Application of electron microscopic analysis and fluorescent in situ hybridization technique for the successful diagnosis of extraskeletal Ewing's sarcoma.

The diagnosis of soft tissue tumors is often challenging. Immunohistochemical investigation, let alone routine histopathological investigation, may not allow definitive diagnosis in some cases. To overcome such difficulties, more advanced techniques need to be adopted. Herein, we report an extremely rare 56-year-old Japanese female case of extraskeletal Ewing's sarcoma (ES), successfully diagnosed by electron microscopy (EM) using formalin-fixed sections and fluorescence in situ hybridization (FISH). The patient had a 2-year history of a tumor growing on the leg. In routine histopathology, invasive proliferation of tumor cells was observed in the dermis. Tumor cells were round and uniform with large hyperchromatic nuclei, which were positively stained for CD56, VS38c, Ki-67, MIC2 and vimentin, but not for pan-keratin AE1 + AE3, cytokeratin 20, chromogranin A, synaptophysin and neuron-specific enolase. As these findings were not conclusive to make the final diagnosis, EM specimens were prepared from formalin-fixed sections and subjected to investigation. Cell surface projections and dense core granules were detected, suggestive of either Merkel cell carcinoma or extraskeletal ES. Subsequent FISH analysis identified reciprocal translocation of the ESWR1 gene, enabling the final diagnosis of extraskeletal ES. This study provides useful information enabling the diagnosis of this uncommon soft tissue tumor.

Plasma hydrogenated cationic detonation nanodiamonds efficiently deliver to human cells in culture functional siRNA targeting the Ewing sarcoma junction oncogene.

The expression of a defective gene can lead to major cell dysfunctions among which cell proliferation and tumor formation. One promising therapeutic strategy consists in silencing the defective gene using small interfering RNA (siRNA). In previous publications we showed that diamond nanocrystals (ND) of primary size 35 nm, rendered cationic by polyethyleneimine-coating, can efficiently deliver siRNA into cell, which further block the expression of EWS/FLI-1 oncogene in a Ewing sarcoma disease model. However, a therapeutic application of such nanodiamonds requires their elimination by the organism, particularly in urine, which is impossible for 35 nm particles. Here, we report that hydrogenated cationic nanodiamonds of primary size 7 nm (ND-H) have also a high affinity for siRNA and are capable of delivering them in cells. With siRNA/ND-H complexes, we measured a high inhibition efficacy of EWS/FLI-1 gene expression in Ewing sarcoma cell line. Electron microscopy investigations showed ND-H in endocytosis compartments, and especially in macropinosomes from which they can escape before siRNA degradation occurred. In addition, the association of EWS/FLI-1 silencing by the siRNA/ND-H complex with a vincristine treatment yielded a potentiation of the toxic effect of this chemotherapeutic drug. Therefore ND-H appears as a promising delivery agent in anti-tumoral gene therapy.

Modeling Ewing sarcoma tumors in vitro with 3D scaffolds.

The pronounced biological influence of the tumor microenvironment on cancer progression and metastasis has gained increased recognition over the past decade, yet most preclinical antineoplastic drug testing is still reliant on conventional 2D cell culture systems. Although monolayer cultures recapitulate some of the phenotypic traits observed clinically, they are limited in their ability to model the full range of microenvironmental cues, such as ones elicited by 3D cell-cell and cell-extracellular matrix interactions. To address these shortcomings, we established an ex vivo 3D Ewing sarcoma model that closely mimics the morphology, growth kinetics, and protein expression profile of human tumors. We observed that Ewing sarcoma cells cultured in porous 3D electrospun poly(ε-caprolactone) scaffolds not only were more resistant to traditional cytotoxic drugs than were cells in 2D monolayer culture but also exhibited remarkable differences in the expression pattern of the insulin-like growth factor-1 receptor/mammalian target of rapamycin pathway. This 3D model of the bone microenvironment may have broad applicability for mechanistic studies of bone sarcomas and exhibits the potential to augment preclinical evaluation of antineoplastic drug candidates for these malignancies.

Ewing sarcoma-primitive neuroectodermal tumor of the uterus: a clinicopathologic, immunohistochemical and ultrastructural study of one case.

