Ewing-like sarcoma: An emerging family of round cell sarcomas.

Ewing-like sarcomas are an emerging subgroup of small round blue cell sarcomas that share various degrees of morphological, immunohistochemical, molecular, and clinical similarity with Ewing sarcoma. Despite these similarities, Ewing-like sarcomas lack the pathognomonic molecular hallmark of Ewing sarcoma: A translocation between a gene of the RNA-binding TET family (EWSR1 or FUS) with a gene of the ETS-transcription family ( FLI1, ERG, ETV1, ETV4, or FEV). Recently, increased use of modern molecular methods based on next-generation sequencing have enabled the identification of distinct subgroups within this previously uncharacterized group of Ewing-like sarcomas based on the discovery of novel molecular driving events. The focus of this review is to provide an update on the main subcategories of Ewing-like sarcomas discovered to date: CIC-rearranged sarcomas, BCOR-rearranged sarcomas, sarcomas with a rearrangement between EWSR1 and a non-ETS family gene, and the substantial fraction of tumors which remain uncharacterized by molecular methods. There is increasing evidence that these tumors represent stand-alone entities with unique characteristics rather than simply a subgroup of Ewing sarcoma; thus, the question of the best therapeutic approach for these often aggressive sarcomas remains of primary importance. Ultimately, large collaborative efforts will be necessary to better determine the characteristics of this rare, heterogeneous family of tumors.

Review with novel markers facilitates precise categorization of 41 cases of diagnostically challenging, "undifferentiated small round cell tumors". A clinicopathologic, immunophenotypic and molecular analysis.

BACKGROUND: Despite extensive immunohistochemical (IHC) and molecular studies combined with morphologic findings, a group of round/ovoid cell tumors histologically similar to Ewing sarcomas (ES) but lacking EWSR1-rearrangements may remain unclassifiable. DESIGN: We retrospectively analyzed 41 Ewing-like tumors (formalin-fixed, paraffin-embedded) previously determined as negative or non-informative for EWSR1-rearrangements by FISH and/or RT-PCR. A new histopathology revision and additional IHC and molecular analyses were carried out in order to investigate whether additional IHC and/or molecular testing in combination with the morphological findings may help in reaching a definitive diagnosis. RESULTS: Almost all the tumors (n=40) involved soft tissue and/or bone and half the patients died of disease. In the archival cases all diagnoses were Ewing sarcoma (ES), Ewing-like sarcoma (ELS), myoepithelial tumor and undifferentiated sarcoma (US). In the new review all the tumors were re-classified as, ES (n=16), Ewing-like tumor with EWSR1 rearrangement and amplification and possible EWSR1-NFATC2 gene fusion (n=1), CIC-rearranged sarcomas or undifferentiated sarcoma, most consistent with CIC-rearranged sarcoma (n=7), sarcoma with BCOR-alteration or undifferentiated sarcoma, consistent with BCOR-associated sarcoma (n=3), neuroblastoma (n=2), unclassifiable neoplasm with neuroblastic differentiation (n=1), malignant rhabdoid tumor (n=2), lymphoblastic lymphoma (n=1), clear cell sarcoma of the gastrointestinal tract (n=1), small cell carcinoma (n=1), sclerosing rhabdomyosarcoma (n=1), desmoplastic small round cell tumor (n=1), malignant peripheral sheath nerve tumor (n=1), poorly-differentiated synovial sarcoma (n=1), Possible gastrointestinal stromal tumor/GIST with predominant round cells (n=1) and possible SMARCA4-deficient-sarcoma (n=1). NKX2.2, ETV4 and BCOR immunoreactivity was observed in all ES, CIC-rearranged sarcomas and sarcomas with BCOR alteration, respectively. CIC-rearrangement by FISH was observed in many of the CIC-rearranged sarcomas. CONCLUSION: Our analysis of 41 Ewing-like tumors confirms that there may be a significant pathological and IHC overlap among Ewing-like tumors, with prognostic and therapeutic impacts. Additional IHC (NKX2.2, ETV4 and BCOR) and molecular studies including FUS, CIC or BCOR analysis may support the final diagnosis when FISH or RT-PCR fail to detect EWSR1-rearrangements. Any molecular findings should always be interpreted in relation to the specific clinical and pathological context.

Identification of Discrete Prognostic Groups in Ewing Sarcoma.

BACKGROUND: Although multiple prognostic variables have been proposed for Ewing sarcoma (EWS), little work has been done to further categorize these variables into prognostic groups for risk classification. PROCEDURE: We derived initial prognostic groups from 2,124 patients with EWS in the SEER database. We constructed a multivariable recursive partitioning model of overall survival using the following covariates: age; stage; race/ethnicity; sex; axial primary; pelvic primary; and bone or soft tissue primary. Based on this model, we identified risk groups and estimated 5-year overall survival for each group using Kaplan-Meier methods. We then applied these groups to 1,680 patients enrolled on COG clinical trials. RESULTS: A multivariable model identified five prognostic groups with significantly different overall survival: (i) localized, age <18 years, non-pelvic primary; (ii) localized, age <18, pelvic primary or localized, age ≥18, white, non-Hispanic; (iii) localized, age ≥18, all races/ethnicities other than white, non-Hispanic; (iv) metastatic, age <18; and (v) metastatic, age ≥18. These five groups were applied to the COG dataset and showed significantly different overall and event-free survival based upon this classification system (P < 0.0001). A sub-analysis of COG patients treated with ifosfamide and etoposide as a component of therapy evaluated these findings in patients receiving contemporary therapy. CONCLUSIONS: Recursive partitioning analysis yields discrete prognostic groups in EWS that provide valuable information for patients and clinicians in determining an individual patient's risk of death. These groups may enable future clinical trials to adjust EWS treatment according to individualized risk.

[Ewing/PNET sarcoma family of tumors: towards a new paradigm?]. / Tumeurs de la famille Ewing/PNET : vers un nouveau paradigme ?

Ewing sarcoma family of tumors are mainly aggressive sarcomas of bone and also arising in soft tissues, which share common features: morphological features of basophilic round cell tumors, immunohistochemical features by expression of membrane CD99 protein, and genetic features with a translocation involving EWS and FLI1 in approximately 90% of cases. The discovery of this translocation has made it possible to unify in a single entity several lesions such as PNET, neuropitheliomas, Askin tumors, Ewing sarcomas… Since then, the extensive use of molecular/genetic methods has helped to identify an increasing number of molecular anomalies in unclassified round cell sarcomas, these sarcomas often harboring an atypical morphology and a less frequent CD99 positivity. Besides the rearrangements between the FET family of genes (EWS or FUS) and the wide ETS family of genes (FLI1, ERG, FEV, ETV…), new partner genes are gradually identified: cases with EWS-non ETS partners are extremely rare, but there are more important groups which are CIC-DUX4 and BCOR-CCNB3 translocation-positive sarcomas. These findings raise the problem of the nosological borders of the Ewing/PNET entity and its links with new "Ewing-like" groups of tumors, and raise the therapeutic problems. The forward-looking identification of new round cell sarcomas should enable studies of wider series to try to answer these questions.

Ewing sarcoma.

Classification of small round cell tumors of bone is often challenging due to overlapping clinicopathologic features. The purpose of this article is to review the clinical, radiological, histologic, and molecular features of Ewing sarcoma and to provide a discussion of the differential diagnosis of small round cell tumors of bone.

Updates on WHO classification for small round cell tumors: Ewing sarcoma vs. everything else.

The WHO Classification of Soft Tissue and Bone Tumours currently recognizes four categories of undifferentiated small round cell sarcoma: Ewing sarcoma, round cell sarcoma with EWSR1-non-ETS fusions including NFATc2 and PATZ1, CIC-rearranged sarcoma, and sarcoma with BCOR genetic alterations. These neoplasms frequently pose significant diagnostic challenges due to rarity and overlapping morphologic and immunohistochemical findings. Further, molecular testing, with accompanying pitfalls, may be needed to establish a definitive diagnosis. This review summarizes the clinical, histologic, immunohistochemical, and molecular features of these neoplasms. In addition, differential diagnosis and areas of uncertainty and ongoing investigation are discussed.

Simultaneous variable selection and class fusion for high-dimensional linear discriminant analysis.

In many high-dimensional microarray classification problems, an important task is to identify subsets of genes that best discriminate the classes. Nevertheless, existing gene selection methods for microarray classification cannot identify which classes are discriminable by these selected genes. In this paper, we propose an improved linear discriminant analysis (LDA) method that simultaneously selects important genes and identifies the discriminable classes. Specifically, a pairwise fusion penalty for LDA was used to shrink the differences of the class centroids in pairs for each variable and fuse the centroids of indiscriminable classes altogether. The numerical results in analyzing 2 gene expression profiles demonstrate the proposed approach help improve the interpretation of important genes in microarray classification problems.

Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks.

The purpose of this study was to develop a method of classifying cancers to specific diagnostic categories based on their gene expression signatures using artificial neural networks (ANNs). We trained the ANNs using the small, round blue-cell tumors (SRBCTs) as a model. These cancers belong to four distinct diagnostic categories and often present diagnostic dilemmas in clinical practice. The ANNs correctly classified all samples and identified the genes most relevant to the classification. Expression of several of these genes has been reported in SRBCTs, but most have not been associated with these cancers. To test the ability of the trained ANN models to recognize SRBCTs, we analyzed additional blinded samples that were not previously used for the training procedure, and correctly classified them in all cases. This study demonstrates the potential applications of these methods for tumor diagnosis and the identification of candidate targets for therapy.

Ewing tumors in infants.

BACKGROUND: Malignancies in infancy are extremely rare. Ewing tumors are hardly ever noted in these children. Since it is generally assumed that malignancies in infancy have an extremely poor outcome, we wanted to investigate whether this was also the case in Ewing tumors. PROCEDURE: We identified in the Munster data bases of CESS81, CESS86, EICESS92 and EuroEwing99 14 children <12 months of age with a tumor of the Ewing family. Numbers of girls and boys were equal. RESULTS: All infants had axial tumors, including pelvic primaries; 80% of the tumors were <200 ml. An uncommon pathology distribution was noted; the majority were peripheral neuroectodermal tumors, only two atypical Ewing, one classical Ewing, and one unspecified PAS positive small round blue cell soft tissue sarcoma was found. Three patients had metastatic disease at initial diagnosis. Treatment modalities were comparable with patients of older age. The number of cytostatic courses ranged from 6 to 15. Dose reductions were limited, ranged from 73% to 90%. Outcome results were similar to those in patients of older ages (5-year EFS 65%, OS 72%). CONCLUSIONS: Infants with Ewing family tumors are in the majority of cases PNETs and are predominantly axial tumors. Outcome is similar to patients with Ewing tumors at older ages. Generally accepted adverse prognostic factors did not influence outcome.

DNA microarrays reveal relationship of Ewing family tumors to both endothelial and fetal neural crest-derived cells and define novel targets.

Ewing family tumors (EFTs) are small round blue cell tumors that show features of neuroectodermal differentiation. However, the histogenetic origin of EFTs is still a matter of debate. We used high-density DNA microarrays for the identification of EFT-specific gene expression profiles in comparison with normal tissues of diverse origin. We identified 37 genes that are up-regulated in EFTs compared with normal tissues and validated expression of these genes in EFTs by both conventional and quantitative reverse transcription-polymerase chain reaction. The expression pattern of EFT-associated genes in normal tissues indicated a high similarity between EFTs and fetal and neuronal as well as endothelial tissues and supports the concept that a primitive neural crest-derived progenitor at the transition to mesenchymal and endothelial differentiation is transformed in EFTs. EFT-associated genes could be used for molecular discrimination between EFTs and other small round blue cell tumors and clearly identified a cell line (SK-N-MC) that was initially established as neuroblastoma as being an EFT. Ectopic expression of the EFT-specific EWS-FLI1 fusion protein in human embryonic kidney (HEK293) cells was not sufficient to induce the complete EFT-specific gene expression signature, suggesting that the EFT-specific gene expression profile is not just a consequence of EWS-FLI1 expression but depends on the histogenetic background of the EFT stem cell.

Type 1 (11;22)(q24:q12) translocation is common in Ewing's sarcoma/peripheral neuroectodermal tumour in south Indian patients.

The Ewing's sarcoma family can present diagnostic difficulties. In the past the basis of diagnosis has been a exclusion. Identification of a specific translocation especially t(11;22) (EWS-FLI 1 fusion gene), which is seen in nearly 85 percent of Ewing's sarcoma cases can help in precise diagnosis. We have carried out a study on twenty patient samples diagnosed to have Ewing's sarcoma/peripheral neuroectodermal tumour (PNET)/small round cell malignant tumour. The study involved RT-PCR analysis for the fusion transcript, followed by sequencing to identify the specific type of fusion. Ninety percent (18/20) of the samples tested were found to be t(11;22) translocations involving EWS-FLI 1 genes. Sixty-one percent (11/18) were found to be type 1 fusion and seven were type 2 (39 percentage). This is the first study in India with quantitative information about the types of EWS-FLI 1 translocations present in Ewing's family of tumours in south Indian patients.

The neuroectodermal tumor of bone.

Four small round cell malignant tumors of bone occurring in children are described. There was no catecholamine secretion and the clinical, radiologic, and biopsy diagnosis in each was Ewing's sarcoma. Glycogen was sparse both on imprints and in tissue sections. The tumors, when extensively sampled, had areas of a lobular growth pattern and Homer Wright rosettes. The rosettes were always focal and varied in complexity from case to case; they were rudimentary in one instance and markedly fibrillar in the most obvious instance. Neuron-specific enolase was demonstrated in tissue sections and in longterm cell cultures from three of the tumors. The cultured cells put out moderately long beaded processes in serum-free medium but had no catecholamine fluorescence. Electron microscopy of the tumor rosettes and the cultured cells showed processes containing aggregates of microtubules and only one case had rare neurosecretory granules. This study suggests that some small round cell tumors of bone and soft tissue in children, which present as Ewing's sarcoma, are neuroectodermal in nature.

Peripheral primitive neuroectodermal tumour and extra-osseous Ewing's sarcoma; a histological, immunohistochemical and DNA flow cytometric study.

Although peripheral primitive neuroectodermal tumour (pPNET) and extra-osseous Ewing's sarcoma (EES) are thought to be closely related neoplasms, their clinical behaviour differs considerably. To determine the clinical relevance of the Schmidt classification scheme for differentiating pPNET and EES, 20 tumour specimens of poorly differentiated round cell tumours were evaluated. In addition, the diagnostic value of several neural markers and the prognostic value of quantitative morphological variables (DNA ploidy, S-phase fraction, and the mitotic activity) were assessed. Homer-Wright rosettes were present in 9 tumours. Neuron specific enolase (NSE) was expressed in 11 tumours, 8 of which expressed a second neural marker (CD57, S100, or neurofilament). According to the Schmidt classification, 11 pPNET and 5 EES were distinguished. HBA-71 was exclusively expressed in pPNET and EES. The remaining tumours were classified as sarcoma not otherwise specified (n = 2), rhabdomyosarcoma (n = 1), and desmoplastic tumour with divergent differentiation (n = 1). EES611 patients fared significantly better than the pPNET patients (100% versus 42% 5-year survival). Neither DNA ploidy nor S-phase fraction assessed in 12 evaluative histograms (9 pPNET and 3 EES), nor mitotic activity yielded information of additional prognostic value. On the basis of this study and the Schmidt classification scheme, it can be concluded that if the diagnosis of EES and pPNET is based on light microscopy (Homer-Wright rosettes) and/or immunohistochemistry (at least two neural markers, i.e. NSE, S-100, CD57, and neurofilament), the classification provides important clinical information. Furthermore, positivity for HBA-71 is helpful in differentiating pPNET and EES from all other small round cell tumours.

Classification and grading of bone sarcomas.

Bone tumors generally are classified according to the cytologic features or products of the lesional cells. In most instances, the classification corresponds to a normal cell or tissue type indigenous to bone. This article focuses on sarcomas of bone and features a classification scheme similar to what has been described at the Mayo Clinic.

Multi-class tumor classification by discriminant partial least squares using microarray gene expression data and assessment of classification models.

High-throughput DNA microarray provides an effective approach to the monitoring of expression levels of thousands of genes in a sample simultaneously. One promising application of this technology is the molecular diagnostics of cancer, e.g. to distinguish normal tissue from tumor or to classify tumors into different types or subtypes. One problem arising from the use of microarray data is how to analyze the high-dimensional gene expression data, typically with thousands of variables (genes) and much fewer observations (samples). There is a need to develop reliable classification methods to make full use of microarray data and to evaluate accurately the predictive ability and reliability of such derived models. In this paper, discriminant partial least squares was used to classify the different types of human tumors using four microarray datasets and showed good prediction performance. Four different cross-validation procedures (leave-one-out versus leave-half-out; incomplete versus full) were used to evaluate the classification model. Our results indicate that discriminant partial least squares using leave-half-out cross-validation provides a more realistic estimate of the predictive ability of a classification model, which may be overestimated by some of the cross-validation procedures, and the information obtained from different cross-validation procedures can be used to evaluate the reliability of the classification model.

Peripheral and central primitive neuroectodermal tumors. A nosologic concept seeking a consensus.

The term primitive neuroectodermal tumor is widely used in the literature for a group of small, round-cell tumors in the central and sympathetic nervous systems and soft tissues as well as a specific diagnostic term for individual neoplasms; however, the contention that these various clinicopathologic entities (neuroblastoma, medulloblastoma, and peripheral neuroepithelioma) are histogenetically related is an unproved hypothesis. Morphologic, cytogenetic, immunohistochemical, biochemical, and in vitro studies have established phenotypic similarities among these putatively related neoplasms whether they originate in the brain, adrenal gland, or soft tissues. Because one tumor resembles another in terms of its phenotypic expression, that does not necessarily imply a common histogenesis. This point has been made by previous investigators. The purpose of this review is to evaluate and discuss the present status of our understanding and some of the controversial aspects of this enigmatic category of neoplasms, mainly occurring in children, known as the primitive neuroectodermal tumors.

Immunophenotypic profile and FISH analysis in a case of Ewing's sarcoma.

Ewing's sarcoma arising in the right fibula was diagnosed in a 12 years old female, considering clinical presentation, radiological findings and histological features. Our work points towards a possible neuroectodermal origin of Ewing's sarcoma, since MIC2/12E7 marker was found to be positive. Translocation t (11;22) (q24;q12) was found as the main cytogenetic change in this tumor, by means of FISH analysis.

Ewing tumor: tumor biology and clinical applications.

The Ewing tumor family includes classical Ewing's sarcoma of bone and soft tissues, peripheral primitive neuroectodermal tumors (pPNET), Askin tumor, and other less frequent variants. This group of tumors is defined by the consistent presence of chromosomal translocations resulting in gene fusions between EWS gene and a member of the ETS family of transcription factors, mainly FLI1 and ERG. Analogous fusions are seen in other solid developmental tumors, like desmoplastic small round cell tumor. These fusions, which are consistently present and tumor-specific, control transcription of several target genes, largely unknown but critical to cell proliferation and differentiation. Therefore, gene fusions are useful to diagnose and classify small round cell tumors, have prognostic significance, are probably useful to detect micrometastasis and monitor minimal residual disease, and are potential therapeutic targets. Secondary molecular alterations, which include mutations of cell cycle regulatory genes, are not tumor-specific but are related to progression and may have prognostic value. The Ewing tumor family represents a paradigm of the application of the knowledge of biology of neoplasia to the clinical management of patients.