Analysis of prognostic factors of extranodal NK/T-cell lymphoma treated with pegaspargase/L-asparaginase: a multicenter retrospective study / 中华血液学杂志

Objective: To explore the prognostic factors of extracellular NK/T cell lymphoma (ENKTL) treated with pegaspargase/L-asparaginase. Methods: The clinical data of 656 ENKTL patients diagnosed at 11 medical centers in the Huaihai Lymphoma Working Group from March 2014 to April 2021 were retrospectively analyzed. The patients were randomly divided into two groups: a training set (460 cases) and a validation set (196 cases) at 7∶3, and the prognostic factors of the patients were analyzed. A prognostic scoring system was established, and the predictive performance of different models was compared. Results: Patients' median age was 46 (34, 57) years, with 456 males (69.5% ) and 561 nasal involvement (85.5% ). 203 patients (30.9% ) received a chemotherapy regimen based on L-asparaginase combined with anthracyclines, and the 5-year overall survival rate of patients treated with P-GEMOX regimen (pegaspargase+gemcitabine+oxaliplatin) was better than those treated with SMILE regimen (methotrexate+dexamethasone+cyclophosphamide+L-asparaginase+etoposide) (85.9% vs 63.8% ; P=0.004). The results of multivariate analysis showed that gender, CA stage, the Eastern Cooperative Oncology Group performance status (ECOG PS) score, HGB, and EB virus DNA were independent influencing factors for the prognosis of ENKTL patients (P<0.05). In this study, the predictive performance of the prognostic factors is superior to the international prognostic index, Korean prognostic index, and prognostic index of natural killer lymphoma. Conclusion: Gender, CA stage, ECOG PS score, HGB, and EB virus DNA are prognostic factors for ENKTL patients treated with pegaspargase/L-asparaginase.

The Efficacy and Safety of Daratumumab-Based Regimen in Treatment of Multiple Myeloma Patients with Renal Impairment / 中国实验血液学杂志

OBJECTIVE@#To investigate the efficacy and safety of daratumumab in treatment of multiple myeloma (MM) patients with renal impairment (RI).@*METHODS@#The clinical data of 15 MM patients with RI who received daratumumab-based regimen from January 2021 to March 2022 in three centers were retrospectively analyzed. Patients were treated with daratumumab or daratumumab combined with dexamethasone or daratumumab combined with bortezomib and dexamethasone and the curative effect and survival were analyzed.@*RESULTS@#The median age of 15 patients was 64 (ranged 54-82) years old. Six patients were IgG-MM, 2 were IgA-MM,1 was IgD-MM and 6 were light chain MM. Median estinated glomerular filtration rate (eGFR) was 22.48 ml/(min·1.73 M2). Overall response rate of 11 patients with MM was 91% (≥MR), including 1 case of stringent complete response (sCR), 2 cases of very good partial response (VGPR), 3 cases of partial response (PR) and 4 cases of minor response (MR). The rate of renal response was 60%(9/15), including 4 cases of complete response (CR), 1 case of PR and 4 cases of MR. A median time of optimal renal response was 21 (ranged 7-56) days. With a median follow-up of 3 months, the median progression-free survival and overall survival of all patients were not reached. After treatment with daratumumab-based regimen, grade 1-2 neutropenia was the most common hematological adverse reaction. Non-hematological adverse reactions were mainly infusion-related adverse reactions and infections.@*CONCLUSION@#Daratumumab-based regimens have good short-term efficacy and safety in the treatment of multiple myeloma patients with renal impairment.

Effects of Decitabine Combined with Anlotinib on Proliferation and Apoptosis of Multiple Myeloma Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the biological effects and its relative mechanism of decitabine combined with anlotinib on multiple myeloma cells.@*METHODS@#The human MM cell lines and primary cells were treated with different concentrations of decitabine, anlotinib, and decitabine+anlotinib, respectively. The cell viability was detected and combination effect was calculated by CCK-8 assay. The apoptosis rate was measured by flow cytometry and the level of c-Myc protein was determined by Western blot.@*RESULTS@#Both decitabine and anlotinib could effectively inhibit the proliferation and induce the apoptosis of MM cell lines NCI-H929 and RPMI-8226. The effect of combined treatment on the inhibition of cell proliferation and induction of apoptosis was stronger than that of single-drug treatment. The combination of the two drugs also showed strong cytotoxicity in primary MM cells. Decitabine and anlotinib could down-regulate the level of c-Myc protein in MM cells and the c-Myc level in the combination group was the lowest.@*CONCLUSION@#Decitabine combined with anlotinib can effectively inhibit the proliferation and induce apoptosis of MM cells, which provides a certain experimental basis for the treatment of human MM.

