Impact of CSF3R Mutation on Treatment Response and Survival of Patients with Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression of CSF3R mutation in acute myeloid leukemia (AML) and analyze its clinical characteristics and prognosis.@*METHODS@#A retrospective study was conducted in 212 patients with AML who were newly diagnosed in the Second Hospital of Shanxi Medical University from January 1th 2018 to June 30th 2021, including 22 patients with CSF3R mutations as mutation group and 190 patients with CSF3R wild type ［66 cases of them were screened by propensity score matching (PSM), as control group］. The early efficacy and survival between the two groups were compared.@*RESULTS@#The median age of patients in the mutation group was 50(17-73) years old, and the ratio of male to female was 1.2:1 The main types were AML with maturation (11 cases) and acute myelomonocytic leukemia (9 cases). Prognostic stratification was carried out according to the risk stratification system of the European leukemia network in 2017, with 16 cases (72.73%) in the middle and high-risk group. At the initial diagnosis, the median count of white blood cell (WBC) was 44.75(1.30-368.71)×109/L, among which 15 cases (68.18%) were >10×109/L, and the median count of platelet (PLT) was 24(4-55)×109/L. CSF3R T618I (68.18%) was a common mutation site, which had concomitant gene mutations, in which CEBPA mutation was the most common (10 cases, 45.45%), but only existed in CSF3R T618I mutation. The CR/CRi rate was 68.18% and 71.21% in the mutant group and the control group (P >0.05), the median over all survival time was 15 months and 9 months (P >0.05), and the median disease-free survival time was 8 months and 4 months (P >0.05), respectively.@*CONCLUSION@#Most AML patients with CSF3R mutation are middle-aged patients, the main types are AML with maturation and acute myelomonocytic leukemia, and most of them have middle and high-risk prognosis. CSF3R mutation may not be an independent prognostic marker for newly diagnosed AML patients.

Quantitative Analysis on Immunophenotype of CD34 / 中国实验血液学杂志

OBJECTIVE@#To investigate the quantitative expression of immunophenotype of CD34@*METHODS@#Multi-parameter flow cytometry (FCM) was used to detect the proportion and mean fluorescence intensity (MFI) of each antigen of bone marrow CD34@*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@*CONCLUSION@#The immunophenotype of CD34

Clinical Significance of Truncated Mutant ΔJBP of TET2 Gene in Patients with Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To the clinical characteristics and prognostic value of the patients with complete deletion of TET_JBP domain (ΔJBP) in TET2 acute myeloid leukemia (AML).@*METHODS@#Next Generation Sequencing technology was used to determine the mutations of 34 AML-related genes (including TET2 gene). The I-TASSER tool was used to predict the tertiary structure of the full-length TET2 protein and TET_JBP structure deletion.@*RESULTS@#Among 38 AML patients with TET2 mutations, 22(57.9%) showed truncation mutations, of which 16 (72.7%) produced TET2ΔJBP truncation mutants. Protein structure prediction showed that the deletion of TET_JBP domain lead to the significant changes of tertiary structure in TET2 protein. Compared with the patients in non-ΔJBP group, the age of patients in ΔJBP group were older (63 vs 54 years old, P=0.047), and the occurrence rate of CEBPA double mutation (CEBPA@*CONCLUSION@#AML patients with TET2ΔJBP truncation mutant shows lower CR rate, shorter EFS and OS after induction chemotherapy, which may be related to the poor prognosis, and co-mutation with CEBPA

Effect of Signal Transduction Pathway Gene Mutations on the One- course Induced Remission Rate and Analysis of Clinical Characteristics in Patients with CBF-AML / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of other gene mutations outside the fusion gene on the first complete remission (CR) 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 (CR) 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 CR 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 CR (P=0.032) and the RFS of patients who got CR after one course of induction chemotherapy was significantly longer than that of patients without CR [7.6 (2.2-44.1) versus 5.8 (1-19.4), (P=0.048)]. The rate of CR 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.

