EWS/FLI-1 oncoprotein subtypes impose different requirements for transformation and metastatic activity in a murine model

Ewing sarcoma/primitive neuroectodermal tumors (EWS/PNET) are characterized by specific chromosomal translocations most often generating a chimeric EWS/FLI-1 gene. Depending on the number of juxtaposed exons assembled, several fusion types have been described with different incidences and prognoses. To assess the impact of each fusion type on the specific phenotypic, tumorigenic, and metastatic features of EWS/PNET, we developed an amenable system using a murine mesenchymal multipotent C3H10T1/2 cell line. Upon transduction of EWS/FLI-1, cells acquired dramatic morphological changes in vitro, including a smaller size and "neurite-like" membrane elongations. Chimeric fusion proteins conferred oncogenic properties in vitro, including anchorage-independent growth and an increased rate of proliferation. Furthermore, EWS/FLI-1 expression blocked mineralization, with concomitant repression of osteoblastic genes, and induced a dramatic repression of the adipocytic differentiation program. Moreover, EWS/FLI-1 promoted an aberrant neural phenotype by the de novo expression of specific neural genes. The intramuscular injection of transduced cells led to tumor development and the induction of overt osteolytic lesions. Analogously, to what was observed in human tumors, type 2 EWS/FLI-1 cells formed primary tumors in immunodeficient mice with a higher incidence and a lower latency than cells bearing types 1 and 3 fusions. By contrast, cells expressing types 2 and 3 fusions showed specific metastatic activity with a higher number of macroscopic metastases in soft tissues and osteolytic lesions in the limbs as compared to type-1-expressing cells. Therefore, the structure of each oncoprotein strongly influenced its tumorigenicity and metastagenicity. Thus, this model provides a basis for understanding the genetic determinants involved in Ewing tumor development and metastatic activity and represents a cellular system to analyze other oncoproteins involved in human sarcomagenesis.     

Ewing sarcoma/peripheral primitive neuroectodermal tumor: adult abdominal tumors with an Ewing sarcoma gene rearrangement demonstrated by fluorescence in situ hybridization in paraffin sections.

The differential diagnosis of small round cell tumors is exhaustive and requires ancillary studies. Relatively recently, fluorescence in situ hybridization (FISH) using probes for specific gene rearrangements has gained wide acceptance. This technique is particularly useful in the differential diagnosis of Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) and desmoplastic small round-cell tumor (DSRCT). In ES/PNET, the EWS gene is juxtaposed to the FLI-1 gene in 85% of cases and to the ERG gene in another 7% of cases; the EWS gene is juxtaposed to the WTI gene in DSRCT. Documentation of the EWS gene rearrangements in EWS/PNET has previously been demonstrated in frozen tissue. We report 2 unusual cases of EWS/PNET diagnosed in abdominal tumors in adults. Although the immunohistochemical results supported a diagnosis of ES/PNET, 1 case morphologically resembled DSRCT. The diagnosis in these 2 cases was confirmed by the FISH demonstration of EWS/FLI-1 gene fusion in paraffin-embedded tissue. Thus, the usefulness of FISH demonstration of an EWS gene rearrangement with these specific probes in such unusual cases is supported and is demonstrated in paraffin-embedded tissue.     

Newly described translocation (18;19)(q23;q13.2) in abdominal wall soft-tissue tumor resembling Ewing sarcoma/primitive neuroectodermal tumor

From a morphologic standpoint, Ewing sarcoma (EWS) is one of a number of pediatric malignancies that are characterized by sheets of small, round, blue cells. Ewing sarcoma can usually be differentiated from other small round blue cell tumors by the presence of a gene rearrangement having a consistent breakpoint within the Ewing sarcoma gene (EWSR1) at 22q12. Although the most common translocation partner is FLI1, located at 11q24, there is a growing list of alternate rearrangements involving different loci. We describe the first example of a soft-tissue sarcoma morphologically and immunohistochemically similar to Ewing sarcoma, but with a novel t(18;19)(q23;q13.2).     

