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To explore mutational characteristics of acute myeloid leukemia (AML) patients withA total of 62 AML patients withCompared withThe genomic landscape and clinical characteristics of AML patients with探讨采用高通量DNA测序技术检测62例与

To investigate the difference expression of circular RNA (circRNA) in acute myeloid leukemia (AML) by using bioinformatics method.The microarray chip data of AML was searched and downloaded from the Gene Expression Omnibus (GEO) of the National Center for Bioinformatics (NCBI). The differences between AML samples and control samples were analyzed by R software. The interaction between deregulated circRNA, miRNA and mRNA were predicted by miranda software and miRTarBase software. The circRNA-miRNA-mRNA regulatory network was constructed by using the cytoHubba plugin based on the Cytoscape software.A total of 203 differential expression of circRNAs were finally collected, including down-regulated 161 circRNAs and up-regulated 42 circRNAs. CircRNA/miRNA/mRNA interaction network was constructed through software prediction. hsa_circ_0001080, hsa_circ_0004511, hsa_circ_0054211, hsa_circ_0001944 may be positively regulated the gene expression in AML.Abnormal expression of circRNA in AML may become a new target for AML treatment.GEO数据库分析环状RNA在急性髓细胞白血病中的差异表达.采用生物信息学方法探究环状RNA（circRNA）在急性髓细胞白血病（AML）中的差异表达.从美国国家生物信息中心（NCBI）的基因表达综合库（GEO）下载AML的基因芯片数据，利用R语言软件对正常对照及AML样本进行差异分析，然后利用miranda软件和miRTarBase软件对差异表达circRNA、miRNA和mRNA之间的相互作用进行预测。通过Cytoscape插件构建circRNA-miRNA-mRNA关系对相关的ceRNA网络.通过差异分析，最终获得了203个差异表达circRNA，包括161个circRNA下调，42个circRNA上调；通过软件预测构建了circRNA/miRNA/mRNA互相作用网络；通过基因异常表达分析发现，hsa_circ_0001080、hsa_circ_0004511、hsa_circ_0054211、hsa_circ_0001944可能正向调控AML的基因表达.在AML中异常表达circRNA可能成为AML治疗的新靶点.