Establishment of diagnostic model to monitor minimal residual disease of acute promyelocytic leukemia by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry / 中华血液学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Effect of Nrf2 and TrxR on proliferation of chronic myeloid leukemia cell and its mechanism / 中华血液学杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Prenatal diagnosis of anhidrotic ectodermal dysplasia with unconventional loci abnormalities: a case report / 中华医学杂志(英文版)

Anhidrotic ectodermal dysplasia (EDA) is a relatively rare congenital hereditary disease. Because of a reduced number of sweat glands, patients are unable to perspire and consequently suffer from hyperthermia and infection. This is a potential cause of death in childhood. Domestic prenatal diagnosis methods focus on genetic diagnosis. But for some conditions, because of the uncertain molecular pathology, we need other methods to assist to in prenatal diagnosis. Here, we report one case of a new mutation locus which may be associated with EDA and the prenatal diagnosis of EDA by fetal skin biopsy under fetoscopy in mid pregnancy, combined with a review of the literature.

Expression of WT1 gene in CD34(+)CD38(-)CD123(+) AML stem cells and its significance analysis / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate whether WT1 gene overexpressed in leukemic stem cells (LSCs) and its significance.</p><p><b>METHODS</b>Expression of WT1(+17AA) and WT1(+KTS) gene isoforms in CD34(+)CD38(-)CD123(+) cells (LSCs) of 47 AML patients were determined by fluorescence quantitative RT-PCR. The ratio of the four splicing isoforms WT1(+/+), WT1(+/-), WT1(-/+) and WT1(-/-) in LSCs were calculated and compared with that in normal CD34(+)CD38(-)CD123(-) cells (HSCs). The relationship in AML patients between LSCs WT1 expression and remission rate, survival time and relapse rate was analyzed.</p><p><b>RESULTS</b>The expression of WT1 gene was highest (0.034 +/- 0.034) in LSCs, and higher in CD34(+)CD38(-)CD123(-) AML cells as compared with that in HSCs (P < 0.05). The proportion of +17AA isoform was predominant over -17AA in all the three cell subsets with no difference. The proportion of +KTS isoform was the highest in HSCs (0.57 +/- 0.04), while the lowest in CD34(+)CD38(-)CD123(-)AML cells (0.50 +/- 0.12) (P < 0.05). No significant difference in the four isoforms expression ratio was observed among the three groups. WT1 expression in LSCs was not correlated with sex, age, FAB subtype and blast cell ratio, while the ratio of CD34(+) cells in blast cell was significantly higher in the WT1 high expression group than in the low expression group (P < 0.01). The CR rate was significantly lower in WT1 high expression group (21.1%) than in the WT1 low expression group (59.1%) (P < 0.05). The follow-up data were available in 41 patients with a median follow-up duration of 118 (3 - 290) days. The median overall survival (OS) for WT1 high and low expression group were 77\[95% confident interval (CI) 45 - 108\], 158 (95%CI 100 - 215) days respectively (P = 0.041).</p><p><b>CONCLUSION</b>WT1 gene overexpressed in AML LSCs and the ratios of four WT1 isoforms have no difference in LSC compared with HSC. Patients with higher LSC WT1 expression have lower CR rate and shorter survival time.</p>

Significance of B cell activating factor of TNF family promoter polymorphisms in patients with immune thrombocytopenic purpura / 中国实验血液学杂志

