Round cell sarcomas – Biologically important refinements in subclassification

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Contents lists available at ScienceDirect

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Review

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Round cell sarcomas – Biologically important refinements in subclassification夽

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˜ Adrián Marino-Enríquez, Christopher D.M. Fletcher ∗ Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA

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Article history: Received 29 January 2014 Received in revised form 23 April 2014 Accepted 26 April 2014 Available online xxx

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Keywords: Sarcoma Ewing 17 18 Q2 Soft tissue Cancer 19 EWSR1 20 CIC-DUX4 21 15 16

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Contents 1. 2.

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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. © 2014 Published by Elsevier Ltd.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ewing sarcoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Clinical features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Therapeutic considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Morphologic and immunohistochemical features. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Molecular biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . So-called “Ewing-like sarcoma” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Round cell sarcomas with EWSR1-non ETS rearrangement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Round cell sarcomas with non EWSR1-ETS rearrangement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Round cell sarcoma with CIC-DUX4 rearrangement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Round cell sarcoma with BCOR-CCNB3 rearrangement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Desmoplastic small round cell tumor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Undifferentiated round cell sarcoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: EWS, Ewing sarcoma; URCS, undifferentiated round cell sarcoma. 夽 This article is part of a Directed Issue entitled: Rare Cancers. ∗ Corresponding author at: Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA. Tel.: +1 617 732 8558; fax: +1 617 566 3897. E-mail address: cfl[email protected] (C.D.M. Fletcher). http://dx.doi.org/10.1016/j.biocel.2014.04.022 1357-2725/© 2014 Published by Elsevier Ltd.

˜ Please cite this article in press as: Marino-Enríquez A, Fletcher CDM. Round cell sarcomas – Biologically important refinements in subclassification. Int J Biochem Cell Biol (2014), http://dx.doi.org/10.1016/j.biocel.2014.04.022

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ARTICLE IN PRESS

1. Introduction

2. Ewing sarcoma

Round cell sarcomas are among the most aggressive soft tissue tumors and often, but not exclusively, affect children or young adults. The therapeutic options available for these tumors are limited, but certain therapeutic modalities are very effective against specific entities. Consequently, proper classification has great clinical impact and is crucial for appropriate patient management. A variety of sarcomas show round cell morphology and enter the differential diagnosis of so-called malignant small round cell tumors, a descriptive designation referring to a heterogeneous group of non-mesenchymal and mesenchymal neoplasms. Most of these malignant small round cell tumors can be properly diagnosed by integrating clinical and morphologic features, with a parsimonious selection of widely available immunohistochemical markers. Among these, mesenchymal round cell tumors may show features of recognizable cell lineages (like rhabdomyosarcoma or mesenchymal chondrosarcoma) or belong to diagnostic categories with a reproducible phenotype but uncertain histogenesis (such as poorly differentiated synovial sarcoma or Ewing sarcoma). A subset of round cell sarcomas cannot be unequivocally classified based on clinical, morphologic, and immunophenotypic features. These tumors often morphologically resemble Ewing sarcoma, but lack its characteristic immunoprofile and genetic features (Table 1). The term undifferentiated round cell sarcoma provides a useful working category for this heterogeneous group of tumors, which are classified by thoroughly excluding other diagnostic categories that are presently defined (Fletcher et al., 2013). The application of cytogenetics and molecular genetic techniques allows for the identification of an increasing number of genetically defined subgroups within this category, which may represent distinct biologic entities (Fig. 1). In the absence of reproducible clinicopathologic features it is unclear, however, to what extent each of these genetically defined groups represents a distinct entity, rather than just a molecular variant without clinical significance. At present, some undifferentiated round cell sarcomas are thought to represent true variants of Ewing sarcoma, and are classified as such, based on the rearrangement of EWSR1 and a member of the ETS gene family. Less frequent cases share some features with Ewing sarcoma but show different genetic rearrangements, often between EWSR1 and non-ETS genes; the designation Ewing-like sarcoma has been proposed by some authors to acknowledge the morphologic commonalities and certain degree of biologic overlap within this group. As discussed below, this label may be appropriate for tumors biologically related to Ewing sarcoma, but its clinical utility is still controversial. The fact that many poorly differentiated round cell sarcomas morphologically similar to Ewing sarcoma, were originally found to harbor EWSR1 rearrangement variants, led to the initial misperception that every undifferentiated round cell sarcoma would be biologically related to Ewing’s. The designation “atypical Ewing sarcoma” became widely popular and was to some extent utilized indiscriminatingly to designate any bone or soft tissue sarcoma resembling Ewing’s, accepting a wide spectrum of morphologic variation, particularly in the pediatric population. This circumstance has perhaps contributed to the historical difficulties in the morphologic subclassification of undifferentiated round cell sarcomas, and may explain at least partially the perceived advantage of molecular classification in this subgroup of tumors, despite the fact that other tumor types (e.g. myoepithelial lesions) may harbor EWSR1 rearrangement. In this review we will summarize recent advances in the subclassification of undifferentiated round cell sarcomas, including Ewing sarcoma, so-called Ewing-like sarcoma, and morphologically similar entities, discussing biologic, clinical, and therapeutic implications.