INTRODUCTION: Ewing sarcoma-primitive neuroectodermal tumors (ES/PNET) constitute a family of neoplasms characterized by a continuum of neuroectodermal differentiation. ES/PNET of the uterus is rare. There are 43 cases published in the English literature as far as we know. We describe an additional case. CASE REPORT: A 56-year-old woman presented with a 2-month history of irregular menopausal vaginal bleeding. After surgical excision, microscopic, immunohistochemical and electron microscopic examination suggested the diagnosis of ES/PNET. The patient underwent combined chemotherapy consisting of ifosfamide, etoposide, and cisplatin. She was alive with no evidence of recurrence or metastasis after 41 months of the initial operation. DISCUSSION: In spite of the rarity of ES/PNET, we should consider it in the differential diagnosis of small cell neoplasms of the uterus.

Wnt-3a and Dickkopf-1 stimulate neurite outgrowth in Ewing tumor cells via a Frizzled3- and c-Jun N-terminal kinase-dependent mechanism.

Recombinant Wnt-3a stimulated the rapid formation of elongated processes in Ewing sarcoma family tumor (ESFT) cells that were identified as neurites. The processes stained positively for polymerized actin and microtubules as well as synapsin I and growth-associated protein 43. Inhibition of the Wnt receptor, Frizzled3 (Fzd3), with antiserum or by short interfering RNA (siRNA) markedly reduced neurite extension. Knockdown of Dishevelled-2 (Dvl-2) and Dvl-3 also suppressed neurite outgrowth. Surprisingly, disruption of the Wnt/Fzd/lipoprotein receptor-related protein (LRP) complex and the associated beta-catenin signaling by treating cells either with the Wnt antagonist Dickkopf-1 (Dkk1) or LRP5/LRP6 siRNA enhanced neuritogenesis. Neurite outgrowth induced by Dkk1 or with LRP5/LRP6 siRNA was inhibited by secreted Fzd-related protein 1, a Wnt antagonist that binds directly to Wnt. Moreover, Dkk1 stimulation of neurite outgrowth was blocked by Fzd3 siRNA. These results suggested that Dkk1 shifted endogenous Wnt activity from the beta-catenin pathway to Fzd3-mediated, noncanonical signaling that is responsible for neurite formation. In particular, c-Jun amino-terminal kinase (JNK) was important for neurite outgrowth stimulated by both Wnt-3a and Dkk1. Our data demonstrate that Fzd3, Dvl, and JNK activity mediate Wnt-dependent neurite outgrowth and that ESFT cell lines will be useful experimental models for the study of Wnt-dependent neurite extension.

Keratin-positive Ewing's sarcoma: an ultrastructural study of 12 cases.

Ewing's sarcoma/primitive neuroectodermal tumor (EWS/PNET) is an aggressive neoplasm of bone and soft tissue. Histologically, it is characterized by the presence of small round blue cells, which usually express MIC-2 and FLI-1 immunohistochemically. The most specific feature for diagnosis, however, is cytogenetic or molecular evidence of a consistent abnormality, the t(11;22)(q24;q12), or variants thereof. The immunohistochemical expression of keratins in a significant proportion of these cases has been highlighted in several recent studies. The ultrastructural features of these keratin-positive tumors have not, however, been characterized in detail. In this study we analyzed the ultrastructural features of 12 well-documented EWS/PNETs that stained strongly for pankeratin by immunohistochemistry. Ultrastructurally, the tumor cells contained a few organelles, which included a small number of mitochondria, poorly developed Golgi complexes, free ribosomes, and inconspicuous rough-endoplasmic reticulum. Rudimentary cell junctions were seen in 2 tumors while prominent junctions were observed in the remaining 10. Five tumors contained intracytoplasmic filaments, and definite tonofibrils were identified in 2. Well-developed basal lamina around tumor cells were also demonstrated in 2 tumors. Follow-up information was available for all cases. Seven patients died of disease, 2 are alive with disease, and 3 have no current evidence of disease. The cohort includes 5 patients with a type-1 translocation, which has been associated with a better prognosis in some studies; 4 of these patients have died of their disease, and 1 is alive with recurrent disease. This study shows that keratin-positive EWS/PNETs have evidence of epithelial differentiation ultrastructurally, and may possibly represent a more aggressive subset of the EWS/PNET group of tumors.

Primary Ewing's sarcoma of the suodenum: a case report.