The study was aimed to examine the B cell activating factor promoter polymorphism of the TNF family (BAFF)-871 C/T in patients with immune thrombocytopenic purpura (ITP) and to explore its correlation with ITP and the relationship between the blood platelet count of newly diagnosed patients with ITP and genotypes of BAFF-871 C/T polymorphisms. Alleles specific polymerase chain reaction (ASP-PCR) and agarose gel electrophoresis were used to identify polymorphisms -871 C/T of BAFF promotor in 133 ITP patients and 117 healthy controls, and determine the genotype of subjects. Meantime, the frequency of genotype and alleles were analyzed. The results indicated that out of 133 patients with ITP, 33.1% patients exhibited C/C, 42.1% patients were heterozygous C/T, and 24.8% patients were homozygous T/T. The corresponding frequencies in 117 healthy controls were 55.6% C/C, 33.3% C/T and 11.1% T/T. The allele frequency of T in ITP patients and healthy controls were 45.9% and 27.4% respectively. There was significant difference in the BAFF-871 C/T genotypic frequency between the ITP patients and healthy controls (p < 0.05). The allele frequency of T in ITP patients was higher than that in healthy controls. There was no significant difference in the blood platelet counts between the various genotype (p > 0.05). It is concluded that the polymorphism -871 C/T of BAFF promoter is correlated with the pathogenesis of ITP. However, there is no significant difference in blood platelet counts between the various genotype, so the polymorphism -871 C/T of BAFF promoter can not be referred as the analysis index for evaluating the severity of ITP.

Relationship between the long type PML-RAR alpha and the prognosis of patients with acute promyelocytic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the relationship between long (L) type PML-RAR alpha fusion gene and the prognosis of patients with acute promyelocytic leukemia (APL).</p><p><b>METHODS</b>PML-RAR alpha fusion gene was detected by RT-PCR in 33 APL patients. The optical density of three bands including E5 (+) E6 (+) 636 bp (no deletion), E5 (-) E6 (+) 492 bp (exon 5 deleted) and E5 (-) E6 (-) 232 bp (both exon 5 and exon 6 deleted) was measured by a UVP analysis system and their relative proportions were calculated. The relative expression level of each splicing band, initial WBC count and age were statistically (for single and multi-factor analysis) analyzed with prognosis.</p><p><b>RESULTS</b>The relative expressions of E5 (-) E6 (+) and E5 (-) E6 (-) in death group were obviously different from that in first complete remission (CR1) group (P < 0.01), but do not for E5 (+) E6 (+) (P > 0.05). The relative expression levels of E5 (-) E6 (+), E5 (-) E6 (-) and E5 (+) E6 (+) were 0.23 +/- 0.12, 0.58 +/- 0.18, 0.20 +/- 0.09 in death group, and 0.45 +/- 0.16, 0.23 +/- 0.12, 0.31 +/- 0.16 in CR1 group, respectively. Initial WBC count and age was no difference between the two groups (P > 0.05). Logistic regression analysis showed that the expression of E5 (-) E6 (+) had no effect on the prognosis (B = 3.475, P = 0.492), but the expression of E5 (-) E6 (-) showed a negative correlation with prognosis (B = -19.660, P = 0.046).</p><p><b>CONCLUSIONS</b>The high expression of E5 (-) E6 (-) is correlated with the poor prognosis for patients with APL.</p>

WT1 gene expression and its isoform ratio in different cell subsets of normal human bone marrow / 中国实验血液学杂志

The Wilms' tumor gene (WT1) is a transcription factor involved in tumorigenesis, especially in leukemogenesis. However, the role of WT1 expression in nonmalignant hematopoietic cells remains unclear. Furthermore, due to alternative splicing at two sites: 17 amino acid residues of exon 5 (+17AA) and 3 amino acid residues (+KTS) between exons 9 and 10, WT1 gene has four main isoforms (17AA+/KTS+, 17AA+/KTS-, 17AA-/KTS+, 17AA-/KTS-, abbreviation: +/+, +/-, -/+, -/-). The isoforms probably existed in hematopoietic cells, which make the research more complex. The aim of study was to elucidate the expression and its isoforms of WT1 gene in different cell subsets of healthy bone marrow donors. The fluorescence RT-PCR detection system was established to measure the expressions of full-length WT1, WT1 (+17AA) and WT1 (+KTS) in CD34(+)CD38(-) (stem cell), CD34(+)CD38(+) (progenitor cell), CD15(+)CD11b(+) (granulocyte), CD33(+)CD14(+) (monocyte), CD20(+)CD5(-) (B-lymphocyte) and CD20(-)CD5(+) (T-lymphocyte) subsets from 18 normal human bone marrow samples. The results showed that WT1 expressed in CD34(+)CD38(-), CD34(+)CD38(+), CD15(+)CD11b(+) and CD33(+)CD14(+), but not in CD20(+)CD5(-) and CD20(-)CD5(+) subsets. The highest expression was in CD34(+)CD38(-), but decreased gradually in CD15(+)CD11b(+) and CD33(+)CD14(+) subsets. WT1 (+17AA), WT1 (+KTS) and WT1 (+/+) isoforms were predominant in CD34(+)CD38(-) and CD34(+)CD38(+) primitive subsets, while in CD15(+)CD11b(+) and CD33(+)CD14(+) the dominant isoforms were WT1 (-17AA), WT1 (-KTS) and WT1 (-/-). It is concluded that the expression of WT1 in normal bone marrow decreases gradually with cell differentiation. Hematopoietic cells may adjust the ratios of WT1 isoforms to inhibit or promote cell differentiation.

