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Available online at www.sciencedirect.com

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Ewing sarcoma Eun-Young K. Choi, MDa, Jerad M. Gardner, MDb, David R. Lucas, MDa, Jonathan B. McHugh, MDa, Rajiv M. Patel, MDa,n a

Department of Pathology, University of Michigan, 3261G Medical Science I, 1301 Catherine St, SPC 5602, Ann Arbor, Michigan 48109 b Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas

article info

abstra ct

Keywords:

Classification of small round cell tumors of bone is often challenging due to overlapping

Ewing sarcoma

clinicopathologic features. The purpose of this article is to review the clinical, radiological,

Ewing family of tumors

histologic, and molecular features of Ewing sarcoma and to provide a discussion of the

Ewing-like sarcoma

differential diagnosis of small round cell tumors of bone.

Primitive neuroectodermal tumor

& 2014 Elsevier Inc. All rights reserved.

Small round cell sarcoma

Introduction Skeletal Ewing sarcoma, extraosseous Ewing sarcoma, primitive neuroectodermal tumor (PNET), and Askin tumor (Ewing sarcoma arising in chest wall) are thought to represent a clinicopathologic spectrum of the same neoplastic entity, sometimes collectively called “Ewing family of tumors.” Shared immunohistochemical and genetic features support this view. The purpose of this article is to provide an overview of the clinical, radiological, histologic, and molecular features of Ewing sarcoma and to review bone tumors in the histologic differential diagnosis.

Ewing sarcoma Epidemiology, clinical, and radiographic features Ewing sarcoma (ES) is the second most common primary malignant bone tumor in children and adolescents, following osteosarcoma. The highest incidence is in the second decade of life, with approximately 9–10 cases per million per year seen in patients aged 10–19 years compared to an overall incidence of three cases per million per year in the United States population.1,2 It is uncommon in patients younger n

than 5 years or older than 30 years. ES occurs predominantly in Caucasians; it is infrequent in the African American population for reasons unknown.3 A slight male predominance exists (M:F sex ratio ¼ 1.5:1).1,3 The majority of ES arise in bone, and up to 30% in soft tissue.4 Skeletal ES most frequently involves the diaphysis or metadiaphyseal region of long bones (lower 4 upper extremities). The pelvis, ribs, and spine are also commonly involved.2,5 The histogenesis of ES remains debated but is thought to originate from either neural crest stem cells6,7 or mesenchymal stem cells.8 Clinically, patients often present with localized pain and swelling. Patients may also present with a palpable mass, pathologic fracture, or constitutional symptoms such as fever, fatigue, weight loss, or anemia.9 Radiographically, ES is a permeative and predominantly osteolytic lesion that frequently extends through cortex into the periosteum and soft tissue. Intermittent activity of the tumor creates the classic “onion-skin” multilayered periosteal reaction. Erosion of outer cortex by periosteal tumor can result in a concave cortical defect called “saucerization.” A “hair-on-end” vertical form of periosteal reaction and Codman triangles (Fig. 1) may also be seen in ES.10 In the absence of cortical destruction, radiographic findings may be subtle and Ewing sarcoma may not be seen on plain radiographs.

Correspondence to: 3261 G Medical Science I, 1301 Catherine St, SPC 5602, Ann Arbor, MI 48109. E-mail address: [email protected]. (R.M. Patel)

0740-2570/$ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1053/j.semdp.2014.01.002

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Fig. 1 – Ewing sarcoma of bone in a child. Plain radiograph shows a mass in the metadiaphyseal region of the distal humerus. Codman triangles (arrow) and extension of tumor into surrounding soft tissue (arrowhead) are seen.

Imaging modalities such as magnetic resonance imaging and computed tomography help determine the extent of bone and soft tissue tumor involvement.