We report a case of Ewing's sarcoma arising from the duodenum in a 20-year-old woman who presented with a rapidly progressive ulcerative lesion. The surgical specimen obtained via Whipple's operation revealed a small round-cell tumor (SRCT) in the first and second portion of the duodenum. The tumor cells revealed strong immunore-activity for CD 99 and vimentin and focal paranuclear dot-like immunoreactivity for cytokeratin. Electron microscopy showed primitive tumor cells with few cytoplasmic organelles, but neither neurosecretory granules nor specific cell junctions were present. On Western blot study, 68-kDa EWS/FLI1 fusion protein was detected. The occurrence of Ewing's sarcoma in the gastrointestinal hollow viscus has recently been recognized, and this case expands the known anatomic sites that can harbor Ewing's sarcoma by demonstrating primary duodenal involvement.

Ultrastructure of the Ewing's sarcoma family of tumors.

Specimens of 47 tumors diagnosed by routine light microscopy as Ewing's sarcoma of bone, and 5 similar soft tissue tumors (extraskeletal Ewing's sarcomas), were examined by transmission electron microscopy. Immunohistochemical stains were performed on all the tumors, and pre-therapy and post-therapy specimens from 5 of the patients were compared. Cell and nuclear areas were assessed in 41 cases by cytomorphometry by using low-magnification electron micrographs. DNA ploidy was determined by static cytometry on 51 of the tumors. None of the methods revealed differences between the bone and soft tissue tumors. The ultrastructural spectrum extended imperceptibly from the typical forms to markedly irregular variants, and was much broader than could be anticipated from the light microscopy. Neural features were observed but they were not common. Comparison of the Ewing's sarcomas with a group of other small round cell tumors (rhabdomyosarcoma, neuroblastoma, small cell carcinoma) using the same techniques showed that they have similar cell and nuclear areas despite the obvious differences in their immunophenotypes and ultrastructure. The collective findings are in keeping with the currently favored view that Ewing's sarcoma and peripheral primitive neuro-ectodermal tumor are the extremes in a morphologic continuum within which neural differentiation ranges from absent to prominent.

Ewing's sarcoma family of tumor arising in the adrenal gland--possible diagnostic pitfall in pediatric pathology: histologic, immunohistochemical, ultrastructural, and molecular study.

We present an adrenal Ewing's sarcoma family of tumor (ESFT) arising in an 11-year-old Japanese boy. Although intensive chemoradiotherapy and radical surgery were performed, the patient died of obstinate disease 1 year and 3 months after the initial presentation. The primary site (adrenal gland) with radiologic findings (with foci of calcification), high titer of serum neuron specific enolase, and sheets of monotonous primitive rounded cells on histology mostly favored neuroblastoma. However, a diagnosis of ESFT was confirmed by immunohistochemical profile, including MIC2-positivity and molecular study disclosing EWS-FLI1 chimera gene verified by direct sequencing. Recognition of adrenal ESFT and use of newly developed diagnostic techniques are required for differential diagnosis of undifferentiated small round cell tumor of the adrenal gland.

Immunohistochemical and ultrastructural investigation of neural differentiation in Ewing sarcoma/PNET of bone and soft tissues.

The authors evaluated the role of immunohistochemistry and electron microscopy in defining neural differentiation in 28 cases of Ewing sarcoma/PNET. The panel of primary antibodies used included vimentin, MIC-2, NSE, S-100 protein, leu7, neurofilaments, GFAP, and chromogranin A. Cases were considered undifferentiated when neural markers were absent, poorly differentiated if one neural marker was present, and well differentiated if two or more markers were observed. Cases were also evaluated for the presence of cytoplasmic processes, microtubules, and neurosecretory granules as ultrastructural features of neural differentiation: the tumor was classified as well differentiated if two of these features were present; and poorly differentiated if one was evident; all other cases were considered undifferentiated. According to immunohistochemistry, 10 cases (35.7%) were undifferentiated, 12 cases (42.9%) were poorly differentiated, and 6 (21.4%) were well differentiated. According to the ultrastructural analysis, 10 tumors were undifferentiated (35.7%), 14 poorly differentiated (50%), and 4 well differentiated (14.3%). The overall concordance between the two techniques was low (35.7%), and both modalities were concordant in classifying only 1 well-differentiated, 5 poorly differentiated, and 4 undifferentiated tumors. In conclusion, the authors suggest that investigations devoted to test the prognostic significance of neural differentiation in these neoplasms should employ both immunohistochemistry and electron microscopy, separately and in combination, to assess what is the most effective choice for predicting the clinical course.

