化学法提取贮存骨髓涂片DNA检测Fms样酪氨酸激酶3基因突变预测急性髓系白血病患者预后

目的 探讨用化学法(苯酚:氯仿:异戊醇法)从贮存的骨髓涂片中提取微量DNA检测Fms样酪氨酸激酶3内部串联重复(FLT3-ITD)突变情况,分析其对急性髓系白血病(AML)预后的预测价值.方法 采用化学法从58例-20℃贮存1~5年的骨髓涂片(AML 48例和非恶性血液病10例)中提取微量DNA,采用聚合酶链反应(PCR)技术检测患者FLT3-ITD突变情况.结果 48例初治AML患者中6例(12.5%)检测到FLT3-ITD,10例非恶性血液病患者中均未检测到FLT3-ITD.FLT3-ITD阳性AML患者初次诱导完全缓解(CR)率较FLT3-ITD阴性者低[0(0/6)比77.78%(21/27),P=0.001],总生存时间短(x2=7.274,P=0.007).肝、脾大和FLT3突变是影响AML患者初次化疗CR的危险因素(OR=7.2,P=0.12;OR=36.3,P=0.10).FLT3-ITD是初治AML患者不良预后的危险因素(RR=9.088,P=0.029).结论 使用化学法提取贮存的AML患者骨髓涂片微量DNA可在临床中应用,是进行回顾性分子生物学研究的重要实验方法.从骨髓涂片中提取微量DNA检测FLT3-ITD可用于预测AML预后.     

酪氨酸激酶小分子抑制剂在肿瘤治疗中的研究

酪氨酸激酶的过度激活与肿瘤发生、发展、预后、转归密切相关.酪氨酸激酶激活可导致下游信号途径Ras-MAPK和PI3K-AKT的激活,最终抑制细胞凋亡,促进细胞生存.不同的酪氨酸激酶特异性抑制剂对不同的肿瘤具有特异性抑制作用.现综述此领域研究进展.     

酪氨酸激酶小分子抑制剂在肿瘤治疗中的研究

酪氨酸激酶的过度激活与肿瘤发生、发展、预后、转归密切相关。酪氨酸激酶激活可导致下游信号途径Ras-MAPK和P13K-AKT的激活，最终抑制细胞凋亡，促进细胞生存。不同的酪氨酸激酶特异性抑制剂对不同的肿瘤具有特异性抑制作用。现综述此领域研究进展。     

急性髓系白血病患者FMS样酪氨酸激酶3基因内部串联重复突变的检测及其临床意义

目的 分析急性髓系白血病（AML）患者骨髓FMS样酪氨酸激酶3基因内部串联重复（FLT3／ITD）突变情况.方法 采用聚合酶链反应方法检测96例初发AML患者FLT3／ITD突变,分析FLT3／ITD阳性患者的临床特征.结果 96例AML患者中FLT3／ITD突变18例（18.8％）.其中M22例,M3 11例,M42例,M53例.FLT3／ITD阳性AML患者初发时外周血白细胞计数中位数为18.0×109／L（3.6× 109～ 137.6×109/L）,FLT3／ITD阴性AML患者为6.3×109/L（4.5×109 ～ 113.0×109/L）,两组比较差异有统计学意义（t=3.04,P＜0.05）.FLT3／ITD阳性者随访16例,中位随访时间10个月（6～ 15个月）,13例处于第1次完全缓解.诱导缓解率为81.3％（13／16）.结论 FLT3／ITD是AML患者常见的基因突变,FLT3／ITD阳性患者白细胞计数较高.     

富含脯氨酸的酪氨酸激酶2在肿瘤中的研究进展

近年来肿瘤的发病率与病死率均呈持续上升趋势,传统治疗方法包括手术切除、放疗和化疗.由于肿瘤的高复发和转移率,目前病死率仍然较高.大量研究表明,肿瘤的发生和转移与某些致癌基因的过表达有关.因此,有必要寻找新的分子生物标志物和治疗靶点,以改善预后.富含脯氨酸的酪氨酸激酶2(Pyk2)是一种能调节细胞增殖、迁移、侵袭和凋亡的...     

FMS样酪氨酸激酶3基因内部串联重复序列突变阳性急性髓系白血病研究进展

FMS样酪氨酸激酶3基因内部串联重复序列(FLT3-ITD)突变是急性髓系白血病(AML)中常见的突变类型,其突变与AML的发生、发展及不良预后关系密切.FLT3-ITD突变阳性AML患者常规化学治疗预后差,复发率高,异基因造血干细胞移植对该类患者疗效不一,索拉非尼等靶向治疗及不同治疗方式的联合治疗的临床前及临床试验已经开展且已初见成效.文章就FLT3-ITD突变在AML中的临床意义及对该类患者治疗的最新进展作一综述.     

