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BackgroundImmunity after SARS-CoV-2 infection or vaccination has been threatened by recently emerged SARS-CoV-2 variants. A systematic summary of the landscape of neutralizing antibodies against emerging variants is needed.MethodsWe systematically searched PubMed, Embase, Web of Science, and 3 pre-print servers for studies that evaluated neutralizing antibodies titers induced by previous infection or vaccination against SARS-CoV-2 variants and comprehensively collected individual data. We calculated lineage-specific GMTs across different study participants and types of neutralization assays.FindingsWe identified 56 studies, including 2,483 individuals and 8,590 neutralization tests, meeting the eligibility criteria. Compared with lineage B, we estimate a 1.5-fold (95% CI: 1.0-2.2) reduction in neutralization against the B.1.1.7, 8.7-fold (95% CI: 6.5-11.7) reduction against B.1.351 and 5.0-fold (95% CI: 4.0-6.2) reduction against P.1. The estimated neutralization reductions for B.1.351 compared to lineage B were 240.2-fold (95% CI: 124.0-465.6) reduction for non-replicating vector platform, 4.6-fold (95% CI: 4.0-5.2) reduction for RNA platform, and 1.6-fold (95% CI: 1.2-2.1) reduction for protein subunit platform. The neutralizing antibodies induced by administration of inactivated vaccines and mRNA vaccines against lineage P.1 were also remarkably reduced by an average of 5.9-fold (95% CI: 3.7-9.3) and 1.5-fold (95% CI: 1.2-1.9).InterpretationOur findings indicate that the antibody response established by natural infection or vaccination might be able to effectively neutralize B.1.1.7, but neutralizing titers against B.1.351 and P.1 suffered large reductions. Standardized protocols for neutralization assays, as well as updating immune-based prevention and treatment, are needed.FundingChinese National Science Fund for Distinguished Young ScholarsResearch in contextEvidence before this studySeveral newly emerged SARS-CoV-2 variants have raised significant concerns globally, and there is concern that SARS-CoV-2 variants can evade immune responses that are based on the prototype strain. It is not known to what extent do emerging SARS-CoV-2 variants escape the immune response induced by previous infection or vaccination. However, existing studies of neutralizing potency against SARS-CoV-2 variants are based on limited numbers of samples and lack comparability between different laboratory methods. Furthermore, there are no studies providing whole picture of neutralizing antibodies induced by prior infections or vaccination against emerging variants. Therefore, we systematically reviewed and quantitively synthesized evidence on the degree to which antibodies from previous SARS-CoV-2 infection or vaccination effectively neutralize variants.Added value of this studyIn this study, 56 studies, including 2,483 individuals and 8,590 neutralization tests, were identified. Antibodies from natural infection or vaccination are likely to effectively neutralize B.1.1.7, but neutralizing titers against B.1.351 and P.1 suffered large reductions. Lineage B.1.351 escaped natural-infection-mediated neutralization the most, with GMT of 79.2 (95% CI: 68.5-91.6), while neutralizing antibody titers against the B.1.1.7 variant were largely preserved (254.6, 95% CI: 214.1-302.8). Compared with lineage B, we estimate a 1.5-fold (95% CI: 1.0-2.2) reduction in neutralization against the B.1.1.7, 8.7-fold (95% CI: 6.5-11.7) reduction against B.1.351 and 5.0-fold (95% CI: 4.0-6.2) reduction against P.1. The neutralizing antibody response after vaccinating with non-replicating vector vaccines against lineage B.1.351 was worse than responses elicited by vaccines on other platforms, with levels lower than that of individuals who were previously infected. The neutralizing antibodies induced by administration of inactivated vaccines and mRNA vaccines against lineage P.1 were also remarkably reduced by an average of 5.9-fold (95% CI: 3.7-9.3) and 1.5-fold (95% CI: 1.2-1.9).Implications of all the available evidenceOur findings indicate that antibodies from natural infection of the parent lineage of SARS-CoV-2 or vaccination may be less able to neutralize some emerging variants, and antibody-based therapies may need to be updated. Furthermore, standardized protocols for neutralizing antibody testing against SARS-CoV-2 are needed to reduce lab-to-lab variations, thus facilitating comparability and interpretability across studies.

Isochromosome 20q- (i(20q-)), as a rare reproducible chromosomal anomaly formed on the basis of 20q-, has not been commonly reported. Due to the rarity of this karyotypic anomaly, the bone marrow morphological characteristics of the patients with i(20q-) have not been clarified until now. In this study, the bone marrow cell morphology from MDS patients with isolated i(20q-), isolated 20q-, and normal karyotype was retrospectively compared and statistically analyzed. The results indicated that the isolated i(20q-) was mostly detected in MDS-MLD patients. The frequency and proportion dysplasia of cytoplasmic vacuolization in erythoid cells and small or unusually large size in myeloid cells of isolated i(20q-) MDS patients were significantly higher than those of normal karyotype MDS patients respectively (P

To analyze the effect of clinical features, routine laboratory examination and related gene mutation on the OS of patients with myelodysplastic syndrome (MDS) after hematopoietic stem cell transplantation (HSCT).121 patients diagnosed as MDS and underwent hematopoietic stem cell transplantation in the First Affiliated Hospital of Soochow University from October 2013 to August 2018 were selected. Basic information of the patients was collected, and blood cells, bone marrow blasts at initial diagnosis, chromosomal karyotypes and gene mutations of the patients were detected.The effect of different factors on overall survival (OS) was analyzed by statistical method.Kaplan-Meier univariate analysis shows that OS was significanly different among different age groups. The 3-year OS rate of patients aged 0-29 years was (83.3±7.7) %, the 3-year OS rate in patients aged 30-49 years was (58.1±7.7 %), and the 3-year OS rate of patients aged 50-69 years was (31.0±22.6) %, which was statistically different (P＜0.05) between different groups. There were also significant differences in OS among patients with different transplantation types. 3-year OS rate: HLA-matched sibling HSCT＞unrelated HLA-matched HSCT＞haploidentical HSCT＞micro HSCT. The OS rate of patients with bone marrow blasts≥10% seems lower than blasts＜10%, but there was no statistical difference.The 3-year OS rate of patients with chromosomal karyotype complex abnormality was (47.7±11.5) %, and that of patients without complex abnormality was (80±4.2) % which was statistical difference (P＜0.05). Patients with DNMT3A, NRAS, TP53 and GATA2 mutations had shorter OS time compared with patients without mutation of these genes, which shows statistically significant (P＜0.05). COX multivariate analysis showed that age, chromosome karyotype, DNMT3A, TET2, GATA2 and NRAS were the independent factors influencing OS of patients after HSCT, with statistically significant difference.age of patients, donor selection of HSCT, chromosome karyotype, DNMT3A, NRAS, TP53, GATA2 and TET2 gene mutations are all independent factors affecting the OS of patients after HSCT. Therefore, the assessment of the OS of MDS patients with transplantation requires comprehensive consideration.影响造血干细胞移植的骨髓增生异常综合征患者 生存期的因素分析.分析进行造血干细胞移植的骨髓增生异常综合征（myelodysplastic syndrome，MDS）患者的临床特点、常规实验室检查及相关基因突变对总生存期（overall survival，OS）的影响.选取并回顾性分析2013年10月至2018年8月在苏州大学附属第一医院诊断MDS并进行造血干细胞移植（hematopoietic stem cell transplantation，HSCT）的患者121例，收集患者基本信息，并检测患者血细胞、初诊骨髓原始细胞、染色体核型及基因突变情况。用统计学方法分析不同因素对生存期的影响.Kaplan-Meier单因素分析发现，不同年龄段之间OS有明显差异，患者年龄越高，3年OS率越低，0-29岁患者3年OS率为（83.3±7.7）% ；30-49岁患者3年OS率为（58.1±7.7）%；50-69岁患者均3年OS率为（31.0±22.6）%, 且各组之间OS率统计学差异显著（P＜0.05）。不同移植类型对患者OS也有显著影响，3年OS率：同胞全相合HSCT＞非血缘全相合HSCT＞单倍体相合HSCT＞微移植。骨髓原始细胞≥10%的患者OS率低于＜10%的患者，但差异没有统计学意义。染色体核型复杂异常患者3年OS率为（47.7±11.5）%，非复杂异常患者的3年OS率为（80±4.2）%（P＜0.05）。DNMT3A、NRAS、TP53、GATA2基因非突变患者比基因突变的患者移植后OS期长，差异有统计学意义（P＜0.05）。COX多因素分析结果显示，年龄、染色体核型、DNMT3A、TET2、GATA2、NRAS对MDS移植后OS有独立影响，且差异具有统计学意义.患者年龄，造血干细胞移植供体选择，患者初诊染色体核型，DNMT3A、NRAS、TP53、GATA2、TET2基因突变均是影响MDS移植后生存期的独立因素。因此对MDS移植患者生存期的评估需要综合多方面考虑.