Lovastatin induces apoptosis in a primitive neuroectodermal tumor cell line in association with RB down-regulation and loss of the G1 checkpoint.

To develop a new approach to the treatment of primitive neuroectodermal tumors we evaluated the effect of the HMG-CoA reductase inhibitor lovastatin on the Ewing's sarcoma cell line CHP-100. Lovastatin induced neural morphology and markers including neuron-specific enolase and neurofilament protein. The acquisition of neural morphology required new mRNA synthesis, and cDNA microarray analysis confirmed that lovastatin altered the program of gene expression. After morphologic differentiation the cells underwent rapidly progressive apoptosis. In normal development of neuronal progenitors, differentiation signals trigger p21WAF1 accumulation, RB hypophosphorylation, enhanced RB-E2F-1 association, and G1 arrest, and these events have been shown to protect from apoptosis. In contrast, in the Ewing's sarcoma cells lovastatin triggered differentiation without causing cell cycle arrest: p21WAF1 was not induced, RB remained hyperphosphorylated, and RB protein expression and RB-E2F-1 association were markedly downregulated, suggesting that loss of an RB-regulated G1 checkpoint promoted apoptosis. Consistent with this hypothesis, adenoviral p21WAF1 decreased DNA synthesis and partially protected from lovastatin-induced cytotoxicity. The data demonstrate a new model for examining the genetic regulation of cell fate in a neural progenitor tumor and suggest a new approach to the treatment of this neoplasm.

EWS/ETS fusion genes induce epithelial and neuroectodermal differentiation in NIH 3T3 fibroblasts.

Ewing's sarcoma is the least differentiated member of the peripheral primitive neuroectodermal (pPNET) tumor family. Chromosomal translocations involving the EWS gene and five different Ets family transcription factor genes create fusion genes encoding aberrant transcription factors and are implicated in the vast majority of Ewing's sarcoma cases. Here, NIH 3T3 fibroblasts were infected with control (tk-neo or RAS) and two different EWS/ETS-expressing retroviruses. In vitro studies of established polyclonal lines expressing the two EWS/ETS genes, either EWS/FLI1 or EWS/ETV1, showed induction of cytokeratin 15 gene expression. Both fusion genes also caused characteristic gross morphologic, histologic, and ultrastructural changes in NIH 3T3 cells when transformed cell lines were injected into CB-17-scid mice. Native NIH 3T3 cells with a spindled cell morphology were converted to polygonal cells with high nucleo-cytoplasmic ratios that continued to express abundant cytokeratin. Extracellular collagen deposition was abolished, rough endoplasmic reticulum was markedly diminished, and rudimentary cell-cell attachments appeared. Most strikingly, neurosecretory-type dense core granules like those seen in pPNET were now evident. This murine model, created in mesenchyme-derived NIH 3T3 cells, demonstrated new characteristics of both neuroectodermal and epithelial differentiation and resembled small round cell tumors microscopically.

Ovarian small cell tumors: an electron microscopic review.

Small cell tumors of the ovary are uncommon but represent an important group to recognize in the differential diagnosis of primary and metastatic ovarian neoplasms. In some cases the correct diagnosis cannot be confidently made on the basis of clinical setting, routine light microscopy, and immunohistochemistry, and electron microscopy may be supportive or definitive in establishing cell type. The cell type is often important in choosing optimal therapy and in predicting prognosis. The authors performed electron microscopy on a moderate number of ovarian small cell tumors and here describe and illustrate the diagnostic features of representative examples of various types. The ultrastructural features of the metastatic tumors, such as embryonal rhabdomyosarcoma, neuroblastoma, and melanoma, are identical to those of their respective primary tumors, are well known, and usually pose no problem in diagnosis. On the other hand, the ultrastructural features of some primary ovarian small cell tumors may present a more difficult differential diagnosis, because they have features that are subtle and/or in common. Exemplary of tumors in this category are diffuse adult granulosa cell tumor, endometrial stromal sarcoma, and small cell carcinomas of the hypercalcemic and pulmonary (oat cell) types. Distinguishing among them may be difficult but is possible, and electron microscopy may be a valuable supplement to the diagnostic information obtained from the clinical presentation, light microscopy, immunohistochemistry and, in some tumors, cytometric analysis of these neoplasms.

Primary endocervical extraosseous Ewing's sarcoma/PNET.

