[Quantitative Analysis on Immunophenotype of CD34+ Myeloid Precursor Cells in Myelodysplastic Syndrome and Its Correlation with Clinical Features].

OBJECTIVE: To investigate the quantitative expression of immunophenotype of CD34+ myeloid precursor cells in myelodysplastic syndrome (MDS) patients and its correlation with clinical characteristics, and understand the effect of quantitative expression of CD7 and CD117 on the prognosis of low-risk MDS patients. METHODS: Multi-parameter flow cytometry (FCM) was used to detect the proportion and mean fluorescence intensity (MFI) of each antigen of bone marrow CD34+ myeloid precursor cells in 79 MDS patients. The correlation between the expression level of each immune marker and clinical characteristics was compared. The effects of quantitative expressions of CD7 and CD117 on the overall survival rate of low-risk patients were explored. RESULTS: Bone marrow blast cell proportion (P<0.01), RBC level (P<0.01), and Hb level (P<0.05) of high-risk MDS patients were higher, while EPO level (P<0.05) was lower than those of low-risk patients. The proportion of CD34+ blast cells (P<0.01), the proportion of CD117 (P<0.05) and the MFI of CD7 (P<0.05) were higher in high-risk patients than those in low-risk patients, but the MFI of CD123 was lower (P<0.05). In high-risk MDS patients, CD15/CD34 (MFI) and CD19/CD34 (MFI) positively correlated with the proportion of total T cells (r=0.458; r=0.505), while CD19/CD34 (%) and CD19/CD34 (MFI) negatively correlated with WBC levels (r=-0.469; r=-0.503). In low-risk MDS patients, CD34+ (%) positively correlated with bone marrow erythrocyte proportion, PLT level and neutrophil level (r=0.426; r=0.486; r=0.495), but negatively correlated with LDH level (r=-0.421); WT1 expression level was positively correlated with CD10/CD34 (%), CD10/CD34 (MFI) and CD117/CD34 (MFI) (r=0.745; r=0.800; r=0.434), while negatively correlated with CD11b/CD34 (%)(r=-0.457); CD19/CD34 (%) and CD71/CD34 (MFI) negatively correlated with NK cell proportion (r=-0.786; r=-0.514); CD10/CD34 (%) positively correlated with Th/Ts, while CD7/CD34 (MFI) negatively correlated with the proportion of Th cells (r=0.738; r=-0.513); HLADR/CD34 (%) and HLADR/CD34 (MFI) negatively correlated with PLT level (r=-0.461; r=-0.445), while HLADR/CD34 (MFI) positively correlated with bone marrow NAP fraction (r=0.552). The quantitative expression of CD7 and CD117 had no significant effect on the overall survival rate of low-risk MDS patients. CONCLUSION: The immunophenotype of CD34+ myeloid precursor cell in different risk groups in MDS patients is related to clinical characteristics. Bone marrow cell morphology, clinical and laboratory features and immunophenotype will be of great significance to the diagnosis, clinical classification and prognosis evaluation of MDS patients.

Predictive Value of Small Dense Low-Density Lipoprotein in Coronary Heart Disease in the Chinese Population.

BACKGROUND: Coronary heart disease (CHD) is a global public health concern, and CHD risk assessment remains a major challenge. Therefore, in this study, the ability of small dense low-density lipoprotein (sdLDL) alone to sufficiently predict CHD risk in the Chinese population was evaluated. METHODS: Patients with CHD (139) and healthy controls (58) were included in this study. Serum sdLDL was measured using the peroxidase method. Other lipid parameters were also determined. RESULTS: The sdLDL level in the CHD group was significantly higher than that in the control group (p < 0.001). Logistic regression analysis revealed that sdLDL was an independent risk factor for CHD. Based on the receiver operating characteristic curves, the area under the curve (AUC) of sdLDL alone for CHD was 0.722. The AUC of triglycerides (TG), high-density lipoprotein (HDL), and sdLDL combined was 0.763, which was larger than that of the independent ones or combinations of any two; however, the value was not significant. CONCLUSIONS: sdLDL alone can predict CHD risk efficiently similar to the combination of TG, HDL, and sdLDL. This finding suggests that sdLDL can be considered as an ideal parameter for the preliminary diagnosis of CHD in Chinese people.