FLI-1在小圆细胞肿瘤鉴别诊断中的应用

目的研究FLI-1和CD99在小圆细胞肿瘤中的表达情况,探讨二者在小圆细胞肿瘤鉴别诊断中的应用价值。方法对46例小圆细胞肿瘤进行FLI-1和CD99免疫组化标记,并结合临床与病理组织学进行对比研究。结果FLI-1在Ewing肉瘤／外周原始神经外胚叶肿瘤（EWS／PNET）中阳性表达率为90．5％（19／21）,在分化差的滑膜肉瘤、横纹肌肉瘤分别为14．3％（1／7）、22．2％（2／9）,而在嗅神经母细胞瘤和间叶软骨肉瘤均无表达。CD99在EWS／PNET中阳性表达率为95．2％（20／21）,在分化差的滑膜肉瘤、横纹肌肉瘤、嗅神经母细胞瘤和间叶软骨肉瘤分别为71．4％（5／7）、66．7％（6／9）、75．0％（3／4）和60％（3／5）。FLI-1标记在EWS／PNET的敏感性为90．5％,特异性为88％;而CD99在EWS／PNET的敏感性为95．0％,特异性为32％。FLI-1在EWS／PNET中的特异性明显高于CD99（P〈0．05）。结论FLI-1在EWS／PNET诊断中的价值优于CD99,并且可用于小圆细胞肿瘤的鉴别诊断。     

Friend leukaemia integration-1 expression in malignant and benign tumours: a multiple tumour tissue microarray analysis using polyclonal antibody

BACKGROUND: Friend leukaemia integration-1 (FLI-1) antibody is a useful marker for Ewing's sarcoma/primitive neuroectodermal tumour (EWS/PNET) and vascular tumours. However, it is also expressed in subsets of lymphoblastic lymphoma, Merkel cell carcinoma (MCC) and desmoplastic small round cell tumour (DSRCT). AIM: To determine expression of FLI-1 in various benign and malignant neoplasms, by immunohistochemical analysis on 4323 tumours using multiple tumour microarrays, as well as on whole sections. RESULTS: FLI-1 was expressed in 46/62 EWS/PNETs, 2/3 olfactory neuroblastomas, 7/102 small cell carcinomas of the lung, 10/34 MCCs, 1/14 rhabdomyosarcoma, 19/132 non-Hodgkin's lymphomas, 2/3 DSRCTs, and in 53/74 benign and malignant vascular tumours. In addition, 27/508 squamous cell carcinomas, 19/837 adenocarcinomas, 10/400 urothelial bladder cancers, 1/40 basal cell carcinomas, 3/29 liposarcomas, 1/40 glioblastoma multiforme and 9/29 medullar carcinomas of the breast expressed FLI-1. The sensitivity and specificity of FLI-1 to distinguish EWS/PNET from all types of malignancies were 74.2% and 96.0%, respectively. Finally, the sensitivity and specificity of FLI-1 to distinguish EWS/PNET from other small round cell tumours (SRCTs) were 74.2% and 91.6%, respectively. CONCLUSION: This study was the first to show that FLI-1 can be seen in a variety of solid tumours, some of which had never been explored before. This finding should be kept in mind, especially when using FLI-1 as a marker for finding the primary origin of poorly differentiated metastatic tumour. Finally, despite the expression of FLI-1 in numerous malignancies, it is still considered to be highly sensitive and specific in distinguishing EWS/PNET from other tumour types in general and from other SRCTs in particular.     

Ewing sarcoma/primitive neuroectodermal tumor of the kidney: a case report. Diagnosed by immunohistochemistry and molecular analysis

BACKGROUND: Ewing sarcoma/primitive neuroectodermal tumor (EWS/PNET) of the kidney is a rare and aggressive tumor. It has a rapid clinical progression with early metastasis and death. Few cases with documented t(11;22) have been reported in the literature. CASE PRESENTATION: We report a case of EWS/PNET of the kidney in a 26-year-old woman with widespread metastasis at initial presentation. The tumor cells showed strong expression for CD99 and FLI-1 monoclonal antibodies and polyclonal antibodies and were negative for WT1 and numerous other markers. The diagnosis was subsequently confirmed by demonstrating t(11;22)(q24;q12) using cytogenetic karyotyping and fluorescence in situ hybridization. CONCLUSIONS: Due to the different prognosis and management between EWS/PNET and other primary renal neoplasms with similar morphology, a histopathologic diagnosis with extreme accuracy should be made. Cytogenetic analysis is an important supportive tool to immunohistochemistry in making the final diagnosis.     