SummaryBeego is a traditional Chinese complete water-only fasting practice initially developed for spiritual purposes, later extending to physical fitness purposes. Beego notably includes a psychological induction component that includes meditation and abdominal breathing, light body exercise, and ends with a specific gradual refeeding program before returning to a normal diet. Beego has regained its popularity in recent decades in China as a strategy for helping people in subhealthy conditions or with metabolic syndrome, but we are unaware of any studies examining the biological effects of this practice. To address this, we here performed a longitudinal study of beego comprising fasting (7 and 14 day cohorts) and a 7-day programmed refeeding phase. In addition to detecting improvements in cardiovascular physiology and selective reduction of blood pressure in hypertensive subjects, we observed that beego decreased blood triacylglycerol (TG) selectively in TG-high subjects and increased cholesterol in all subjects during fasting; however, the cholesterol levels were normalized after completion of the refeeding program. Strikingly, beego reduced platelet formation, activation, aggregation, and degranulation, resulting in an alleviated thrombosis risk, yet maintained hemostasis by sustaining levels of coagulation factors and other hemostatic proteins. Mechanistically, we speculate that downregulation of G6B and MYL9 may influence the observed beego-mediated reduction in platelets. Fundamentally, our study supports that supervised beego reduces thrombosis risk without compromising hemostasis capacity. Moreover, our results support that beego under medical supervision can be implemented as noninvasive intervention for reducing thrombosis risk, and suggest several lines of intriguing inquiry for future studies about this fasting practice (http://www.chictr.org.cn/index.aspx, number, ChiCTR1900027451).

Relapse is the major cause of mortality in patients with acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Effective preventive intervention in high-risk AML may be crucial. In this study, we investigated the clinical efficacy and safety of low dose decitabine (DAC) as part of a modified Busulfan-Cyclophosphamide (Bu-Cy) regimen for high-risk AML patients undergoing allo-HSCT to reduce relapse rate. Fifty-nine patients received DAC (20 mg/m2/d, i.v.) for 5 days, followed by modified Bu-Cy (DAC group). A matched-pair control (CON) group of 177 patients (matched 1:3) received modified Bu-Cy only. The differences were more substantial among patients with active disease: 2-year OS, 80.7% (DAC) versus 43.5% (CON), P = 0.011 and 2-year LFS, 64.9% (DAC) versus 39.2% (CON), P = 0.024. Median time to relapse was 8 months (DAC) versus 5 months (CON) for the entire groups and 6.5 months (DAC) versus 3.5 months (CON) for patients with active disease. In summary, our data indicated that the conditioning regimen containing low dose DAC may confer a survival advantage in high-risk AML patients with active disease undergoing allo-HSCT, and a prospective randomized trial is warranted to confirm these observations.

ObjectiveThe outbreak of novel coronavirus disease 2019 (COVID-19) imposed a substantial health burden in mainland China and remains a global epidemic threat. Our objectives are to assess the case fatality risk (CFR) among COVID-19 patients detected in mainland China, stratified by clinical category and age group.MethodsWe collected individual information on laboratory-confirmed COVID-19 cases from publicly available official sources from December 29, 2019 to February 23, 2020. We explored the risk factors associated with mortality. We used methods accounting for right-censoring and survival analyses to estimate the CFR among detected cases.ResultsOf 12,863 cases reported outside Hubei, we obtained individual records for 9,651 cases, including 62 deaths and 1,449 discharged cases. The deceased were significantly older than discharged cases (median age: 77 vs 39 years, pConclusionsOur estimates of CFR based on laboratory-confirmed cases ascertained outside of Hubei suggest that COVID-19 is not as severe as severe acute respiratory syndrome and Middle East respiratory syndrome, but more similar to the mortality risk of 2009 H1N1 influenza pandemic in hospitalized patients. The fatality risk of COVID-19 is higher in males and increases with age. Our study improves the severity assessment of the ongoing epidemic and can inform the COVID-19 outbreak response in China and beyond.

The physiological hematopoiesis depends on the programmed expression of a series of gene regulated by mechanisms at various levels. Currently, the epigenetic regulation has been considered as the most important mechanism during hematopoietic differentiation, resulting in a specific epigenomic landscape in the hematopoietic stem/progenitor cells. We try to concisely review the epigenetic mechanisms, including the genomic methylation, the histone modifications and the expression profiles of noncoding RNA, illustrating briefly the differentiation from the hematopoietic stem/progenitor cells to to the erythroid, myeloid and lymphoid cells.正常生理造血的表观遗传学调控和表观基因组景观.造血干细胞向各系列终末造血细胞的发育分化由一系列基因的有序表达所主导，当前对基因表达的调控机理的研究和认识已深入到表观遗传学和表观基因组层面。本文从基因组甲基化水平、染色质组蛋白修饰标识和非编码RNA的表达态势等方面，对正常造血系统从造血干/祖细胞向红系、髓系和淋系发育分化过程中的表观遗传学调控机理和表观基因组景观作一简明的综述。.

Due to chemoresistance and metastasis, the overall prognosis of osteosarcoma (OS) has not improved over the last two decades. Exploring novel therapeutic agents that can circumvent theses malignant phenotypes of OS would be essential to improve the survival of OS patients. Triptolide is a unique diterpene triepoxide that possesses potent antitumor activities.However, the effects and mechanism of triptolide on OS cells remain unknown. The effects of triptolide on viability, apoptosis, cell cycle distribution and migratory ability of OS cells were measured using MTT, flow cytometry and wound healing and transwell invasion assays. And an OS tumor xenograft mouse model was produced to further study the in vivo antitumor effects of triptolide. The expression of DUSP1 at the protein and mRNA level in OS cells was detected by western blot and qPCR. We report that triptolide exhibits multidimensional antitumor activities in OS cells, including the induction of apoptosis and G1 phase accumulation, inhibition of cell viability, migration, and invasion. We further demonstrate that triptolide inhibits the expression of dual-specificity protein phosphatase1 (DUSP1) through inhibiting its promoter activity, which causes sustained activation of three subfamilies of mitogen-activated protein kinase (MAPK). And the modulation of DUSP1/MAPK cascade is associated with the apoptosis of OS cells, since the ectopic expression of DUSP1 or the inhibition of MAPK using specific inhibitors can counteract triptolide-induced apoptosis. In addition, triptolide enhances doxorubicin-induced apoptosis. In summary, our study suggests that DUSP1 is an important cellular target of triptolide, and triptolide may be a promising treatment option for OS as a single agent or combined with other chemotherapeutics.