[Effect of Signal Transduction Pathway Gene Mutations on the One- course Induced Remission Rate and Analysis of Clinical Characteristics in Patients with CBF-AML].

OBJECTIVE: To investigate the effect of other gene mutations outside the fusion gene on the first complete remission (CR1) induced by one course of induction chemotherapy in patients with core binding factor-associated acute myeloid leukemia (CBF-AML). METHODS: DNA was extracted from bone marrow or peripheral blood samples of newly diagnosed CBF-AML patients admitted to the Hematology Department of the Second Hospital of Shanxi Medical University from January 2015 to January 2019. Next-generation sequencing was used for detection of 34 kinds of hematologic malignancy-related gene mutations in patients with CBF-AML, the effect of related gene mutations on the first complete remission (CR1) rate in one course of induction chemotherapy was analyzed by combineation with clinical characteristics. RESULTS: 34 kinds of genes in bone marrow or peripheral blood of 43 patients were detected by high throughput sequencing and the gene mutations were detected in 16 out of 34 genes. The mutation rate of KIT gene was the highest (48.8%), followed by NRAS (16.3%), ASXL1 (16.3%), TET2 (11.6%), CSF3R (9.3%), FLT3 (9.3%), KRAS (7.0%). The detection rates of mutations in different functional genes were as follows: genes related with signal transduction pathway (KIT, FLT3, CSF3R, KRAS, NRAS, JAK2, CALR, SH2B3, CBL) had the highest mutation frequency (72.1% (31/43); epigenetic modification gene mutation frequency was 30.2% (13/43), including ASXL1, TET2, BCOR); transcriptional regulation gene mutation frequency was 7.0% (3/43), including ETV6, RUNX1, GATA2). Splicing factor related gene mutation frequency was 2.3% (1/43), including ZRSR2). The CR1 rate was 74.4% after one course of induction chemotherapy. At first diagnosis, patients with low expression of WT1 (the median value of WT1 was 788.9) were more likely to get CR1 (P=0.032) and the RFS of patients who got CR1 after one course of induction chemotherapy was significantly longer than that of patients without CR1 [7.6 (2.2-44.1) versus 5.8 (1-19.4), (P=0.048)]. The rate of CR1 in the signal transduction pathway gene mutation group was significantly lower than that in non-mutation group (64.5% vs 100%) (P=0.045), while the level of serum hydroxybutyrate dehydrogenase (HBDH) was significantly higher than that in non-mutation group [(418 (154-2702) vs 246 (110-1068)] (P=0.032). There was no difference in CD56 expression between the two groups (P=0.053), which was limited to the difference between (≥20%) expression and non-expression. (P=0.048). CONCLUSION: CBF-AML patients with signal transduction pathway gene mutation are often accompanied by high HBDH level and CD56 expression, moreover, the remission rate induced by one course of treatment is low.

Droplet digital PCR for BCR/ABL(P210) detection of chronic myeloid leukemia: A high sensitive method of the minimal residual disease and disease progression.

OBJECTIVE: This study intended to establish a droplet digital PCR (dd-PCR) for monitoring minimal residual disease (MRD) in patients with BCR/ABL (P210)-positive chronic myeloid leukemia (CML), thereby achieving deep-level monitoring of tumor load and determining the efficacy for guided clinically individualized treatment. METHODS: Using dd-PCR and RT-qPCR, two cell suspensions were obtained from K562 cells and normal peripheral blood mononuclear cells by gradient dilution and were measured at the cellular level. At peripheral blood (PB) level, 61 cases with CML-chronic phase (CML-CP) were obtained after tyrosine kinase inhibitor (TKI) treatment and regular follow-ups. By RT-qPCR, BCR/ABL (P210) fusion gene was undetectable in PB after three successive analyses, which were performed once every 3 months. At the same time, dd-PCR was performed simultaneously with the last equal amount of cDNA. Ten CML patients with MR4.5 were followed up by the two methods. RESULTS: At the cellular level, consistency of results of dd-PCR and RT-qPCR reached R2  ≥ 0.99, with conversion equation of Y = 33.148X1.222 (Y: dd-PCR results; X: RT-qPCR results). In the dd-PCR test, 11 of the 61 patients with CML (18.03%) tested positive and showed statistically significant difference (P < .01). In the follow-up of 10 CML patients who were in MR4.5. All patients were loss of MR4.0, and 4 were tested positive by dd-PCR 3 months earlier than by RT-qPCR. CONCLUSION: In contrast with RT-qPCR, dd-PCR is more sensitive, thus enabling accurate conversion of dd-PCR results into internationally standard RT-qPCR results by conversion equation, to achieve a deeper molecular biology-based stratification of BCR/ABL(P210) MRD. It has some reference value to monitor disease progression in clinic.