Ewing sarcoma is a distinctive round cell sarcoma of bone and soft tissues with (usually minimal) neuroectodermal differentiation, driven by a chimeric fusion gene involving EWSR1 and an ETS gene family member. At present, Ewing sarcoma refers to a spectrum of lesions described independently and which historically were considered distinct entities, based on particular clinical features and a different degree of neuroectodermal differentiation (Fletcher et al., 2013). These include Ewing sarcoma, Askin tumor, and peripheral primitive neuroectodermal tumor (PNET) or peripheral neuroepithelioma, which 20 years ago were designated collectively Ewing sarcoma Family of Tumors (Dehner, 1993; Delattre et al., 1994). Over the years, with the benefit of studies including larger numbers of well-characterized cases, experiments inducing neural differentiation in both Ewing sarcoma and neuroepithelioma in vitro models, and a better understanding of the genetic basis of the disease, it has become apparent that these lesions fall within a continuous spectrum that is sufficiently homogeneous biologically, clinically and, most importantly, therapeutically, to be grouped under a unifying designation. 2.1. Clinical features Ewing sarcoma arises most often in the diaphysis of long bones of the lower extremity, in pelvic bones, ribs, or spine, in children and young adults; about 80% of patients are younger than 20 at diagnosis (Cotterill et al., 2000; Jawad et al., 2009). Soft tissue lesions are comparatively more common in older patients, and often affect deep soft tissues in central locations (Baldini et al., 1999). Skin and visceral lesions, particularly renal, have been well documented (Parham et al., 2001; Collier et al., 2011). Ewing sarcoma shows a slight male predominance, and a striking predilection for Caucasian patients, in comparison to other ethnic groups (Jawad et al., 2009). The primary tumors are typically painful, not uncommonly being mistaken for an inflammatory or infectious process until a mass is detected clinically or radiologically (Widhe and Widhe, 2000). About 25% of Ewing sarcoma cases are metastatic at diagnosis (Esiashvili et al., 2008). 2.2. Therapeutic considerations Advances in therapeutic regimens have significantly improved the prognosis of Ewing sarcoma and thus highlight the need for accurate diagnosis (Esiashvili et al., 2008). The use of specific multimodality protocols that combine aggressive systemic chemotherapy with local control measures using surgery and/or radiation therapy has improved the overall survival to approximately 70% and 30% at 5-years for localized and metastatic disease, respectively (Ladenstein et al., 2010). Unlike other round cell sarcomas, Ewing sarcoma is particularly sensitive to regimens that alternate VDC (vincristine, doxorubicin, and cyclophosphamide) with ifosfamide and etoposide (Grier et al., 2003). At present, patients should ideally be managed in multidisciplinary teams in order to be promptly diagnosed and rapidly receive preoperative chemotherapy, followed, where possible, by surgical resection and additional cycles of chemotherapy to maximize therapeutic response (Grier et al., 2003). Treatment guidelines take into account the site, size and stage of the tumor, and extent of response to neoadjuvant therapy to adapt the treatment and minimize the risk of relapse (Picci et al., 1993; Rodriguez-Galindo et al., 2007). 2.3. Morphologic and immunohistochemical features Ewing sarcoma shows a range of histologic appearances corresponding to the variable degree of neuroectodermal differentiation.

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Table 1 Gene fusions, chromosomal rearrangements and basic clinicopathological features of the undifferentiated round cells sarcomas discussed in this review. Fusion

Karyotype

Characteristic anatomic location

Morphology

CD99 expression

Other notable markers

Seminal references

Ewing sarcoma EWSR1-FLI1

t(11;22)(q24;q12)

Bone or soft tissues

Ewing sarcoma or so-called ‘atypical Ewing sarcoma’

+++

FLI1

Delattre et al. (1994)

EWSR1-ERG EWSR1-ETV1

t(21;22)(q22;q12) t(7;22)(p22;q12)

+++ +++

ERG NSE, S100, DES, EMA

Sorensen et al. (1994) Jeon et al. (1995)

EWSR1-ETV4 EWSR1-FEV

t(17;22)(q21;q12) t(2;22)(q35;q12)

Extraosseous Extraosseous

Round cell sarcoma with EWSR1-non ETS rearrangement t(20;22)(q13;q12) Bone EWSR1-NFATC2

+++ +++

Kaneko et al. (1996) Peter et al. (1997)

So-called ‘atypical Ewing sarcoma’ So-called ‘atypical Ewing sarcoma’ PNET So-called ‘atypical Ewing sarcoma’ Myoepithelial tumor

+++

None

Szuhai et al. (2009)

+++

NSE

Wang et al. (2007)

+++ +++

Mastrangelo et al. (2000) Sumegi et al. (2011)

+++

DES, keratins NSE, Synaptophysin NSE, S100

Ewing sarcoma Ewing sarcoma

+++ +++

ERG, NSE None

Shing et al. (2003) Ng et al. (2007)

URCS or so-called ‘atypical Ewing sarcoma’

Weak focal

WT1

Kawamura-Saito et al. (2006)

Round cell sarcoma with BCOR-CCNB3 rearrangement BCOR-CCNB3 inv(X)(p11) Bone

URCS

50% +++

CCNB3

Pierron et al. (2012)

Desmoplastic small round cell tumor EWSR1-WT1 t(11;22)(p13;q12)

Intraabdominal

URCS with desmoplasia

–

WT1

Gerald et al. (1995)

Undifferentiated round cell sarcoma Non recurrent None (yet!)

Variable

URCS

–

Variable

Fletcher (2008)

EWSR1-SP3

t(2;22)(q31;q12)

Bone or soft tissues

EWSR1-PATZ1 EWSR1-SMARCA5

inv(22) in t(1;22) t(4;22)(q31;q12)

Chest wall Lumbar spine

(EWSR1-POU5F1)

t(6;22)(p21;q12)

Bone

Round cell sarcoma with non EWSR1-ETS rearrangement FUS-ERG t(16;21)(p11;q22) Chest wall FUS-FEV t(2;16)(q35;p11) Bone (clavicle) Round cell sarcoma with CIC-DUX4 rearrangement t(4;19)(q35;q13) Soft tissues CIC-DUX4 t(10;19)(q26;q13)

Yamaguchi et al. (2005)

Abbreviations: PNET, peripheral neuroectodermal tumor; URCS, undifferentiated round cell sarcoma. +++, strong membranous expression; −, no expression.

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The tumors are highly cellular, with a vaguely lobular or diffuse architecture and little or no stroma beyond thin fibrovascular septa (Fig. 2A). Necrosis is common, occasionally confluent. Less differentiated tumors (typical Ewing sarcoma) are monotonous, sheet-like proliferations of cells with scant clear or pale cytoplasm, inconspicuous cell borders, and round to ovoid nuclei with smooth nuclear contours. The chromatin is characteristically fine and evenly distributed, occasionally with small nucleoli. There are scattered small clusters of more hyperchromatic cells, with angulated nuclear contours (so-called dark cells, in contrast with the more numerous light cells), likely reflecting degenerative changes. Occasionally, the cells are larger with more abundant clear cytoplasm, due to abundance of glycogen that can be detected by PAS histochemistry. Tumors composed of larger cells, showing increased cytologic pleomorphism, larger nuclei with irregular contours and prominent nucleoli have been designated atypical or large-cell Ewing sarcoma and may show higher mitotic rates, although they do not behave differently (Fig. 3) (Folpe et al., 2005). Tumors with other peculiar histologic features (such as spindle cytomorphology, fascicular growth pattern, epithelial adamantinoma-like differentiation, or abundant sclerosis or hyalinized stroma) also have been variably called atypical Ewing sarcoma (Folpe et al., 2005; Llombart-Bosch et al., 2009). Better differentiated tumors (PNET), which are much less common, are composed of cells with more eosinophilic cytoplasm, ovoid or angulated nuclei with coarser chromatin and more prominent nucleoli, forming characteristic Homer Wright rosettes (cores of radial fibrillary projections surrounded by nuclei in a circular arrangement). The immunohistochemical profile of Ewing sarcoma reflects the degree of neuroectodermal differentiation. Expression of NSE

(neuron-specific enolase), neurofilament, synaptophysin, PGP9.5 (UCHL1) and Leu-7 (CD57 or B3GAT1) is variable, and more prominent in differentiated tumors. Detection of the glycoprotein CD99, with antibodies like O-13 or MIC-2, provides the most useful diagnostic information: Ewing sarcoma cells express CD99 strongly and diffusely, in a linear membranous pattern, regardless the degree of differentiation (Fig. 2B) (Ambros et al., 1991). Expression of Fli-1 can be detected in most cases, although with less diagnostic value (Folpe et al., 2000). Rare cases express glial or ganglionic antigens, desmin (Parham et al., 1992), and keratins (Srivastava et al., 2005). 2.4. Molecular biology The prototypical biologic driver of Ewing sarcoma is the EWSR1FLI1 fusion oncoprotein, an extensively studied fusion that results from a t(11;22)(q24;q12) translocation and which functions as an aberrant transcription factor (Turc-Carel et al., 1988; Delattre et al., 1992). EWSR1 encodes a ubiquitously expressed RNA-binding protein involved in DNA transcription, and FLI1 is a member of a large family of DNA transcription factors that contain the highly conserved, 85 amino acid-long ETS domain. In the context of the gene fusion, 5 sequences from EWSR1 contribute a strong transcriptional regulatory domain, and 3 sequences of FLI1, or an alternative ETS gene family member, contribute a DNA binding domain (May et al., 1993). About 10% of Ewing sarcoma cases show analogous translocations and gene fusions involving EWSR1 and an alternative member of the ETS transcription factor family, such as ERG, ETV1, ETV4 or FEV (Sorensen et al., 1994; Jeon et al., 1995; Kaneko et al., 1996; Peter et al., 1997). The EWSR1-FLI1 oncogenic fusion is considered the initiating event in Ewing sarcoma; its sustained expression

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Fig. 1. Chromosomal rearrangements described in undifferentiated round cell sarcomas, including Ewing sarcoma (red), round cell sarcomas with EWSR1-non ETS rearrangement (orange), round cell sarcomas with non EWSR1-ETS rearrangement (gray), round cell sarcomas with CIC-DUX4 rearrangement (green), round cell sarcomas with BCOR-CCNB3 rearrangement (blue), and desmoplastic small round cell tumor (purple). The graphical representation is a Circos plot (Krzywinski et al., 2009).

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is essential for Ewing sarcoma cells’ survival and proliferation, and its downstream targets contribute to tumorigenesis (Arvand and Denny, 2001). The EWSR1-FLI1 oncoprotein binds to specific DNA sites containing the GGAA or GGAT core sequences, in particular ACCGGAAGTG (Szymczyna and Arrowsmith, 2000), and modulates the abnormal expression of a network of over 1000 genes (Smith et al., 2006). Transcriptional activation occurs through interactions with transcriptional machinery or direct binding to promoter or microsatellite sequences; genes relevant to transformation, like hTERT or VEGF, have been shown to be directly upregulated by EWSR1-FLI1 in Ewing sarcoma samples or heterologous cellular models (Ohali et al., 2003; Fuchs et al., 2004). The major effect of EWSR1-FLI1 is, however, transcriptional repression, which also occurs through direct interactions, like repression of the histone acetyl-transferase p300 and CDKN1A (Nakatani et al., 2003), TGFBR2 (Hahm et al., 1999), IGFBP-3 (Prieur et al., 2004), or indirectly by upregulation of transcriptional repressors like NKX2-2, NR0B1 or EZH2 (Smith et al., 2006; Mendiola et al., 2006; Riggi et al., 2008). The most common fusion oncoprotein in Ewing sarcoma fuses EWSR1 exon 7 to FLI1 exon 6, accounts for ∼55% of cases and is known as type I fusion. Type II fusion involves exon 7 of EWSR1 and FLI1 exon 5, and is present in ∼25% of cases. Further rare EWSR1FLI1 variants, each accounting for <3% of the cases, also occur. Despite slightly different biologic properties (Lin et al., 1999), the phenotypic differences between tumors with different fusion variants are minimal and, most importantly, the clinical differences are

negligible. Substantial excitement was initially generated by studies describing prognostic differences between the specific fusion variant in Ewing sarcoma, with tumors driven by type I fusion showing a better outcome (Zoubek et al., 1996; de Alava et al., 1998; Fletcher, 1998). Subsequent studies, however, failed to validate this finding and currently information about the fusion type is not routinely used in clinical practice (van Doorninck et al., 2010; Le Deley et al., 2010). Similarly, alternative ETS fusion partners function as molecular equivalents of FLI1 and the resulting tumors are phenotypically indistinguishable (Fig. 4) (Ginsberg et al., 1999). EWSR1-ETS oncoproteins are necessary but insufficient for cellular transformation; in fact, expression of EWSR1-FLI1 in many human cell lines results in cell death or growth arrest (Deneen and Denny, 2001; Lessnick et al., 2002). The cellular context seems critical for transformation by Ewing’s oncoproteins, and significant efforts have been addressed to identify the cell of origin of Ewing sarcoma; this search has been hampered, however, by the undifferentiated phenotype of these tumors and the lack of identifiable precursor lesions (Toomey et al., 2010). Several lines of evidence indicate that a mesenchymal progenitor or stem cell, likely neuralcrest derived, are most likely the cell of origin of Ewing sarcoma (Tirode et al., 2007; Riggi et al., 2008). The massive transcriptional reprogramming driven by EWSR1FLI1 in the appropriate cellular context induces oncogenic pathways that operate in Ewing sarcoma. Activation of polycomb repressors maintains an undifferentiated cellular state (Riggi et al.,