Pathology Grossly, the mass is gray-tan with infiltrative borders. Areas of hemorrhage and necrosis are often present, resulting in soft or partially liquefied areas that may resemble purulent exudate. Microscopically, ES is a histologically diverse group of tumors with varying degrees of neural differentiation. Traditionally, ES is divided into three major histologic subtypes: classical ES, primitive neuroectodermal tumor (PNET), and atypical ES. Classical ES, which constitutes the majority of cases, is comprised of solid sheets or vague lobules of uniform small cells with round or oval nuclei, smooth nuclear contours, fine chromatin, inconspicuous nucleoli, scant amount of lightly eosinophilic or clear cytoplasm, and indistinct cell borders (Fig. 2A). Cytoplasm clearing is due to accumulation of cytoplasmic glycogen, which can be highlighted by Periodic acid–Schiff (PAS) stain. Areas of necrosis and hemorrhage are common. Perivascular cuffing may be seen in areas of geographic necrosis (Fig. 2C). Mitotic count is typically low and extracellular matrix absent. Tumors with evidence of neural differentiation (Fig. 2D) are classically termed primitive neuroectodermal tumor (PNET). Homer Wright rosettes (clusters of cells with a solid neurofibrillary core formed by tangled cytoplasmic processes) or immunohistochemical evidence of neural differentiation support a diagnosis of PNET. Various definitions have been proposed to define the minimal criteria for PNET,11 but a widely accepted diagnostic criterion is not available. Atypical ES (large cell ES) refers to tumors with features that deviate from those described in classical ES such as nuclear enlargement and pleomorphism, irregularity of nuclear membrane, vesicular or course chromatin texture, and prominent nucleoli (Fig. 3A).12–14

Other histologic patterns have been observed in genetically confirmed cases of ES such as extensive spindling (Fig. 3B), abundant hyaline sclerosis, hemagioendothelial features, and adamantinoma-like ES (Fig. 3C and D). More typical ES histomorphology is usually seen, at least focally, within these variants.12,14,15 The adamantinoma-like ES is an interesting variant with a nested, epithelioid growth pattern. Histologic features that have been described include prominent desmoplasia, peripheral nuclear palisading, hyperchromatic nuclei, and dense eosinophilic matrix.12,16 Adamantinoma-like ES usually has strong cytokeratin staining. Suspected ES cases with unusual histologic features require ancillary studies to confirm the diagnosis. While knowledge of histologic heterogeneity is important for recognizing ES, determination of exact histologic subtypes may not be critical given shared genetic abnormalities that characterize the Ewing family of tumors.

Immunophenotype CD99 is a highly sensitive and useful immunohistochemical marker for ES, usually showing a diffuse, strong, membranous pattern of distribution (Fig. 2B).12,14 In tumors that are CD99 negative or have unusual staining pattern, further workup with cytogenetic or molecular studies should be done to confirm ES. Although sensitive, CD99 is not specific for ES. Many other neoplasms with small round cell morphology can be positive for CD99 including lymphomas, mesenchymal chondrosarcoma, small cell osteosarcoma, synovial sarcoma, and desmoplastic small round cell tumor (DSRCT).17–22 Markers of neural differentiation (e.g., NSE, S-100 protein, and CD57) may be expressed even in the absence of histologic evidence of neural differentiation.11,23 FLI1 is touted as a sensitive marker for ES, with positive cases showing nuclear staining. FLI1, however, is not specific for ES and has been observed in other neoplasms including lymphoblastic lymphomas (LBL), DSRCT, Merkel cell carcinoma, and synovial sarcoma. Background endothelial cells

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Fig. 2 – Ewing sarcoma. (A) Classical Ewing sarcoma. High-power view shows a monotonous population of small round cells with fine chromatin, inconspicuous nucleoli, and cytoplasmic clearing due to accumulation of glycogen. (B) Immunohistochemical stain for CD99 typically shows a diffuse, strong, membranous staining pattern. (C) Geographic areas of necrosis and perivascular cuffing are commonly seen. (D) Primitive neuroectodermal tumor (PNET) with area of confluent Homer Wright rosettes, indicative of neural differentiation.

Fig. 3 – Histologic variation in Ewing sarcoma. (A) Atypical (large cell) Ewing sarcoma. High-power magnification showing enlarged, mildly pleomorphic, overlapping nuclei with coarse chromatin and large nucleoli. (B) Areas with extensive spindle cell features may be seen. (C and D) Adamantinoma-like Ewing sarcoma with striking areas of hyalinized sclerosis juxtaposed to distinct nests of tumor cells with focal peripheral palisading of nuclei. This case had unusual areas of myxoid change. The case was CD99 and EWSR1 break-apart FISH positive confirming the diagnosis.

