Insulin-like growth factor I expression by tumors of neuroectodermal origin with the t(11;22) chromosomal translocation. A potential autocrine growth factor.

Expression of insulin-like growth factor I (IGF-I) mRNA by some tumor cell lines of neuroectodermal origin has been described. To further explore the significance of IGF-I mRNA expression in these tumors, a more extensive analysis was performed. Most (9 of 10) neuroectodermal tumor cell lines with a t(11;22) translocation (primitive neuroectodermal tumor [PNET], Ewing's sarcoma, esthesioneuroblastoma) expressed IGF-I mRNA, whereas 0 of 15 cell lines without the translocation (PNET, neuroblastoma) expressed IGF-I. Furthermore, inasmuch as all neuroblastoma (12 of 12) cell lines examined expressed IGF-II RNA, the pattern of IGF expression could distinguish between these closely related tumors. CHP-100, a PNET cell line with the t(11;22) translocation, was shown to secrete both IGF-I protein and an IGF binding protein, IGFBP-2. This cell line also expressed the type I IGF receptor mRNA, and blockade of this receptor by a monoclonal antibody (alpha IR3) inhibited serum-free growth. These data demonstrate that IGF-I expression is a property of neuroectodermal tumors with a t(11;22) translocation and that interruption of an IGF-I autocrine loop inhibits the growth of these tumor cells.     

Targeting Lyn inhibits tumor growth and metastasis in Ewing's sarcoma

Src family tyrosine kinases (SFK) play an important role in growth and metastasis of many types of human malignancies. However, their significance in Ewing's sarcoma remains to be elucidated. The purpose of this study was to evaluate the role of Lyn, one member of the SFK, in Ewing's sarcoma growth and metastasis and to determine whether a SFK inhibitor can induce Ewing's tumor regression. Lyn was expressed and activated in TC71, A4573, and SK-ES human Ewing's sarcoma cells. Lyn expression was seen in 13 of 15 patient tumor samples, 6 of which showed Lyn activation. Specific inhibition of Lyn using small interfering RNA significantly decreased primary tumor growth and lytic activity, and also reduced lung metastases in vivo. Down-regulation of Lyn resulted in decreased invasive capacity of tumor cells in vitro. AP23994, a small-molecule SFK inhibitor, decreased Lyn kinase activity and suppressed TC71 cell growth in vitro in a dose-dependent manner. Furthermore, treatment of mice bearing s.c. TC71 tumors with AP23994 or with polyethylenimine/Lyn-small interfering RNA gene therapy resulted in reduced Lyn kinase activity and significant tumor growth suppression. EWS/FLI-1, which is translocation fusion protein associated with Ewing's sarcoma, regulated Lyn gene expression and kinase activity. These data suggest that targeting Lyn may be a new therapeutic approach in treatment of Ewing's sarcoma.     

Establishment and characterization of a clonal human extraskeletal Ewing's sarcoma cell line, EES1

Ewing's sarcoma, a small round cell sarcoma arising in soft tissue as well as the bone, is one of the most malignant tumors in children and young adults. Few established cell lines of extraskeletal Ewing's sarcoma (EES) have been reported, which made it difficult to examine the biological features of EES. Therefore, we have established a new clonal cell line of EES. We report its morphological characters, results of chromosomal and immunohistochemical analysis. A piece of tumor obtained from the 18-year-old female patient with EES was xenografted in a nude mouse. In vitro subcultured cells were then obtained from this xenograft. A clonal cell line was subsequently established by limiting dilution and designated EES1. EES1 cells had a doubling time of 24 hours. In the xenografted tumor, the cells expressed vimentin, CD99 (MIC2), neuron specific enolase (NSE) and cytokeratin. The original tumor cells also expressed vimentin, CD 99, and NSE, but was negative for cytokeratin. The morphological and immunohistochemical features of this cell line established, except for cytokeratin expression, were consistent with those of the primary tumor. Cytogenetic analysis of EES1 revealed chromosomal translocation of t(11; 12)(q24;ql2). The chimeric fusion of the Ewing's sarcoma gene in band 22q12 with the Friend leukemia virus integration-1 gene in band 11q24 was also demonstrated. Fluorescence in situ hybridization further confirmed the presence of translocation involving the Ewing's sarcoma gene in both the primary tumor and EES1 cells. In conclusion, we have established a human EES cell line EES1, which will provide a useful model for studying various aspects of human EES.     