Association of Next Generation Sequencing Based Genotypic Profiling with MICM Characteristics in NPM1 Mutated Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explain the clinicobiological heterogeneity of NPM1 mutated (NPM1mut) acute myeloid leukemia (AML) by analyzing the association between next-generation sequencing (NGS) profiles and MICM characteristics in patients with this AML subtype.@*METHODS@#Data of 238 NPM1mut patients with available NGS information on 112 genes related to blood disease was collected, and χ2 test and nonparametric test were used to analyze the distribution association between NGS-detecting mutations and conventional MICM parameters.@*RESULTS@#In entire NPM1mut cohort, totaling 240 NPM1 mutation events were identified, of whom 10 (10/240, 4.2%) were missense mutations, which did not involve any W288 or W290 locus and were found exclusively in NPM1mut/FLT3-ITD- group. All but one of these missense mutations (9/10, 90%) were accompanied by AML subtype-defining recurrent cytogenetic or molecular abnormalities, of which 7 cases were in the low risk and 2 in the high risk. NPM1mut occurred solely as an insertion/deletion (indel) type in the NPM1mut/FLT3-ITD+ group. The incidence of favorable plus unfavorable karyotypes in NPM1mut/FLT3-ITD- group was higher than in NPM1mut/FLT3-ITD+ group (6.4% vs. 0, P=0.031). The positive rates of CD34 and CD7 in NPM1mut/FLT3-ITD+ group were significantly higher than in NPM1mut/FLT3-ITD- group (CD34: 47.9% vs. 20.6%, P<0.001; CD7: 61.5% vs. 29.9%, P<0.001). Logistic analysis showed that FLT3-ITD independently predicted for CD34+ and CD7+ [odds ratio (OR)=5.29, 95%CI: 2.64-10.60, P<0.001; OR=3.47, 95%CI: 1.79-6.73, P<0.001; respectively]. Ras-pathway mutations independently predicted for HLA-DR+ (OR=4.05, 95%CI: 1.70-9.63, P=0.002), and KRAS mutation for MPO- (OR=0.18, 95%CI: 0.05-0.62, P=0.007). TET2/IDH1 mutations independently predicted for CD34- and CD7- (OR=0.26, 95%CI: 0.11-0.62, P=0.002; OR=0.30, 95%CI: 0.14-0.62, P=0.001; respectively), and MPO+ (OR=3.52, 95%CI: 1.48-8.38, P=0.004). DNMT3A-R882 independently predicted for CD7+ and HLA-DR+ (OR=3.59, 95%CI: 1.80-7.16, P<0.001; OR=13.41, 95%CI: 4.56-39.45, P<0.001; respectively), and DNMT3A mutation for MPO-(OR=0.35, 95%CI: 1.48-8.38, P=0.004).@*CONCLUSION@#Co-existing FLT3-ITD in NPM1mut AML independently predicts for CD34+ and CD7+, co-existing Ras-pathway mutation for HLA-DR+ and MPO-, co-existing TET2/IDH1 mutation for CD34-, CD7-, and MPO+, and co-existing DNMT3A mutation for HLA-DR+, CD7+, and MPO-, thereby providing a new mechanism explanation for the immunophenotypic heterogeneity of these AML patients.