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and lymphocytes show normal expression of FLI1.14,24–26 Antibodies against ERG show sensitivity for ES with EWSR1– ERG fusion but are not specific for ES.27 NKX2.2, which was recently proposed as a sensitive marker for ES (sensitivity 93% and specificity 89%), was notably negative in the tested lymphoma cases (including LBL) and small cell osteosarcomas, but positive in mesenchymal chondrosarcoma.28 ES may express epithelial markers. Immunoreactivity to keratins is seen in up to a third of cases but is usually only focal when present.12,29,30 Machado et al.29 also observed EMA and CEA expression in 6.6% and 20.8% of ES, respectively. Rarely, desmin can be expressed.12,31 Keratin immunoreactivity may be particularly strong and diffuse in adamantinoma-like ES.

Genetic rearrangements ES is genetically defined by a balanced translocation that involves the EWSR1 gene (locus 22q12) and a member of the ETS family of transcription factors, most frequently FLI1 or ERG.32,33 The EWSR1–ETS chimeric protein functions as an aberrant transcription factor that promotes oncogenesis by dysregulation of downstream target genes.34 Approximately 85% of ES harbor a t(11;22)(q24;q12) resulting in EWSR1–FLI1 gene fusion.35 The second most common translocation in ES is t(21;22)(q22;q12), resulting in EWSR1–ERG fusion.36 Alternative ETS fusion partners for EWSR1 are infrequent and include ETV,37 ETV4,38 or FEV.39 Additional chromosomal abnormalities may also be present in ES. For example, gains in chromosome 8, chromosome 12, and 1q are common copy number changes seen in ES.40

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Rarely, FUS is rearranged with an ETS gene (Table)41–43 in undifferentiated sarcomas that are morphologically and immunophenotypically similar to ES but lack an EWSR1 rearrangement. FUS belongs to the same TET/FET family of RNA-binding proteins as EWSR1 and is thought to have functionality that is interchangeable with EWSR1, providing an alternative mechanism for oncogenesis in ES.41 Conventional cytogenetic and molecular studies are useful diagnostic aids for tumors that cannot be classified based on histology and immunohistochemistry alone. Fresh tissue is required for cytogenetic studies while formalin-fixed, paraffinembedded tissue can be used for RT-PCR and fluorescence in situ hybridization (FISH). Identification of a characteristic translocation by cytogenetic studies of the EWSR1–ETS fusion transcript by RT-PCR confirms a diagnosis of ES. A negative RTPCR result, however, does not exclude ES as the tumor could have a variant breakpoint or fusion partner that is not detectable by the assay. Detection of EWSR1 split signal by FISH (Fig. 4) supports a diagnosis of ES but does not provide information about its fusion partner. Therefore, complementary studies are needed to rule out other tumors in the differential that could also have EWSR1 rearrangements (e.g., DSRCT). Non-random gene rearrangements other than TET–ETS fusions have also been identified in small round cell sarcomas that have histologic and immunophenotypic features that overlap with ES. Most involve EWSR1 rearrangements with non-ETS family genes (Table 1). The WHO 2013 classification system includes these in a category termed “Ewinglike sarcoma.” Given the small number of cases with these chromosomal abnormalities, it is presently unclear if these

Table – Small round cell sarcoma-associated recurrent gene rearrangements recognized in the 4th edition WHO Classification of Tumours of Soft Tissue and Bone (modified table 19.01). Tumor

Fusion gene

Chromosomal abnormality

EWSR1–FLI1 EWSR1–ERG EWSR1–ETV1 EWSR1–ETV4 EWSR1–FEV

t(11;22)(q24;q12) t(21;22)(q22;q12) t(7;22)(p22;q12) t(17;22)(q21;q12) t(2;22)(q35;q12)

FUS–ETS fusions

FUS–ERG FUS–FEV

t(16;21)(p11;q22) t(2;16)(q35;p11)

EWSR1–non-ETS fusions

EWSR1–NFATc2 EWSR1–POU5F1 EWSR1–SMARCA5 EWSR1–PATZ EWSR1–SP3

t(20;22)(q13;q12) t(6;22)(p21;q12) t(4;22)(q31;q12) Submicroscopic inv(22) in t(1;22)(p36.1;q12) t(2;22)(q31;q12)

Non-TET–non-ETS fusion

BCOR–CCNB3

inv(X)(p11.4p11.22)

Mesenchymal chondrosarcoma

HEY1–NCOA2

Possibly del(8)(q13.3q21.1) resulting in HEY1 (8q21.1) and NCOA2 (8q13.3) fusion

Alveolar rhabdomyosarcoma

PAX3–FOXO1 PAX7–FOXO1

t(2;13)(q35;q14) t(1;13)(p36;q14)