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Fig. 2. (A) Ewing sarcoma is composed of a uniform proliferation of round cells with scant pale cytoplasm, inconspicuous cell borders, and round to ovoid nuclei with smooth nuclear contours (H&E); (B) Characteristic diffuse and strong CD99 membranous expression in Ewing sarcoma.

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2008) in which the PI3K/AKT pathway is essential. Different mechanisms mediate PI3K/AKT activation in Ewing sarcoma, including signaling through IGF1R (Yee et al., 1990), E-Cadherin and ERBB4 (Kang et al., 2007; Mendoza-Naranjo et al., 2013), and CAV1 (Tirado et al., 2006). Important signaling intermediates like PKC-␤ and PRAS40 regulate this pathway in Ewing sarcoma (Huang et al., 2012; Surdez et al., 2012) and may provide additional therapeutic targets. The biologic functions of CD99 are unclear, but recent data suggest relevant roles in Ewing sarcoma oncogenesis by blocking neuroectodermal differentiation (Rocchi et al., 2010); a mechanistic connection between CD99 and EWS-FLI1 transcripts has been described through a shared regulatory microRNA, miR-30a-5p (Franzetti et al., 2013). Secondary genetic aberrations and copy number alterations include cell cycle alterations, such as TP53 mutation and CDKN2A deletions, which are found in 15–25% of cases, and are more frequent in clinically aggressive and poorly chemoresponsive cases (Deneen and Denny, 2001; Huang et al., 2005). Similarly, gain of 1q, resulting in overexpression of the ubiquitin ligase CTD2, is strongly associated with relapse and poor outcome (Mackintosh et al., 2012). Despite remarkable advances in our understanding of Ewing sarcoma biology, biology-based therapies are only just starting to be evaluated in the clinical setting. The initial phase II clinical trials evaluating IGF1R inhibition did not fulfill the high expectations they had generated (Juergens et al., 2011; Pappo et al., 2011), perhaps due to lack of molecular stratification of the patient population or lack of efficacy of the IGF1R blocking antibodies used (Ho and Schwartz, 2011). Additional small molecule inhibitors and combination approaches are being tested, given the compelling preclinical evidence supporting this therapeutic intervention. Recent unbiased pharmacogenomic studies have identified an unexpected sensitivity of Ewing sarcoma cells to PARP inhibition (Barretina et al., 2012; Garnett et al., 2012). Although the biologic mechanisms underlying this sensitivity are only partially understood, several preclinical observations provide a strong rationale to test PARP1 inhibition as a therapeutic strategy in Ewing sarcoma (Brenner et al., 2012).

3. So-called “Ewing-like sarcoma”

Fig. 3. (A) ‘Atypical Ewing sarcoma’ with marked cellular heterogeneity and nuclear pleomorphism. This tumor arose in the thigh of a 27 year old female. The tumor cells have abundant clear cytoplasm and irregularly shaped nuclei, with prominent nucleoli (H&E). The diagnosis in this case was confirmed by FISH.

Fig. 4. Ewing sarcoma with EWSR1-FEV rearrangement, showing morphologic features characteristic of classic Ewing sarcoma, including cellular monomorphism and homogeneous chromatin. This lesion was a pulmonary metastasis in a 7 year old patient ((A) H&E; (B) EWSR1 break apart FISH).

Some authors consider the designation “Ewing-like sarcoma” useful to collectively designate two types of undifferentiated round cell sarcomas: those with EWSR1 rearrangement, with a fusion partner other than an ETS family member; and those without EWSR1 rearrangement, but the biology of which is similar to that of conventional Ewing sarcoma with an EWSR1 fusion oncoprotein (mainly by gene expression profiling). The advocates of this approach consider that there are enough similarities within this group, mainly regarding patient management and treatment purposes, to warrant a collective designation (Antonescu, 2014). As discussed below, even though Ewing-type therapeutic regimens may be the only option available to treat these patients, the clinical setting and the therapeutic responses are heterogeneous and much less predictable than those of conventional Ewing sarcoma. The low frequency of these cases and the limited information available confounds their appropriate classification. From an academic perspective, we regard tumor classification as an evolving tool, and would recommend using analytical terminology that captures as much morphologic and biologic information as possible, to enable stratification and provide the working construct upon which clinical nuances would manifest themselves, over time. The question about how to define an entity in face of clinical, morphologic and molecular heterogeneity is actually a philosophical one. From a clinical perspective, however, a pragmatic approach is preferred and descriptive terms useful for the clinical team treating the

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patient in each particular context are appropriate (Fletcher, 2008). Ongoing investigations will clarify the actual clinical relevance of these subtypes of round cell sarcoma and the most appropriate terminology.

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Rare round cell sarcomas harbor rearrangements of EWSR1 and an alternative fusion partner gene, which is not an ETS transcription factor family member (Fig. 1). These genetic subsets may represent discrete entities, but the small number of cases reported so far make it difficult to delineate their clinicopathologic characteristics; they share, however, clinical and histopathologic features with Ewing sarcoma to a variable extent, in some cases including diffuse membranous CD99 staining (Table 1). The non-ETS fusion partners described so far include other transcription factors – like PATZ1 (Mastrangelo et al., 2000), SP3 (Wang et al., 2007), or NFATC2 (Szuhai et al., 2009) – or non-transcription factors – like the chromatin remodeling gene SMARCA5 (Sumegi et al., 2011). Clinically, round cell sarcomas with EWSR1-NFATC2 and EWSR1-SMARCA5 were bone tumors, while those with EWSR1PATZ1 and EWSR1-SP3 were extraosseous lesions, all of them affecting young or middle-aged adults. Histopathologically, only a case associated with EWSR1-PATZ1 was described as morphologically typical Ewing sarcoma, while most showed morphologic features of so-called ‘atypical Ewing sarcoma’ – atypical nuclear features including prominent nucleoli and nuclear pleomorphism in cases with EWSR1-SP3 and EWSR1-NFATC2 (Wang et al., 2007; Szuhai et al., 2009); abundant mitoses were noted in a case associated with EWSR1-SMARCA5 (Sumegi et al., 2011). Diffuse membranous expression of CD99 by immunohistochemistry was detected in two cases with EWSR1-NFATC2 and EWSR1-SMARCA5 fusion genes, while the other cases were negative or focally positive. Biologically, these tumors fall within a spectrum between Ewing sarcoma and other aggressive EWSR1-driven sarcomas, such as desmoplastic small round cell tumor. To some extent, the structure and function of the fusion oncoproteins driving these tumors provide insights into the oncogenic mechanisms underlying Ewing sarcoma and, in a more general sense, EWSR1-driven neoplasia. All the fusions described include the 5 end of EWSR1, including the promoter region and exons 1 to 6–8, similarly to EWSR1-ETS gene fusions. The 3 partner gene provides the carboxy-terminal domain and the specificity of the oncogenic fusion protein, which in some cases recapitulates biologic functions of EWSR1-ETS proteins. For example, the EWSR1-NFATC2 chimeric protein identified in four bone round cell sarcomas includes exons 3–11 of the transcription factor NFATC2, losing the regulatory calcineurin-binding domain encoded by exons 1 and 2 while preserving its DNA binding domain (Szuhai et al., 2009). Despite differences with the ETS-family transcription factors, the NFATC2 core domain recognizes the same GGAA sequence variant, and like EWSR1-ETS cooperates with the AP1 complex to bind to specific promoter sequences. In fact, in silico analysis identified a potential NFATC2 binding site at the promoter region of all the genes targeted by FLI1 and ERG in combination with the AP1 complex, suggesting that the downstream effects of EWSR1-NFATC2 may be due to activation of ETS transcription factors which results in deregulation of a similar set of genes (Szuhai et al., 2009). Interestingly, EWSR1-NFATC2 is recurrently amplified in these tumors, which may indicate a weaker transforming potential of this particular EWSR1 oncoprotein (Szuhai et al., 2009). The biologic relationship of EWSR1-SP3- and EWSR1-PATZ1associated round cell sarcomas to Ewing’s is less immediate. Both EWSR1 fusion partners are zinc-finger proteins that contribute to the fusion a DNA-binding domain devoid of negative regulatory domains (an inhibitory domain in SP3 and strong transcriptional repression POZ domain in PATZ1) (Mastrangelo et al., 2000; Wang

et al., 2007). This structure is identical to that of the EWSR1WT1 fusion oncoproteins observed in desmoplastic small round cell tumor; in fact, a round cell sarcoma associated with EWSR1PATZ1 showed polyphenotypic differentiation, demonstrated by desmin and keratin expression (Mastrangelo et al., 2000), while the EWSR1-SP3-associated sarcoma was poorly responsive to therapy and clinically very aggressive (Wang et al., 2007). Both facts probably place these tumors nosologically closer to desmoplastic small round cell tumor than to Ewing sarcoma. Finally, EWSR1-SMARCA5-associated round cell sarcoma demonstrates little biologic overlap with other round cell sarcomas. SMARCA5 participates in chromatin remodeling and hence belongs to an area of biology unrelated to ETS transcription factors. SMARCA5 is a member of the SWI/SNF family of proteins and contributes ATPase and helicase activity to the RSF chromatin remodeling and nucleosome spacing complex (Bochar et al., 2000). Little is known about the biology of the EWSR1-SMARCA5 fusion oncoprotein, beyond its transforming potential (Sumegi et al., 2011). A major structural difference with most other EWSR1 fusions is that EWSR1-SMARCA5 does not contain a site-specific DNA binding domain, so that the function of this oncoprotein must depend on epigenetic deregulation of gene expression. Parallels can easily be drawn with the oncogenic mechanisms underlying other SWI/SNF-driven tumors, like the loss of a dominant negative effect observed in rhabdoid tumor (Wilson et al., 2010; Sankar and Lessnick, 2011), but there is no experimental evidence in this regard. It may be pertinent to mention here two independent reports of undifferentiated tumors, most likely myoepithelial tumors, with EWSR1-POU5F1 rearrangements that were approached as undifferentiated round cell sarcomas by their authors (Yamaguchi et al., 2005; Deng et al., 2011). The EWSR1-POU5F1 fusion protein reported includes basically the full length POU5F1 (also known by its alternate name OCT4), which retains its DNA-binding abilities and acquires transforming potential by the presence of the chimeric N-terminal EWSR1 domain (Lee et al., 2007; Kim et al., 2009). POU5F1 is a homeodomain transcription factor that regulates pluripotency of stem cells, and participates in early embryonic development (Rosner et al., 1990). POU5F1 physiologic expression is restricted to germ cells, as well as embryonic and somatic stem cells, and regulates a very different set of genes than ETS transcription factors. Those two EWSR1-POU5F1-associated “sarcomas” showed heterogeneous morphology, alternating between diffuse proliferation of round cells and areas of nested polygonal and spindle-cells (Yamaguchi et al., 2005; Deng et al., 2011). Such morphologic features, in addition to an immunophenotype characterized by strong S100 expression and focal keratin expression, makes the diagnosis of myoepithelial tumor much more likely (Antonescu et al., 2010). 3.2. Round cell sarcomas with non EWSR1-ETS rearrangement A small number of EWSR1-rearrangement negative tumors resembling Ewing sarcoma have been found to harbor rearrangements of FUS, a gene closely related to EWSR1 located at 16p11 (Shing et al., 2003; Ng et al., 2007). FUS and EWSR1 belong to the FET family of RNA-binding proteins and share highly homologous sequences (Crozat et al., 1993). In addition to their structural similarities, the NH2-terminal transactivation domains of FUS and EWSR1 appear to be functionally interchangeable both in vitro and in vivo, at least in terms of transforming potential in a variety of cellular contexts (Zinszner et al., 1994; Shing et al., 2003). In consequence, the fusion proteins that result from rearrangements of EWSR1 or FUS with a given fusion partner are considered biologically equivalent and the resulting tumors are phenotypically indistinguishable. Certain structural considerations

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may explain the recurrent breakpoints observed in these fusion genes (Panagopoulos et al., 1997; Shing et al., 2003). Interestingly, and for unknown reasons, the oncogenic fusions described to date in Ewing-like sarcoma with FUS rearrangements have involved the ETS genes ERG and FEV, but not FLI1, the most common fusion partner of EWSR1 (Shing et al., 2003; Ng et al., 2007). The biology of FUS-ETS oncoproteins has been better studied in acute myelogenous leukemia with t(16;21)(p11;q22), a hematologic malignancy driven by FUS-ERG oncoproteins (Panagopoulos et al., 1994). FUS-ERG acts as an aberrant transcription factor, inhibits apoptosis and has transforming activity in murine and human myeloid and fibroblastic cellular models (Yi et al., 1997; Pereira et al., 1998). There are some potentially critical differences, however, between FUS-ERG oncoproteins in AML and in Ewing sarcoma, the most significant one being the variation in the FUS breakpoints: the FUS-ERG transcripts in AML always include exons 1–7 of FUS, while the fusion in Ewing sarcoma often lacks all or part of exons 6 and 7, which encode a domain essential for leukemic transformation (the so-called ‘transforming regulation domain 2’ or TR2) and thus may engage different oncogenic pathways (Ichikawa et al., 1999). This structural feature may define the striking phenotypic differences between these two entities, which may also be determined by the cellular context in which the fusion oncoprotein occurs. One case of a FUS-FEV oncoprotein described is structurally unusual and incorporates exons 1–10 of FUS (preserving most of the FUS RNA-binding domain) with unknown significance (Ng et al., 2007). Phenotypically, FUS-ERG- and FUS-FEV-associated round cell sarcomas are indistinguishable from Ewing sarcoma, including clinical presentation, histomorphology and diffuse membranous CD99 expression (Shing et al., 2003; Ng et al., 2007). Given the genetic and phenotypic similarities, it seems reasonable to consider these tumors molecular variants of Ewing sarcoma and, in fact, to incorporate the possibility of FUS or FET family genes as molecular equivalents to EWSR1 in the definition of Ewing sarcoma. Perhaps the most relevant consideration regarding this group of tumors is the resulting difficulty for molecular diagnosis, given that most laboratories utilize EWSR1-centered tests (either RT-PCR or breakapart FISH probes) that will not identify these rare variants.

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3.3. Round cell sarcoma with CIC-DUX4 rearrangement

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A subgroup of recently identified soft tissue sarcomas with undifferentiated round cell morphology are characterized by CIC-DUX4 gene fusion, resulting from recurrent t(4;19) or t(10;19) chromosomal translocations (Kawamura-Saito et al., 2006; Yoshimoto et al., 2009). These are extraskeletal lesions that affect young adults (median age at diagnosis: 24 years), with a slight male predominance, most often arising in the limbs, and which appear to pursue an aggressive clinical course with frequent early metastasis (Italiano et al., 2012). Morphologically, CIC-DUX4associated sarcomas are poorly differentiated tumors, composed of sheets or solid groups of small, round to ovoid cells, occasionally spindled (Fig. 5A). The tumor cells have scant clear or slightly amphophilic cytoplasm, and vesicular nuclei with irregular nuclear shape and coarse chromatin, with frequent nucleoli. Although similar to Ewing sarcoma, a greater degree of morphologic heterogeneity, the presence of nucleoli and more abundant cytoplasm, combined with prominent mitotic activity are features suggestive of CIC-DUX4-associated round cell sarcoma. Staining for CD99 is variable, usually in a membranous pattern but often weak in intensity and focal in distribution (Graham et al., 2012; Italiano et al., 2012) (Fig. 5B). Nuclear WT-1 positivity is frequent (Specht et al., 2014) (Fig. 5C). CIC is a high mobility group box transcription factor gene located on chromosome 19q13, physiologically expressed in the CNS, while

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Fig. 5. (A) Round cell sarcoma with CIC-DUX4 rearrangement composed of round cells with clear and irregular nuclei, with coarse chromatin and frequent nucleoli (H&E). This tumor arose in the right tibia of a 20 year old male. CD99 expression is typically focal and weak, with some cells showing linear membranous stain (B). The tumor cells often demonstrate nuclear WT1 staining (C).

DUX4 is a gene that encodes for a double homeobox transcription factor and for which two genomic copies are located within polymorphic macrosatellite repeats on 4q35 and 10q26. CIC-DUX4 seems to act as an aberrant transcription factor with transforming capacity (Kawamura-Saito et al., 2006). As a consequence of the fusion, which preserves most of CIC, the resulting chimeric protein includes the HMG box of CIC, a putative binding site for TLE proteins, and the C-terminal end of DUX4, providing neomorphic DNA-binding capacity. A large part of N-terminal DUX4 is lost, including the DNA-binding double homeodomain (Fig. 6A). The net result appears to be enhanced transcriptional activity of CIC, with subsequent deregulation of downstream targets. Interestingly, the CIC-DUX4 fusion oncoprotein leads to upregulation of

Fig. 6. Domain structure of the oncoproteins CIC-DUX4 (A) and BCOR-CCNB3 (B), characteristic of two recently described genetic types of undifferentiated round cell sarcoma distinct from Ewing sarcoma.

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Fig. 7. Morphologic features of a round cell sarcoma with BCOR-CCNB3 rearrangement. This was a large pelvic lesion in a 13 year old boy. Tumor cells are round-to-ovoid, focally spindled ((A) H&E). There was intense but heterogeneous CD99 expression, not membranous, with some cells showing a “dot-like” staining pattern ((B) CD99).

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a several ETS family genes, including ETV1, ETV4 and ETV5, similar to the genes upregulated directly or indirectly in Ewing sarcoma, suggesting that there may be a very close biologic relationship between these tumor types. Molecular variants of CIC-DUX4 including CIC and alternative fusion partners are likely to be functional, since similar tumors have been described with t(18;19)(q23;q13.2) and der4t(4;?8)(q35;q22) karyotypes (Alaggio et al., 2009; Riccardi et al., 2010). Despite the apparent biologic relationship between CIC-DUX4associated round cell sarcoma and Ewing’s, preliminary data suggest that CIC-DUX4-associated round cell sarcoma is not as sensitive to Ewing sarcoma-type chemotherapeutic regimens. One complete response and two partial responses have been reported so far using such therapies (Italiano et al., 2012), and anecdotal personal experience certainly suggests a less predictable response. Since CIC-DUX4-associated tumors seem particularly common amongst EWSR1 rearrangement-negative round cell sarcomas (up to two thirds of the cases) (Italiano et al., 2012), molecular testing for this aberration is highly recommended in undifferentiated round cell sarcomas resembling Ewing’s, to better understand the behavior of these tumors and hopefully to improve patient management.

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Another subset of very recently identified round cell sarcomas is characterized by a BCOR-CCNB3 gene fusion, resulting from a paracentric inversion of chromosome X. A single study has identified 24 tumors of this type, which are rare lesions (4%, 24 out of 594 Ewing-like sarcomas negative for EWSR1 rearrangements), usually arising in bone, that resemble Ewing sarcoma clinically and morphologically (Fig. 7) (Pierron et al., 2012). The oncogenic BCOR-CCNB3 fusion includes most of the sequence of BCOR, a strong epigenetic transcriptional repressor. BCOR expression regulates the homeostasis of mesenchymal tissues during early embryogenesis, and contributes to normal laterality determination (Hilton et al., 2007; Fan et al., 2009). Germ-line inactivating mutations of BCOR lead to the development of oculofaciocardiodental syndrome, a rare inherited skeletal dysplasia characterized by an increase in the osteogenic potential of mesenchymal stem cells (Ng et al., 2004). Similar somatic mutations contribute to the development of several malignancies, including cytogenetically normal acute

myeloid leukemia (Grossmann et al., 2011), myelodysplastic syndromes (Damm et al., 2013), and a subset of medulloblastoma (Pugh et al., 2012). In addition, chromosomal translocations involving BCOR have been identified in a t(X;17)(p11;q12) variant of acute promyelocytic leukemia (Yamamoto et al., 2010), in endometrial stromal sarcomas carrying a t(X;22) translocation (Panagopoulos et al., 2013) and, more recently, in ossifying fibromyxoid tumors (Antonescu et al., 2014). The mechanisms by which BCOR contributes to such a remarkably heterogeneous group of tumors are not fully understood, but most of the underlying genetic events result in loss of function of BCOR, presumably leading to epigenetic deregulation (Yamamoto et al., 2010). In BCOR-CCNB3-associated round cell sarcoma, the C-terminal part of the fusion corresponding to exons 5–12 of cyclin B3 is sufficient on its own to drive cell-cycle progression, an effect that is amplified at least 2-fold by the fusion event (Pierron et al., 2012). Little is known about the specific functional contributions of BCOR and CCNB3 domains to the fusion oncoprotein (Fig. 6B). Interestingly, the description of a Ewing-like undifferentiated round cell sarcoma with an ins(4;X) involving BCOR underscores the relevance of this part of the fusion (Surace et al., 2005), and suggests that molecular variants of this entity may exist. Despite remarkable clinicopathologic similarities with Ewing sarcoma, BCOR-CCNB3-associated tumors are biologically distinct by gene expression profiling and SNP array analyses. In particular, neither the EWSR1-ETS expression signature, nor most of the well-known EWSR1-FLI1 target genes, such as NR0B1, CAV1, NKX22 or IGFBP3 are expressed in the BCOR-CCNB3-positive tumors. In addition, copy-number abnormalities frequent in Ewing sarcoma, like 8+, 1q+, and 16q−, were not detected in these tumors (Pierron et al., 2012). Given all these features, round cell sarcoma with BCOR-CCNB3 rearrangement is currently considered a type of bone sarcoma unrelated to Ewing’s. The high expression levels of BCOR-CCNB3 fusion oncoprotein can be detected with CCNB3specific antibodies, which show strong nuclear staining and provide a simple and reliable immunohistochemical marker to diagnose this subset of tumors (Pierron et al., 2012).

4. Desmoplastic small round cell tumor Desmoplastic small round cell tumor (DSRCT) is a better known aggressive mesenchymal malignancy that primarily affects children and young adults, who usually present with widespread involvement of the abdominal or peritoneal-lined cavities (Gerald et al., 1991; Cummings et al., 1997). DSRCT is composed of nests of small round tumor cells with polyphenotypic differentiation that most often are sharply delineated by prominent stromal desmoplasia. Cytogenetically, the tumors are characterized by a recurrent t(11;22)(p13;q12) chromosomal translocation (Rodriguez et al., 1993) that forms an in-frame fusion between WT1 and EWSR1 (Ladanyi and Gerald, 1994; Gerald et al., 1995). The most common fusion in DSCRT occurs between exon 7 of EWSR1 and exon 8 of WT1, but several translocation variants have been described (Antonescu et al., 1998). The structure and function of EWSR1-WT1 variants have been well studied and are partially characterized (Gerald and Haber, 2005). In all cases, the fusion oncoprotein retains the N-terminal transcriptional regulatory domain of EWSR1, and the three carboxyl-terminal DNA-binding zinc fingers of WT1, while losing the RNA-binding activity of EWSR1 and transcriptional repression activity of WT1. A splicing event in the WT1 region creates an alternative isoform of the fusion, including three additional amino acids (KTS), which has different biologic properties due to altered DNA-binding specificity (Reynolds et al., 2003; Gerald and Haber, 2005). The chimeric EWSR1-WT1 protein functions as a strong transcription factor that directly binds to the promoter

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Fig. 8. Nests of poorly differentiated cells sharply circumscribed by desmoplastic stroma characteristic of desmoplastic small round cell tumor ((A) H&E). This was a large intraabdominal lesion in a 32 year old male, and expressed keratins (B), and desmin (C), in a characteristically irregular pattern.

Fig. 9. Undifferentiated round cell sarcoma. This lesion affected a 54 year old male in the right posterior knee fossa. Despite focal membranous CD99 expression, tumor cells were negative for EWSR1 rearrangement by FISH, while RT-PCR did not detect CIC-DUX4 and BCOR-CCNB3 transcripts.

regions of targets genes relevant to its oncogenic role, such as BAIAP3, ENT4 and EGR1 (Liu et al., 2000; Palmer et al., 2002; Li et al., 2008). EWSR1-WT1 also induces upregulation of tyrosine kinase receptor and ligand genes, such as IGF1R, KDR and PDGFA, which contribute to the biology of DSRCT (Karnieli et al., 1996; Lee et al., 1997). Interestingly, rare truncated variants of EWSR1-WT1 that lack WT1 sequences are still able to potently transactivate IGFR1, raising questions regarding the exact mechanism underlying IGFR1 overexpression in DSRCT and the mode of action of the EWSR1-WT1 oncoproteins. The morphology of DSRCT sets it apart from other round cell sarcomas (Fig. 8). In addition to the characteristic stromal desmoplasia, which is occasionally absent, the tumor cells are arranged in anastomosing nests of irregular shapes and variable sizes. The cells are usually small, with hyperchromatic nuclei of irregular shape and scant cytoplasm. Some tumors exhibit focal epithelial differentiation, glands, or rosette formation. Necrosis and cystic degeneration are common, as well as prominent abnormal vascularization. The immunoprofile of DSRCT is variable and distinctively complex. The tumor cells express proteins associated with multiple lineages, including epithelial, muscular, and neural. Most cases express keratins, EMA, NSE and desmin, the latter often showing a distinctive ‘dot-like’ staining pattern. CD99 may be positive, usually in a weak membranous fashion but occasionally with a strong diffuse pattern comparable to Ewing sarcoma. Of note, exceptional cases of molecularly-confirmed Ewing sarcoma may show polyphenotypic differentiation, complicating the differential diagnosis to the point that some cases may fulfill criteria for both diagnoses (Ordi et al., 1998). DSRCT is a very aggressive malignancy, most often lethal, and only rarely responds to aggressive multimodality therapy (Kushner et al., 1996; Gerald et al., 1998). As a remarkable exception, two reported DSRCT with EWSR1-WT1 fusions and spindle cell morphology behaved in an indolent manner (Alaggio et al., 2007),

although their morphology was highly unusual making the diagnosis uncertain; those tumors probably represent extraordinary outliers. The dependency of DSRCT on receptor tyrosine kinase signaling likely explains the modest clinical activity of sunitinib observed in some patients with DSRCT (Italiano et al., 2013). 5. Undifferentiated round cell sarcoma Even after expert histopathologic evaluation, and pertinent immunophenotypic and molecular characterization, a group of round cell sarcomas morphologically similar to Ewing remain unclassifiable according to current criteria, and can be designated undifferentiated round cell sarcomas (Fig. 9). This is a heterogeneous category that should be considered a diagnosis of exclusion, for undifferentiated tumors that lack specific morphologic features, identifiable line of differentiation, or recurrent molecular events (Fletcher, 2008). Thanks to a better recognition of distinct morphologic subgroups, an improved understanding of tumor biology and the concomitant development of molecular and immunohistochemical markers, the proportion of undifferentiated sarcomas has decreased remarkably over the years (Alaggio et al., 2009). According to the largest series analyzing this issue, based on systematic centralized review of pediatric rhabdomyosarcoma by the Intergroup Rhabdomyosarcoma Study, about 3% of pediatric sarcoma cases remain unclassified (34–63 in a series of 1527 cases, in a study that did not apply molecular techniques) (Pawel et al., 1997). This proportion may well diminish further in upcoming years with the recognition of novel subgroups with reproducible features. Despite the lack of insight into the biology of these tumors, it seems that tumor cytomorphology provides a useful predictor of tumor behavior: indeed, undifferentiated round cell sarcomas should be distinguished from undifferentiated sarcomas composed mainly of spindle cells in fascicular growth, which vaguely resemble congenital/infantile fibrosarcoma and usually pursue a less

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aggressive clinical course (Pawel et al., 1997; Alaggio et al., 2009). Undifferentiated round cell sarcomas are invariably aggressive, often present at advanced clinical stage at diagnosis, and response to therapy is unpredictable. In consequence, these tumors pose a diagnostic and clinical challenge, and result in great uncertainty regarding patient management.

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Significant advances have been made in recent years in the pathologic classification of seemingly undifferentiated round cell sarcomas. Following the model of rhabdomyosarcoma and Ewing sarcoma, for which the establishment of strict and reproducible diagnostic criteria has enabled the successful use of well-defined therapeutic regimens, best efforts have to be made to accurately subclassify these round cell sarcomas and to minimize the use of the nondiscriminatory label ‘undifferentiated’. The main value will be the reproducible stratification of lesions with different clinical behavior and prognosis, which in turn will facilitate rational planning of treatment. At present, there are few therapeutic alternatives for undifferentiated round cell sarcomas other than Ewing-type treatments. Often times the designation ‘Ewing-like sarcoma’ is utilized in particular clinical settings, in agreement with the clinical team, when such a therapeutic option seems the most appropriate. The use of cytogenetics and molecular genetic analysis in undifferentiated round cell sarcomas enables the identification of an ever increasing number of entities within this group, for which morphology and immunohistochemistry usually provide preliminary diagnostic orientation. 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 unquestionable impact in terms of biologic understanding and coherent classification schemes, and will help to guide the potential development of rational new therapies.

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Brisset et al. (2001) and Greco et al. (1988). Acknowledgments

˜ Adrián Marino-Enríquez is supported by a Career Development Award from The Sarcoma Alliance for Research and Collaboration 748 Q5 (SARC). 749 747

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