[Expression of CD25 in acute B cell lymphoblastic leukemia and its clinical significance].

This study was purposed to investigate the relation of CD25 with the acute B cell lymphoblastic leukemia (B-ALL) and its clinical significance. A totol of 88 newly diagnosed B-ALL patients were enrolled in this study. The immunophenotype of leukemic myeloblasts were detected by flow cytometry, including interleukin 2 receptor α chain (CD25), ß chain (CD122), γ chain (CD132), CD19, CD20, CD10, CD34, CDIgM, CD79a, CD22 and CDTDT. The expression of BCR/ABL fusion gene was detected by qualitative PCR. The expression of IL2RA (CD25 gene) was detected by real-time qualitative RT-PCR. The results showed that there was no significant statistical difference in WBC count, Hb level, PLT count, marrow blast rate, peripheral blast rate, hepato-lienal infiltration, lymph node infiltration, levels of CD10, CD20, CD22, CD34, CD79a, CDTDT, CDIgM expression between B-ALL patients with CD25(+) and B-ALL patients with CD25(-), while the CD19 expression level in B-ALL patients with CD25(+) was higher than that in B-ALL patients with CD25(-). Out of 88 B-ALL patients, 21 patients showed BCR/ABL(+)(21/88) and their CD25(+) expression level was 66.7% (14/21); 67 patients showed BCR/ABL(-) and their CD25(+) expression level was 4.5% (3/67), there was statistical difference between these two groups (P < 0.05), but the expression level of IL2RA mRNA was not statistical different between CD25(+) and CD25(-) groups (P > 0.05). Among 21 BCR/ABL(+) B-ALL patients the remission rate and relapsed rate were not statistical different between CD25(+) an CD25(-) groups.In BCR/ABL(+) B-ALL patients 8 patients relapsed, the relapsed rate was 38.1% (8/21). In BCR/ABL(-) B-ALL patients 9 patients relapsed, the relapse rate was 13.4% (9/67), there was statistical difference between BCR/ABL(+) and BCR/ABL(-) two groups (P < 0.05). In BCR/ABL(+) group the RFS (relapse free survival) was 21 months, in BCR/ABL(+) CD25(+) patients the RFS was 15 months, while in BCR/ABL(+) CD25(-) patients the RFS was 21 months, in BCR/ABL(-) CD25(-) patients, the RFS was 24 months. It is concluded that the CD25 expresses at high level in B-ALL patients with BCR/ABL(+), which may serve as a predictive marker for the presence of BCR/ABL fusion gene, and relates with relapse, CD25(+) may serve as a adjuvant indicator for poor prognosis.

[Effect of Nrf2 and TrxR on proliferation of chronic myeloid leukemia cell and its mechanism].

OBJECTIVE: To explore the effect of nuclear factor erythroid-2 related factor 2 (Nrf2) and thioredoxin reductase (TrxR) gene on proliferation of chronic myeloid leukemia (CML) line cells and its mechanism. METHODS: Four interfering sequences of Nrf2 and one negative control sequence were designed and synthesised based on the principle of target sequence of siRNA, then constructed lentivirus vectors, which were transfected into K562 cell lines. The transfection effect was observed by laser scanning confocal microscope (LSCM) and flow cytometer (FCM); The depressing effect of siRNA was analyzed by real-time PCR. The cell proliferation inhibiting rate was measured with CCK-8 assay, the apoptotic rate by Annexin V-PE/PI with FCM and the apoptotic morphology of cells by LSCM. RESULTS: The transfection efficiency of lentivirus was 65%. One cell line K562-C3 which significantly inhibited Nrf2 mRNA was obtained by real-time PCR, Nrf2 relative quantitation (RQ) expressions were 1.003±0.093 and 0.344±0.032 in the control group and K562-C3 respectively; TrxR expression also decreased with RQ as 1.090±0.549 and 0.395±0.029 respectively. The cellular proliferation inhibition rates of K562-C3 were (4.74±0.39)%, (6.13±1.78)% and (25.36±3.77)%, respectively at 24, 48 and 72 h. The apoptotic rate induced by K562-C3 (29.9%) at 72 hours was obviously higher than in the control group (7.9%). The Annexin V-PE positive K562-C3 cells presented the following apoptotic characteristics, such as karyopyknosis, nuclear fragmentation and apoptotic bodies observed by LSCM. CONCLUSION: Nrf2 specific siRNA could repress its expression at the cellular level and down-regulate the expression of its downstream antioxidant enzyme, such as TrxR, which lead to increased apoptotic rate and decreased cell proliferation.

