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.     

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。     

Primary extraskeletal Ewing’s sarcoma of the lumbar nerve root: A case report

BACKGROUNDEwing’s sarcoma (ES) is a highly aggressive bone malignancy. Extraskeletal ES (EES) originating in the spinal canal is extremely rare. Herein, we report on a rare case of EES with a primary lumbar spinal nerve root including the complete diagnosis and treatment.CASE SUMMARYA young female patient presented with a complaint of right lower limb pain for 1 mo. Magnetic resonance imaging (MRI) revealed an 11 mm × 14 mm × 31 mm mass in the lumbar epidural region extending at the fifth lumbar spine (L5) level toward the right L5 neural foramen. Our initial diagnosis was an epidural schwannoma. The patient underwent total laminectomy, tumor resection and pedicle screw internal fixation and the L5 root tumor was found to have been completely removed intraoperatively. Histopathological examination of the lesion showed a typical ES with a large number of small, round cells. Immunohistochemistry analysis indicated positive CD99 and S100. After surgery, the patient received chemotherapy and radiotherapy with a 1 year of follow-up and no recurrent tumors or new lesions were found upon spine MRI and positron emission tomography/computed tomography reexamination.CONCLUSIONClinically, ES outside the bone should be considered when nerve root tumors are encountered inside and outside the spinal canal and the diagnosis should be determined by pathological biopsy. After surgical resection, chemotherapy and radiotherapy should be performed. After treatment, active follow-up and regular review should be completed.     

EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells.

The translocation t(11;22) is a common chromosomal abnormality detected both in Ewing's sarcoma and in primitive neuroectodermal tumor cells. The translocation results in an EWS-Fli1 fusion gene, made up of the 5' half of the EWS gene on chromosome 22 fused to the 3' half of the Fli1 gene on chromosome 11. Recent studies have evaluated possible roles of the fusion gene products. However, the biological significance of EWS-Fli1 is still unknown. Using a competitive polymerase chain reaction technique, we show here that there might be a correlation between the expression levels of the EWS-Fli1 fusion gene and the proliferative activities of Ewing's sarcoma and primitive neuroectodermal tumor cells. When the EWS-Fli1 expression is inhibited by antisense oligodeoxynucleotides against the fusion RNA, the growth of the tumor cells is significantly reduced both in vitro and in vivo. The data further indicate the growth inhibition of the cells by the antisense sequence might be mediated by G0/G1 block in the cell cycle progression. These results suggest that EWS-Fli1 may play an important role in the proliferation of the tumor cells, and the EWS-Fli1 fusion RNA could be used as a target to inhibit the growth of Ewing's sarcoma and primitive neuroectodermal tumor with the specific antisense oligonucleotide.     

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.     

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.     

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）。     

长骨造釉细胞瘤样尤文肉瘤临床病理观察

目的探讨长骨造釉细胞瘤样尤文肉瘤的诊断和鉴别诊断。方法应用常规组织病理和免疫组化观察1例造釉细胞瘤样尤文肉瘤并复习相关文献。结果造釉细胞瘤样尤文肉瘤与造釉细胞瘤有相似的组织学形态,keratin均（＋）,但造釉细胞瘤样尤文肉瘤vimentin、CD56、CD99和Syn也是（＋）。结论长骨造釉细胞瘤样尤文肉瘤是一种高度恶性的肿瘤,它与长骨造釉细胞瘤具有不同性质。     

CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis

Ewing sarcoma (EWS) is an aggressive bone tumor of uncertain cellular origin. CD99 is a membrane protein that is expressed in most cases of EWS, although its function in the disease is unknown. Here we have shown that endogenous CD99 expression modulates EWS tumor differentiation and malignancy. We determined that knocking down CD99 expression in human EWS cell lines reduced their ability to form tumors and bone metastases when xenografted into immunodeficient mice and diminished their tumorigenic characteristics in vitro. Further, reduction of CD99 expression resulted in neurite outgrowth and increased expression of β-III tubulin and markers of neural differentiation. Analysis of a panel of human EWS cells revealed an inverse correlation between CD99 and H-neurofilament expression, as well as an inverse correlation between neural differentiation and oncogenic transformation. As knockdown of CD99 also led to an increase in phosphorylation of ERK1/2, we suggest that the CD99-mediated prevention of neural differentiation of EWS occurs through MAPK pathway modulation. Together, these data indicate a new role for CD99 in preventing neural differentiation of EWS cells and suggest that blockade of CD99 or its downstream molecular pathway may be a new therapeutic approach for EWS.     

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.     

Treatment approaches for metastatic Ewing's sarcoma: a review of the literature.

The Ewing's sarcoma family of tumors (ESFT) is an aggressive group of neoplasms that represent approximately 3% of all pediatric malignancies. The overall survival rates in patients with localized disease are approaching 75%. The outcome for the 25% of patients who present with metastatic disease, however, remains poor, with long-term survival rates of less than 30%. This review will explore the natural history of ESFT including clinical presentation, molecular pathology, and high-risk features of the disease. Outcomes of metastatic treatment protocols to date will be examined as well as the rationale for current and future therapies. Nursing considerations in caring for patients with metastatic ESFT will be discussed. A case scenario will be reviewed to highlight treatment and supportive care issues in the management of the disease. Cancer therapy in general is becoming more complex; treatment approaches involve different ways of targeting tumor cells. It is crucial that nurses caring for these patients understand the rationale behind treatment strategies so that appropriate patient education and support may be given.     