The Effects of Decitabine Combined with All-Trans Retinoic Acid on the Number of Immune Cells in Myeloid Neoplasms / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of decitabine (DEC) combined with all-trans retinoic acid (ATRA) on the number of immune cells, efficacy and adverse reactions in the treatment of myeloid neoplasms patients.@*METHODS@#Eighty-four patients with myeloid tumors, including AML, MDS-EB-1 or MDS-EB-2 treated by the regimen containing decitabine in our hospital from January 2009 to October 2019 were enrolled and retrospectively analyzed, among the patients, 21 patients treated with DEC alone, 24 patients treated with DEC combined with ATRA (DEC/ATRA) and 39 patients treated with DEC combined with G-CSF priming regimen (DEC/priming). The changes of peripheral blood immune cell levels before and after treatment of the patients between the three groups were compared, and the differences in clinical efficacy and adverse reactions of the patients between the three groups were also compared.@*RESULTS@#There was no statistical differences in the number of immune cells among the patients in the three groups before treatment (P>0.05). NK cell levels decreased significantly in the patients in DEC and DEC/ATRA group after treatment (P<0.05); After treatment, the levels of CD8+ and CD3+T cells in the patients treated by DEC /priming regimen significantly increased (P<0.05), while the levels of CD3-HLA-DR+ B cells significantly decreased (P<0.05). The overall response rate (ORR) of the patients in DEC/ATRA group (75%) and DEC/priming group (74.36%) was significantly higher than 42.86% in DEC monotherapy group, and the differences showed statistically significant (P<0.05), while the ORR between the patients in DEC/ATRA and DEC/priming group showed no statistic differences (P>0.05). There were no statistical differences in overall survival (OS) and incidence of bleeding between the patients in the three groups (P>0.05). The incidences of grade 3 to 4 bone marrow suppression and the infection rate of the patients in DEC monotherapy and DEC/ATRA group were significantly lower than that in DEC/priming regimen group after treatment (all P<0.05), however, there was no statistical difference between DEC monotherapy and the DEC/ATRA group.@*CONCLUSION@#The efficacy of DEC/ATRA on myeloid neoplasms is comparable to that of DEC/priming regimen, and the anti-myeloid tumor effect of DEC/ATRA regimen may be related to the regulation of NK cells and T cells.

Expression and Prognostic Value of MiR-155 in Patients with Chronic Lymphocytic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explore the expression of miR-155 in patients with chronic lymphocytic leukemia (CLL) and its correlation with the clinical and biological features in CLL.@*METHODS@#The expression of miR-155 was detected by quantitative real time polymerase chain reaction (qRT-PCR) in the peripheral mononuclear cells collected from 73 CLL patients and CD19 positive B cells collected from 60 healthy controls, respectively. The expression of miR-155 in CLL patients was compared with the healthy controls, and the correlation of miR-155 expression with the clinical characteristics of CLL patients such as age, sex, Binet stage, cytogenetic and molecular genetic, as well as the relationship of miR-155 expression level with time to treatment (TTT) and overall survival (OS) was further analysed.@*RESULTS@#The expression of miR-155 was significantly elevated in CLL patients compared with the healthy controls. Further analysis showed that miR-155 expression decreased in the patients with the mutated immunoglobulin heavy chian variable region (IGHV) as compared with the patients unmutated (P＜0.05), and its expression was significant higher in the IGHV-39 family (P＜0.05). Fluorescence in situ hybridization (FISH) showed that the expression of miR-155 increased in patients with P53 mmutation/deletion and ATM deletion (P＜0.05). However, OS and TTT were not different between the patients with high and low expression of miR-155.@*CONCLUSION@#MiR-155 is increasingly expresses in CLL patients and correlates with poor prognosis, suggesting its important role in the genesis and progress of CLL.

Comparison of the efficacy and safety between decitabine combined with all-trans retinoic acid or half dose priming regimen in the treatment of elderly patients with myelodysplastic syndromes and acute myeloid leukemia / 医学研究生学报

Objective Decitabine (DAC) combined with the half dose priming regimen (HDPR) is a common treatment of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) in the elderly. This study was to compare the clinical effect and safety of DAC combined with all-trans retinoic acid (ATRA) versus DAC plus HDPR in the treatment of MDS with excess of blasts (MDS-EB) or AML in elderly patients. Methods We retrospectively analyzed 48 elderly patients (≥60 years) with myeloid neoplasms (AML, MDS-EB-1 or MDS-EB-2) ineligible for standard chemotherapy treated in our hospital from January 2014 to October 2018, 22 by DAC+ATRA (group A) and the other 26 by DAC+HDPR (group B). We compared the overall response rate (ORR), overall survival (OS) and adverse events between the two groups of patients. Results No statistically significant difference was observed between groups A and B in ORR (86.4% vs 76.9%, P = 0.643) or median OS (26.2 vs 24.9 mo, P = 0.920). The median time to response was significantly longer in group A (2 courses) than in B (1 course) (P = 0.006). Compared with group A, group B showed remarkably lower incidence rates of grade-3 to -4 cytopenia (54.5% vs 84.6%, P = 0.029) and infection (45.5% vs 76.9%, P = 0.037), longer duration of neutropenia (P < 0.05), and higher volumes red blood cell infusion and platelet infusion (P < 0.05). There was no statistically significant difference in the incidence rate of bleeding between the two groups (P = 0.643). Conclusion DAC+ATRA and DAC+HDPR have comparable clinical effects on myeloid neoplasms in elderly patients, but the former is safer and better tolerated while the latter can achieve a more rapid response.