Expression and clinical significance of peripheral blood CD4~+CD25~+T cells in patients with systemic lupus erythematosis / 中华风湿病学杂志

Objective To investigate the expression and clinical significance of peripheral blood CD4~+, CD25~+ and CD4~+CD25~+ T subpopulations in patients with systemic lupus erythematosis.Methods The per- centage and fluorescence intensities of peripheral blood CD4~+,CD25~+ and CD4~+CD25~+ subpopulations from 34 SLE and 18 normal controls were measured with flow cytometry assay,then the correlation with clincal data was analyzed.The CD25~+ cells were defined as the CD25~(high) cells if their fluorescence intensity was higher than 10. Results The percentage of CD4~+CD25~+,CD4~+CD25~(high) T lymphocytes in active SLE patients[(4.80?1.21)% and (0.25?0.10)%]was lower than that in normal controls[(8.92?3.21)% and(0.44?0.22)% and non-active SLE patients(11.28?2.09)% and(0.59?0.34)%](P＜0.05).However,as for the CD25~+ cells in the CD4~+ T cells,there was no difference between SLE patients and normal control group.Peripheral blood CD4~+CD25~+,CD4~+CD25~(high) cells in SLE were reversely correlated with SLEDAI(r=-0.74,P=0.004 and r=-0.614,P=0.026),but not with others such as complements,ANA titers etc.Peripheral blood CD4~+ and CD25~+ lymphocytes in active SLE pa- tients were also lower than those in normal controls[(23?7)vs(34?7)and(7.4?1.8)vs(13.9?3.4),P＜0.05]. CD25 fluorescence intensities were higher in the SLE patients those in the normal controls,but CD4 fluores- cence intensities were not.Conclusion CD4~+CD25~+ may play a role in the pathogenesis of SLE.

Acquired hemophilia A in patients with systemic lupus erythematosus:report of a case and review of literature / 中华风湿病学杂志

Objective To summarize the manifestation and treatment of acquired hemophilia A in pa- tients with systemic lupus erythematosus(SLE).Method A case was investigated retrospectively and the lit- erature was reviewed.Results A 25-year-old woman with a 5 year history of SLE was admitted to hospital due to abdominal pain.She was diagnosed with acquired factorⅧinhibitor deficiency based on a prolonged activated partial-thromboplastin time(APTT,135.3 s),reduced factorⅧactivity(0.9%)and factorⅧin- hibitor(26.1 BU/ml).Sonography and magnetic nuclear resonance of the abdomen confirmed the presence of a retro-uterine hematoma.The patient was initially treated with intravenous pulse and oral corticosteroids,factorⅧplasma concentrated and intravenous immunoglobulin.Clinical and biological improvement was promptly obtained.Conclusions Attention should be paid to the association between SLE and acquired hemophilia A. Combination therapy may be recommended as initial therapy for the management of acquired hemophilia A in patients with SLE.But no standardized treatment can be recommended at present.