Interphase cytogenetics for the detection of the t(11;22)(q24;q12) in small round cell tumors.

Among the small round cell tumors differential diagnosis is particularly difficult for their undifferentiated or primitive character. In this mixed group of tumors, only the primitive neuroectodermal tumors, which include Ewing's sarcoma (ES), show the unique and consistent feature of the (11;22)(q24;q12) translocation, which can therefore be considered a hallmark of these neoplasias. We analyzed four primitive neuroectodermal tumor cell lines, one osteosarcoma cell line, and 11 patients by fluorescent in situ hybridization with cosmid clones 23.2 and 5.8, bracketing the t(11;22) at 11q24. Metaphase spreads from tumor cell lines, and from biopsy specimens of three patients with ES were analyzed. In the remaining eight patients comprising five ES, two small cell osteosarcomas and one chronic osteomyelitis, only nuclei preparations were available for analysis. We detected the t(11;22) in interphase nuclei of the four primitive neuroectodermal tumor cell lines, of three patients in which the karyotype demonstrated the translocation and in five cases of ES in which cytogenetic analysis had not been possible. Two cases of small cell osteosarcoma and one chronic osteomyelitis were also analyzed and were both normal with respect to the t(11;22). By analyzing cell lines and small round cell tumor samples by fluorescent in situ hybridization, we established that interphase cytogenetics is a rapid alternative to chromosomal analysis for the detection of the t(11;22) and represents an invaluable tool for the differential diagnosis of small round cell tumors.     

RT-PCR法检测EWS-FLI1融合基因诊断尤文肉瘤/外周原始神经外胚层瘤

目的探讨尤文肉瘤/外周原始神经外胚层瘤(EWS/pPNET)石蜡包埋组织中EWS-FLI1融合基因表达的临床病理意义。方法采用免疫组化对1例纵隔尤文肉瘤/外周原始神经外胚层瘤进行观察,应用反转录-聚合酶链反应(RT-PCR)检测融合基因EWS-FLI1的表达。结果肿瘤由小圆细胞、卵圆形细胞及短梭形细胞构成,巢状、片状或列兵样排列,间质显著增生,未见典型菊形团结构。免疫组化显示CK、EMA和CD99弥漫强( )。RT-PCR检测出EWS-FLIl融合基因的表达。结论EWS/pPNET与促结缔组织增生性小圆细胞肿瘤(DSRCT)具有重叠的形态学和免疫组化特点,石蜡包埋组织中检测EWS-FLI1融合基因的表达可作为诊断EWS/pPNET的可靠指标。     

儿童肿瘤及急性白血病中ICAM-1(CD54)的表达及在CIK细胞免疫治疗中的临床意义

本研究旨在探讨细胞间黏附分子1(ICAM-1,CD54)在初诊儿童肿瘤及急性白血病细胞上的阳性表达率,以了解其分布规律及其临床意义。采用免疫组织化学方法检测46例儿童实体瘤的病理组织切片ICAM-1的阳性率,通过流式细胞仪检测60例儿童急性白血病细胞上ICAM-1的阳性率。儿童实体瘤包括淋巴瘤10例,肝母细胞瘤3例,神经母细胞瘤6例,横纹肌肉瘤2例,尤文氏肉瘤6例,纤维肉瘤2例,原始神经外胚层肿瘤5例,肾母细胞瘤11例,骨肉瘤1例;急性白血病包括急性淋巴细胞白血病(ALL)20例,急性非淋巴细胞白血病(ANLL)40例(M1 6例、M2 7例、M3 7例、M4 15例、M5 5例)。结果表明,儿童肿瘤组中3例肝母细胞瘤ICAM-1全部阳性,而在淋巴瘤、横纹肌肉瘤、神经母细胞瘤及尤文氏肉瘤阳性率不高,纤维肉瘤、肾母细胞瘤及原始神经外胚层肿瘤患儿中未见ICAM-1表达。急性白血病组中ALL的ICAM-1阳性率为55%,ANLL的M1、M2、M3型ICAM-1的阳性率为65%,M4、M5型为50%。结论:ICAM-1在儿童肿瘤、急性白血病细胞上的表达呈一定变异性,其在肝母细胞瘤及ANLL(M1、M2和M3)上阳性率高,而在纤维肉瘤、肾母细胞瘤及原始神经外胚层肿瘤等中不表达。     

