Phytochemistry and Pharmacological Activities of the Diterpenoids from the Genus Daphne.

There are abundant natural diterpenoids in the plants of the genus Daphne from the Thymelaeaceae family, featuring a 5/7/6-tricyclic ring system and usually with an orthoester group. So far, a total of 135 diterpenoids has been isolated from the species of the genus Daphne, which could be further classified into three main types according to the substitution pattern of ring A and oxygen-containing functions at ring B. A variety of studies have demonstrated that these compounds exert a wide range of bioactivities both in vitro and in vivo including anticancer, anti-inflammatory, anti-HIV, antifertility, neurotrophic, and cholesterol-lowering effects, which is reviewed herein. Meanwhile, the fascinating structure-activity relationship is also concluded in this review in the hope of providing an easy access to available information for the synthesis and optimization of efficient drugs.

[Survival and Prognosis of Patients with Acute Myeloid Leukemia with Myelodysplasia-Related Changes Transformed from Myelodysplastic Syndrome].

OBJECTIVE: To explore the risk factors affecting the survival and efficacy of patients with acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) transformed from myelodysplastic syndrome (MDS). METHODS: The clinical data of 60 patients with AML-MRC transformed from MDS who hospitalized in The Third Affiliated Hospital of Soochow University from January 2010 to December 2021 were retrospectively analyzed. The demographic data and laboratory parameters, cytogenetic karyotypes, target genes of AML detected by next generation sequence, risk stratification, treatment regimen, therapeutic efficacy and survival outcome were documented. Rank sum test and Chi-square test or Fisher exact test were used to compare the survival and efficacy. The effects of clinical parameters, risk stratification and treatment regimens on the survival and efficacy of the AML-MRC patients were analyzed by univariate and multivariate analysis. RESULTS: The median overall survival (OS) of the AML-MRC patients was 4.5 months, the 1-year OS rate was 28.3%, and the complete remission (CR) rate after treatment was 33.3%. The univariate analysis showed that age≥60 years, leukocytosis, severe thrombocytopenia, poor-risk group and only accepted hypomethylating agents(HMAs) or supportive therapy were the risk factors affecting OS. COX multivariate analysis showed that thrombocytopenia ( HR=4.46), HMAs therapy (compared to transplantation, HR=10.47), supportive therapy (compared to transplantation, HR=25.80) and poor-risk group (compared to medium-risk group, HR=13.86) were independent hazard factors for median OS of patients with AML-MRC. The univariate analysis showed that the risk factors affecting 1-year OS in patients with AML-MRC were age≥60 years, thrombocytopenia, time of transformation from MDS to AML (TTA)≥3 months, fibrinogen-albumin ratio index (FARI)≥0.07, CONUT score≥5, poor-risk group and supportive therapy. Binary logistic regression analysis showed that the independent risk factors for 1-year OS in AML-MRC patients were age≥60 years ( HR=11.23), thrombocytopenia ( HR=8.71), FARI≥0.07 ( HR=5.19) and poor-risk group ( HR=14.00). The risk factors affecting CR of AML-MRC patients in univariate analysis were age≥60 years, thrombocytopenia, FARI≥0.1, CONUT score≥5, poor-risk group and supportive therapy, while binary logistic regression analysis showed that age≥60 years( HR=7.35), CONUT score≥5 ( HR=9.60), thrombocytopenia ( HR=12.05) and poor-risk group ( HR=32.5) were independent risk factors affecting CR of the patients. CONCLUSION: The OS of AML-MRC patients is poor, old age(≥60 years old), supportive therapy, HMA therapy, poor-risk, thrombocytopenia, FARI≥0.07 and CONUT score≥5 may be associated with poor prognosis.

[The Efficacy and Safety of Daratumumab-Based Regimen in Treatment of Multiple Myeloma Patients with Renal Impairment].

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.

[Clinical Characteristics and Prognostic Factors of Patients with Primary Parotid Gland Lymphoma].