A 36-year-old woman presented with intermenstrual spotting and was found to have a cystic mass involving the uterine cervix on a pelvic ultrasound examination. A necrotic and hemorrhagic tumor was excised by hysterectomy and processed for light and electron microscopic investigation and molecular analysis. Microscopic examination revealed a small round cell tumor that immunohistochemical studies (including staining for the highly restricted surface antigen p30/32MIC2) and ultrastructural studies indicated was an extraosseous Ewing's sarcoma (EES)/primitive neuroectodermal tumor (PNET). This diagnosis was established by detection of EWS/ERG fusion transcript through reverse transcription polymerase chain reaction (RT-PCR) with nested primers. Full body computed tomography failed to detect any extrauterine tumor, and the patient is clinically free of disease 18 months after hysterectomy. This case represents the first report of a primary EES/PNET arising in the uterine cervix.

[Clinico-pathologic study of Ewing's sarcomas of bone and soft tissue and peripheral primitive neuroectodermal tumors].

OBJECTIVE: To study the diagnosis and differentiation of EW and PNET. METHODS: Fourteen cases of Ewing's sarcomas (EW) and peripheral primitive neuroectodermal tumors (PNET) were studied by light microscopy, immunohistochemistry and electron microscopy (EM). Twelve cases were followed up. Schmidt criterion was used for the differential diagnosis of EW and PNET. RESULTS: There were 6 cases of EW and 8 cases of PNET. Six of 8 PNET cases had Homer-Wright (H-W) rosettes. In this series, 12/14 cases were positive for O13 (HBA71) staining. NSE was positive in 3 cases of EW. All PNET cases were positive for neural markers, and 5 of them were positive for more than two of these markers. Electronmicroscopically, there were neurosecretory granules (4/4 cases), nerve-like protrusions and microfilaments (1/4 case) in PNET. In 3 of 6 EW and 1 of 6 PNET, PAS staining was positive. During the follow-up period from 2 months to 5 years, 3 cases with intraosseous EW remain alive and free of tumor. The remaining patients are dead or having their tumors metastasized. CONCLUSION: EW is more primitive in cell differentiation, while PNET has more neural differentiation. The presence of H-W rosettes is an important morphologic feature of PNET. To differentiate EW from PNET is of clinical significance O13 is a useful marker for the diagnosis of EW/PNET.

Appraisal of the comparative utility of immunohistochemistry and electron microscopy in the diagnosis of childhood round cell tumors.

To provide an objective assessment of the comparative utility of fluorescence- and peroxidase-based immunohistochemistry and electron microscopy, an observer blinded study was conducted under realistic study conditions utilizing a large sampling of poorly differentiated pediatric round cell tumors. Working independently, using a single ancillary technique of particular expertise, each of three investigators attempted to render a specific diagnosis with regard to 50 diagnostically challenging tumors. The results were compared against the subsequent "file diagnosis" established by consensus with all relevant information made available. A grading scheme was applied wherein points were awarded based on the accuracy and confidence of diagnosis. A comparative efficiency rating, expressed as a percentage, was formulated by dividing the number of points awarded each technique by the total number of points theoretically available. Electron microscopy proved superior overall, with an efficiency rating of 89%. Immunoperoxidase and immunofluorescence studies yielded efficiency ratings of 71 and 61%, respectively. Used in combination, the techniques achieved an efficiency rating of 95%. Application of these ancillary techniques resulted in a revision of the provisional diagnosis in 11 of 50 cases, and left only two cases without a firm specific diagnosis.

Differentiating small round cell sarcomas of the soft parts by an innovative immunogold labeling method: an ultrastructural study.

A new immunoelectron microscopy procedure was developed by remaking the fixed-frozen tissue specimens into LR White resin blocks suitable for postembedding colloidal gold immunolabeling, and used to examine 16 cases of small round cell soft tissue sarcomas. In rhabdomyosarcoma, ultrastructural double-immunogold staining demonstrated a coexpression of muscle specific actin and desmin in the same tumor cell. In both Ewing's sarcoma and peripheral neuroepithelioma, the heterogeneous expression of MIC2 gene product (p30/32MIC2) in each tumor cell was demonstrated as well. In peripheral neuroepithelioma, the colloidal gold immunolabeling for neurofilament demonstrated the intermediate filaments surrounding microtubules. The procedure for ultrastructural colloidal gold immunolabeling using fixed-frozen tissue is thus considered to be useful not only for tumor diagnosis, but also for investigating various subcellular structures.