胸膜外孤立性纤维瘤的CT和MRI表现：10例报告及文献复习

目的 探讨胸膜外孤立性纤维瘤(ESFT)的CT和MRI表现.方法 回顾性分析10例经手术病理证实的ESFT的影像学资料,其中4例接受CT平扫加增强扫描,6例接受MRI平扫加增强扫描.结果 10例ESFT中,位于腹部3例,盆腔、枕骨大孔区SFT各2例,右侧颈根部、左眼眶、左颞部皮下SFT各1例;7例肿瘤呈圆形或卵圆形,3例呈分叶状;8例边界清楚,2例部分边界不清;肿瘤直径2.3~20.6cm,平均(7.78±6.82)cm.CT表现:1例呈囊实性,3例呈等密度实性,其中2例可见坏死、1例见多发钙化;增强后3例肿瘤实性部分渐进性强化,另1例为早期轻度强化.MRI表现:T1WI、T2WI均呈低信号1例,T1WI等信号、T2WI低信号1例,T1WI等信号、T2WI高信号3例,T1WI及T2WI均呈高信号1例;3例T2WI可见线样或片状低信号;增强后肿瘤均明显强化.结论 ESFT的影像学表现有一定特征;T2WI肿瘤主体呈低信号或肿瘤内部多发线样或片状低信号对诊断ESFT具有较高价值.     

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.     

宫颈粒细胞肉瘤临床病理观察

目的探讨宫颈粒细胞肉瘤的临床病理学特征、诊断及鉴别诊断：方法结合文献对1例罕见的宫颈粒细胞肉瘤的临床表现、组织形态学特点、免疫组化及治疗和预后进行探讨。结果宫颈粒细胞肉瘤以宫颈肿块为首要表现，形态学上肿瘤细胞弥漫分布，小至中等大小，胞质少，核圆形、椭圆形，染色质细腻，核分裂易见。免疫组化肿瘤细胞MPO、CD15、CD68和溶菌酶（＋）。结论宫颈粒细胞肉瘤是一种罕见肿瘤，仅凭形态学易误诊为淋巴瘤、ES／PNET、小细胞癌等。了解其本质有助于早期诊断和进行恰当的治疗。     

EWS/ATF1 expression induces sarcomas from neural crest–derived cells in mice

Clear cell sarcoma (CCS) is an aggressive soft tissue malignant tumor characterized by a unique t(12;22) translocation that leads to the expression of a chimeric EWS/ATF1 fusion gene. However, little is known about the mechanisms underlying the involvement of EWS/ATF1 in CCS development. In addition, the cellular origins of CCS have not been determined. Here, we generated EWS/ATF1-inducible mice and examined the effects of EWS/ATF1 expression in adult somatic cells. We found that forced expression of EWS/ATF1 resulted in the development of EWS/ATF1-dependent sarcomas in mice. The histology of EWS/ATF1-induced sarcomas resembled that of CCS, and EWS/ATF1-induced tumor cells expressed CCS markers, including S100, SOX10, and MITF. Lineage-tracing experiments indicated that neural crest–derived cells were subject to EWS/ATF1-driven transformation. EWS/ATF1 directly induced Fos in an ERK-independent manner. Treatment of human and EWS/ATF1-induced CCS tumor cells with FOS-targeted siRNA attenuated proliferation. These findings demonstrated that FOS mediates the growth of EWS/ATF1-associated sarcomas and suggest that FOS is a potential therapeutic target in human CCS.     

婴儿胸膜肺母细胞瘤临床病理观察

目的探讨胸膜肺母细胞瘤临床病理学特点及鉴别诊断。方法对1例胸膜肺母细胞瘤进行组织病理学观察并行免疫组化标记,同时复习相关文献。结果患儿男性,4个月。咳喘伴紫绀3天。巨检:右肺肿物,组织大小9 cm×6 cm×4 cm,切面灰白色,实性、质软;肿物与肺界限尚清,并占据大部分肺。镜检:肿瘤主要由原始胚胎性的圆形或卵圆形细胞组成,细胞中等大小,胞质少,核仁清,核分裂易见;部分区域主要由梭形及短梭形细胞构成的梭形细胞肉瘤或恶性纤维组织细胞瘤结构区,其间可见分化尚好的软骨岛或软骨结节。免疫组化:瘤细胞vimentin(+),软骨结节S-100(+),残留上皮成分CK(AE1/AE3)、EMA和TTF-1(+),NSE局灶(+),间质细胞SMA局灶少许(+),CD99和bcl-2弱(+),desmin、myoglobin、calretinin、calponin、FLI、MyoD-1和CD34(-)。结论胸膜肺母细胞瘤较为罕见,但恶性程度相对较高,组织病理学最典型表现为原始恶性小细胞聚集。临床上需要与肺母细胞瘤、肉瘤样间皮瘤、胚胎性横纹肌肉瘤、滑膜肉瘤、原始神经外胚叶肿瘤等相鉴别。