Desmoplastic small round cell tumor

EWSR1–WT1

t(11;22)(p13;q12)

Poorly differentiated synovial sarcoma

SYT–SSX1 SYT–SSX2 SYT–SSX4

t(X;18)(p11;q11)

Ewing sarcoma and “Ewing-like sarcoma” EWSR1–ETS fusions

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Fig. 4 – EWSR1 dual-color, break-apart FISH performed using formalin-fixed, paraffin-embedded tissue specimens. The assay consists of two DNA probes that flank the EWSR1 gene breakpoint region (22q12), seen as red and green signals in these images. Intact cells have two red and two green signals. (A) Negative result. Two adjacent or overlapping red and green signals indicate that both EWSR1 alleles remain intact. (B) Positive result. When a EWSR1 allele becomes rearranged during a translocation, the probes flanking the breakpoint region are split apart and result in separated red and green signals. Therefore, cells positive for EWSR1 rearrangement have one red signal, one green signal, and one overlapping red and green signal. (Photographs courtesy of Dr. Bryan L. Betz.) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

represent a new sarcoma subtype or should be classified as members of the ES family of tumors; these are currently treated in a similar manner to ES.33 Of note, BCOR–CCNB3 fusion is a recurrent genetic abnormality that was recently described by Pierron et al.44 in a subset of sarcomas that resemble ES but lack an EWSR1–ETS translocation. In this entity, an X-chromosome paracentric inversion, inv(X)(p11.4p11.22), results in an intrachromosomal fusion between BCOR and CCNB3, resulting in an oncogenic fusion protein. Clinical characteristics are similar to ES: median age 13 years, slight male predilection, and preferential involvement of long bones, spine, and pelvis. About 20% tumors were located in soft tissue. The tumors were reported to have morphology similar to ES; many had small round cell morphology while others had spindle cell features. Strong membranous CD99 staining was reported in less than half of the tumors. Secondary chromosomal abnormalities commonly seen in ES, such as gain of chromosome 8, were not observed. Furthermore, geneexpression profiling studies strongly suggested that sarcomas with BCOR–CCNB3 fusion represent a new sarcoma subtype, distinct from ES.44

Prognosis Prognosis has markedly improved with the advent of current multimodal therapy with a 5-year survival rate around 65%.45 One of the most important adverse prognostic factors is the presence of metastatic disease at time of diagnosis or early tumor recurrence; 5-year survival for this group is 25–30%.46 Other important prognostic factors include anatomic site of involvement, tumor size, and degree of treatment-induced tumor necrosis.5,47 Neuroectodermal differentiation does not appear to have prognostic significance.11

Differential diagnosis Other primary bone sarcomas, lymphomas, and metastatic disease may mimic skeletal ES. Primary bone lymphomas (PBLs) are rare and tend to occur in older patients. Secondary involvement of bone by a systemic lymphoma is much more common. PBL often involves the diaphysis of long bone. Radiographic findings are non-specific but may show a permeative, destructive, mixed lytic, and sclerotic lesion. Classification of PBL is the same as for their extraosseous counterparts. In the west, PBLs are predominantly B-cell lymphomas, most commonly diffuse large B-cell lymphomas (DLBCLs). Large cell lymphomas are often larger in size than ES cells. Irregular nuclear contours, background mature small lymphocytes, and sclerosis, along with crush artifact, are often seen in primary bone lymphomas (Fig. 5). Broad panels of immunohistochemical stains with inclusion of B- and T-cell markers help differentiate lymphoma from ES. CD45 positivity rules out ES. Intraosseous lymphoblastic lymphoma (LBL) can mimic ES both histologically and immunophenotypically.17,26 LBL can be negative for CD45, CD20, and CD3 by immunohistochemistry but positive for CD99 and FLI1. The expression of CD43, CD79a, and TdT supports a diagnosis of LBL. Molecular studies for EWSR1 rearrangement and immunoglobulin heavy chain and gamma T-cell receptor gene rearrangement may also be helpful. Mesenchymal chondrosarcoma is an uncommon tumor that can arise in either bone or soft tissue. It affects a broad age range, but most frequently occurs in adolescents and young adults. In contrast to the distribution of skeletal ES, there is a predilection for craniofacial bones such as the jaw. Ribs, vertebrae, pelvis, and long bones may also be affected. Radiographically, mesenchymal chondrosarcomas are predominately lytic, destructive lesions that often extend