OBJECTIVE: To explore the clinical characteristics and prognostic factors of patients with primary parotid gland lymphoma, and construct a prognostic model nomogram for patients with primary diffuse large B-cell lymphoma (DLBCL) of parotid gland. METHODS: Primary parotid gland lymphoma and primary DLBCL of parotid gland patients from 1984 to 2016 were identified from the Surveillance, Epidemiology, and End Results (SEER) database. Univariate and multivariate Cox regression analysis were conducted to determine the independent prognostic factors of primary parotid gland lymphoma and primary DLBCL of parotid gland, respectively. According to the established independent prognostic factors of primary DLBCL of parotid gland, nomogram was built to predict 3- and 5-year survival, and the discrimination and calibration of the model were evaluated by concordance index (C-index) and calibration plots. RESULTS: A total of 2 610 patients with primary parotid gland lymphoma were identified. Their median age was 66(15-99) years old, the male to female ratio was 1∶1.8, and 20.5% of them was primary DLBCL of parotid gland, which was the most common histological subtype in aggressive lymphomas. Multivariate Cox regression analysis showed that sex, age, Ann Arbor stage, years of diagnosis, marital status, histological subtype, surgery, and radiation were the independent prognostic factors of primary parotid gland lymphoma, while age, marital status, surgery, and chemotherapy were the independent prognostic factors of primary DLBCL of parotid gland. The C-index of the prediction model was 0.702(95%CI: 0.696-0.768), reflecting a good discrimination ability. The predicted value probability of the calibration plots was close to the actual value probability, reflecting a good accuracy ability. CONCLUSIONS: Sex, age, Ann Arbor stage, years of diagnosis, marital status, histological subtype, surgery, and radiation were the independent prognostic factors of primary parotid gland lymphoma. The nomogram survival prediction model for primary DLBCL of parotid gland patients can assist clinical decision effectively.

[Effects of Decitabine Combined with Anlotinib on Proliferation and Apoptosis of Multiple Myeloma Cells].

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].

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 Clinical Value of Neutrophil CD64 Index in Hematological Malignancies with Pulmonary Infection].

OBJECTIVE: To investigate the clinical value of neutrophil CD64 index in hematological malignancies with pulmonary infection. METHODS: The cohort study method was used to retrospectively analyze the clinical data of 125 patients with hematological malignancies and pulmonary infections who were treated in The Third Affiliated Hospital of Soochow University. All the patients were divided into four stages according to the diagnosis and treatment process: non-infected stage (T1), the symptoms of infection had appeared before using antibiotics (T2), one week after anti-infective treatment (T3), and after stopping antibiotics (T4). CD64 index, C-reactive protein (CRP), blood cell count, and immune cell level were compared before and after infection (T1 vs T2), the correlation between CD64 index and other indicators were explored, the change trends of the significantly different indicators in the course of the disease were observed, and the diagnostic efficacy of CD64 index and CRP were compared. The surviving patients were followed up for whether reinfection occurred within 30 days after discharge, and the re-examination results of indices before discharge (in stage of T4) between reinfected and non-reinfected patients were compared to find the risk factors of reinfection. RESULTS: Before and after infection, the CD64 index, CRP, CD14+HLA-DR+, CD4+, and lymphocyte counts were significantly different (all P<0.05). There was a negative correlation of CD64 index with CD14+HLA-DR+ (r=-0.395, P<0.001), a negative correlation with CD3+ (r=-0.1.87, P=0.047), and a negative correlation with lymphocyte count (r=-0.230, P=0.006), while a positive correlation with CRP(r=0.313, P<0.001). The area under the curve of CD64 index, CRP, and CD64 index combined with CRP was 0.790 (95%CI: 0.711-0.868), 0.754(95%CI: 0.667-0.841), and 0.835(95%CI: 0.762-0.907), respectively; the sensitivity was 59.6%, 72.7%, and 74.7%, the specificity was 89.2%, 73.0%, and 78.4%, and the cut-off value was 0.488, 0.457, and 0.531, respectively. There were only two re-examination indexes showed significantly different before discharge between reinfected patients and non-reinfected patients: CD14+HLA-DR+ (F=8.524, P=0.004) and CD64 index (F=9.993, P=0.002). The increase of CD64 index was an independent risk factor for reinfection within 30 days after discharge from the hospital (HR=1.790, 95%CI: 1.343-2.386, P<0.001). CONCLUSION: CD64 index has diagnostic value in patients with hematological malignancies and pulmonary infection, and its specificity is higher than that of CRP. The combination of the two indicators can improve the diagnostic sensitivity. CD64 index has a predictive value for reinfection within 30 days after infection treatment.

[The Effects of Decitabine Combined with All-Trans Retinoic Acid on the Number of Immune Cells in Myeloid Neoplasms].

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.

[Efficacy Analysis of Bendamustine-Based Combination Regimen in Treatment of Patients with Relapsed/Refractory Non-Hodgkin Lymphoma].

OBJECTIVE: To investigate the efficacy and safety of bendamustine combined with gemcitabine, vinorelbine，glucocorticoids (BeGEV)±X regimen in treatment of patients with relapsed/refractory non-Hodgkin lymphoma. METHODS: A total of 18 relapsed/ refractory non-Hodgkin lymphoma patients at the age of 18 years or older hospitalized in the First People's Hospital of Changzhou from March 2020 to March 2021 were selected. They received two or more cycles of BeGEV±X regimen. X could be anti-CD20 monoclonal antibody, PD-1-blocking antibodies, lenalidomide, BTK inhibitor, Bcl-2 inhibitor and so on according to patients' disease feature. The clinical efficacy and adverse effects were observed. RESULTS: In total, 18 patients completed two or more cycles of BeGEV±X regimen, including 14 with diffuse large B-cell lymphoma, one with low-grade follicular lymphoma, one with follicular lymphoma grade 3b, one with angioimmunoblastic T-cell lymphoma and one with peripheral T-cell lymphoma, not otherwise specified. 11 patients were male. The median age of the patients was 64 years old. 17 patients had modified Ann Arbor stage Ⅲ/Ⅳ disease. 13 patients had high- intermediate risk or high risk IPI score, while 15 patients had high-intermediate high risk or high risk NCCN-IPI score. 14 cases had extranodal sites of disease. And 6 cases had bulky disease. 12 patients experienced refractory disease, while 8 patients had received 3 line or more prior treatment. After two or three cycles of chemotherapy, the complete response rate was 6/18, the partial response rate was 3/18, and the objective response rate was 9/18. From the beginning of salvage chemotherapy to the end of follow-up, the median progression-free survival time was 130 days, and the median overall survival was 152 days. The most common grade 3 to 4 adverse events were hematologic toxicities, infection and febrile neutropenia. CONCLUSION: BeGEV±X is an effective salvage regimen in treatment of patients with relapsed/refractory non-Hodgkin lymphoma, while adverse events such as hematologic toxicities and infection should be closely monitored.

[Effects of simvastatin plus all-trans retinoic acid on WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4].

OBJECTIVE: To investigate the effects of simvastatin (SV) plus all-trans retinoic acid (ATRA) on the proliferation, differentiation, apoptosis and WT1/hDMP1 gene expression profiles of human promyelocytic leukemia cell line NB4. METHODS: The NB4 cell was incubated with simvastatin and ATRA alone or in combination. And the NB4 cell without any treatment was adopted as a normal control. The cells of different groups were collected at 24, 48 and 72 h post-incubation. Their morphological changes were observed after Wright staining. The method of MTT was employed to assay the growth inhibition rate and flow cytometry was used to detect the early-stage ratios of apoptosis and cell necrosis. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the WT1/hDMP1 gene expression levels. RESULTS: The cell inhibition rates increased gradually (F = 7.15, P = 0.000) at 15, 10 and 5 µmol/L SV respectively. And so did the expression levels of CD11b (F = 3.41, P = 0.014) and Annexin-V (F = 43.38, P = 0.000). However the expression levels of WT1 decreased gradually (F = 5.35, P = 0.001) reversely with the elevated levels of hDMP1 (F = 22.61, P = 0.000). Furthermore the NB4 cell exhibited the most significant changes at 15 µmol/L SV. After a 72-hour incubation, the expression levels of CD11b (89.46% ± 9.13%)and hDMP1 (626.9 ± 56.9) in NB4 cells at 15 µmol/L SV plus 0.5 µmol/L ATRA were significantly higher than those with ATRA(71.27% ± 7.27%, P = 0.000 and 421.8 ± 38.3, P = 0.003 in each) and SV alone(62.41% ± 6.37%, P = 0.003 and 241.4 ± 21.9, P = 0.003 in each). A combination of 15 µmol/L SV with 0.5 µmol/L ATRA displayed obvious interactions with the expressions of CD11b and hDMP1 (F = 4.09, P = 0.025 and F = 29.58, P = 0.000 in each). And there was no significant interaction for cell inhibition rates and Annexin-V expression. CONCLUSION: Simvastatin in vitro inhibits the proliferation of NB4 cell, induces its differentiation and promotes its apoptosis. And the lowered expression of WT1 has a dose-dependent correlation with the elevated expression of hDMP1. It indicates that simvastatin has the synergistic in vitro anti-promyelocytic potency.

The significance of detecting WT1 expression in childhood acute leukemias.

WT1 (Wilms' tumor gene 1) overexpression is implicated in the prognosis of acute leukemia. The purpose of this study was to investigate WT1 expression and its clinical implication in childhood acute leukemia (AL) in Chinese population. Bone marrow specimen from 200 children at different stages of acute leukemia and from 21 children without leukemia were studied. The WT1 expression at diagnostic marrow specimen in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) was higher than control group, whereas WT1 expression in AML was higher than in ALL, and WT1 expression level in relapse in ALL increased more significantly than in AML. The WT1 expression level showed positive correlation with the hypodiploidy and BCR-ABL fusion gene in acute leukemia. A rapidly decrease of WT1 expression level predicted a good response to the induction therapy and low expression of WT1 correlates with remission status. This study suggested that WT1 expression levels in acute leukemia can potentially be a marker for evaluating therapeutic efficacy, correlating with monitoring minimal residue disease, and predicting hematological relapse in children acute leukemia.

[Expression and Prognostic Value of MiR-155 in Patients with Chronic Lymphocytic Leukemia].

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.

A randomized phase II, open-label and multicenter study of combination regimens of bortezomib at two doses by subcutaneous injection for newly diagnosed multiple myeloma patients.

PURPOSE: Combinations of bortezomib (Velcade), cyclophosphamide and dexamethasone have shown significant efficacy and safety for patients of newly diagnosed multiple myeloma (NDMM). In this study, we compared the efficacy and safety of modified VCD regimens with novel changes in bortezomib dose and schedule for NDMM. METHODS: Eighty-five NDMM patients from multiple centers were randomly assigned to a high-dose (1.6 mg/m2) (group A) or a low-dose (1.3 mg/m2) (group B) bortezomib, administrated on days 1, 6, 11, and 16 subcutaneously in a 4-week cycle for nine cycles, combined with 40 mg dexamethasone on bortezomib days and cyclophosphamide 300 mg/m2 on days 1-3 intravenously. RESULTS: After four cycles, complete response (CR) or better in group A (43.6%) was higher than that in group B (12.8%) (P = 0.002). During induction, for patients with R-ISS stage III, the CR or better rate in group A was superior to that in group B (P = 0.01). Of patients < 65, the CR or better rate of group A was superior to that of group B (P = 0.004). Rapid onset of CR occurred in group A (P < 0.01). Meanwhile, rate of 3-4 diarrhea was higher in group A (P = 0.03), which caused higher rate of dose reduction for patients ≥ 65 (P = 0.041). No significant difference between the two groups in PFS and OS. CONCLUSIONS: The studied high-dose VCD as induction regimen had an improved CR rate, especially in patients < 65 or with R-ISS stage III, and is feasible for young and high-risk patients. Trial registration ClinicalTrials.gov: NCT02086942.

[Elimination of leukemic CD34+ progenitor cells by using cytotoxic T lymphocytes specifically targeting WT1-derived peptide: an experimental study in vitro].