To investigate the mechanisms of anti-apoptosis and immune evasion in drug-resistant leukemia cells mediated by STAT3, further to explore the possible mechanism of leukemia relapse caused by minimal residual.Drug-resistance leukemia cell line was established by transfecting pcDNA3.1-STAT3 into K562 cells (K562/STAT3). The expression of STAT3, BAX and NKG2D ligands (MICA and ULBP1) in K562/-cells, K562/STAT3 were detected by Western blot and/or RQ-PCR. Cells apoptosis and the killing effect of NK cells on leukemia cells were detected by flow cytometry.The expression of the total STAT3, STAT3 phosphorylation in K562/STAT3 was significantly increased, and P-gp mRNA expression was increased also significantly (P0.005). In K562/STAT3 cells, the expression of pro-apoptotic BAX (P=0.005) was significantly lower, and the number of apoptotic cells (P=0.002) induced by adriamycin was significantly decreased as compared with those in K562/- cells. After K562/STAT3 cells were treated by STAT3 inhibitor (SH-4-54), the expression of BAX mRNA (P=0.017) was significantly higher and the number of apoptotic cells (P=0.005) was significantly increased. The MICA and ULBP1 mRNA expression in K562/STAT3 cells was significantly lower than that in K562/- cells, and also for MICA and ULBP1 protein (MICA and ULPB1 mRNA: P0.0001, MICA protein: P=0.001, ULPB1 protein: P=0.022). After K562/STAT3 cells were treated with STAT3 inhibitor (SH-4-54), the expression of MICA mRNA and protein was increased (mRNA: P=0.001, protein: P=0.002), but ULBP1 mRNA and protein showed no significantly change (mRNA: P=0.137, protein: P=0.1905). The cytotoxicity of NK cells to K562/STAT3 cells was susceptible as compared with K562/- (P=0.002), but the cytotoxicity of K562/STAT3 cells to NK cell could be recovered by STAT3 inhibitor (P=0.006).STAT3 phosphorylation can inhibits cell apoptosis and promotes cell immune escape. STAT3 inhibitors can promote the apoptosis of leukemia cells and increase their sensitivity to NK cells.STAT3介导耐药白血病细胞抗凋亡和免疫逃逸机制的研究.研究STAT3在耐药白血病细胞凋亡及免疫逃逸中的作用，并探讨白血病微小残留病引起的白血病复发的可能机制.用pcDNA3.1-STAT3质粒转染K562细胞，建立耐药白血病细胞株K562/STAT3细胞。RQ-PCR和（或）Western blot检测空载质粒转染的对照细胞株K562/-细胞和K562/STAT3细胞中STAT3、BAX以及NKG2D配体的表达。流式细胞术检测K562/-细胞和K562/STAT3细胞的凋亡情况及NK细胞对K562/-细胞和K562/STAT3细胞的杀伤作用.K562/STAT3细胞中总STAT3和STAT3磷酸化水平均明显升高，而且其耐药蛋白P-gp mRNA的表达明显升高（P0.005）。与K562/-细胞相比，K562/STAT3细胞促凋亡基因BAX mRNA的表达水平明显下降（P=0.005），且多柔比星引起的K562/STAT3细胞凋亡明显减少（P=0.002）；用STAT3抑制剂（SH-4-54）处理K562/STAT3细胞后，促凋亡基因BAX mRNA的表达水平明显升高（P=0.017），同时细胞凋亡也明显增加（P=0.005）。与K562/-细胞相比，K562/STAT3细胞NKG2D配体（MICA和ULBP1）mRNA的表达水平明显下降（P0.0001），MICA和ULBP1蛋白的表达水平亦明显下降（MICA：P=0.001，ULBP1：P=0.022）；用STAT3抑制剂（SH-4-54）处理K562/STAT3细胞后，MICA mRNA和蛋白表达水平升高（mRNA：P=0.001，蛋白：P=0.002），但ULBP1 mRNA和蛋白无明显改变（mRNA：P=0.137，蛋白：P=0.1905）。NK细胞对耐药K562/STAT3细胞的杀伤能力较K562/-细胞下降（P=0.002），但是STAT3抑制剂恢复了K562/STAT3细胞对NK细胞的杀伤敏感性（P=0.006）.STAT3磷酸化介导了耐药白血病细胞抗凋亡，降低其对NK细胞的杀伤敏感性。STAT3抑制剂可以促进耐药白血病细胞凋亡并增加其对NK细胞的杀伤敏感性.

To investigate the regulatory effects of Olaparib on natural killer cell activating receptor (NKG2D) ligands expression on human acute myeloid leukemia (AML) cell line HL-60, and to explore the molecular mechanism of Olaparib on HL-60 cells.After HL-60 cells in logarithmic growth phase were treated with Olaparib at different concentrations for different times (24, 48 h), the expression of NKG2D ligand on the surface of HL-60 cells was detected by flow cytometry. Western blot was used to dectect the expression of ERK expression in HL-60 cells. The killing effect of NK cells to HL-60 cells was detected by CFSE/PI method.10 μmol/L Olaparib could upregulate the expression of NKG2D ligand on the surface of HL-60 cell at 24 and 48 hours, while 5 μmol/L Olaparib could induce up-regulation of the expression of ULBP-2 and ULBP-3 at 48 hours. Western blot analysis showed that ERK phosphorylation of HL-60 cells was enhanced after treating with Olaparib. The killing effect of NK cells to HL-60 cells could be enhanced by Olaparib, however, ERK inhibitor could suppress the killing effect of NK cells to HL-60 cells.Olaparib can upregulate NKG2D ligands expression on the surface of HL-60 cells and enhance the cytotoxicity of NK cell to HL-60 cells. The mechanism may be related to Olaparib promoting ERK phosphorylation expression.Olaparib对HL-60细胞NKG2D配体表达的调节作用及相关机制研究.研究Olaparib对人急性髓系白血病细胞株HL-60细胞表面NKG2D配体表达的调节作用, 并初步探索其内在调控机制。.对数生长期的HL-60细胞经不同浓度Olaparib（1.25、2.5、5、10 μmol/L）作用24、48 h后, 采用流式细胞术检测细胞表面NKG2D配体表达情况；Western blot检测HL-60细胞内ERK蛋白表达变化情况；CFSE/PI法检测NK细胞对HL-60细胞的杀伤作用。.10 μmol/L Olaparib作用24和48 h均可上调HL-60细胞表面NKG2D配体的表达, 5 μmol/L Olaparib作用48 h可诱导ULBP-2、ULBP-3表达上调；Western blot检测结果显示, Olaparib处理后的HL-60细胞内ERK磷酸化水平增强。Olaparib可增强NK细胞对HL-60细胞的杀伤作用, 但ERK抑制剂可下调NK细胞对HL-60细胞的杀伤作用。.Olaparib可上调HL-60细胞表面NKG2D配体表达, 增强NK细胞对其的杀伤作用, 其机制可能与Olaparib促进ERK磷酸化表达有关。.

To investigate the mutation of RUNX1 gene in patients with myelodysplastic syndrome (MDS) and its correlation with other gene mutations and some clinical parameters.The mutations of RUNX1, DNMT3A, TET2, IDH1/2, NPM1, FLT3-ITD and C-KIT in 170 patients with MDS were detected by direct and indirect sequencing of genomic DNA-PCR amplification products.The RUNX1 mutation was found in 23 patients (13.5 %, 23/170). Among the 170 patients, other most frequent mutation was TET2 (11.2%, 19/170), followed by mutations in DNMT3A (9.4%, 16/170), NPM1 (8.2%, 14/170), IDH2 (4.1%, 7/170)、FLT3-ITD (2.9%, 5/170), IDH1 (1.7%, 3/170) and c-KIT (0.58%, 1/170). The most common coexisting mutations were TET2 (5/23). The RUNX1-mutated group showed significantly higher leukocyte levels, higher percentages of blast cells, higher incidences of leukemia transformation and lower platelet counts in comparison with RUNX1 non-mutation group (P＜0.05). whereas there were no statistically significant difference in age, MDS subtype, karyotype and hemoglobin level between 2 groups (P＞0.05). Seventeen patients harboring RUNX1 mutations were followed up and almost 47.05% (8/17) of the patients progressed into acute myeloid leukemia (AML). The rates of transformation into AML in ASXL1-mutation group was significantly higher than that in ASXLL- non-mutation group (47.05% vs 11.7%) (P=0.001).The incidence of RUNX1 mutation is high in MDS patients. The RUNX1-mutated patients have higher leukocyte level, higher percentages of blast cells, higher incidences of leukemia transformation and lower platelet count.骨髓增生异常综合症患者RUNX1基因突变分析.探讨骨髓增生异常综合症（MDS）患者中RUNX1基因突变情况，以及与其他基因突变和部分临床参数间的相关性.采用基因组DNA-PCR扩增产物直接测序法检测170例MDS患者中RUNX1、DNMT3A、TET2、IDH1/2、NPM1、FLT3-ITD、C-KIT突变情况.170例患者中RUNX1基因突变检测率为13.5%（23/170），其他基因突变率依次为：TET2（11.2%，19/170）、DNMT3A（9.4%，16/170）、NPM1（8.2%， 14/170）、IDH2（4.1%，7/170）、FLT3-ITD（2.9%，5/170）、IDH1（1.7%，3/170）、c-KIT（0.58%，1/170）。最常见的RUNX1突变共存基因为TET2（5/23）。RUNX1突变组与未突变组患者相比，具有更高的外周白细胞水平及更低的血小板水平，差异均具有统计学意义（P＜0.05）；但在中位年龄、MDS亚型、染色体核型、血红蛋白水平等方面2组间均无统计学差异。对17例RUNX1突变患者及111例未突变患者进行了有效随访，其中8例RUNX1突变的MDS患者进展为急性髓系白血病（AML），白血病转化率为47.05%（8/17），13例RUNX1未突变的MDS患者进展为AML，白血病转化率为11.7%（13/111），差异有统计学意义（P=0.001）.RUNX1突变在MDS患者中有较高的发生率，伴有该突变的MDS患者具有更高白细胞水平、更低的血小板水平、更高的骨髓原始细胞比例及白血病转化率.

To explore the coexisting mutations in IDH-mutated acute myeloid leukemia(AML) and its relation with partial clinical parametrs.The exon 4 mutation of IDH1/2 gene was screened by using genome DNA-PCR combined with sanger sequencing, 51 targeted gene mutations in the patients with IDH1/2 mutation were detected by using high throughput DNA sequencing combined with sanger sequencing.Among 358 patients, the IDH1/2 mutation was found in 46 cases including IDH1 mutation in 35 cases and IDH2 mutation in 11 cases, 97.87%(45/46) patients with IDH1/2 mutation simultaneously carried other gene mutations including 8(17.8%) cases with mutation of double gene, 17(37.8%) cases with mutation of 3 genes and 20(44.4%) cases with mutation of ≥ 4 genes. The mutation frequency of each patient averaged 3.52 times. In mutation of accompanied genes, the common genes were NPM1(n=29, 63.0%), next DNMT3A(n=25, 54.3%), FLT3-ITD(n=7, 15.2%), TET2(n=5, 10.9%) and NRAS(n=5, 10.9%). The average WBC level of patients with NPM1 mutation in IDH1 mutation group was higher than that of patients in wild type group(P0.05). The complete remission (CR) rate of patients with DNMT3A mutation was significant lower than that of patients with wild type (30% vs 80%, P0.01). The presence of ≥ 4 mutations was found to be significantly associated with higher white blood level than that in the patients with double mutations(P0.05).More than 95% AML patients with IDH1/2 mutation commonly show additional mutations. The number and the type of IDH coexisting mutations have certain effect on the clinical features and CR rate.急性髓系白血病中与IDH1/2突变共存的基因突变分析.探讨急性髓系白血病（AML）患者中与IDH1/2突变共存的基因突变及其与部分临床参数的相关性。.采用基因组DNA-PCR联合Sanger测序法筛选IDH1/2基因4号外显子突变；采用高通量DNA测序联合Sanger测序法检测IDH1/2突变患者51种肿瘤靶基因突变.358例患者中共检出46例IDH1/2突变，其中35例IDH1突变，11例IDH2突变。97.8%（45/46）IDH1/2突变患者同时携带其他基因突变，其中双基因突变8例，3个基因突变共存17例，≥4个基因突变共存20例。每例患者平均突变3.52次。伴随基因突变中最常见的基因为NPM1（n=29，63.0%），其他依次为： DNMT3A（n=25，54.3%）、FLT3-ITD(n=7，15.2%)、TET2（n=5，10.9%）及NRAS（n=5，10.9%）。在IDH1突变组中，伴NPM1突变者的平均外周血白细胞水平高于正常者，伴DNMT3A突变患者的CR率明显低于正常者，差异均有统计学意义（P=0.034，0.003）；≥4个基因突变患者的白细胞水平明显高于双基因突变者，差异显著（P=0.037）.95%以上伴IDH1/2突变的AML患者同时存在额外基因突变，基因突变个数及突变类型对患者的临床特征及CR率有一定的影响.

Leukemic cells can survive after chemotherapy by acquisition of multidrug resistance genes, but other phenotypes related to escape from immune recognition remain elusive. Adriamycin-resistant K562/AO2 cells are less susceptible to elimination by NK cells compared with wild type K562 cells due to lower expression of NKG2D ligands. Treatment of K562/AO2 cells with STAT3 inhibitor VII resulted in reduced expression of multidrug resistance gene P-glycoprotein, and up-regulation of NKG2D ligands on K562/AO2 cells. Meanwhile, K562/AO2 cells treated with STAT3 inhibitor proliferated less and were more susceptible to killing by NK cells than untreated K562/AO2 cells. The enhanced cytotoxicity of NK cells against K562/AO2 cells was partly blocked by treatment of NK cells with anti-NKG2D antibodies. These data suggest that STAT3 contributes to NK cell recognition by modulating NKG2D ligands in K562/AO2 cells, which may a mechanism by which cells survive and cause relapse of leukemia.

To explore the coexistence of ASXL1 and CALR gene mutations in patients with essential thrombocytheima (ET) and with primary myelofibrosis(PMF), and to compare the differences of clinical characteristics between ET and PMF patients carrying ASXL1 and CALR mutations, and ET and PMF patients carrying solitary gene mutation, and ET and PMF patients without any mutations.The mutations of ASXL1 gene at exon 12, CALR gene at exon 9 and MPL gene at exon 10 in 263 essential ET patients and 29 PMF patients were detected by PCR amplification followed by direct sequencing of genomic DNA. The JAK2V617F mutations were used by allele specific PCR detection.72.6%(212/292)of patients harbored at least one mutation. The incidences of ASXL1 and CALR mutations were 5.8% and 30.5%, respectively. The frequencies of JAK2V617F and MPL mutations were 39.0% and 2.4%, respectively. 5.1%(15/292) of patients had double mutations, including ASXL1 and CALR(n=11), ASXL1 and JAK2V617F(n=2), MPL and CALR(n=1) and ASXL1 and MPL(n=1). The frequency of concurrent ASXL1 and CALR mutations was found to be high. Significant difference was found on hemoglobin levels and platelet counts between CALR and ASXL1 mutations and single mutation (P0.05),however, the difference on leukocyte counts and median age was not found. Compared with negative patients, the presence of ASXL1 and CALR mutations was found to be significantly correlative with lower hemoglobin level (P=0.045), lower leukocyte count (P=0.002) and with higher platelet counts(P=0.001), but the difference of median age was not found.The frequency of concurrent ASXL1 and CALR mutations is higher in ET patients. The coexistence of ASXL1 and CALR gene mutations significantly associated with lower hemoglobin level and higher platelet count.

The cytokine-induced killer cells (CIK) have been reported to have potent cytotoxicity against a variety of tumor cells including multiple myleoma (MM) cells. The mechanisms that CIK cell recognizing MM cells remain unknown. Recent studies indicated that the interaction between NKG2D receptor and NKG2D ligands plays an important role in inducing cytotoxicity against various target cells by natural killer cells (NK). We suspect whether NKG2D receptor and NKG2D ligands interaction is also responsible for the killing of MM cells by CIK as the same way did NK cells. We expanded CIK cells from healthy controls with interferon (IFN)-γ, CD3 monoclonal antibodies (mAb) and interleukin-2 (IL-2), and checked expression of NK cell receptors on CIK cells by flow cytometry. About 86% bulk CIK cells expressed NKG2D receptor but not other NK receptors, such as CD158a, CD158b and NCRs. We analyzed NKG2D ligands expression in MM patients by flow cytometry, primary plasma cells from 8 out of 13 (62%) MM patients expressed different levels of ULBPs or MICA/B on the cell surface. Interestingly, when stimulated with MM cell line U266 that expressed some levels of MICA/B, only NKG2D expressing CIK cells released IFN-γ. CIK cells showed cytotoxicity against NKG2D ligands expressing U266 and primary MM cells, and the cytotoxicity was partially blocked by treating CIK with anti-NKG2D antibody. We conclude that NKG2D-NKG2D ligand interaction may be one of the mechanisms by which CIK cells kill MM cells.

Summary Introduction: Gene mutations play an important role in acute myeloid leukemia (AML) pathogenesis. Several genes have been identified in AML, such as FLT3, KIT, NPM1, and JAK2. This study investigated the frequency of novel mutations in IDH1 (amino acid R132) and IDH2 (R140 and R172) and analyzed their impact on disease biology and interaction with other mutations in Chinese patients with de novo AML. Methods: A total of 195 patients were screened for mutations in the IDH1, IDH2, JAK2 V617F, NPM1, FLT3, and KIT genes, using polymerase chain reaction (PCR)-based and direct sequencing assays. Results: IDH mutations occurred at a considerable frequency of 15.89% in Chinese AML cases; IDH2 R140Q was the most frequent genetic alteration and was associated with older age, normal karyotype, and French-American-British classification M2 at diagnosis. There was a strong association of IDH2 mutation with NPM1 mutations and a trend with FLT3-internal-tandem duplication. Conclusion: IDH mutations may be a novel genetic marker in cytogenetically normal AML and may cooperate in leukemogenesis.

Breast cancer patients with positive epidermal growth factor receptor (EGFR) expression have significantly worse post-relapse prognosis than patients with negative EGFR expression. Vinorelbine (NVB) is usually reserved as a salvage therapy after anthracyclines and taxanes in patients with breast cancer. To see whether EGFR expression has a predictive value in NVB-mediated effect on human breast cancer cells, we examined 50 primary breast cancer samples. Of these, 42% were found to be NVB sensitive by ATP-tumour chemosensitivity assay. Sensitivity was correlated with EGFR expression level (p = 0.001). To dynamically examine EGFR's effect on NVB sensitivity in breast cancer cells, we used the real-time cell electronic sensing system with EGFR-positive and EGFR-negative breast cancer cell lines, MCF-7 and MDA-MB-435s, respectively. MCF-7 is NVB sensitive, while MDA-MB-435 is NVB resistant. NVB-induced cytotoxicity to MCF-7 can be partly reversed with inhibitory anti-EGFR antibody. NVB up-regulated EGFR expression in MCF-7 cells, which affects ERK1/2 phosphorylation. This cellular response mechanism may cause greater input to non-lethally damaged cells. These data suggest that EGFR expression can be used as a prognostic factor for breast cancer sensitivity to NVB, which could help identify appropriate treatments for breast cancer.

This study was aimed to analyze the expression of NKG2D ligands in human leukemic cells and to investigate the effects of matrine on NKG2D ligand expression. The expressions of NKG2D ligand MICA/B, ULBP1-3 in several human leukemia cell lines (K562, OUN-1, U937 and K562/AO2), as well as primary leukemic cells isolated from malignant leukemia patients were analyzed by flow cytometry. After treatment with different doses of matrine, the expression level of NKG2D ligands in these leukemic cells was detected by FCM. The results indicated that NKG2D ligand expression was detected in both the leukemia cell lines and primary malignant leukemic cells. Generally, the expression of ULBP was high or obviously higher than that of MICA/B in leukemia cell lines and primary leukemic cells. The expression pattern of NKG2D ligands was different among these cells, possibly due to the different types of leukemia. Not all the expression of NKG2D ligands was upregulated after matrine treatment. Much higher expressions of ULBP2 and ULBP3 were found in K562 cells, compared to the other cell lines, which partly contributes to the higher sensitivity of K562 cells to NK cytotoxicity as target cells. It is concluded that there is universal expression of NKG2D ligand in leukemia cells. The high ULBP expression is prevalent in human leukemia cells. Matrine has the potential to induce the expression of NKG2D ligands in leukemia cells.

This study was purposed to investigate the CIK cell cytotoxicity to hematological malignant cell lines by interaction NKG2D receptors and corresponding ligands. The CIK cells was expanded from healthy individual with interferon (IFN)γ, CD3 monoclonal antibodies (mAb) and interleukin-2 (IL-2). The subset of lymphocyte and the expression of NK cell receptors on CIK cells was detected by flow cytometry; NKG2D ligand expression on hematological malignant cell lines was also analyzed by flow cytometry, the calcein acetoxymethyl ester (CAM) was used for labeling target cells, then the cytotoxicity of CIK cells to hematological malignant cell lines was detected by flow cytometry. The results showed that most of CIK cells expressed CD3 (97.85 ± 1.95%) , CD3(+)CD8(+) cells and CD3(+)CD56(+) cells increased significantly as compared with un-cultured cells (P0.001;P = 0.033). About 86% CIK cells expressed NKG2D receptor but no other NK receptors such as CD158a, CD158b and NCR. Different levels of NKG2D ligands were detected in hematological malignant cell lines U266, K562 and Daudi. CIK cells showed high cytotoxicity to these three different cell lines, and this cytotoxicity was partially blocked by treating CIK cells with anti-NKG2D antibody (U266 52.67 ± 4.63% vs 32.67 ± 4.81%, P = 0.008;K562 71.67 ± 4.91% vs 50.33 ± 4.91%, P = 0.007;Daudi 68.67 ± 5.04 vs 52.67 ± 2.60%, P = 0.024) . It is concluded that most of CIK cells express NKG2D receptor, interaction of NKG2D-NKG2D ligands may be one of the mechanisms, by which CIK cells kill hematological malignant cells.

To investigate the enhanced cytotoxicity against leukemia cells of natural Killer (NK) cells from cord blood (CB) after expansion in vitro.NK cells was expanded on a layer of trophoblast cells with irradiated K562-mb15-41BBL cell line for 21 days. The levels of receptors on NK cells were detected by flow cytometry. Cytotoxicity of expanded NK cells against leukemia cells and specific ligand of immunoglobulin like(Ig- liKe)receptors were assessed using 51Cr released assay.There were no differences of inhibitory receptors expression between fresh NK cells and expanded NK cells [CD158a:(16.77±11.65)% vs(14.37±11.12)%, P0.05; CD158b: (42.48±18.11)% vs (40.92±19.02)%, P0.05; NKG2A: (70.20±18.43)% vs (78.90±13.69)%, P0.05], but higher activated receptors expression on expanded NK cells [NKp30: (54.10±13.27)% vs (4.14±2.05)%, P0.05; NKp44: (72.10±17.30)% vs (0.52±1.16)%, P0.05; NKp46: (80.63±14.01)% vs (44.19±6.19)%, P0.05; NKG2D: (97.50±2.55)% vs (72.25±14.35)%, P0.05]. Expanded NK cells showed higher cytotoxicity against leuKemia cell lines than fresh NK cells [K562: (74.3±3.6)% vs (55.3±4.2)%, P0.05; Raji: (60.6±5.0)% vs (12.0±3.6)%, P0.05]. CD158a⁻ CD158b⁻ NK cells had higher cytotoxicity on four types of target cells, but CD158a⁺CD158b⁻ CB-NK cell had lower cytotoxicity on 221-Cw4 and 221-Cw3Cw4 cells. CD158a⁻ CD158b⁺ CB- NK cells had lower cytotoxicity on 221-Cw3 and 221-Cw3Cw4, but CD158a⁺CD158b⁺ CB-NK cells had higher cytotoxicity on 721- 221 cells.Expression of activated receptors of expanded NK cells were up-regulated, but no changes of inhibitory receptors. Expanded NK cells showed high cytotoxicity against leukemia cells and kept the specificity of ligand of Ig-like receptors, which could be beneficial to cell-therapy for tumor.

To investigate NK cell cytotoxicity to leukemic cell by NKG2D receptors and NKG2D ligands interaction upregulated by hydroxyurea (HU).Leukemic cell lines OUN-1 and primary leukemic cells were cultured for 24 hours in the presence of HU, then the NKG2D ligands expressions were analyzed by flow cytometry (FCM). Isolated NK cells from healthy individual cultured for 72 hours in presence of IL-2 were used as effect cell, and leukemic cell line OUN-1 treated with HU was used as target cell, NK cell cytotoxicity against leukemic cell line was assessed using chromium-51 release assay.Leukemic cell lines showed upregulation of MIC A/B (MFI: 8.9 ± 0.9 vs 23.5 ± 3.4, P = 0.01) and ULBP2 (MFI: 14.5 ± 0.6 vs 33.5 ± 4.8, P = 0.03) following incubation with HU. HU also upregulated the NKG2DLs on primary leukemia cells from patients with acute myeloid leukemia. Treatment of OUN-1 with HU significantly increased the cytotoxicity of NK cells isolated from healthy individual \[(62.0 ± 5.6)% vs (76.0 ± 5.3)%, P = 0.02\], and the enhancing effect of HU was partly blocked by anti-NKG2D Abs \[(76.0 ± 5.3)% vs (46.0 ± 4.5)%, P = 0.00\].HU selectively upregulated NKG2D ligand expression on leukemic cell lines, and enhanced NK cell cytotoxicity against leukemic cells through NKG2D receptors and NKG2D ligands interaction.

To analyze the correlation between clinical features and cytogenetic finding of 45 adult patients with acute lymphoblastic leukemia (ALL), and to assess the value of chromosomal examination for the diagnosis and prognosis.Fluorescence in situ hybridization (FISH) was utilized for detecting the BCR/ABL fusion gene and P53 gene. Median survival time (MST) of patients was compared using Log-rank test.Respectively, the MST of patients with white blood cell count (WBC) ≤30 × 10(9)/L, normal karyotype, or without a Philadelphia chromosome were significantly greater than those with WBC30 × 10(9)/L, abnormal karyotype or Philadelphia chromosome (P0.05).WBC, karyotype abnormalities and presence of Philadelphia chromosome are independent factors for the prognosis of ALL in adult patients.

To investigate the prognostic value of t(11; 18) (q21; q21) in gastric mucosa-associated lymphoid tissue lymphoma.A cohort of thirty-six gastric mucosa-associated lymphoid tissue lymphoma patients who were pathologically identify diagnosis from January 1994 to June 2004 were followed up retrospectively and studied using fluorescence in situ hybridization(FISH) technique to detect t(11; 18) (q21; q21) chromosomal translocation on preservative paraffin specimen.Among thirty-six patients, fifteen (41.67%) were positive for t (11; 18) (q21; q21). All but one were followed up to March 2010, general median survival time (MST) was 87 months. The MST were 43 and 130 months for t(11; 18) positive and negative patients, respectively. The MST between these two groups was notably different (chi-square=29.57, P0.01).t(11; 18) (q21; q21) is important prognostic factor for gastric mucosa-associated lymphoid tissue lymphoma.

Breast cancer patients with positive epidermal growth factor receptor (EGFR) expression have significantly worse post-relapse prognosis than patients with negative EGFR expression. Vinorelbine (NVB) is usually reserved as a salvage therapy after anthracyclines and taxanes in patients with breast cancer. To see whether EGFR expression has a predictive value in NVB-mediated effect on human breast cancer cells, we examined 50 primary breast cancer samples. Of these, 42% were found to be NVB sensitive by ATP-tumour chemosensitivity assay. Sensitivity was correlated with EGFR expression level (p = 0.001). To dynamically examine EGFR's effect on NVB sensitivity in breast cancer cells, we used the real-time cell electronic sensing system with EGFR-positive and EGFR-negative breast cancer cell lines, MCF-7 and MDA-MB-435s, respectively. MCF-7 is NVB sensitive, while MDA-MB-435 is NVB resistant. NVB-induced cytotoxicity to MCF-7 can be partly reversed with inhibitory anti-EGFR antibody. NVB up-regulated EGFR expression in MCF-7 cells, which affects ERK1/2 phosphorylation. This cellular response mechanism may cause greater input to non-lethally damaged cells. These data suggest that EGFR expression can be used as a prognostic factor for breast cancer sensitivity to NVB, which could help identify appropriate treatments for breast cancer.

Abstract 5119 Cytokine-induced killer (CIK) cells are T lymphocytes enriched in CD3+CD56+ cells, which can be easily and rapidly expanded in vitro from human peripheral blood, bone marrow or cord blood mononuclear cells with the sequential addition of interferon (IFN)-{gamma}, OKT-3 and high doses of interleukin (IL)-2. The cytokine-induced killer (CIK) cells have been reported to have potent cytotoxicity against a variety of tumor cells including multiple myleoma (MM) cells. The mechanism of CIK cell recognizing MM cells remains unknow. Recent studies indicated that ligation of NKG2D on immunological cells directiy induce cytotoxicity. We suspect whether NKG2D receptor induction on CIK cells by cytokines is responsible for the killing of MM cells by CIK. We expended CIK cells from healthy controlswith interferon (IFN)-γ, CD3 monoclonal antibodies (mAb) and IL-2, and checked expression of NK cell receptors on CIK cells by flow cytometry. We found higher expression of NKG2D receptor and lower other NK receptors, such as CD158a,CD158b and NCRs on expanded CIK. These CIK cells showed higer cytotoxicity to multiple myleoma cell line U266 expressing NKG2D ligands. Interestingly, when cocultured with U266 cells, only NKG2D expressing CIK cells released IFN-γ detected by flow cytometry. We next analyzed NKG2D ligands expression on primary plasma cells in 22 MM patients by flow cytometry, the primary plasma cells in 16/22 (72.7%) MM patients expressed different levels of ULBPs or MICA/B on the cell surface. CIK cells showed higher cytotoxicity (12.5%) to NKG2D ligands expressing primary plasma cells compared to those did not express NKG2D ligands. The killing of CIK against MM cells were partially blocked by treatment of CIK with anti-NKG2D antibody. We conclude that NKG2D-NKG2D ligangd interaction may be one of the mechanisms by which CIK cells kill MM cells. Disclosures: No relevant conflicts of interest to declare.