Background: miRNAs are a class of small noncoding RNA molecules that regulate gene expression by degradation of target mRNAs or inhibition of translation. Metastasis is one of the major problems that give obstacles to improve the clinical outcome of breast cancer. However, the underlying molecular mechanism of breast cancer metastasis remains largely unclear. Emerging studies have demonstrated that miRNAs play an important part in tumor invasion and metastasis. In the present study, we aimed to investigate the role and mechanism of miR-200c-3p in regulating the migration and invasion of paclitaxel-resistant breast cancer cells, and identify potential targets for breast cancer treatments. Material and methods: Paclitaxel-resistant breast cancer cell line MCF-7/Tax was established by stepwise selection in increasing concentration of paclitaxel. The 50% inhibitory concentration and cell viability were measured by the MTT assay. The expression level of miR-200c-3p in MCF-7/Tax and the parental MCF-7 cells was assessed by quantitative real-time RT-PCR. Gain-of-function and loss-of-function study on cell migration and invasion abilities were carried out by transfection of miR-200c-3p mimics or inhibitors respectively. The molecular target of miR-200c-3p was verified by dual-luciferase reporter assay. Results: Paclitaxel-resistant breast cancer cell line MCF-7/Tax was established. Compared with parental MCF-7 cells, expression of miR-200c-3p determined by real-time RT-PCR was significantly down-regulated in paclitaxel-resistant MCF-7/Tax cells. The ability of migration and invasion of paclitaxel-resistant MCF-7/Tax cells was obviously increased as compared to parental MCF-7 cells. In addition, we found that up-regulation of miR-200c-3p expression inhibited the migration and invasion of MCF-7/Tax cells, while down-regulation of miR-200c-3p expression increased the migration and invasion of MCF-7/Tax cells. Dual-luciferase reporter assay demonstrated that SOX2 was one of the direct targets of miR-200c-3p in breast cancer. Conclusions: Our results indicate that reduced expression of miR-200c-3p plays a crucial role in cell migration and invasion of paclitaxel-resistant breast cancer cell, and miR-200c-3p may suppress the cell migration and invasion possibly partially through SOX2. Our findings suggest that miR-200c-3p may serve as a promising therapeutic target for breast cancer metastasis. Keywords miRNA · Breast cancer ·Invasion Acknowledgments: The project supported by the grant of the Natural Science Foundation of Zhejiang Province (No.LQ13H160016), and the Medical Sciences and Technology of Zhejiang Province (No.2013KYA026, No.2015DTA004). Citation Format: Wang X, Chen J, Chen Z, Huang J, Ye W. Reduced expression of miR-200c-3p contributes to cell migration and invasion in paclitaxel-resistant breast cancer cell [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-07-07.

Breast cancer, especially triple-negative breast cancer, is one of the deadliest cancers in women. To date, there is a lack of a good therapeutic regimen for it. PPARγ has been reported to be a tumor suppressor and could be activated by many agonists involved in cancer inhibition. Therefore, the expression of PPARγ in breast cancer was analyzed by online software UALCAN whose data were from the TCGA database. The results revealed that the PPARγ expression was reduced in breast cancer tissues. Furthermore, the methylation in the PPARγ promoter was also assayed and the results indicated that the methylation level in the PPARγ promoter in breast cancer tissue was higher than that in normal tissue. In order to verify the methylation in promoter involved in the regulation of gene PPARγ expression, the 5'-Aza and fluorescence assays were performed and the results proved that methylation in promoter participated in gene PPARγ expression regulation. Pioglitazone, a PPARγ agonist, still was not investigated in breast cancer. Therefore, the effects of pioglitazone on breast cancer cells were tested by cell viability, scratch and transwell assays, and results indicated that the pioglitazone has the inhibition effect on the proliferation and migration of breast cancer cells by PPARγ which was correlated with the JAK2/STAT3 pathway. In order to further confirm the inhibition effect of pioglitazone on breast cancer in vivo, the nude mice model was administrated by gavage with pioglitazone. And the results indicated that pioglitazone could inhibit the growth of breast cancer in the PPARγ overexpression group in vivo. In summary, the expression of gene PPARγ was decreased in breast cancer tissues, which was correlated with its methylation in the promoter region. Moreover, pioglitazone could exert its inhibition on breast cancer proliferation and migration by the JAK2/STAT3 pathway.

Background BCL11A encodes a C2H2 type zinc-finger protein. During normal haematopoietic cell differentiation BCL11A expression is down-regulated. Data in mice suggest up-regulation of BCL11A is involved in the pathogenesis of myeloid leukaemias. BCL11A expression in persons with acute myeloid leukaemia (AML) is not systematically studied. Objective Interrogate associations between BCL11A expression at diagnosis and clinical and laboratory valuables and outcomes in newly-diagnosed persons with AML. Methods We determined BCL11A mRNA levels in bone marrow and blood mononuclear cells in 292 consecutive newly-diagnosed subjects with AML by reverse transcript and real-time polymerase chain reaction. Data were compared to mRNA levels in bone marrow cells of normals. Results Subjects with BCL11A transcript levels at diagnosis exceeding the median value of 2.434 (±3.423 SD; 25th–75th inter-quartile range, 1.33–4.29) had higher WBC levels, a greater proportion of bone marrow myeloblasts, were more likely to be FAB M0 subtype, less likely to be FAB M3 subtype, more likely to be in the intermediate cytogenetic risk cohort, less likely to have a complex karyotype and more likely to have DNMT3AR882 and FLT3-ITD mutations than subjects with transcript levels below the median value. In 89 subjects receiving conventional induction chemotherapy the complete remission rate was 54% (95% confidence interval [CI]; 33, 75%) in the lower BCL11A cohort and 65% (45, 85%; P = 0.26) in the higher BCL11A cohort. 3 year survival was 33% (2, 65%) in the lower BCL11A cohort and 15% (0, 39%; P = 0.35) in the high BCL11A cohort. Conclusion BCL11A transcript levels at diagnosis was significantly associated with several clinical and laboratory variables. There were also non-significant associations with complete remission rate and survival. These data suggest a possible role for BCL11A expression in AML biology.

The aim of the present study was to investigate the effect of 5-aza-2′-deoxycytidine (decitabine; DAC) and all-trans retinoic acid (ATRA) on Wilms' tumor 1 (WT1) in acute myeloid leukemia (AML) in vitro. The methylation status of the WT1 promoter was analyzed using methylation-specific polymerase chain reaction (MSP). The expression level of WT1 was detected by reverse transcription-quantitative polymerase chain reaction. The effect of DAC and ATRA on cell differentiation was evaluated by flow cytometry. The WT1 gene was methylated in U937 cells, but unmethylated in SHI-1 and K562 cells; the U937 cells did not express the WT1 gene, but the SHI-1 and K562 cells highly expressed the WT1 gene. DAC and ATRA, alone or in combination, exhibited no effect on the expression level of WT1 in the U937 cells and on the differentiation of the K562 cells. The combined treatment of DAC and ATRA markedly decreased the WT1 expression levels of the SHI-1 and K562 cells, and induced the differentiation of the SHI-1 and U937 cells. In the SHI-1 cells, WT1 expression changed inversely to the dynamic changes of cluster of differentiation 11b-positive rates. In conclusion, the combined treatment of DAC and ATRA has clinical therapeutic potential in acute monocytic leukemia patients with high WT1 expression and a poor response to standard induction chemotherapy.

Previous studies on the pathogenesis of myelodysplastic syndrome (MDS) have identified multiple associated gene mutations, including mutations of tetmethylcytosinedioxygenase 2, isocitrate dehydrogenase [NADP(+)] 1 cytosolic, isocitrate dehydrogenase [NADP(+)] 2 mitochondrial and additional sex combs like 1 transcriptional regulator, all of which may be considered epigenetic regulators. Furthermore, mutations of RAS type GTPase family genes have been identified in 10–15% patients with MDS. The authors' previous study on the gene expression profile of cluster of differentiation 34+ cells using microarray analysis identified elevated expression of RAP1GTPase activating protein 1 (Rap1GAP) in patients with MDS compared with that in non-malignant blood diseases (NM) control group. To further investigate the mechanism of increased Rap1GAP expression, the methylation pattern of the promoter of this gene was determined in 86 patients with MDS (n=29), acute myeloid leukemia (AML) (n=31) or NM (n=26) using bisulfite-specific polymerase chain reaction and DNA sequencing. The results demonstrated that the methylation of Rap1GAP occurred in all 29 patients with MDS at multiple CpG sites. The methylation level of Rap1GAP in patients with MDS was decreased compared with that in patients with NM. Significant differences at 4CpG sites (5,7,8 and 12) of Rap1GAP promoter were identified between MDS and NM. Furthermore, based on the present clinical records of the patient cohort, the methylation status of Rap1GAP promoter did not appear to be associated with the clinicopathological characteristics of patients with MDS, including age, gender and International Prognosis Score System. The difference in methylation level at CpG site 8 of Rap1GAP promoter was identified to be significantly increased in patients with MDS-refractory anemia with ring sideroblasts compared with that in the MDS-refractory cytopenia with multilineage dysplasia or MDS-unclassified groups. The results of the present study suggest that patients with MDS exhibit a lower overall methylation level within Rap1GAP promoter compared with patients with NM or AML. In addition, the methylation level at the four identified CpG sites can distinguish between MDS and NM.