Ewing's sarcoma family of tumors: an overview from diagnosis to survivorship

The Ewing's sarcoma family of tumors (ESFT) is a malignant primary bone tumor often involving soft tissue that affects not only children but also young adults. Since 1992, with the addition of ifosfamide and etoposide to standard chemotherapy for primary tumors, much improvement has been made in the treatment of ESFT, with a primary focus on children. Though often recognized as a childhood cancer, it can affect individuals into the middle years of their lives, but little is known about the outcomes of adults with ESFT. ESFT, which includes Ewing's sarcoma, extraosseous Ewing's sarcoma, Askin tumor, and primitive neuroectodermal tumor, is the second most common primary malignant bone tumor in children and adolescents. It accounts for 10% of primary malignant bone tumors in children and 3% of all childhood malignancies. The most common presenting symptoms of ESFT are pain or swelling. Treatment for ESFT consists of a multimodal approach, including chemotherapy, radiation therapy, and surgery. Children and young adults with Ewing's sarcoma face many physical challenges from their illness and the complications of their treatments. Nurses play an instrumental role in assessment techniques, which lead to prompt evaluation and intervention. Nurses are vital in the education and reinforcement of supportive care needs for this patient population.     

阴道尤文肉瘤/原始神经外胚层瘤临床病理观察

目的报道1例起源于阴道的骨骼外尤文肉瘤／原始神经外胚层瘤（ES/PNET），结合文献资料，复习其临床、病理、超微结构、免疫表型、细胞基因、鉴别诊断、治疗及预后等。方法该病例选白天津市病理会诊中心2001-06--2005-06间的1998例疑难病理标本，经常规制片，HE染色，又辅以免疫组化染色。结果阴道肿物呈带蒂息肉，由幼稚核深染小圆细胞组成，免疫组化呈神经及肌源性双向免疫表型，病理诊断阴道原发性ES/PNET。结论 ES/PNET诊断标准除原始小细胞外常向神经分化，必须具备免疫组化CD99及神经内分泌标记2项以上阳性，细胞遗传学分析有染色体易位t（11；22）（q24；q12）。     

采用 DCDF-FISH 早期监测 BCR / ABL( +) ALL 患者耐药简

本研究探讨双色双融合荧光原位杂交技术（DCDF-FISH）对BCR/ABL阳性伴复杂染色体易位的急性淋巴细胞白血病（ALL）患者的应用价值.通过骨髓细胞形态学检查、染色体分析、流式细胞术和DCDF-FISH技术等方法观察1例急性淋巴细胞白血病的临床特征,并通过DCDF-FISH技术动态观察患者对治疗的反应及病情演变.结果表明：患者发病时呈现急性淋巴细胞白血病表现,流式细胞术发现患者表达幼稚B淋巴细胞分子标志CD10、CD19和CD34,染色体分析显示,患者骨髓细胞有46,XY,i（8）,ider（9）t（9;22）[23]/47,idem,＋der（22） t（9;22）[7]核型;FISH显示,患者初发病时83％细胞含有BCR/ABL融合基因,其中5％的肿瘤细胞显示1R1G2F信号模式、14％显示1R1G3F、64％显示1R1G4F;患者经格列卫联合VTLP化疗而完全缓解时,FISH显示肿瘤细胞降19％,但是1R1G2F信号模式的细胞却增加到18％;患者经过巩固治疗后复发,1R1G2F信号模式的细胞增加到38％,最后患者因耐药而死亡.结论：复杂易位的BCR/ABL（＋）的急性淋巴细胞白血病患者存在多个肿瘤细胞亚群,且不同的亚群对药物的反应性可能不同,因此通过DCDF-FISH技术的信号模式以及对不同亚群细胞动态变化观察,可以在早期监测患者对治疗的反应性以及耐药情况.     

Basic fibroblast growth factor as a growth inhibitor for cultured human tumor cells.

Basic fibroblast growth factor (bFGF) stimulates the proliferation of many cells and it is found in a wide variety of normal or transformed tissues. As demonstrated here, bFGF is also present in cultured human Ewing's sarcoma cells. Unexpectedly, however, bFGF isolated from these cells inhibits their own proliferation, indicating that bFGF can act as an endogenous (autocrine) growth inhibitor for cultured Ewing's sarcoma cells. Since bFGF also inhibits the proliferation of some further tumor cells, but stimulates that of others, it can be considered a bifunctional regulator of tumor cell proliferation. The autocrine growth-inhibitory effect of bFGF in Ewing's sarcoma cells may explain the low mitotic activity of Ewing's sarcomas.     

Ewing's sarcoma: transplantation in nude rat

Eight Ewing's sarcoma, primary tumor or metastasis, have been transplanted in Nude Rats. These tumors grow slowly and only in female rats. One of them has been maintained for 13 months with 5 passages. It has conserved all the characteristics of the primary tumor, histologic and ultramicroscopic morphology, glycogen secretion and cytogenetic modification (11.22 translocation). The graft of Ewing's sarcoma to Nu/Nu rats is a valuable system to get more material in good condition to study the nature and the origin of Ewing's cells, to test the new chemotherapy trials and to prepare and test the monoclonal antibodies.     

环腺苷酸拟似物8-CPT-cAMP诱导M2b型急性髓系白血病细胞系Kasumi-1细胞分化的研究

为了解环腺苷酸拟似物8-对氯苯硫基环腺苷酸（8-CPT-cAMP）对M2b型急性髓系白血病（AML-M2b）细胞的作用,以AML-M2b细胞株Kasumi-1细胞为模型,通过观察细胞生长、形态、表面分化抗原、细胞周期分布以及对四氮唑蓝的还原能力的改变,研究8-CPT-cAMP对Kasumi-1细胞增殖及分化的影响,并应用半定量RT-PCR和Western blot检测药物处理前后Kasumi-1细胞内AML1-ETO融合蛋白的变化。结果发现,8-CPT-cAMP（200μmol/L）可明显抑制Kasumi-1细胞增殖而促使细胞趋向分化,但这种分化不是典型的完全终末性分化,8-CPT-cAMP对Kasu-mi-1细胞内AML1-ETO融合基因及其编码蛋白的表达无显著影响。结论：8-CPT-cAMP对AML-M2b细胞具有诱导分化效应。     

E1A gene therapy inhibits angiogenesis in a Ewing's sarcoma animal model

We assessed vascular endothelial growth factor (VEGF) expression in four different human Ewing's sarcoma cell lines (TC71, SK-ES, RD, and A4573) and in tumors in nude mice induced following s.c. injection of TC71 cells. Three of the four cell lines (TC71, SK-ES, and A4573) expressed significantly higher levels of VEGF than did normal human osteoblasts. Transfection of the adenovirus type 5 early region 1A (E1A) gene into TC71 cells down-regulated VEGF expression in vitro. In the mice bearing TC71 cell tumors, intratumoral injections of an adenoviral vector containing the E1A gene (Ad-E1A) decreased VEGF expression, inhibited tumor growth, and increased the survival rates in comparison with the mice given injections of PBS or an adenoviral vector containing beta-galactosidase (Ad-beta-gal). E1A gene therapy also significantly reduced blood vessel density and induced cell apoptosis in the tumors. These results demonstrate that E1A gene therapy inhibits angiogenesis, most likely by suppression of VEGF expression. Thus, E1A gene therapy may be a new therapeutic approach for Ewing's sarcoma.     

恶性外胚层间叶瘤临床病理观察

目的报道1例罕见的发生于项背部的恶性外胚层间叶瘤。方法复习相关文献,讨论恶性外胚层间叶瘤的临床表现、组织学特征、治疗和预后。结果患儿男性,6岁。肿块组织学显示神经源标记的小圆形、卵圆形细胞和肌源标记的梭形细胞。治疗以手术切除为主,辅以化疗。结论恶性外胚层间叶瘤常发生于小儿,可能起源于外胚层、中胚层或迁徙而来的神经嵴组织。肿瘤由间叶和神经外胚层两种成分组成,需与横纹肌肉瘤、Ewing肉瘤/PNET、神经肌迷芽瘤、恶性蝾螈瘤、畸胎瘤、肾母细胞瘤及小儿腹腔内纤维组织增生性小细胞肿瘤相鉴别,综合治疗可改善预后。     