雷公藤甲素对急性髓系白血病伴FMS样酪氨酸激酶3内部串联重复细胞株MV411增殖和凋亡的影响及其机制研究

目的 探讨雷公藤甲素(TP)对急性髓系白血病伴FMS样酪氨酸激酶3内部串联重复(FLT3-ITD)的细胞株MV411增殖和凋亡的影响,及其对PI3K-Akt-mTOR通路的作用.方法 四甲基偶氮唑盐(MTT)法测定不同浓度TP作用24、48、72 h时MV411细胞的增殖抑制率;流式细胞术检测48、72 h的细胞凋亡率;实时荧光定量聚合酶链反应(PCR)检测PI3K-Akt-mTOR通路相关基因FLT3、PTEN、PI3K、Akt、mTOR mRNA的表达.结果0、5、10、20、40、80 nmol/L TP作用24、48、72 h,MV411细胞增殖受到抑制,呈现时间-剂量依赖性.0、10、20 nmol/L TP作用48 h后,MV411细胞平均早期凋亡率分别为(3.30±0.20)%、(17.10±0.36)%、(35.67±0.61)%,作用72 h后分别为(7.37±0.32)%、(49.33±0.40)%、(68.92±0.11)%,同一时间各浓度组间差异均有统计学意义(均P=0.000).TP能明显降低FLT3、PI3K、Akt、mTOR mRNA的表达,升高PTEN mRNA的表达.结论TP可能通过影响PI3K-Akt-mTOR通路相关基因的表达发挥抑制MV411细胞增殖、诱导其凋亡的作用.     

卵巢浆液性癌中受体酪氨酸激酶EphB3蛋白表达及临床意义

目的探讨受体酪氨酸激酶EphB3蛋白在正常输卵管上皮组织、卵巢浆液性肿瘤组织和卵巢浆液性癌组织中的表达及其与卵巢浆液性癌临床病理特征的关系。方法采用免疫组化法检测9例正常输卵管、12例卵巢浆液性囊腺瘤和53例卵巢浆液性癌组织中EphB3蛋白的表达情况,进一步分析EphB3蛋白表达与卵巢癌临床病理特征的关系。结果正常输卵管上皮和卵巢良性肿瘤中EphB3蛋白的强阳性(+++)表达率分别为100.0%(9/9)、75.0%(9/12),明显高于卵巢浆液性癌的18.9%(10/53),差异有统计学意义(P<0.001)。EphB3蛋白表达与肿瘤分级(r=-0.606,P<0.001)和FIGO分期(r=-0.463,P<0.001)呈负相关,而与患者年龄、大网膜转移、腹腔转移和盆腔淋巴结转移无关(P>0.05)。结论 EphB3可能成为区分高、低级别浆液性癌的辅助指标,帮助卵巢浆液性癌病理诊断及制定治疗方案。     

保罗样激酶PLK1与肿瘤预后及治疗研究进展

保罗样激酶1(PLK1)在启动、维持及完成有丝分裂中扮演重要角色.已发现PLK1基因在多种恶性肿瘤中存在过表达,并与某些肿瘤的恶性生物学行为及预后密切相关,有望成为恶性肿瘤的又一新标记物及肿瘤治疗的新靶点.本文对PLK1与肿瘤预后及治疗相关研究进展做一综述.     

PKC 412 small-molecule tyrosine kinase inhibitor: single-compound therapy for pancreatic cancer

BACKGROUND: PKC412 is a kinase inhibitor that blocks protein kinase C (PKC), vascular endothelial growth factor receptors, platelet-derived growth factor receptor FLT3, and other class III receptor tyrosine kinases. The enthusiasm for this compound is based on its inhibitory effect even in the case of FLT3 mutations. The aim of this study was to analyze the role of FLT3 in pancreatic cancer and to study the biological activity of combined inhibition of neovascularization and mitogenesis in this disease. METHODS: FLT3 expression was analyzed in 18 pancreatic cancer specimens by real-time quantitative polymerase chain reaction (RTQ-PCR) and immunohistochemistry. Sixteen pancreatic cancer cell lines were screened for ITD and D835 point mutations of the FLT3 gene. MTT assays and anchorage-independent growth assays were used to study cell growth. Flow cytometry was used for cell cycle analysis and apoptosis quantification. In vivo AsPC-1 and HPAF-II cells were used for orthotopic tumor modeling. Immunohistochemistry was used to quantify tumor angiogenesis. RESULTS: FLT3 expression is down-regulated in pancreatic cancer. Activating FLT3 mutations (ITD, D835) were not detectable in any of the pancreatic cancer cell lines. Cell growth was significantly inhibited as cell-cycle progression was reduced and programmed cell death increased. In vivo PKC412 therapy resulted in a significant inhibition of orthotopic tumor growth with abrogation of tumor angiogenesis. CONCLUSIONS: These data highlight that PKC412 may be a new compound in target therapy of inoperable pancreatic cancer patients and suggest a potential role for the combined use of broad spectrum kinase inhibitors in the management of these patients.     

Immuno-gene therapy with adenoviruses expressing fms-like tyrosine kinase 3 ligand and CD40 ligand for mouse hepatoma cells in vivo

Introduction of genes encoding immuno-stimulatory cytokines into cancer cells is known to enhance antitumor immunity. CD40 ligand (CD40L, CD154) and fms-like tyrosine kinase 3 ligand (Flt3L) are recently identified cytokines, which have been demonstrated to stimulate antitumor immunity in several cancer models. However little is known about antitumor activity of Ftl3L and CD40L against hepatocellular carcinoma (HCC). In the present study, we constructed replication-defective adenoviruses expressing Flt3L and CD40L and examined their therapeutic efficacy on mouse HCC, MH134 cells. Subcutaneous injection of MH134 cells genetically engineered to express Flt3L and/or CD40L developed tumors in all the syngeneic immunocompetent mice, but tumor growth was significantly delayed as compared to control mice. Partial inhibition of this antitumor effect in athymic nude mice suggests that both innate and adaptive immunity appear to play a role. It was shown by immunodepletion of NK cells with anti-asialo-GM1 antibody that the effector cells involved in innate immunity are NK cells. In a therapeutic setting, however, injection of adenovirus expressing Flt3L or CD40L into pre-established MH134 tumors exhibited no efficacy. These data demonstrate that Flt3L and CD40L induce significant, but only weak, antitumor immunity against MH134 cells presumably through both innate and adaptive immunity. Our results suggest that immuno-gene therapy with Flt3L and CD40L may need adjuvant modalities to achieve strong immune response.     

A novel tumor suppressor function of Kindlin-3 in solid cancer

Kindlin-3 (FERMT-3) is known to be central in hemostasis and thrombosis control and its deficiency disrupts platelet aggregation and causes Leukocyte Adhesion Deficiency disease. Here we report that Kindlin-3 has a tumor suppressive role in solid cancer. Our present genetic and functional data show that Kindlin-3 is downregulated in several solid tumors by a mechanism involving gene hypermethylation and deletions. In vivo experiments demonstrated that Kindlin-3 knockdown in 2 tumor cell models (breast cancer and melanoma) markedly increases metastasis formation, in accord with the in vitro increase of tumor cell malignant properties. The metastatic phenotype was supported by a mechanism involving alteration in β3-integrin activation including decreased phosphorylation, interaction with talin and the internalization of its active form leading to less cell attachment and more migration/invasion. These data uncover a novel and unexpected tumor suppressor role of Kindin-3 which can influence integrins targeted therapies development.     

A novel cancer immunotherapy based on the combination of a synthetic carbohydrate-pulsed dendritic cell vaccine and glycoengineered cancer cells

Immune tolerance to tumor-associated carbohydrate antigens (TACAs) has severely restricted the usefulness of most TACAs. To overcome this problem, we selected a sialylated trisaccharide TACA, GM3, as a target antigen, and tested a new immunotherapeutic strategy by combining metabolic bioengineering with dendritic cell (DC) vaccination. We engineered cancer cells to express an artificial structure, N-phenylacetyl-D-neuraminic acid, in place of the natural N-acetyl-D-neuraminic acid of GM3 by using N-phenylacetyl-D-mannosamine (ManNPhAc) as a biosynthetic precursor. Next, we selectively targeted the bioengineered cancer cells by vaccination with DCs pulsed with the GM3 N-phenylacetyl derivative. Vaccination with GM3NPhAc-KLH-loaded DCs elicited robust GM3NPhAc-specific T cell-dependent immunity. The results showed that this strategy could significantly inhibit FBL3 tumor growth and prolong the survival of tumor-bearing mice; B16F10 lung metastases could also be reduced. These findings lay out a new strategy for overcoming immune tolerance to TACAs, such as GM3, for the development of effective tumor immunotherapies.     

Myeloablative chemotherapy followed by autologous stem cell infusion may overcome the adverse prognostic impact of FLT3 (foetal liver tyrosine kinase 3) mutations in patients with acute myeloid leukaemia and normal karyotype

In this study, we analysed the prognostic relevance of foetal liver tyrosine kinase 3 (FLT3) mutations in 73 patients with acute myeloid leukaemia (AML) with normal karyotype, who survived induction and consolidation and received autologous stem cell transplantation (ASCT) after successful mobilization of peripheral blood stem cell (PBSC). There were 44 males and 29 females with a median age of 54 years (range 20-77). Overall, 16 out of 73 autografted patients (22%) had FLT3 mutations. More in detail, FLT3/ITDs were detected in 10 out of 73 patients (14%), while FLT3 D835 mutations were detected in five cases (7%). One patient (1%) was found as having both abnormalities. White blood cell count (p=0.009), serum concentration of lactate dehydrogenase (p=0.01), and percentages of peripheral blood (p=0.002) and bone marrow blasts (p=0.03) were significantly higher in patients showing the FLT3 mutations. On the contrary, overall survival and disease-free survival were similar between patients with or without FLT3 mutations (p=0.73 and 0.78, respectively). In conclusion, our data suggest that myeloablative chemotherapy supported by auto-PBSCT may overcome the adverse prognostic implications of FLT3 mutations in AML. However, it is to consider that autografted patients are highly selected for best response to induction, consolidation and mobilization, as well as for minor non-haematologic toxicity.     

肝动脉栓塞化疗、三维适形放疗与斯普林联合治疗中晚期肝癌的临床探索

目的:评价肝动脉栓塞化疗、三维适形放疗及联合斯普林治疗中晚期肝癌的安全性及疗效。方法:35例不能手术的原发性肝癌先行肝动脉栓塞化疗1~2次,在此基础上行病灶的三维适形放疗(3-dimension comformal radiation therapy,3DCRT),单次剂量2 Gy,每日1次,总剂量40~56 Gy(平均50.5 Gy)。上述治疗开始即配合斯普林注射液10 mL/d静脉滴注,连续治疗6~8周。结果:随访6~45个月(中位随访期24个月),随访率97.1%;35例患者中CR 5例(14.3%),PR20例(57.1%),NC 8例(22.9%),PD 2例(5.7%),总有效率(CR PR)为71.4%。1、2、3年生存率分别为65.7%、48.6%、31.4%;43.3%(13/30)患者的血清肿瘤标志物(AFP)降至正常;85.7%的患者治疗后生活质量较治疗前明显改善;本组治疗后比治疗前CD3 、CD4 、CD4 /CD8 比值升高,CD8 明显下降(P<0.01)。主要毒性反应为胃肠道反应和发热,按WHO的不良反应分级标准均为Ⅰ、Ⅱ级,患者均能顺利完成全部治疗。结论:肝动脉栓塞化疗、三维适形放疗及联合斯普林治疗无手术机会的肝癌患者具有创伤小,安全性高的特点,能提高生活质量,改善自身细胞免疫功能。     

A novel serum protein signature associated with resistance to epidermal growth factor receptor tyrosine kinase inhibitors in head and neck squamous cell carcinoma

BackgroundAcquired resistance to tyrosine kinase inhibitors (TKIs) is becoming a major challenge in the treatment of many cancers. Epidermal growth factor receptor (EGFR) is overexpressed in squamous carcinomas, notably those of the head and neck (HNSCC), and can be targeted with several TKIs. We aimed to identify soluble proteins suitable for development as markers of EGFR TKI resistance in cancer patients to aid in early and minimally invasive assessment of therapeutic responses.MethodsResistant HNSCC cell lines were generated by exposure to an EGFR TKI, gefitinib, in vitro. Cell lines were characterised for their biological behaviour in vitro (using growth inhibition assays, flow cytometry, western blots, antibody arrays and/or immunoassays) and in vivo (using subcutaneous tumour xenografts). Sera from EGFR-treated and -untreated HNSCC patients were analysed by immunoassay.ResultsTwo independent sublines of CAL 27 and a PJ34 subline with acquired resistance to EGFR TKIs (gefitinib, erlotinib and afatinib) were developed. Resistant cells grew as highly aggressive xenografts leading to reduced host survival rates compared with EGFR-TKI sensitive cells. This suggested a link between resistance in vitro and poor prognosis in vivo. A significant upregulation of proteins linked to tumour angiogenesis and invasion was identified in resistant cells. This ‘resistance-associated protein signature’ (RAPS) was detected in the sera of a small cohort of HNSCC patients and was associated with reduced survival.ConclusionWe have identified a protein signature associated with EGFR-TKI resistance that may also be linked to poor prognosis and warrants further investigation as a potential clinical biomarker.     

A novel approach for identification of tumor-associated antigens expressed on the surface of tumor cells

To improve tumor targeting in a subset of patients, where tumor cells do not express the well-known tumor antigens widely used in immunotherapy, we have developed a novel biotechnological tool. It is useful for tumors of various origins for the identification of tumor-associated proteins, which are differentially expressed in tumor cells with respect to normal tissue, and exposed on the cell surface. For this purpose, a combination of techniques, such as "suppression subtractive hybridization" and "transmembrane trapping," was employed. In applying this novel approach to breast cancer, we identified a large panel of cDNA fragments encoding for the well-known tumor-associated surface antigens, such as erb-B2, erbB3 and the urokinase receptor and, more importantly, for several clones overexpressed in breast cancer, whose cDNA fragments match the sequences of hypothetical transmembrane proteins with unknown function. The latter may represent novel tumor-specific targets.