Background Increasing evidence suggests that neutrophils play a critical role in tumorigenesis, tumour cell proliferation and metastasis. The prognostic significance of such inflammation-associated markers has been explored in different cancers. Aim To evaluate the prognostic effect of baseline neutrophil counts and nadir neutrophils on advanced gastric cancer (AGC) patients who were treated with two different chemotherapy regimens in our institution. Methods Data were collected retrospectively for 260 AGC patients treated between 1 February 2009 and 31 December 2011. The prognostic effect of baseline neutrophil counts and nadir neutrophils on AGC patients was evaluated. Results Approximately 79% of the patients experienced neutropenia during chemotherapy. The median survival was 369 days for patients with neutrophil counts ≤7.5 × 109/L and 326 days for patients with neutrophil counts >7.5 × 109/L (P < 0.001).The median survival was 340 days for patients with no neutropenia (grade 0), 422 days for patients with mild neutropenia (grade 1–2) and 339 days for patients with severe neutropenia (grade 3–4) (P < 0.001).The adjusted hazard ratios (HR) for mild and severe neutropenia compared with absent neutropenia were 0.572 (P = 0.002) and 1.246 (P = 0.219) respectively. Furthermore, it was suggested that pretreatment baseline neutrophil counts ≤7.5 × 109/L may be an independent predictor (HR = 0.683; P = 0.005). We also observed that other factors were independently associated with worse survival, such as higher performance status, stage IV and the presence of ascites. Conclusion Our findings suggest that baseline neutrophil count and chemotherapy-induced neutropenia can be conveniently available as clinical biomarkers in AGC. Mild myelosuppression in patients with AGC most likely leads to better overall survival, whereas a high baseline neutrophil count may be associated with a worse prognosis.

Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem/progenitor cells. As bone marrow cells are extremely diverse in these disorders, the origin and evolution of MDS clones are difficult to identify and trace. Cellular dysplasia is a distinct morphologic feature; however, whether the dysplastic cells represent abnormal clones or only nonspecific superficial phenomena remains to be clarified. To address this question, 97 patients were examined for dysplasia features, among them bone marrow slides of 16 patients with chromosomal abnormalities were subjected to fluorescence in situ hybridization (FISH) to determine the karyotype of these dysplastic cells. Furthermore, the emerging frequencies of abnormal karyotypes in various differentiated stages of each lineage were also evaluated by a combination of morphological evaluation and FISH karyotyping. Our results indicate that the overall percentage of dysplastic cells does not differ significantly among the WHO subtypes, while the megakaryoid lineage presents the most frequent dysplasia in all subtypes. A positive correlation between dysplastic cells and FISH-detectable abnormal clones was observed, but the dysplastic morphology was not a specific feature of FISH-detectable abnormal clones. FISH-detectable abnormal clones can differentiate into mature granulocytes and erythrocytes, in coexistence with cells originating from the normal clones.

Background: This study was conducted to determine the antineoplastic activities of 5-aza-2′-deoxycytidine (decitabine; DAC) and all-trans retinoic acid (ATRA), administered either alone or in combination, on in vitro cultured SHI-1 cells as well as their effects on the expression of the tumor suppressor gene p16INK4a (p16) and the retinoic acid receptor (RAR)-β. Methods: Cell growth inhibition, differentiation and apoptosis were determined in SHI-1 cells treated with DAC and/or ATRA, and the combination index of the two compounds was calculated. Methylation of the p16 and RAR-β genes in SHI-1 cells was detected by methylation-specific polymerase chain reaction (PCR). Real-time quantitative reverse transcriptase PCR was used to detect mRNA expression of the p16 and RAR-β genes, and Western blot analysis was performed for protein expression. Results: The drug combination had a synergistic effect on growth inhibition, differentiation and apoptosis of SHI-1 cells, and the effects of DAC and ATRA were dependent on time. DAC, either alone or in combination with ATRA, induced demethylation of the genes p16 and RAR-β, whereas ATRA alone had no effect on methylation. The RAR-β gene was reexpressed following DAC-ATRA combination treatment, and both agents had no effect on p16 expression. Conclusion: The results revealed that DAC used in combination with ATRA has significant clinical potential in the treatment of acute monocytic leukemia. © 2014 S. Karger AG, Basel

Numerous acquired molecular and cytogenetic abnormalities are strongly associated with hematological malignancies. The breakpoint cluster region-ABL proto-oncogene 1 (BCR-ABL) rearrangement leads to a p210 chimeric protein in typical chronic myeloid leukemia (CML), whereas 17–25% of patients with acute lymphocytic leukemia and 0.9–3% patients with de novo acute myeloid leukemia (AML) carry a p190BCR-ABL fusion protein. Cases of patients with AML/CML carrying two specific primary molecular changes, BCR-ABL and core binding factor-β-myosin heavy chain 11 (CBFβ-MYH11) fusion genes have been rarely reported. The present study aimed to understand the nature and mechanism of this particular type of leukemia through case reports and literature review. A total of four patients who were diagnosed as AML/CML with BCR-ABL and CBFβ-MYH11 fusion genes in the First Affiliated Hospital of Soochow University (Suzhou, China) between January 2004 and December 2012 were examined. Morphological analysis of bone marrow cells, flow cytometry, quantitative polymerase chain reaction of p210BCR-ABL and CBFβ-MYH11 transcripts as well as cytogenetic and fluorescence in situ hybridization analyses were performed. A total of 4 patients who exhibited fusion of p210BCR-ABL and CBFβ-MYH11 were identified. A single patient (case 1) was first diagnosed CML-acute phase (AP), which progressed rapidly to CML-blast crisis (BC), and three patients (cases 2, 3 and 4) were diagnosed with AML with bone marrow eosinophilia at first presentation with no evidence of previous onset of CML. All cases achieved remission following conventional chemotherapy/hematological stem cell transplantation combined with the inhibitor of tyrosine kinase (TKI) maintenance therapy. The patients with CML carrying and expressing BCR-ABL and CBFβ-MYH11 fusion genes appeared more likely to rapidly progress to AP or BC. Therefore, the product of the CBFβ-MYH11 fusion gene may serve an important role in the transformation of CML. The co-expression of p210BCR-ABL and CBFβ-MYH11 fusion genes in myeloid leukemia may be a molecular event occurring not only during the development of CML, but also in AML.

The aim of the current study was to investigate the antineoplastic activities of 5-aza-2′-deoxycytidine (also known as decitabine; DAC) and all-trans retinoic acid (ATRA), administered alone or in combination, in K562 cells in vitro, as well as the effects on the expression of the tumor suppressor genes, p16INK4a (p16) and retinoic acid receptor β (RAR-β). Cell growth inhibition, differentiation and apoptosis in K562 cells treated with DAC and/or ATRA were detected. The methylation of the p16 and RAR-β genes in the K562 cells was detected using the methylation-specific polymerase chain reaction (PCR) method. Quantitative PCR was used for the detection of the mRNA expression of the p16 and RAR-β genes, and western blot analysis was used to detect protein expression. DAC and ATRA, alone or in combination, had no effect on the growth inhibition, differentiation and apoptosis of the K562 cells. DAC alone induced the demethylation of the p16 gene, and combination of DAC and ATRA demonstrated more evident demethylation of the p16 gene, however, ATRA alone had no effect on methylation. The RAR-β promoter region was not methylated in the K562 cells. DAC in combination with ATRA appeared to produce a greater activation of the RAR-β gene, which led to the upregulation of the RAR-β expression level. ATRA enhanced the effect of DAC on p16 demethylation, and the combination of the two drugs was found to activate RAR-β expression, which indicated that DAC used in combination with ATRA has clinical potential in the treatment of human erythroleukemia.