Core-Shell type lipid/rPAA-Chol polymer hybrid nanoparticles for in vivo siRNA delivery

Our previous study had reported that cholesterol-grafted poly(amidoamine) (rPAA-Chol polymer) was able to self-assemble into cationic nanoparticles and act as a potential carrier for siRNA transfection. In this study, the core–shell type lipid/rPAA-Chol hybrid nanoparticles (PEG-LP/siRNA NPs and T7-LP/siRNA NPs) were developed for improving in vivo siRNA delivery by modifying the surface of rPAA-Chol/siRNA nanoplex core with a lipid shell, followed by post-insertion of polyethylene glycol phospholipid (DSPE-PEG) and/or peptide (HAIYPRH, named as T7) modified DSPE-PEG-T7. The integrative hybrid nanostructures of LP/siRNA NPs were evidenced by dynamic light scattering (DLS), confocal laser scanning microscope (CLSM), cryo-transmission electron microscope (Cryo-TEM) and surface plasmon resonance (SPR) assay. It was demonstrated that the T7 peptide modified LP/siRNA NPs (T7-LP/siRNA NPs) exhibited uniform and spherical structures with particle size of 99.39 ± 0.65 nm and surface potential of 42.53 ± 1.03 mV, and showed high cellular uptake efficiency and rapid endosomal/lysosomal escape ability in MCF-7 cells. Importantly, in vitro gene silencing experiment demonstrated that both of pegylated and targeted LP/siEGFR NPs exhibited significantly stronger downregulation of EGFR protein expression level in MCF-7 cells, compared to that of the physical mixture of siRNA lipoplexes and rPAA-Chol/siRNA nanoplexes. In vivo tumor therapy on nude mice bearing MCF-7 tumors further confirmed that the targeted T7-LP/siEGFR NPs exhibited the greatest inhibition on tumor growth via transferrin receptor-mediated targeting delivery, without any activation of immune responses and significant body weight loss following systemic administration. These findings indicated that the core-shell type T7-LP/siRNA nanoparticles would be promising siRNA delivery systems for in vivo tumor-targeted therapy.     

PEGylated carboxymethyl chitosan/calcium phosphate hybrid anionic nanoparticles mediated hTERT siRNA delivery for anticancer therapy

Lack of safe and effective delivery vehicle is the main obstacle for siRNA mediated cancer therapy. In this study, we synthesized a pH-sensitive polymer of PEG grafted carboxymethyl chitosan (PEG-CMCS) and developed anionic-charged hybrid nanoparticles of PEG-CMCS and calcium phosphate (CaP) for siRNA delivery through a single-step self-assembly method in aqueous condition. The formed nanoparticles with charge of around −8.25 mv and average diameter of 102.1 nm exhibited efficient siRNA encapsulation and enhanced colloidal and serum stability. The test in vitro indicated that the nanoparticles entered into HepG2 cells by endocytosis, and achieved endosomal escape of siRNA effectively due to the pH-responsive disassembly of nanoparticles and dissolution of CaP in the endosome. Reporter gene silencing assay showed that luciferase siRNA delivered by the anionic nanoparticles could achieve gene silencing efficacy comparable to that of conventional Lipofectamine 2000. Additionally, dramatic hTERT knockdown mediated by the anionic nanoparticles transfection induced significant apoptosis of HepG2 cells in vitro. After intravenous injection in tumor-bearing BALB/c nude mice, the nanoparticles specifically accumulated into tumor regions by EPR effect, leading to efficient and specific gene silencing sequentially. Most importantly, the nanoparticles carrying hTERT siRNA inhibited tumor growth significantly via silencing hTERT expression and inducing cells apoptosis in HepG2 tumor xenograft. Moreover, comprehensive safety studies of the nanoparticles confirmed their superior safety both in vitro and in vivo. We concluded that the PEG-CMCS/CaP hybrid anionic nanoparticles possessed potential as a safe and effective siRNA delivery system for anticancer therapy.     

Solid pseudopapillary neoplasms of the pancreas are associated with FLI-1 expression, but not with EWS/FLI-1 translocation.