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Fig. 5 – Primary bone lymphoma. (A) Polymorphous population of atypical lymphocytes with irregular nuclear contours and admixed with non-neoplastic small lymphocytes. (B) Crush artifact and fibrosis are often present in biopsy specimens of primary bone lymphomas and make interpretation challenging.

through cortex into soft tissue; mottled chondroid calcification may be apparent in the mass. Microscopically, they are biphasic tumors comprised of islands of differentiated cartilage surrounded by sheets of undifferentiated small cells (Fig. 6). The amount of cartilage matrix present can vary considerably between tumors. Spindling in the small cell component may be seen. Cartilage matrix may be focally calcified. Hemangiopericytoma-like vessels are often observed.48 Neoplastic cartilage distinguishes mesenchymal chondrosarcoma from ES. On a limited biopsy, however, only the small cell component may be sampled. CD99 can show diffuse and strong staining in the small cell component. Immunopositivity for SOX9 and the absence of FLI1 and ERG immunoreactivity help support a diagnosis of mesenchymal chondrosarcoma. HEY1–NCOA2 fusion has been reported in mesenchymal chondrosarcomas; EWSR1 rearrangement is absent. Small cell osteosarcoma is a very rare form of osteosarcoma that often affects adolescents and commonly occurs in the metaphysis of long bones.49 Radiographic features are similar to conventional osteosarcomas and may present as a destructive, mixed lytic and blastic mass with extension into soft tissue. Microscopically, tumors are comprised of small cells with scant cytoplasm, round to oval nuclei, and variable amounts of lace-like osteoid production (Fig. 7). Spindle cell variants have also been described. The presence of osteoid

differentiates small cell osteosarcoma from ES. CD99 may be positive in the small cell component. FLI1 is negative. No recurrent chromosomal abnormality has been identified; small cell osteosarcoma lacks EWSR1 rearrangements. Metastases or direct extension to adjacent bone by a soft tissue mass is much more common than primary bone tumors and should be considered in the differential of small round cell tumors of bone. Metastatic tumors with small round cell morphology that can mimic ES include neuroblastoma, small cell carcinoma (Fig. 8A and B), alveolar rhabdomyosarcoma (ARMS) (Fig. 8C and D), poorly differentiated synovial sarcoma (PDSS), and DSRCT. CD99 immunoreactivity in a small round blue cell tumor essentially rules out a diagnosis of neuroblastoma. Virtually all other small round cell tumors that may metastasize to bone have been reported to demonstrate membranous CD99 immunoreactivity to some degree. Thus, occasionally, other ancillary immunohistochemical or molecular studies are required to distinguish these tumors from ES. In patients older than 45 years, small cell carcinomas need to be considered in the differential diagnosis, particularly metastatic pulmonary small cell carcinoma and cutaneous neuroendocrine (Merkel cell) carcinoma. Clinical history and imaging studies are usually sufficient to rule out the former. By immunohistochemistry, pulmonary small cell carcinoma is typically TTF-1 positive, while ES is negative for this marker (Fig. 8A and B).

Fig. 6 – Mesenchymal chondrosarcoma is a biphasic tumor. (A) Small round cells are admixed with islands of cartilage. (B) The undifferentiated small cell component has histologic features similar to that of other small round blue cell tumors, including ES.

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Fig. 7 – Small cell osteosarcoma. (A) The presence of lace-like osteoid matrix differentiates this tumor from ES. (B) Higherpower view showing lace-like osteoid production.

Merkel cell carcinoma (MCC) typically arises in the dermis or subcutis in patients older than 60 years. In contrast to ES, MCC is positive for cytokeratin 20 and CAM5.2 in a characteristic perinuclear dot pattern. The majority of pulmonary small cell and Merkel cell carcinomas are negative for CD99. ARMS typically arise in soft tissue, but may metastasize to bone. These tumors have a distinct alveolar pattern and are often discohesive in areas. In addition to round cells, multinucleated giant cells and rhabdomyoblasts are often present. Muscle-specific immunohistochemical markers such as myogenin and MyoD1 are positive in ARMS and negative in ES (Fig. 8C and D). ARMS is also positive for desmin, which may rarely be positive in ES as well. ARMS is distinguished from ES at the molecular level by recurrent translocations involving either PAX3 or PAX7 in the majority of cases. Unlike ES, there

