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.

Important Recently Characterized Non-Ewing Small Round Cell Tumors.

Round cell sarcomas morphologically similar to Ewing sarcoma, but lacking the classic immunohistochemical features, EWSR-ETS family fusions, and other signs of differentiation, are classified as Ewing-like sarcomas. Recent molecular advances led to the discovery and characterization of two recurrent oncogenic fusion rearrangements, CIC-DUX4 and BCOR-CCNB3, in a significant subset of Ewing-like sarcomas. Uncovered alternate fusion partners broadened the proposed classification of these tumors to CIC-rearranged sarcomas and BCOR-rearranged sarcomas. This article summarizes the clinicopathologic and molecular features of these entities, with particular attention paid to those features that overlap with and distinguish these sarcomas from other round cell sarcomas.

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.

Array-based DNA-methylation profiling in sarcomas with small blue round cell histology provides valuable diagnostic information.

Undifferentiated solid tumors with small blue round cell histology and expression of CD99 mostly resemble Ewing sarcoma. However, they also may include other tumors such as mesenchymal chondrosarcoma, synovial sarcoma, or small cell osteosarcoma. Definitive classification usually requires detection of entity-specific mutations. While this approach identifies the majority of Ewing sarcomas, a subset of lesions remains unclassified and, therefore, has been termed "Ewing-like sarcomas" or small blue round cell tumors not otherwise specified. We developed an approach for further characterization of small blue round cell tumors not otherwise specified using an array-based DNA-methylation profiling approach. Data were analyzed by unsupervised clustering and t-distributed stochastic neighbor embedding analysis and compared with a reference methylation data set of 460 well-characterized prototypical sarcomas encompassing 18 subtypes. Verification was performed by additional FISH analyses, RNA sequencing from formalin-fixed paraffin-embedded material or immunohistochemical marker analyses. In a cohort of more than 1,000 tumors assumed to represent Ewing sarcomas, 30 failed to exhibit the typical EWS translocation. These tumors were subjected to methylation profiling and could be assigned to Ewing sarcoma in 14 (47%), to small blue round cell tumors with CIC alteration in 6 (20%), to small blue round cell tumors with BCOR alteration in 4 (13%), to synovial sarcoma and to malignant rhabdoid tumor in 2 cases each. One single case each was allotted to mesenchymal chondrosarcoma and adamantinoma. 12/14 tumors classified as Ewing sarcoma could be verified by demonstrating either a canonical EWS translocation evading initial testing, by identifying rare breakpoints or fusion partners. The methylation-based assignment of the remaining small blue round cell tumors not otherwise specified also could be verified by entity-specific molecular alterations in 13/16 cases. In conclusion, array-based DNA-methylation analysis of undifferentiated tumors with small blue round cell histology is a powerful tool for precisely classifying this diagnostically challenging tumor group.

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.