尿激酶受体基因反义RNA转移体系的建立及其在白血病研究中的应用

尿激酶受体 (uPAR)表达与肿瘤 /白血病细胞的侵袭与转移能力明显相关。为探讨逆转录病毒载体介导的uPAR基因反义RNA转移体系在抑制白血病细胞表达uPAR中的价值 ,构建了表达反义uPAR基因的逆转录病毒载体LaCD87SN ,用脂质体转染 交互感染策略建立uPAR基因反义RNA转移体系 ,并以双嗜型aCD87病毒转导U937白血病细胞 ;用PCR分析反义uPAR基因的整合和表达 ;用流式细胞术和明胶酶谱分别检测白血病细胞CD87表达和基质金属蛋白酶 (MMP)活性。结果显示 :通过转染 交互感染获得上清中病毒滴度为 6 .3× 10 5cfu/ml的双嗜型病毒产生细胞Am12 /aCD87;aCD87病毒感染的U937/aCD87细胞内存在aCD87原病毒的整合 ,并高水平表达uPAR基因反义RNA。此外 ,与载体对照的U937/NeoR细胞相比 ,U937/aCD87细胞表面CD87分子表达并无明显降低 ,但其分泌MMP 9的能力显著下降。结论 :逆转录病毒载体介导的反义RNA转移不能有效地下调白血病细胞表面uPAR表达 ,但可能干扰CD87分子与MMP的相互作用。     

t（8;21）急性髓系白血病发病机制的研究进展

近50%的急性髓系白血病可以找到染色体易位,常见的染色体易位是t（8;21）（q22;q22）。这种易位使21号染色体上的aml1（runx1）基因与8号染色体上的eto（mtg8,runx1t1）基因相互融合形成aml1/eto融合基因。最初对于t（8;21）急性髓系白血病发病机制的研究一直着重在造血转录激活因子AML1转化为白血病抑制因子,在靶基因调控水平上阻碍髓系细胞分化,aml1/eto融合基因在造血分化过程关键点抑制作为肿瘤抑制因子的造血转录因子。目前认为,t（8;21）染色体易位及二次突变引起造血干细胞减少的系别限制性及基因组不稳定性是引发aml1/eto融合基因阳性白血病的主要原因。本文就aml1/eto融合基因及其剪接变异体在调控干细胞更新、阻断造血分化及与各造血系统特异的转录因子相互作用的分子机制进行综述。     

Regional drug delivery with radiation for the treatment of Ewing's sarcoma. In vitro development of a taxol release system.

Recently, several studies have suggested the radiosensitizing effect of taxol, a microtubular inhibitor. Our overall hypothesis is that a combination of radiation and taxol may demonstrate therapeutic efficacy over doses of either individually. Studies examining taxol use have mostly focused on systemic administration, which can lead to undesired effects. To circumvent these side effects, we propose a locally administered polymeric microsphere delivery system combined with radiation therapy for the treatment of Ewing's sarcoma. The present study focuses on the in vitro ability of taxol when present as a microencapsulated drug delivery system, and delivered locally at the site of the sarcoma/tumor, to block cells in the G2/M phase of the cell cycle and potentially enhance the radiation sensitivity of cells. Using the bioresorbable poly(anhydride-co-imide), poly[pyromellityl-imidoalanine-1,6-bis(carboxy-phenoxy)hexane] (PMA-CPH), and the radiosensitizing agent taxol, a microsphere based delivery system was fabricated. A solvent evaporation technique was used to encapsulate taxol at doses of 1%, 5%, and 10% in PMA-CPH microspheres. Release kinetics studies demonstrated that the total amount of taxol released and the release rate were directly dependent on loading percentage. Taxol's bioactivity and radiosensitizing ability were measured using flow cytometry. Co-culture of Ewing's sarcoma cells with and without taxol-loaded microspheres demonstrated that released taxol retained its bioactivity and effectively blocked cells in the radiosensitive G2/M phase of mitosis. The taxol-radiation delivery system studied achieved an 83% decrease in tumor cell count compared to control. Taxol effectively sensitized Ewing's sarcoma cells to radiation with radiosensitivity shown to be independent of radiation dose at levels of dosages studied. This work has demonstrated that taxol can be effectively released from a biodegradable PMA-CPH microsphere delivery system while maintaining potent combined cytotoxic and radiosensitizing abilities.