Background: Everolimus has been testified to be effective among postmenopausal women with hormone receptor-positive human epidermal growth factor receptor-2 negative (HR+/HER2-) metastatic breast cancer (MBC). However, everolimus at dose of 10mg was accompanied by a higher incidence of mTOR-inhibitor class-effect adverse events (AEs) which usually leading to dose reduction or interruption. Additionally, some studies suggested that there was no correlation between dose intensity (5 vs. 10 mg labeled dose) and efficacy. Consequently, we conducted the present study aiming to explore the efficacy and safety of everolimus at dose of 5 mg plus endocrine therapy in Chinese population. Methods: 68 HR+/HER2- MBC patients were included in this exploratory study who received everolimus at the dose of 5mg in Zhejiang Cancer Hospital between September 2014 and September 2017. Progression free survival(PFS) and overall survival(OS) were estimated by the Kaplan- Meier method, and the hazard ratios(HRs) and corresponding 95% confidence intervals(CIs) were estimated using the Cox proportional harzard model. Besides, objective response rate(ORR), clinical benefitrate(CBR) and safety profile were also evaluated. Results: After a median follow up of 14 months, PFS was 5.3 months (95%CI 4.2-6.4), and OS was immature. 16(23.5%) were at the first or second-line, 52(76.5%) were at third-line or later. PFS for the first and second-line is significantly longer than that for the third-line or later (12.9 months vs. 4.6 months, P=0.009, HR=0.395, 95% CI=0.192-0.811). 11(16.2%) achieved partial response (PR), 42(61.7%) had stable disease (SD), and 15(22.1%) reported progressive disease (PD). The ORR and CBR were 16.2%, 35.2%, respectively. Most common all grade adverse events were stomatitis(26.5%), fatigue(10.0%), infection(11.8%), thrombocytopenia(5.9%), anemia(4.4%), hyperglycemia(4.4%). The most common ≥3 grade adverse events were stomatitis(4.4 %), infection(2.2%) and thrombocytopenia(1.1%). Conclusions: The combination of low-dose everolimus and endocrine therapy was highly effective especially at the earlier line in Chinese population. And the safety profiles were similar to previous studies but the incidences were greatly lower. The combination of low-dose everolimus and endocrine therapy may become a proper option for HR+/HER2- MBC. Citation Format: Shao X, Wang X, Chen Z, Zheng Y. Efficacy and safety of low-dose everolimus in Chinese HR-positive, HER2-negative metastatic breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-29.

The role of tissue inhibitor of metalloproteinase-2 (TIMP-2) in extramedullary infiltration of acute leukemia is unclear. We demonstrated in our previous study that the up-regulation of TIMP-2 promoted SHI-1 cell invasion in vitro. We investigated in the present study whether TIMP-2 would have the same effect in vivo. A retroviral vector carrying human TIMP-2 cDNA was constructed and transfected into SHI-1 cells. Three subclone cells (S1, S2 and S3) that highly expressed TIMP-2 were selected to establish nude mouse models of acute leukemia. Times of leukemic onset in mice of S1, S2 and S3 groups were all earlier than that of the SHI-1 group, whereas the survival times of S1, S2 and S3 groups were all shorter than that of the SHI-1 group (p0.05). Histopathological results demonstrated severe leukemic infiltration in numerous organs in each group. Reverse transcription polymerase chain reaction (RT-PCR) assay showed that several organs expressed the MLL/F6 fusion gene. Moreover, the numbers of organs infiltrated by leukemic cells in S1, S2 and S3 groups were more than those in the SHI-1 group (p0.05). Up-regulating TIMP-2 expression enhanced SHI-1 cell invasion in nude mice and resulted in more severe leukemia infiltration. This phenomenon suggests that targeted therapy with TIMP-2 for acute leukemia should be performed with prudence.

This study aimed to determine the clinical characteristics and prognostic significance of the meningioma 1 (MN1) gene and MN1-associated microRNA expression in Chinese adult de novo acute myeloid leukemia (AML) patients. The expression level of MN1, microRNA-20 (miR-20a), and microRNA-181b (miR-181b) in bone marrow mononuclear cells was measured in 158 newly diagnosed AML patients and 20 cases of normal healthy donors by real-time quantitative reverse transcriptase polymerase chain reaction. All AML patients significantly overexpressed MN1 at the level of 0.01983 (P

Xiao-yan Yang,1,* Ming-ying Zhang,1,* Qi Zhou,1 Shui-yan Wu,1 Ye Zhao,2 Wei-ying Gu,3 Jian Pan,1 Jian-nong Cen,2 Zi-xing Chen,2 Wen-ge Guo,4 Chien-shing Chen,5 Wen-hua Yan,6 Shao-yan Hu1 1Department of Haematology and Oncology, Children’s Hospital of Soochow University, 2Department of Haematology, The First Affiliated Hospital of Soochow University, Suzhou, 3Department of Haematology, The Third Affiliated Hospital of Soochow University, Changzhou, People’s Republic of China; 4Department of Mathematical Sciences, New Jersey Institute of Technology, Newark, NJ, 5Department of Internal Medicine, Division of Hematology and Medical Oncology and Biospecimen Laboratory, Loma Linda University, Loma Linda, CA, USA; 6Department of Cardiology, Children’s Hospital of Soochow University, Suzhou, People’s Republic ofChina *These authors contributed equally tothis work Abstract: 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

To explore the autophagy activity of CD34+ cells in bone marrow of MDS patients and its clinical significance.The activity of autophagy in bone marrow CD34+ cells from 20 MDS patients, 20 non-malignant anemia patients and 5 AML patients admitted in our hospital from October 2012 to March 2014 was detected by flow cytometry (FCM).The autophagy activity in low risk MDS patients and non-malignant anemia patients were both significantly higher than that in both high risk MDS and AML patients (P0.05), and more interestingly, the autophagy activity in MDS negatively correlated with World Health Organization classification-based prognostic system (WPSS) score (r=-0.877) .The autophagy activity CD34+ cells in the patients with MDS is higher than that in AML patients, and negatively correlated with WPSS scores, indicating that the decrease of autophagy activity maybe accelerate the genesis and development of MDS and relate with the prognosis of MDS patients.

Myelodysplastic syndrome (MDS) is a stem cell disease that has a characteristic morphological dysplasia. Adhesion molecules and the Wnt signaling pathway are mostly involved with the self-renewal, proliferation and differentiation of hematopoietic stem cells (HSCs) while Rho GTPases are closely correlated with the cytoskeleton and therefore cell morphology. To gain insight into the poorly understood pathophysiology of MDS, the present study focused on analyzing the gene expression profiles of these molecules with whole genomic array using CD34(+) cells from MDS patients. These profiles showed that N-cadherin, E-cadherin and c-myc binding protein tended to be downregulated, whereas β-catenin, Ras-proximate-1 GTPase-activating protein (Rap1GAP), c-myc promoter binding protein, Rac1, Rac2 and CDC42 tended to be upregulated. However, no change in the expression of genes involved in the canonical Wnt signaling pathway, with the exception of β-catenin, was observed. The array results were confirmed by real-time quantitative polymerase chain reaction (RQ-PCR) using CD34(+) cells from a cohort of patients with MDS-refractory anemia (RA) [WHO (2008) RCUD, RCMD and MDS-U] who had normal karyotypes. Only Rap1GAP and Rac2 showed higher expression levels when mononuclear cells were used from another group of patients with MDS-RA [WHO (2008) RCUD, RCMD and MDS-U] who also had normal karyotypes. We believe that the cadherin-β-catenin-c-myc signaling axis is crucial in the hematopoiesis of HSCs in the early stages of MDS. In addition, Ras-proximate-1 (Rap1), which is negatively regulated by Rap1GAP, may serve as an initiator of this axis through interplay with cadherin. This pathway is strengthened by the upregulation of Rac2, which may allow the nuclear translocation of β-catenin. The aberrant expression of Rho GTPases may also be responsible for the dysplasia characteristics observed in MDS. This study provides vital and new insights into the pathophysiology of MDS. The two small G proteins, Rap1GAP and Rac2, may act as new molecular markers for the diagnosis of MDS.

We report the fabrication of Al-doped ZnO thin-film transistors (FeFETs) on the ferroelectric Pb(Zr0.3Ti0.7)O3 (PZT) gate insulator for the application of nonvolatile random access memory. The results demonstrate the basic principle of Al-doped ZnO resistive switching between the high and low resistive states upon the polarization switching of ferroelectric layer. Own to the good ferroelectric property and high reliability of PZT, such as fatigue, high speed of signal reading and writing, low coercive electric field, etc., this device has an excellent electrical performance. The memory device exhibits a source-drain current modulation with an ON/OFF current ratio close 103.

The KIR2DS4 variants differ in exon 5 and play a role in hematopoietic stem cells transplantation (HSCT). A sequence-based testing (SBT) and TOPO TA cloning system identifying and distinguishing alleles of the KIR2DS4 gene was established and applied to a total of 150 Chinese-Han individuals: 75 patients received T-cell-depleted HSCT and their unrelated donors. The majority (139) of the 150 samples (92.7%) were positive for KIR2DS4. Four of the nine known KIR2DS4 alleles, KIR2DS4 *00101, *003,*004, and *007, were identified. In the haplotype A/A group, a higher risk of acute graft-versus-host disease (aGVHD) was seen when the donor carried two full-length KIR2DS4 alleles (RR 9.0 [95% CI 1.2-66.9], P = 0.010). Our findings suggested that the expression of full-length 2DS4 (*001) in A/A group may contribute to a worse clinical outcome after URD-HSCT. These data would be beneficial for the selection of suitable donors.

This abstract was not presented at the symposium.

The fragile histidine triad (FHIT) gene plays an important role in anti-cancer and the abnormal methylation of FHIT gene is found in many carcinomas. The epigenetic changes of tumor suppressor genes (TSG) are now recognized as an abnormal mechanism contributing to the development of myelodysplastic syndrome (MDS). To clarify the role of FHIT in MDS, we examined the methylation status of FHIT in patients with MDS. Methylation-specific polymerase (MSP) chain reaction was performed to detect the aberrant promoter methylation of FHIT gene in 55 patients with MDS. The abnormal methylation of the FHIT gene was found in 26 of 55 (47.2%) MDS cases, but it was not in normal control. No relationship was found between FHIT gene methylation and sex, hematologic parameters, chromosomal abnormalities of MDS patients. However, the significant difference was observed in the frequencies of FHIT gene hypermethylation among patients with RA/RARS (7/25, 28.0%), RAEB (11/18, 61.1%) and RAEBt (8/11, 72.7%) (chi2 value=7.938, P=0.019). Furthermore, there was a positive correlation between the frequency of FHIT gene hypermethylation and different IPSS groups (chi2 value=10.110, P=0.018). The MDS patients with FHIT gene methylation had significantly shorter survival time than those without FHIT methylation (20.0 months vs. 40.0 months, P=0.025). These results suggested that aberrant methylation of the FHIT gene might be one of molecular events involved in the disease progression of MDS and be an adverse prognostic factor in MDS.

Dear Editor: AML1-ETO fusion transcription factor is one of the most frequent chromosomal abnormalities detected in human acute myeloid leukaemia subtype 2 (M2). However, AML1-ETO alone is insufficient to develop leukaemia [1, 2]. To search for putative novel leukaemia associated genes, we therefore studied the gene expression profiles in AML-M2. BAALCas one of the genes found may be crucial in the pathogenesis of AML-M2. Some studies have reported that overexpression of BAALC gene was seen in patients with AML and ALL, and more strikingly, in a subset of AML with normal karyotype while predicting a poor prognosis [3–9]. However, its importance in AML-M2 patients with t(8;21) translocation had never been mentioned. To explore the function of BAALC in its possible role in the pathogenesis of human myeloid leukaemia subtype 2(M2) in a hope of finding one more useful biomarker for molecular diagnosis, we have examined the expression level of BAALC’m bone marrow mononuclear cells (MNCs) of patients with M2 by using quantitative real-time RT-PCR. Compared to 15 samples of normal bone marrow cells, the expression level of BAALC was significantly up-regulated in 18 pretreated M2 patients. The median level of BAALC transcripts in pretreated patients with AML-M2 and non-malignant blood diseases were 3.93 (ranging 0.71–8.94) and 0.04 (ranging 0.01–0.14), respectively (Fig.​(Fig.1).1). The abundance of BAALC mRNA relative to that of ABL mRNA in the cells from most M2 patients was significantly greater than that in the MNCs from non-malignant blood diseases (P < 0.001) (Fig.​(Fig.1,1, Table 3). Figure 1 Quantification of BAALC mRNAs in the BMNCs bone marrow samples from patients with AML-M2, or non-malignant blood disease. The cDNA prepared from the bone marrow cells of 33 patients, consisting of 18 pretreated AML-M2 cases, and 15 with non-malignant ... Table 3 Sequences of primers and TagMan probes for BAALC, AML1-ET0 and ABL Results from 61 samples of M2 patients at different stages showed that the expression pattern of BAALC was generally parallel to that of AML1-ETO fusion gene in AML-M2 patients with t(8;21) translocation. To evaluate the impact of BAALC expression values on clinical outcome without seeking an optimal cut point, the expanded cohort of AML patients were dichotomized at the median value and divided into two expression groups. In high BAALC group, samples appeared to synthesize more copies of AML-ETO transcript (n = 21; median expression = 9.66, range 0.5–27.5), compared with low BAALC group (n = 40; median expression = 0.45, range 0–12.1). The percentage of myeloid blast cells in bone marrow cells in high BAALC group was markedly higher than that in low BAALC group, namely 53.2%(n = 12) versus 7% (n = 26). In addition, the serum levels of lactic dehydrogenase (LDH) were also significantly different between these two groups. High level of LDH was 698.7 (n = 11) in high BAALC group versus 194.6 (n = 25) in low BAALC group (Fig.​(Fig.22). Figure 2 Statistical analysis on AML1-ETO expression, percentage of myeloid blast in BM and serum level of LDH in AML-M2 patients with different BAALC expression. AML patients with high BAALC expression show significantly higher AML1-ETO (A), percentage of myeloid ... Besides, for each individual patient, BAALC was expressed at a high level, at pretreated stage, while became lower in partial remission or complete remission. In 6 patients, the expression of BAALC was detected high in pretreated phase, and turned lower during remission phase. The same tendency was found in the expression of AML1-ETO (Fig.​(Fig.33). Figure 3 Correlation between BAALC expression and clinical phase BAALC were detected in pretreated phase and remission phase in 6 patients. AML-ETO was detected at the same time. In all patients, we have compared the expression level of BAALC among different clinical outcome groups, demonstrating significant differences (Table 1). In overall samples, the expression level of BAALC was positively correlated with the AML1-ETO transcription level (r = 0.63, P < 0.001). A positive correlation between BAALC expression level and the percentage of myeloid blasts (r = 0.68, P < 0.001), or LDH level (r = 0.39, P < 0.05) in the serum was also detected (Table 2). Table 1 Comparison of BAALC expression during different clinical stages Table 2 Correlation coefficient among the BAALC, AML-ETO, Blast percentage and LDH level Our present study has shown that the expression pattern of BAALC was generally parallel to AML1-ETO fusion gene in AML-M2 patients with t(8;21) translocation. The high BAALC group patients contained more myeloid blast cells in bone marrow, and had higher level of LDH in serum in comparison with low BAALC group. Besides, the high level of BAALC expression in patients at pretreated stage remarkably reduced to a significant low level after achievement of complete remission. Taking all these together, our results suggested that the AML-M2 patients in high BAALC expression group suffered a higher malignant burden and may have a poor prognosis [9]. Some reports showed that allogeneic haematopoietic stem cell transplantation might overcome the adverse prognostic effect of high BAALC expression [9]. This may assist to design a personal therapeutic plan in advance for those who expressed high level of BAALC at diagnose.