[Establishment of diagnostic model to monitor minimal residual disease of acute promyelocytic leukemia by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry].

OBJECTIVE: To screen the potential protein biomarkers in minimal residual disease (MRD) of the acute promyelocytic leukemia (APL) by comparison of differentially expressed serum protein between APL patients at diagnosis and after complete remission (CR) and healthy controls, and to establish and verify a diagnostic model. METHODS: Serum proteins from 36 cases of primary APL, 29 cases of APL during complete remission and 32 healthy controls were purified by magnetic beads and then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The spectra were analyzed statistically using FlexAnalysis(TM) and ClinProt(TM) software. RESULTS: Two prediction model of primary APL/healthy control, primary APL/APL CR were developed. Thirty four statistically significant peptide peaks were obtained with the m/z value ranging from 1000 to 10 000 (P < 0.001) in primary APL/healthy control model. Seven statistically significant peptide peaks were obtained in primary APL/APL CR model (P < 0.001). Comparison of the protein profiles between the two models, three peptides with m/z 4642, 7764 and 9289 were considered as the protein biomarker of APL MRD. A diagnostic pattern for APL CR using m/z 4642 and 9289 was established. Blind validation yielded correct classification of 6 out of 8 cases. CONCLUSIONS: The MALDI-TOF MS analysis of APL patients serum protein can be used as a promising dynamic method for MRD detection and the two peptides with m/z 4642 and 9289 may be better biomarkers.

Identification of a novel circularized transcript of the AML1 gene.

The AML1 gene is an essential transcription factor regulating the differentiation of hematopoietic stem cells into mature blood cells. Though at least 12 different alternatively spliced AML1 mRNAs are generated, three splice variants (AML1a, AML1b and AML1c) have been characterized. Here, using the reverse transcription-polymerase chain reaction with outward-facing primers, we identified a novel non-polyadenylated transcript from the AML1 gene, with exons 5 and 6 scrambled. The novel transcript resisted RNase R digestion, indicating it is a circular RNA structure that may originate from products of mRNA alternative splicing. The expression of the novel transcript in different cells or cell lines of human and a number of other species matched those of the canonical transcripts. The discovery provides additional evidence that circular RNA could stably exist in vivo in human, and may also help to understand the mechanism of the regulation of the AML1 gene transcription.

[Expressions of Th17 cells and interleukin 17 in patients with primary immune thrombocytopenia and their clinical significance].

The aim of this study was to detect the rate of T-helper (Th)17 cells and interleukin (IL)-17 level in peripheral blood of patients with primary immune thrombocytopenia (ITP) and to explore their clinical significance. The proportion of Th17 cells from 48 patients with ITP and 28 healthy controls was detected by flow cytometry, and the IL-17 level was evaluated by enzyme-linked immunosorbent assay (ELISA). The results showed that the percentage of Th17 cells in ITP group was (1.40 ± 1.35)%, which was significantly higher than that in healthy control group (P < 0.05), but in the glucocorticoid hormone-treated group it was significantly lower than that in treated group without glucocorticoid hormone(P < 0.05). The level of IL-17 expressed by Th17 cells in ITP patients was (19.624 ± 5.187) pg/ml, which was higher than that in the healthy control group (P < 0.05), it was lower in the glucocorticoid hormone treated group than that in treated group without glucocorticoid hormone, but there was no statistically significant difference between the glucocorticoid treated and treated group without glucocorticoid hormone (P > 0.05). It is concluded that the Th17 cells may involve in the pathogenesis of ITP, and the glucocorticoid hormone probably plays a therapeutic role through inhibiting Th17 cells.