All-Trans Retinoic Acid and Decitabine Synergistically Induce Anti-Leukemia Effect on U937 Cell Line and Newly Diagnosed Elder AML Patients / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of all transretinoicacid(ATRA) combined with decitabine (5-Aza-2'-deoxycytidine;DAC) on DNA methylation and gene expression of p16INK4a (p16) and retinoic acid receptor β (RARβ), and to explore their combined anti neoplastic effect on U937 cells and newly diagnose delder acute myeloid leukemia(AML) patients.</p><p><b>METHODS</b>The expression levels of p16 and RARβ were determined by qRT-PCR and Western blot. Methylation-specific PCR was used to analyze their methylation status. WST-1 and flow cytometry were performed to detect growth inhibition, differentiation, apoptosis and cell cycle of U937 cells respectively.</p><p><b>RESULTS</b>The expression p16 and RARβ was down-regulated by promoter hypermethylation in newly diagnose delder AML patients and U937 cells. Combination treatment of ATRA and DAC induced DNA hypomethylation as well as gene expression of p16 and RARβ, which contributed to the growth inhibition, differentiation, apoptosis and cell cycle arrest of U937 cells. In addition for elder AML patients intolerable to standard chemotherapy, the combination regimen of ATRA and DAC showed antineoplastic activity accompamied by up-regulation of p16 and RARβ expression and decrease of bone marrow blast, moreover the parients showed good tolerence to the reginen.</p><p><b>CONCLUSION</b>The regimen of ATRA combined with DAC as the combination therapeutic strategy for inducing differentiation and demethylation possesses the anti-AML potency, and contributes to optimizing the therapeutic strategy for elder AML patients and promoting the clinical prognosis.</p>

Prognostic Significance of the Percentage of Blasts with CD34/CD38/CD123Phenotype in Acute Myeloid Leukemias / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the percentage of blasts with the CD34/CD38/CD123phenotype in de novo acute myeloid leukemia (AML) patients and analyse its correlation with prognosis.</p><p><b>METHODS</b>The percentage of CD34/CD38/CD123cells in the blast population of 148 newly diagnosed patients with AML was determined by using flow cytometry and its correlation with complete response, disease-free survival and overall survival were evaluated.</p><p><b>RESULTS</b>The median percentage of CD34/CD38/CD123cells in newly diagnosed patients was 2.8% (ranged from 0.01 to 67%). The high expression of CD34/CD38/CD123in AML patients positively correlated with the NPM1 wild-type (χ=5.194,P<0.05), but did not relate with the positive FLT3-ITD mutations (χ=0.418,P>0.05). Further multivariable analysis showed that the higher expression of the CD34/CD38/CD123was associated with lower complete remission (P<0.05), worse disease-free survival(P<0.01) and shorter overall survival(P<0.01) in AML patients.</p><p><b>CONCLUSION</b>The percentage of CD34/CD38/CD123cells at diagnosis significantly correlates with the response to treatment and survival. This prognostic marker may be used to rapidly identify the risk of treatment failure in clinical practice.</p>

Autologous Peripheral Blood Hematopoietic Stem Cell Transplantation with IBu Precondition Regimen in Treatment for 11 Patients with Low to Intermediate Risk Acute Myeloid Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To evaluate safety and efficacy of autologous peripheral blood hematopoietic stem cell transplantation (auto-PBHSCT) with IBu precondition regimen consisting of high-dose idarubicin (IDA) and busulphan (Bu) for treatment of patients with low and intermediate risk acute myeloid leuekmia (AML).</p><p><b>METHODS</b>A total of 11 patients with AML (5 low and 6 intermediate risk patients) treated with auto-PBHSCT with IBu precondition regimen (IDA 20 mg/m, continuous i.v. from d-13 to d-11, Bu 0.8 mg/kg/q6h i.v. for 2h, from d-5 to d-2) from March 2011 to July 2014 were analyzed retrospectively. Adverse effects and transplantation-related mortality (TRM) were evaluated. Kaplan-Meier analysis was performed to calculate the overall survival (OS), disease-free survival (DFS) and cumulative relapse rate (RR). Cox regression was performed for univariate analysis for DFS.</p><p><b>RESULTS</b>Among the 11 patients, 10 patients obtained hematopoietic reconstitution, 1 patient died during transplantation, thus the TRM was 9.1%. The adverse effects were well tolerated. With median follow-up of 31.6 (8.7-52.5) months, 7 patients (63.3%) were alive, including 6 patients (54.5%) in continuous complete remission (CR). Median OS and DFS were not reached. The 3-year OS, DFS and RR were (57.7±16.3)%, (52.5±17.6)% and 47.5%, respectively. Univartiate analysis indicated that the age, sex, interval between diagnosis and transplantation, white blood cell count at diagnosis, risk-grouping (low or intermediate risk), disease status before transplantation (CR1 or CR2), and count of mononuclear cells for infusion all can not influence DFS(P>0.05, respectively).</p><p><b>CONCLUSION</b>The treatment of auto-PBHSCT with IBu precondition regimen for low to intermediate risk AML patients is safe and effective.</p>

MicroRNA-10a expression in FAB different subtype of acute myeloid leukemia and its relationship with drug resistance / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was to investigate the expression of miR-10a in the different FAB subtype of acute myeloid leukemia (AML) and its relationship with drug resistance.</p><p><b>METHODS</b>Forty de novo patients with AML, 16 patients with non-malignant hematologic disease and three AML cell lines HL-60, U937 and HL-60/ADR were enrolled in this study, the MiR-10a expression in bone marrow mononuclear cells of above-mentioned patients and 3 AML cell lines was detected by TaqMan RT-PCR. The correlation of miR-10a with clinicopathological factors of AML patients was analyzed.</p><p><b>RESULTS</b>The miR-10a expression level in HL-60 cell line was higher than that in U937 cell line (P = 0.039). And its expression level in de novo AML patients was higher than that in patients with non-malignant hematologic disease (P < 0.01). FAB-AML-M3 patients exhibited higher expression of miR-10a than that in M1, M2 and M4 (P < 0.05); HL-60/ADR cell line showed higher miR-10a expression than that in HL-60 cell line (P < 0.01) . Except M3, the patients without CR (non-CR) after the first cycle of chemotherapy showed a higher level of miR-10a as compared with CR patients (P < 0.01).</p><p><b>CONCLUSION</b>The high expression of miR-10a may be closely related to over-proliferation of promyelocyte and drug resistance of acute myeloid leukemia cells, except M3.</p>

Effects of simvastatin on PI3K/AKT signaling pathway in human acute monocytic leukemia cell line SHI-1 / 中国实验血液学杂志

To investigate the effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin (SV) on proliferation, apoptosis and the PI3K/AKT signaling pathway in human acute monocytic leukemia cell line SHI-1. SHI-1 cells were incubated with different concentrations of SV (5, 10, 15 µmol/L). Otherwise, SHI-1 cells without any treatment were used as control. Cells in different groups were collected at 24, 48 and 72 h after incubation for further detection. MTT method was used to assay the growth inhibition rate and flow cytometry was used to detect the early stage apoptosis ratio. The human PI3K-AKT Signaling Pathway RT(2) Profiler(TM) PCR Array was used to detect the expression of 84 genes involved in PI3K-AKT signaling. The results indicated that the SV inhibited the proliferation and inducted the apoptosis of SHI-1 cells in time- and dose-dependent manners significantly. The growth inhibition rates of SHI-1 cells treated with 15 µmol/L SV for 24, 48 and 72 h were 26.82, 47.09 and 63.92, respectively; and their early stage apoptosis ratios were 5.75, 13.25 and 15.59, respectively. Compared with the control group, expression levels of 39 genes were changed in the group of 15 µmol/L SV at 48 h, among them 26 genes were down-regulated and 13 genes were up-regulated. It is concluded that the SV can inhibit proliferation and induce apoptosis of SHI-1 cells, and the mechanism may be associated with the changes of gene expression level in PI3K-AKT signaling pathway regulated by SV.

Transcriptional activity of WT1 gene promoter and enhancer in diverse cell lines / 中国实验血液学杂志

The objective of study was to investigate tissue-specific transcriptional activity of WT1 (Wilms' tumor gene) promoter and enhancer in cell lines with diverse tissue origin for leukemic gene therapy depending on WT1 transcriptional regulation elements. WT1 promoter and enhancer were ligated into pEGFP-1 to construct a recombinant vectors with EGFP gene as a reporter. By using electroporation or lipofectamine, the resultant constructs were transfected into 13 cell lines including WT1-expressing hematopoietic cell lines (K562, NB4, THP-1 and SHI-1), WT1-nonexpressing hematopoietic cell lines (U937 and Jurkat), WT1-expressing nonhematopoietic cell lines (MCF-7, T47D and 293) and WT1-nonexpressing nonhematopoietic cell lines (ECV304, SMMC7721, HT-29 and SHG44). The mean fluorescence intensity (MFI) of EGFP representing the transcriptional activities of promoter and/or enhancer was analyzed by using flow cytometry in the transfected cells which stably expressed EGFP. The results indicated that the vectors, pEWP containing WT1 promoter and pEWPA containing both WT1 enhancer and promoter, were constructed by recombinant DNA technique. Among nonhematopoietic cell lines, pEWP induced the highest EGFP expression in ECV304 (16.54 +/- 2.45 times as high as pEGFP-1), mildly higher in MCF-7 and SHG44 (9.46 +/- 1.10 and 7.29 +/- 0.73 times of pEGFP-1 level), and lowest in HT-29 (0.99 +/- 0.02 times as much as pEGFP-1) respectively. Among hematopoietic cell lines, EGFP expression was highest in K562 cell line (2.93 +/- 0.27 times of pEGFP-1), which was statistically higher than those in Jurkat and SHI-1 cell lines (0.74 +/- 0.03 and 0.84 +/- 0.09 times of pEGFP-1 level) respectively. pEWPA, with WT1 enhancer inserted at Afl II site near SV40 polyA, increased basal transcription levels of the WT1 promoter in HT-29, SHI-1 and K562 cells by 4.81, 3.06 and 1.01-fold respectively. It is concluded that the transcriptional activities of WT1 promoter in the recombinant vector seem unrelated to the constitutional expression level of endogenous WT1 gene. The WT1 enhancer promotes the transcriptional activities of WT1 promoter in some of the cell lines regardless of the hematopoietic tissue origin.

Application of real-time quantitative PCR in selection of transfected cell strains for transgenic overexpression / 中国实验血液学杂志

To explore the feasibility of real-time quantitative PCR (QRT-PCR) for selecting cell strains which overexpress a certain transgene, expression level of RbAp46 was detected in transfected cell strains by using optimal real-time PCR with SYBR Green I. Meanwhile, semi-quantitative RT-PCR and Western blot were performed to compare with the QRT-PCR. The results showed that values of RbAp46(N) were 2064.42 +/- 253.47, 860.94 +/- 291.07, 234.456 +/- 31.08, 18.17 +/- 5.14 and 1.46 +/- 0.54 in K562/RbAp46, K562/CMV, SHG44/RbAp46 monoclone, SHG44/RbAp46 multiclone and SHG44/CMV, respectively. The results were consistent with that determined by semi-quantitative RT-PCR and Western blot. It is concluded that QRT-PCR provides a highly efficient and reproducible method for selection of transfected cell subclones at different level of transgene expression.

Detection of RbAp46 expression in bone marrow cells of leukemia patients by real-time quantitative RT-PCR / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate retinoblastoma (Rb) associated protein 46 (RbAp46) gene expression levels in bone marrow (BM) cells of leukemia patients.</p><p><b>METHODS</b>Real-time quantitative reverse polymerase chain reaction (QRT-PCR) method was used for detecting RbAp46 expression levels in BM cells of 140 patients with acute leukemia (AL), 13 with chronic myelogenous leukemia in chronic phase (CML-CP), 7 with CML in blast crisis (CML-BC) and 32 with non-leukemic disorders.</p><p><b>RESULTS</b>The M-Estimators of RbAp46 were higher in 98 newly diagnosed ALs and 5 relapsed ALs than in 28 ALs in complete remission (CR) and 32 non-leukemic controls (178.23 and 213.65 vs 85.89 and 88.08, respectively). No statistic difference was found between the CR group and control group, or between the newly diagnosed group and relapsed group. The M-Estimators of RbAp46 in patients with CML-CP was 58.27, similar to that in control, but much lower than that in CML-BC (173.24). Among 98 newly diagnosed ALs, the M-Estimators of RbAp46 in M(3) and M(4) were the lowest in all of the subtypes. Furthermore, the RbAp46 expression levels were not correlated with the expression of the fusion genes of bcr/abl, PML-RARalpha, and multidrug resistant gene (mdr1), but were positively correlated with Wilms' tumor gene (WT1) expression levels and negatively with AML1/ETO fusion gene expression.</p><p><b>CONCLUSION</b>RbAp46 expression levels in ALs and CML-BC were strikingly higher than that in non-leukemias and CML-CP, and might participate in leukemogenesis.</p>

Study on DNA methylation status of WT1 gene in its promoter region in hematologic malignancy cell lines / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the DNA methylation status of WT1 gene promoter region in hematologic malignancy cell lines and its correlation with WT1 gene expression.</p><p><b>METHODS</b>1. RT-PCR and methylation-specific PCR were performed for detecting WT1 gene expression and DNA methylation status in its promoter region in 8226, HL-60, Jurkat, K562, KG-1, NB4, Raji, SHI-1, U266 and U937 cell lines. 2. Treatment of U937 cells with 5-aza-CdR, a demethylation inducing agent and the changes in WT1 gene expression level and its promoter region methylation status were determined.</p><p><b>RESULTS</b>1. HL-60, K562, KG-1, NB4 and SHI-1 cells showed higher levels while 8226, Jurkat, Raji, U266 and U937 cells showed extremely low levels of WT1 expression. DNA hypermethylation in WT1 gene promoter region was identified in 8226, Jurkat, Raji, U266 and U937 cells. 2. The WT1 gene expression in U937 was enhanced after treatment with 5-aza-CdR accompanied with the decrease of methylated and the increase of unmethylated levels in its promoter region.</p><p><b>CONCLUSION</b>Modulation of the DNA methylation status in WT1 promoter region is one of the epigenetic mechanisms for regulating its expression.</p>

Detection of WT1 expression in bone marrow of acute leukemia patients with real-time quantitative RT-PCR / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate Wilms' tumor gene (WT1) expression levels in bone marrow (BM) of acute leukemia patients (ALs).</p><p><b>METHODS</b>A real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) method was established for detecting WT1 and internal reference GAPDH expression levels in BM of 108 ALs and 23 non-leukemia controls by Light Cycler.</p><p><b>RESULTS</b>The median expression levels of WT1 in 70 newly diagnosed ALs and 11 relapsed ALs were statistically higher than those in 23 ALs in complete remission (CR) and 23 non-leukemic controls (75.10 and 89.56 vs 2.07 and 1.51 respectively). No statistic differences was found between the CR group and control group, nor between the newly diagnosed group and relapsed group. Of the 70 newly diagnosed ALs, median WT1 expression level of acute granulocytic leukemias was significantly higher than that of acute monocytic leukemias (M(5)), but there was no statistic differences among the M(1), M(2), M(3) and ALL subtypes. Furthermore the WT1 levels were not correlated to peripheral WBC counts, BM blast percentage and multidrug resistant gene (mdr1) expression at presentation, but correlated to chromosome karyotypes. Dynamic analysis of WT1 levels of 2 patients on treatment showed that WT1 expression levels predicted relapse.</p><p><b>CONCLUSION</b>WT1 expression levels in ALs were strikingly higher than that in non-leukemias. WT1 can be a marker for detecting MRD and evaluating therapy efficacy in leukemias.</p>