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.

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.

Round cell sarcomas - biologically important refinements in subclassification.

Round cell sarcomas are a heterogeneous group of tumors that often affect children and young adults and, if untreated, often pursue a very aggressive clinical course. Specific subtypes of round cell sarcoma, like Ewing sarcoma or rhabdomyosarcoma, respond to well-defined therapeutic regimens so that proper classification is crucial for appropriate patient management. A subset of round cell sarcomas, however, lack specific clinical, morphologic, and immunophenotypic features and cannot be unequivocally classified based on such features. Systematic application of cytogenetics and molecular genetic techniques has allowed for the identification of an increasing number of genetically defined subgroups within this category of undifferentiated tumors. Although the clinical relevance of these molecular categories is yet to be proven, the systematic identification of lesions that share reproducible biologic, and often morphologic and immunophenotypic features, has great impact in terms of biologic understanding and coherent classification schemes, and will help to guide the potential development of rational new therapies. In this review we discuss the main categories of undifferentiated round cell sarcoma, in relation to Ewing sarcoma and its molecular variants, with particular emphasis on the genetic and biologic features of recently described entities including desmoplastic small round cell tumor and CIC-DUX4 as well as BCOR-CCNB3-associated round cell sarcomas. This article is part of a Directed Issue entitled: Rare Cancers.

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.

De la patología estructural a la patología molecular: un modelo en el sarcoma de Ewing / From structural pathology to molecular pathology. A model of Ewing sarcoma

La existencia de un grupo distintivo de neoplasias de células redondas de hueso y partes blandas altamente malignas fue reconocida desde el siglo pasado. Este grupo de tumores de células redondas y pequeñas comprenden un conjunto heterogéneo de neoplasias pobremente diferenciadas formadas por células pequeñas, redondas u ovales con alto índice nuclear-citoplasmático, núcleos redondos con cromatina dispersa o agrupada y nucléolo poco evidente. En el año 1921, James Ewing, profesor de Patología de la Universidad de Cornell, USA, describió estas características histológicas en un tumor que denominó endotelioma difuso de hueso. Charles Oberling, en Paris, en el año 1928, denominó la neoplasia como Sarcoma de Ewing. De manera resumida podemos plantear la evolución de las técnicas diagnósticas aplicadas en el estudio de la siguiente forma: 1) Aplicacion de técnicas convencionales histológicas: H&E. 2) Histoquímica convencional: PAS, Carmin de Best. 3) Microscopía Electrónica. 4) Inmunohistoquímica: marcadores mesenquimales, neurales, epiteliales y otros. 5) Cultivos celulares. 6) Xenotrasplante de tumores. 7) Estudios citogenéticos convencionales: traslocaciones cromosómicas principales. 8) FISH. 9) Biología molecular: reordenamiento genético y tipos de genes de fusión. Se presenta una visión global de los distintos aspectos de la morfología, ultra estructura inmunohistoquímica, genética convencional y molecular asi como nuevas aproximaciones a la terapéutica de este tumor óseo.

Ewing tumors that do not overexpress BMI-1 are a distinct molecular subclass with variant biology: a report from the Children's Oncology Group.

PURPOSE: Ewing sarcoma family tumors (ESFT) are aggressive tumors of putative stem cell origin for which prognostic biomarkers and novel treatments are needed. In several human cancers, high expression of the polycomb protein BMI-1 is associated with poor outcome. We have assessed the potential clinical significance of BMI-1 expression level in ESFT. EXPERIMENTAL DESIGN: BMI-1 expression was assessed in 130 tumors by immunostaining and associations with clinical features and outcome determined. The molecular signatures of BMI-1-low and BMI-1-high tumors were compared using microarrays and differentially activated canonical pathways identified by gene-specific enrichment analysis. Automated quantitative analysis of phosphoproteins was used to assess relative levels of pathway activation. Sensitivity to IGF1-R inhibition was determined using MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assays. RESULTS: BMI-1 is overexpressed by the vast majority of ESFTs. However, in 20% of cases, BMI-1 levels are low to undetectable. Significantly, although clinical presentation and outcome were similar between BMI-1-high and BMI-1-low tumors, whole genome expression array analysis showed marked differences in their respective gene expression profiles. Gene-specific enrichment analysis identified that several cancer-associated canonical biological pathways, including IGF1, mTOR, and WNT, are significantly downregulated in BMI-1-low compared with BMI-1-high tumors. Consistent with these in vivo data, the response to IGF1-R inhibition in vitro was diminished in BMI-1-low compared with BMI-1-high ESFT cells. CONCLUSION: ESFT that do not overexpress BMI-1 represent a novel subclass with a distinct molecular profile and altered activation of and dependence on cancer-associated biological pathways.

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.

Tumor classification by combining PNN classifier ensemble with neighborhood rough set based gene reduction.

Since Golub applied gene expression profiles (GEP) to the molecular classification of tumor subtypes for more accurately and reliably clinical diagnosis, a number of studies on GEP-based tumor classification have been done. However, the challenges from high dimension and small sample size of tumor dataset still exist. This paper presents a new tumor classification approach based on an ensemble of probabilistic neural network (PNN) and neighborhood rough set model based gene reduction. Informative genes were initially selected by gene ranking based on an iterative search margin algorithm and then were further refined by gene reduction to select many minimum gene subsets. Finally, the candidate base PNN classifiers trained by each of the selected gene subsets were integrated by majority voting strategy to construct an ensemble classifier. Experiments on tumor datasets showed that this approach can obtain both high and stable classification performance, which is not too sensitive to the number of initially selected genes and competitive to most existing methods. Additionally, the classification results can be cross-verified in a single biomedical experiment by the selected gene subsets, and biologically experimental results also proved that the genes included in the selected gene subsets are functionally related to carcinogenesis, indicating that the performance obtained by the proposed method is convincing.

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.

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.

Ewing's sarcoma family of tumors

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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.

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.

Primitive neuroectodermal tumor of the gallbladder.

Ewing sarcoma/primitive neuroectodermal tumor (ES/ PNET) most commonly occurs in the soft tissues of the extremities and the paravertebral areas. To our knowledge, no case of PNET has been reported in the gallbladder to date. We report a case of PNET of the gallbladder occurring in a 53-year-old woman. Microscopically, the monotonous small round cells with a formation of Homer-Wright rosettes showed intense membranous positive immunoreactivity for MIC2/CD99, as well as for neuron-specific enolase and synaptophysin. Other small round cell tumors, such as lymphoma, rhabdomyosarcoma, or melanoma, were excluded in light of negative immunoreactivity for leukocyte common antigen, desmin, and S100. The diagnosis of PNET was rendered based on the characteristic morphology and immunohistochemical findings, despite an absence of chromosomal translocations, such as t(11; 22)(q24;q12) and t(21;22)(q22;q12).