In this study, tissue-specific transcriptional activity of WT1 promoter and enhancer was investigated in a hope to design gene therapy strategy based on the transcriptional regulatory elements of WT1 gene. WT1 promoter and/or enhancer were ligated into pEGFP-1 to construct recombinant vectors with EGFP gene as a reporter and analyzed using Flow cytometry. The results showed that pEWP containingWT1 promoter induced the highest EGFP expression in ECV304 at 16.54 +/- 2.45 times, mildly higher in MCF-7 and SHG44 at 9.46 +/- 1.10 and 7.29 +/- 0.73 times, and in K562 cell line at 2.93 +/- 0.27 times, compared to that of pEGFP-1. However, pEWPA, with WT1 enhancer inserted at Afl II site, 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. pEWPD inserted at NotI and pEWPE inserted at BamI had no ability to increase the transcriptional activity of WT1 promoter. Moreover, reporter gene silenced was observed in transfected host cells by flow cytometryt and real-time PCR. These results suggested that transcriptional activities of WT1 promoter in the recombinant vector seemed not correlated to the constitutional expression level of endogenous WT1 gene. The WT1 enhancer could promote the transcriptional activities of WT1 promoter in some of the cell lines regardless of the hematopoietic tissue origin. The inserted site of enhancer in vector influenced the transcriptional activity of promoter and the extent of reporter gene silencing exerted the influence on the analysis of transcriptional activity.

BACKGROUND Bladder cancer is the most common type of urinary system tumours. It is frequently associated with genetic mutations that deregulate the cell cycle and render these tumours resistant to apoptosis. Survivin, a newly discovered member inhibitor of apoptosis protein (IAP) family in several human cancers, by inducing cell proliferation and inhibiting apoptosis is frequently activated in bladder cancer. We studied the influence of small interfering RNA (siRNA) targeting survivin on the biological behaviour of bladder cancer cells. METHODS A double strand survivin target sequence specific siRNA was designed and synthesized. After transfection of bladder cancer cell line T24 by siRNA/liposome complex with increasing concentrations (50200 nmol/L), the transfectant cells were intratumourally injected at different doses (5 microg or 50 microg). The effects were measured in vitro and in vivo. RESULTS The selected siRNA efficiently down-regulated survivin mRNA expression in a dose and time dependent manner. The maximal effect was achieved at the concentration of 100 nmol/L, at which survivin expression level was down-regulated by 75.91%. The inhibition rate of cell growth was 55.29% (P < 0.01) and the markedly increased apoptotic rate was 45.70% (P < 0.01). In vivo intratumoural injection of 50 microg siRNA-survivin could notably prevent the growth of bladder cancer (P < 0.01) in xenografted animals. CONCLUSION The application of siRNA-survivin could markedly inhibit survivin expression in bladder cancer cell line by inducing apoptosis and inhibiting the growth of the tumour. It may become a new gene therapy tool for bladder cancer.

We report the effect of rapid thermal annealing (RTA) on the optical properties of GaN0.01As0.99 samples grown by molecular beam epitaxy. In particular, a blueshift of the PL peak energy is observed when annealing the samples at 650–900 °C. Samples annealed showed pronounced enhancement in PL intensity as compared to the as-grown sample, and 850 °C is proposed as the optimum RTA temperature. The results are examined as a consequence of RTA-induced nitrogen diffusion inside the GaN0.01As0.99 material rather than diffusion out of the alloy, which homogenizes initial nitrogen composition fluctuations. In addition, for photo-modulated reflectance (PR) spectra of RTA samples, the fundamental band gap transition (E0) and the transition from the spin–orbit split-off valence band ( E 0 + Δ 0 ) are observed. Both of the two transitions increase with increasing annealing temperature.

We examined the effect of simvastatin (SV) alone and in combination with all-trans retinoic acid (ATRA) on proliferation, differentiation, apoptosis and apolipoprotein M (apoM) expression in the human promyelocytic leukemia cell line NB4. The NB4 cells were incubated with 10 μM Simvastatin (10SV) and 0.5 μM ATRA alone or in combination, taking NB4 cells without any treatment as normal controls. The cells of different groups were collected at 24, 48 and 72 h post-incubation for further detection. Their morphological changes were observed after Wright stain. MTT method was used to assay the growth inhibition rate and flow cytometry to detect CD11b expression level and the early stage apoptosis ratio. Real-time quantitative reverse transcriptase-polymerase chain reaction was used to detect the apoM gene expression levels. As expected 0.5 μM ATRA did not affect proliferation or apoptosis, strongly induced differentiation and decreased apoM expression. 10SV inhibited proliferation, increased apoptosis, induced differentiation and increased apoM expression in a time-dependent manner. The addition of ATRA to SV did not increase the effect of SV on proliferation and apoptosis, but increased the effect of SV on differentiation. And completely abrogated the effect of SV on apoM expression. Together these results show that SV has anti-leukemic properties by itself and that combined therapy may have a place in the current anti-leukemic arsenal.

Refractory thrombocytopenia (RTC) is an unusual subtype of myelodysplastic syndrome (MDS) that initially presents as chronic pure thrombocytopenia. Because of the lack of distinguishable dysplasia, RTC has often been misdiagnosed as idiopathic thrombocytopenic purpura. We describe the case of a patient with RTC and trisomy 8 for whom a bone marrow mononuclear cell (BMNC) gene expression profile was obtained by means of a complementary DNA microarray analysis. Compared with the healthy control subject, the RTC patient differentially expressed 105 genes, of which 88 were down-regulated and 17 were up-regulated. The expression pattern of 16 genes, including those for RNA helicase-related protein (RNAHP), heat shock 105kD (HSP105B), interferon-related developmental regulator 1 (IFRD1), cyclin C (CCNC), and DNA-damage-inducible transcript 3 (DDIT3), which are usually seen in BMNCs from typical MDS patients, was observed in this case. However, this RTC patient exhibited an expression pattern distinct from that of other MDS patients. We suggest that RTC be classified as a subtype of MDS on the basis of its characteristic clinical-hematologic features and specific molecular basis.

Purpose: The aim of this study was to develop a viable strategy to integrate photon plan and proton/carbon ion plan based on deformable registration. Methods: Two prostate cancer patients were enrolled in this study. Each patient has 2 CTs, which were input in the Raystation radiotherapy treatment planning system (TPS). CT1 was deformed to the second CT2 using the Hybrid deformation method. The dice similarity coefficient (DSC) parameter was used to evaluate the difference between the actual structures (bladder, rectum and CTV) and the corresponding deformed structures on CT2. The prescription dose was 63.02GyE to CTV, which included 49.32GyE for CTV1 with carbon and boost 13.7Gy for CTV2 with photon. The carbon plan was made first in Syngo TPS (Syngo PT Planning system, version VB10. Siemens, Germany) on CT1 and transferred to Raystation TPS. Selected Isodoses (23.5Gy, 36.8Gy, 39.1Gy, 47.0Gy and 49.3Gy) of carbon plan were converted to contours and then deformed to CT2, which was used as normal tissues for photon plan optimization on CT2. The final plan was the combination of photon plan and the carbon deformation plan on the CT2. The plan from this strategy was compared with direct optimization of the photon plan on CT2 added some clinical endpoints from carbon plan on CT1. Results: The new strategy with deformable registration is tested and combined plans were successfully obtained for the 2 patients. This strategy obtained both integrated DVH and dose distribution information. For patient 1, the rectum V30, V60 and bladder V63 were 45.8, 10.3 and 9.7 for the combined plan with deformation and 48.1, 11.0 and 12.0 for the direct photon plan. Conclusion: The new strategy for combining photon and carbon/proton is feasible. However, the clinical accuracy is still need more evaluation.

Objective: To investigate the feasibility of marking the human tumor cells with enhanced green fluorescent protein (EGFP) in vitro. Methods: The retroviral vector LGSN encoding EGFP was constructed and three human tumor cell lines were infected with LGSN amphotropic virus. Tumor cell lines that stably express EGFP were selected with G418. The integration and expression of EGFP gene were analyzed by polymerase chain reaction, and flow cytometry (FCM). Results: After gene transfection and ping-pong transduction, amphotropic producer line Am12/LGSN was generated with a stable green fluorescence signal readily detectable by FCM in up to 97% of examined cells. The viral titer in the supernatants was up to 8.2×105CFU/ml. After transduction and selection, G418-resistant leukemia K562, mammary carcinoma MCF-7, and bladder cancer 5637 cells were developed, in which the integration of both EGFP and neomycin resistance gene was confirmed by DNA amplification. In comparison with uninfected cells, FCM analysis revealed EGFP expression in up to 90% (range 85.5%–90.0%) of tumor cells containing LGSN provirus. Conclusion: The retroviral vector LGSN can effectively mark the human tumor cells with a stably EGFP expression which may be in studying tumor growth, metastasis and angiogenesis.

Most Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) patients often show rapid recurrence and development of ABL kinase domain (KD) mutation after tyrosine kinase inhibitor (TKI) treatment. To further investigate the mechanism of Ph(+) ALL fast relapse after TKI treatment, ABL KD mutation in 35 Chinese Ph(+) ALL with TKI resistance was detected by direct sequencing. The results showed that 77.1% (27/35) Ph(+) ALL patients with TKI resistance had ABL KD mutation and 55.6% (15/27) Ph(+) ALL patients with ABL KD mutation had T315I. Interestingly, 77.8% (21/27) Ph(+)ALL showed ABL mutation G: C→A:T, including T315I, E255K and E459K. Furthermore, all the Ph(+) ALL patients with two or more ABL KD mutations collaborated with complex chromosome abnormality and all the TKI-resistant Ph(+) ALL patients, whose karyotype progressed from simple t (9;22) into complex, developed ABL KD mutation. Moreover, the expression level of uracil-DNA glycosylase UNG2, which inhibits G:C→A:T transition in genomic DNA, decreased in Ph(+) ALL with TKI-resistance compared to that in newly diagnosis Ph(+) ALL. It is concluded that there is a high frequent ABL KD G:C→A:T mutation and a high genomic instability in Chinese TKI-resistant Ph(+) ALL. In addition, the decreased UNG2 expression in TKI-resistant Ph(+) ALL probably contributes to their high rate of ABL KD G:C→A:T mutation.

Essential thrombocythemia (ET) is a chronic myeloproliferative neoplasm (MPN) characterized by an increased number of platelets in the peripheral blood. Recently, somatic frameshift mutations at ex...

This study was aimed to investigate the effect of AML1-ETO fusion protein on the anti-apoptotic gene BCL-2 in leukemic cells and to explore its role in leukemogenesis. The apoptotic levels of U937-WT, U937-Mock and U937-A/E1-4 cells were examined by flow cytometry. And cleaved caspase-3 protein expression was detected by Western blot. BCL-2 gene expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and BCL-2 promoter in AML1-ETO positive leukemia cell line. The results indicated that in U937-A/E cells but not in U937-WT or U937-Mock cells, apoptotic cells statistically significantly increased, and AML1-ETO expression also significantly enhanced activation of caspase-3. AML1-ETO-expressing cell subclones displayed significantly low levels of BCL-2 mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, levels of BCL-2 mRNA were markedly lower as compared with other acute myeloid leukemias lacking this translocation. The enriched regions in transfected cells were located within BCL-2 promoter. It is concluded that BCL-2 is the direct target gene of AML1-ETO. AML1-ETO can down-regulate the expression of BCL-2.

Objective: To investigate retroviral-mediated transfer and expression of human multidrug resistance (MDR) gene MDR1 in leukemic cells. Methods: Human myeloid cells, K562 and NB4, were infected by MDR retrovirus from the producer PA317/HaMDR, and the resistant cells were selected with cytotoxic drug. The transfer and expression of MDR1 gene was analyzed by using polymerase chain reaction (PCR), flow cytometry (FCM) and semisolid colonies cultivation. Results: The resistant cells, K562/MDR and NB4/MDR, in which integration of the exogenous MDR1 gene was confirmed by PCR analysis, displayed a typical MDR phenotype. The expression of MDR1 transgene was detected on truncated as well as full-length transcripts. Moreover, the resistant cells were P-glycoprotein postiive at 78.0% to 98.7% analyzed with FCM. The transduction efficieny in K562 cells was studied on suspension cultures and single-cell colonies. The transduction was more efficient in coculture system (67.9%-72.5%) than in supernatant system (33.1%~46.8%), while growth factors may improve the efficiency. Conclusion: Retrovirus could allow a functional transfer and expression of MDR1 gene in human leukemia cells, and MDR1 might act as a dominant selectable gene for coexpression with the genes of interest in gene therapy.

Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O6-methylguanine-DNA-methyltransferase (MGMT) gene could increase resistance to 1,3-Bis(2- Chloroethyl)-l-Nitrosourea (BCNU). Methods: The eDNA encoding the MGMT was isolated by using RTPCR method from total RNA of fresh human liver, the fragment was cloned into pGEM-T vector and further subcloned into GINa retrovirus vector. Then the G1Na- MGMT was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, high titer amphotropic PA317 producer clone with the highest titer up to 5.8×105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMTcDNA under stimulation of hemopoietic growth factors. Results: The retrovirus vector construction was verified by restriction endonuclease analysis and DNA sequencing. PCR, RT-PCR, Southern Blot, Western Blot and MTT analyses showed that MGMT drug resistance gene has been integrated into the genomic DNA of cord blood CD34 + cells and expressed efficiently. The transgene cord blood CD34 + cells conferred 4-folds stronger resistance to BCNU than untransduced cells. Conclusion: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34+ cells and its expression provided an experimental foundation for gene therapy in clinical trial.

Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (NeoR) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1. The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells. Results: The titer of DOSPER directly transfected GP+envAm12 cells determined on NIH3T3 cells was 8.0×105 CFU/ml, while that of producer infected with retrovirus was 1.6×107 CFU/ml. Integration of NeoR gene into all leukemia cells was confirmed by polymerase chain reaction (PCR). Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated NeoR gene on colonies after 7 days’ culture. Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.

This study was aimed to explore the contribution of autophagy associated gene Beclin1 in the prognosis of myelodysplastic syndrome (MDS) by detecting the expression level of Beclin1 in bone marrow mononuclear cells (BMNC) from 40 MDS patients, 14 non-malignant anemia patients and 25 AML patients. The expression of Beclin1 mRNA was detected by real-time quantitative polymerase chain reaction (qRT-PCR). At the same time, the Western blot was used to analyze the expression of Beclin1 proteins. The results showed that the expression of Beclin1 in low risk MDS patients and non-malignant anemia patients was both significantly higher than that in acute myeloid leukemia patients (P0.01). And more interestingly, the Beclin1 mRNA expression in MDS group was negatively correlated with World Health Organization classification-based prognostic system (WPSS) score (r = -0.495). It is concluded that the expression of Beclin1 in the patients with MDS is higher than that in AML patients, and negatively correlated with WPSS scores. Beclin1 is a potential biomarker for predicting prognosis of the patients with MDS.