OBJECTIVE: To investigate the effects of targeting elimination of the leukemic CD34+ progenitor cells by using cytotoxic T lymphocytes (CTLs) specifically against Wilms tumor gene (WT1)-derived peptide. METHODS: A 9-mer WT1 peptide (CMTWNQMNL) containing HLA-A * 0201-binding anchor motifs was synthesized. The suspended cells were collected from the culture of the peripheral blood mononuclear cells and divided into 2 groups: Group D (pure T cells) and Group C (IL2 + T cells). The dendritic cells (DCs) generated from the peripheral blood mononuclear cells of an HLA-A* 0201-positive healthy donor were repeatedly loaded with WT1 peptide so as to elicit cytotoxic T cells (CTLs) specifically for WT1 peptide, and restricted by HLA-A * 0201 (Group A). The specific lysis effects of WT1 peptide specific CTLs upon leukemic bone marrow CD34+ progenitor cells positively expressing WT1 (3 being HLA-A * 0201(+) and 3 being HLA-A*0201(-)), peripheral blood CD34+ cells from healthy persons (2 being HLA-A * 0201(+) and 1 being HLA-A * 0201(-)), and leukemia cells of the lines of NB4 (HLA-A * 0201(+)/WT1(+)), U937 (HLA-A * 0201(+)/WT1(-)), and K562 (HLA-A * 0201(-)/WT1(+)) were measured by methyl thiazolyl tetrazolium (MTT) chromatometry assay. The colony-forming unit-granulocyte macrophage (CFU-GM) forming capability of the leukemic bone marrow CD34+ progenitor cells and peripheral blood CD34+ cells from healthy persons treated with WT1 peptide specific CTLs were also determined by methylcellulose medium colony-forming unit assay. The CTLs elicited by DCs without WT1 peptide loading(Group B)and T cells cultured with IL-2 (Group C) were taken as controls. RESULTS: The CTLs specific for WT1 peptide and restricted by HLA-A * 0201 (Group A) exerted a specific lysis upon the 3 cases with HLA-A * 0201(+)/WT1(+) leukemic bone marrow CD34+ progenitor cells and NB4 leukemia cells, the specific lysis levels were 55.3% +/- 2.8%, 67.1% +/- 3.2%, 49.4% +/- 3.8% and 55.0% +/- 3.7% respectively, all significantly higher than those upon the 3 cases with HLA-A * 0201(-)/WT1(+) leukemic bone marrow CD34+ progenitor cells, normal healthy peripheral blood CD34+ cells of the healthy persons, and leukemia cell of the lines K562 and U937 (the specific lysis levels were all < 20%). The specific lysis level of Group A CTLs was significantly higher than those of Group B and Group C CTLs (both P < 0.01). The relative colony formation rates of WT1-CTLs in Group A upon the 2 cases with HLA-A * 0201(+)/WT1(+) leukemic CD34+ progenitor cells were 17.8% +/- 4.0% and 20.8% +/- 3.41% respectively, both significantly lower than those of the none WT1-CTLs in Group B (88.9% +/- 3.4% and 91.8% +/- 5.7% respectively, both P < 0.01). There was no significant difference in the relative colony formation rate upon HLA-A * 0201(+)/WT1(-) leukemic bone marrow CD34+ progenitor cells and the HLA-A * 0201(-)/WT1(-) or HLA-A * 0201(+)/WT1(-) normal peripheral blood CD34+ progenitor cells between the CTLs of Groups A and B. CONCLUSIONS: The CTLs specific for WT1 and restricted by HLA-A * 0201 can exert specific lysis upon leukemic CD34+ progenitor cells highly expressing WT1 gene, and can inhibit the CFU-GM colony formation of such leukemic CD34+ progenitor cells. The product of expression of WT1 gene may be used as a target in the leukemic CD34+ cells.

[All-Trans Retinoic Acid and Decitabine Synergistically Induce Anti-Leukemia Effect on U937 Cell Line and Newly Diagnosed Elder AML Patients].

OBJECTIVE: 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 ß (RARß), and to explore their combined anti neoplastic effect on U937 cells and newly diagnose delder acute myeloid leukemia(AML) patients. METHODS: The expression levels of p16 and RARß 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. RESULTS: The expression p16 and RARß 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 RARß, 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 RARß expression and decrease of bone marrow blast, moreover the parients showed good tolerence to the reginen. CONCLUSION: 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.

[Bone marrow WT1 gene expression and clinical significance in chronic myelogenous leukemia].

OBJECTIVE: To investigate the clinical significance of Wilms tumor gene (WT1) expression levels in the bone marrow (BM) of chronic myelogenous leukemia patients. METHODS: Real-time quantitative retroversion polymerase chain reaction (RQ-RT-PCR) method was established for detecting WT1 and GAPDH expression levels in the BM of 109 CML and 23 non-leukemic patients with Light Cycler. Normalized WT1 expression level (WT1(N)) was determined as a ratio between WT1 and GAPDH expression times 10(4). RESULTS: The median expression levels of WT1(N) in 23 CML patients of accelerate phase (CML-AP) and 22 CML patients of blast crisis (CML-BC) were statistically higher than those of 64 CML patients of chronic phase (CML-CP) and 23 non-leukemic controls (103.71 and 129.44 vs 3.44 and 1.47). No statistic differences were found between the CML-CP group and control group, nor between the CML-AP group and CML-BC group. Furthermore, the WT1(N) levels were correlated to the BCR/ABL fusion gene expression levels. Dynamic change of WT1(N) levels in 7 CML patients before or after allogeneic bone marrow transplantation showed that WT1 expression levels could predict relapse of leukemia. CONCLUSION: WT1 expression levels in CML patients in accelerate phase or blast crisis were strikingly higher than those in non-leukemic patients or CML patients in chronic phase, in whom WT1 expression was undetectable or showed low expression. WT1 gene could be an useful marker for detecting MRD and evaluating therapeutic efficacy in CML.