is no rearrangement involving EWSR1. PDSS may have a predominantly small cell morphology making its distinction from ES difficult at times. These tumors show immunoreactivity for cytokeratins such as AE1/AE3, EMA, and cytokeratins 7 and 19 to a greater degree than most ES. In the differential diagnosis of ES, TLE-1 is a sensitive and specific marker of synovial sarcoma, including PDSS. Synovial sarcomas have a recurrent t(X;18) translocations involving SYT but not EWSR1. DSRCT has a propensity to present in young males as a large intraabdominal mass with peritoneal implants. There is an extensive desmoplastic stromal response surrounding islands of atypical small round blue cells. By immunohistochemistry, there is expression of markers of epithelial and mesenchymal differentiation, including immunoreactivity for cytokeratins, vimentin, and neuroendocrine

Fig. 8 – Metastatic tumors to bone with small round cell morphology can mimic Ewing sarcoma. Shown here are (A and B) metastatic small cell carcinoma with positive TTF-1 nuclear staining by immunohistochemistry and (C and D) alveolar rhabdomyosarcoma with immunoreactivity for myogenin.

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markers such as neuron-specific enolase and synaptophysin, as well as membranous CD99 positivity on occasion. A unique perinuclear dot pattern of desmin is seen in this tumor. DSRCTs have a characteristic translocation t(11;22)(p13;q12), which results in an EWSR1–WT1 fusion transcript that can be detected by RT-PCR. EWSR1 rearrangement by FISH is positive in DSRCT and does not help differentiate it from ES. In addition, undifferentiated small round cell tumor with CIC (19q13.2) rearrangements is an emerging sarcoma subtype that has been reported to metastasize to bone.50 To our knowledge, all 30 CIC-rearranged sarcomas were primary soft tissue tumors.50–59 The histologic features of these tumors may overlap with atypical ES, but an extracellular myxoid matrix may be present. CD99 immunohistochemical staining is often focal, weak, or negative, and these tumors lack an EWSR1 rearrangement. Case reports of primary bone rhabdomyosarcoma, DSRCT, and synovial sarcoma exist,60–62 but evidence of a soft tissue primary should be sought if these sarcomas are suspected given the rarity of such events. Correlation with clinical, imaging, immunohistochemical, and genetic findings is essential for correct classification of small round cell tumors of bone.

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Conclusion Ewing sarcoma is a histologically heterogeneous family of tumors with varying degrees of neural differentiation that are characterized by an EWSR1–ETS translocation. Integration of clinical, radiographic, immunohistochemical, and molecular information is essential for diagnosis, particularly in tumors with atypical histologic features. In addition, new sarcoma subtypes are emerging as previously unrecognized gene rearrangements such as BCOR–CCNB3 are discovered. Rapid advances in molecular methodologies will undoubtedly continue to facilitate discovery of novel genetic events that help refine diagnostic criteria and shed light on the biology of undifferentiated small round cell sarcomas.

r e f e r e n c e s

14.

15.

16.

17.

18.

19. 1. Esiashvili N, Goodman M, Marcus RB Jr. Changes in incidence and survival of Ewing sarcoma patients over the past 3 decades: Surveillance Epidemiology and End Results data. J Pediatr Hematol Oncol. 2008;30(6):425–430. 2. Mascarenhas L, Siegel S, Spector L, Arndt C, Femino D, Malogolowkin M. Malignant bone tumors. In: Bleyer A, O0 Leary M, Barr R, et al., eds. Cancer Epidemiology in Older Adolescents and Young Adults 15 to 29 Years of Age, Including SEER Incidence and Survival: 1975–2000. Bethesda, MD: National Cancer Institute; 2006:97–109. 3. Jawad MU, Cheung MC, Min ES, Schneiderbauer MM, Koniaris LG, Scully SP. Ewing sarcoma demonstrates racial disparities in incidence-related and sex-related differences in outcome: an analysis of 1631 cases from the SEER database, 1973–2005. Cancer. 2009;115(15):3526–3536. 4. Applebaum MA, Worch J, Matthay KK, et al. Clinical features and outcomes in patients with extraskeletal Ewing sarcoma. Cancer. 2011;117(13):3027–3032. 5. Cotterill SJ, Ahrens S, Paulussen M, et al. Prognostic factors in Ewing0 s tumor of bone: analysis of 975 patients from the

20.

21.

22.

23.

24.

31 (2014) 39–47

European Intergroup Cooperative Ewing0 s Sarcoma Study Group. J Clin Oncol. 2000;18(17):3108–3114. Lawlor ER, Lim JF, Tao W, et al. The Ewing tumor family of peripheral primitive neuroectodermal tumors expresses human gastrin-releasing peptide. Cancer Res. 1998;58(11): 2469–2476. O0 Regan S, Diebler MF, Meunier FM, Vyas S. A Ewing0 s sarcoma cell line showing some, but not all, of the traits of a cholinergic neuron. J Neurochem. 1995;64(1):69–76. Tirode F, Laud-Duval K, Prieur A, Delorme B, Charbord P, Delattre O. Mesenchymal stem cell features of Ewing tumors. Cancer Cell. 2007;11(5):421–429. Widhe B, Widhe T. Initial symptoms and clinical features in osteosarcoma and Ewing sarcoma. J Bone Joint Surg Am. 2000; 82(5):667–674. Stoker DJ, Saifuddin A. Bone tumours (2): malignant lesions. In: Adam A, Dixon AK, et al., eds. Grainger & Allison0 s Diagnostic Radiology: A Textbook of Medical Imaging. Edinburgh: Churchill Livingstone; 2008:1059–1081. Parham DM, Hijazi Y, Steinberg SM, et al. Neuroectodermal differentiation in Ewing0 s sarcoma family of tumors does not predict tumor behavior. Hum Pathol. 1999;30(8):911–918. Folpe AL, Goldblum JR, Rubin BP, et al. Morphologic and immunophenotypic diversity in Ewing family tumors: a study of 66 genetically confirmed cases. Am J Surg Pathol. 2005;29(8): 1025–1033. Nascimento AG, Unii KK, Pritchard DJ, Cooper KL, Dahlin DC. A clinicopathologic study of 20 cases of large-cell (atypical) Ewing0 s sarcoma of bone. Am J Surg Pathol. 1980;4(1):29–36. Llombart-Bosch A, Machado I, Navarro S, et al. Histological heterogeneity of Ewing0 s sarcoma/PNET: an immunohistochemical analysis of 415 genetically confirmed cases with clinical support. Virchows Arch. 2009;455(5):397–411. Machado I, Noguera R, Mateos EA, et al. The many faces of atypical Ewing0 s sarcoma. A true entity mimicking sarcomas, carcinomas and lymphomas. Virchows Arch. 2011;458 (3):281–290. Fujii H, Honoki K, Enomoto Y, et al. Adamantinoma-like Ewing0 s sarcoma with EWS–FLI1 fusion gene: a case report. Virchows Arch. 2006;449(5):579–584. Ozdemirli M, Fanburg-Smith JC, Hartmann DP, Azumi N, Miettinen M. Differentiating lymphoblastic lymphoma and Ewing0 s sarcoma: lymphocyte markers and gene rearrangement. Mod Pathol. 2001;14(11):1175–1182. Granter SR, Renshaw AA, Fletcher CD, Bhan AK, Rosenberg AE. CD99 reactivity in mesenchymal chondrosarcoma. Hum Pathol. 1996;27(12):1273–1276. Lae ME, Roche PC, Jin L, Lloyd RV, Nascimento AG. Desmoplastic small round cell tumor: a clinicopathologic, immunohistochemical, and molecular study of 32 tumors. Am J Surg Pathol. 2002;26(7):823–835. Devaney K, Abbondanzo SL, Shekitka KM, Wolov RB, Sweet DE. MIC2 detection in tumors of bone and adjacent soft tissues. Clin Orthop Relat Res. 1995(310):176–187. Riopel M, Dickman PS, Link MP, Perlman EJ. MIC2 analysis in pediatric lymphomas and leukemias. Hum Pathol. 1994;25(4): 396–399. Machado I, Alberghini M, Giner F, et al. Histopathological characterization of small cell osteosarcoma with immunohistochemistry and molecular genetic support. A study of 10 cases. Histopathology. 2010;57(1):162–167. Carter RL, al-Sams SZ, Corbett RP, Clinton S. A comparative study of immunohistochemical staining for neuron-specific enolase, protein gene product 9.5 and S-100 protein in neuroblastoma, Ewing0 s sarcoma and other round cell tumours in children. Histopathology. 1990;16(5):461–467. Folpe AL, Hill CE, Parham DM, O'Shea PA, Weiss SW. Immunohistochemical detection of FLI-1 protein expression: a

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study of 132 round cell tumors with emphasis on CD99positive mimics of Ewing0 s sarcoma/primitive neuroectodermal tumor. Am J Surg Pathol. 2000;24(12):1657–1662. Rossi S, Orvieto E, Furlanetto A, Laurino L, Ninfo V, Dei Tos AP. Utility of the immunohistochemical detection of FLI-1 expression in round cell and vascular neoplasm using a monoclonal antibody. Mod Pathol. 2004;17(5):547–552. Lin O, Filippa DA, Teruya-Feldstein J. Immunohistochemical evaluation of FLI-1 in acute lymphoblastic lymphoma (ALL): a potential diagnostic pitfall. Appl Immunohistochem Mol Morphol. 2009;17(5):409–412. Wang WL, Patel NR, Caragea M, et al. Expression of ERG, an Ets family transcription factor, identifies ERG-rearranged Ewing sarcoma. Mod Pathol. 2012;25(10):1378–1383. Yoshida A, Sekine S, Tsuta K, Fukayama M, Furuta K, Tsuda H. NKX2.2 is a useful immunohistochemical marker for Ewing sarcoma. Am J Surg Pathol. 2012;36(7):993–999. Machado I, Navarro S, Lopez-Guerrero JA, Alberghini M, Picci P, Llombart-Bosch A. Epithelial marker expression does not rule out a diagnosis of Ewing0 s sarcoma family of tumours. Virchows Arch. 2011;459(4):409–414. Gu M, Antonescu CR, Guiter G, Huvos AG, Ladanyi M, Zakowski MF. Cytokeratin immunoreactivity in Ewing0 s sarcoma: prevalence in 50 cases confirmed by molecular diagnostic studies. Am J Surg Pathol. 2000;24(3):410–416. Parham DM, Dias P, Kelly DR, Rutledge JC, Houghton P. Desmin positivity in primitive neuroectodermal tumors of childhood. Am J Surg Pathol. 1992;16(5):483–492. Delattre O, Zucman J, Plougastel B, et al. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature. 1992;359(6391):162–165. de Alava E, Lessnick SL, Sorensen PH. Ewing Sarcoma. In: Fletcher CDM, Bridge JA, Hogendoorn PC, et al., eds. WHO Classification of Tumours of Soft Tissue and Bone. Lyon: IARC Press; 2013:306–309. de Alava E, Pardo J. Ewing tumor: tumor biology and clinical applications. Int J Surg Pathol. 2001;9(1):7–17. Delattre O, Zucman J, Melot T, et al. The Ewing family of tumors—a subgroup of small-round-cell tumors defined by specific chimeric transcripts. N Engl J Med. 1994;331(5):294–299. Sorensen PH, Lessnick SL, Lopez-Terrada D, Liu XF, Triche TJ, Denny CT. A second Ewing0 s sarcoma translocation, t(21;22), fuses the EWS gene to another ETS-family transcription factor, ERG. Nat Genet. 1994;6(2):146–151. Jeon IS, Davis JN, Braun BS, et al. A variant Ewing0 s sarcoma translocation (7;22) fuses the EWS gene to the ETS gene ETV1. Oncogene. 1995;10(6):1229–1234. Kaneko Y, Yoshida K, Handa M, et al. Fusion of an ETS-family gene, EIAF, to EWS by t(17;22)(q12;q12) chromosome translocation in an undifferentiated sarcoma of infancy. Genes Chromosomes Cancer. 1996;15(2):115–121. Peter M, Couturier J, Pacquement H, et al. A new member of the ETS family fused to EWS in Ewing tumors. Oncogene. 1997;14(10):1159–1164. Armengol G, Tarkkanen M, Virolainen M, et al. Recurrent gains of 1q, 8 and 12 in the Ewing family of tumours by comparative genomic hybridization. Br J Cancer. 1997;75(10):1403–1409. Shing DC, McMullan DJ, Roberts P, et al. FUS/ERG gene fusions in Ewing0 s tumors. Cancer Res. 2003;63(15):4568–4576. Berg T, Kalsaas AH, Buechner J, Busund LT. Ewing sarcomaperipheral neuroectodermal tumor of the kidney with a FUS– ERG fusion transcript. Cancer Genet Cytogenet. 2009;194(1):53–57. Ng TL, O0 Sullivan MJ, Pallen CJ, et al. Ewing sarcoma with novel translocation t(2;16) producing an in-frame fusion of FUS and FEV. J Mol Diagn. 2007;9(4):459–463. Pierron G, Tirode F, