.</p>