[Prognostic Significance of the Percentage of Blasts with CD34+/CD38low/-/CD123+ Phenotype in Acute Myeloid Leukemias].

OBJECTIVE: To investigate the percentage of blasts with the CD34+/CD38low/-/CD123+ phenotype in de novo acute myeloid leukemia (AML) patients and analyse its correlation with prognosis. METHODS: The percentage of CD34+/CD38low/-/CD123+ cells 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. RESULTS: The median percentage of CD34+/CD38low/-/CD123+ cells in newly diagnosed patients was 2.8% (ranged from 0.01 to 67%). The high expression of CD34+/CD38low/-/CD123+ in AML patients positively correlated with the NPM1 wild-type (χ2=5.194,P<0.05), but did not relate with the positive FLT3-ITD mutations (χ2=0.418,P>0.05). Further multivariable analysis showed that the higher expression of the CD34+/CD38low/-/CD123+ was 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. CONCLUSION: The percentage of CD34+/CD38low/-/CD123+ cells 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.

[Autologous Peripheral Blood Hematopoietic Stem Cell Transplantation with IBu Precondition Regimen in Treatment for 11 Patients with Low to Intermediate Risk Acute Myeloid Leukemia].

OBJECTIVE: 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). METHODS: 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/m2, 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. RESULTS: 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). CONCLUSION: The treatment of auto-PBHSCT with IBu precondition regimen for low to intermediate risk AML patients is safe and effective.

Decitabine enhances bortezomib treatment in RPMI 8226 multiple myeloma cells.

The present study investigated the interactions between decitabine (DAC) and bortezomib (BTZ) in RPMI 8226 multiple myeloma (MM) cells. Cells were exposed to DAC alone and in combination with BTZ for 48 h. A Cell Counting Kit­8 assay was performed to assess the rate of proliferation inhibition in the cells. Cell apoptosis was investigated by Annexin V-fluorescein isothiocyanate and propidium iodide staining. Flow cytometry was used to detect the different cell cycle stages. Western blotting was performed to analyze the protein expression levels of poly(ADP­ribose) polymerase 1 (PARP­1), caspase­3, ­9 and DNA (cytosine­5­)­methyltransferase 1 (DNMT1). Reverse transcription­quantitative polymerase chain reaction was used to assess DNMT1 gene expression. The combination of DAC and BTZ increased the proliferation inhibition, apoptotic rate and G0­G1 arrest compared with use of a single therapeutic agent. In addition, the combination treatment enhanced PARP­1 cleavage, caspase­3 and caspase­9 activation and downregulated the protein and mRNA expression levels of DNMT1. Therefore, the current study determined that the combination of BTZ and the epigenetic agent DAC may be a novel therapeutic strategy to improve the efficacy of BTZ in patients with MM.

High expression of S100A8 gene is associated with drug resistance to etoposide and poor prognosis in acute myeloid leukemia through influencing the apoptosis pathway.

S100A8 has been increasingly recognized as a biomarker in multiple solid tumors and has played pivotal roles in hematological malignancies. S100A8 is potentially an indicator for poor survival in acute myeloid leukemia (AML) in retrospective studies. However, the mechanisms of S100A8 are diverse in cancers. In this study, we investigated the correlation of S100A8 at the transcription level with clinical parameters in 91 de novo AML patients and explored its mechanisms of chemoresistance to etoposide in vitro. The transcription level of S100A8 was significantly lower at initial and relapse stages of AML samples than at complete remission (P<0.001) and than in the control group (P=0.0078), while no significant difference could be found between initial and relapse stages (P=0.257). Patients with high transcription levels of S100A8 exhibited a shorter overall survival (P=0.0012). HL-60 cells transfected with S100A8 showed resistance to etoposide with a higher level IC50 value and lower apoptosis rate compared with HL-60 cells transfected with empty vector. Thirty-six genes were significantly downregulated and 12 genes were significantly upregulated in S100A8 overexpression group compared with control group in which 360 genes involved in apoptotic genes array were performed by real-time reverse transcriptase polymerase chain reaction. Among them, the caspase-3, Bcl-2, and Bax were verified by Western blot analysis which indicated that the role of S100A8 in resistance to chemotherapy was closely related with antiapoptosis. In conclusion, critical S100A8 provided useful clinical information in predicting the outcome of AML. The main mechanism of S100A8 which promoted chemoresistance was antiapoptosis.