Solid pseudopapillary neoplasms of the pancreas are rare pancreatic tumors with mostly benign behavior, affecting almost exclusively women. Their histogenetic origin is still unsolved, but a recently reported EWS/FLI-1 translocation t(11;22)(q24;q21) and the consistent expression of CD56 and the progesterone receptor, both genes located on the long arm of chromosome 11, point to chromosome 11q as a potential locus of gene aberration in solid pseudopapillary neoplasms. To further elucidate this issue, we studied 30 cases of solid pseudopapillary neoplasms by comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH) and immunohistochemistry. Immunohistochemically, 38% showed nuclear expression of FLI-1 and all cases revealed positivity for CD56 and the progesterone receptor, whereas no solid pseudopapillary neoplasm expressed CD34. No translocation of the EWS gene was found by FISH and no gross chromosomal gain or loss was detected by CGH. It is concluded that FLI-1 expression in solid pseudopapillary neoplasms is not associated with an EWS/FLI-1 translocation. In addition, there are no chromosomal gains or losses, especially on chromosome 11, where the FLI-1 gene is located adjacent to the gene for CD56 (NCAM). These data add another feature to the complex phenotypic appearance of solid pseudopapillary neoplasms.     

Diagnostic utility of FLI-1 monoclonal antibody and dual-colour, break-apart probe fluorescence in situ (FISH) analysis in Ewing's sarcoma/primitive neuroectodermal tumour (EWS/PNET). A comparative study with CD99 and FLI-1 polyclonal antibodies

AIMS: To compare the sensitivity and specificity of the recently commercially available FLI-1 monoclonal (FLI-1m) antibody with the currently used antibodies [CD99 and FLI-1 polyclonal (FLI-1p)] in the diagnosis of Ewing's sarcoma/primitive neuroectodermal tumour (EWS/PNET) and to determine the diagnostic value of the EWSR1 (22q12) dual-colour, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique. MATERIALS AND METHODS: Forty-three cases of well-documented EWS/PNET and 15 non-EWS/PNET cases were retrieved from the archival files. Immunohistochemistry (IHC) for FLI-1p, FLI-1m and FISH analysis was performed. RESULTS: The most sensitive and specific test panel for the diagnosis of EWS/PNET is the combination of CD99 and FLI-1p. FISH had a very high specificity (100%) but only a moderate sensitivity (50%). CONCLUSION: The combination of CD99 and FLI-1p is the method of choice for the diagnosis of EWS/PNET. EWRS1 (22q12) dual-colour, break-apart rearrangement probe FISH should be used as a confirmatory test in addition to CD99 and FLI1-p due to its high specificity.     

Multifunctional,self-assembling anionic peptide-lipid nanocomplexes for targeted siRNA delivery

Formulations of cationic liposomes and polymers readily self-assemble by electrostatic interactions with siRNA to form cationic nanoparticles which achieve efficient transfection and silencing in vitro. However, the utility of cationic formulations in vivo is limited due to rapid clearance from the circulation, due to their association with serum proteins, as well as systemic and cellular toxicity. These problems may be overcome with anionic formulations but they provide challenges of self-assembly and transfection efficiency. We have developed anionic, siRNA nanocomplexes utilizing anionic PEGylated liposomes and cationic targeting peptides that overcome these problems. Biophysical measurements indicated that at optimal ratios of components, anionic PEGylated nanocomplexes formed spherical particles and that, unlike cationic nanocomplexes, were resistant to aggregation in the presence of serum, and achieved significant gene silencing although their non-PEGylated anionic counterparts were less efficient. We have evaluated the utility of anionic nanoparticles for the treatment of neuronal diseases by administration to rat brains of siRNA to BACE1, a key enzyme involved in the formation of amyloid plaques. Silencing of BACE1 was achieved in vivo following a single injection of anionic nanoparticles by convection enhanced delivery and specificity of RNA interference verified by 5′ RACE-PCR and Western blot analysis of protein.     

Small cell osteosarcoma with Ewing sarcoma breakpoint region 1 gene rearrangement detected by interphase fluorescence in situ hybridization

Because of its characteristic morphologic appearance, small cell osteosarcoma (SCO) can be confused with other small round cell malignancies of the bone, most importantly with Ewing sarcoma, making this distinction difficult. A specific tool used in separating SCO from Ewing sarcoma has been the detection of Ewing sarcoma breakpoint region 1 (EWSR1) gene rearrangements in Ewing sarcoma and their absence in SCO. However, there are rare case reports that have documented the existence of EWSR1 gene rearrangement in SCO. In this report, we describe another case of SCO with an EWSR1 gene rearrangement detected by interphase fluorescence in situ hybridization. Our finding adds support to the existing evidence that SCO is a tumor that can be characterized by EWSR1 gene arrangements. Therefore, we caution the pathology community not to rely solely on molecular studies in distinguishing SCO from Ewing sarcoma.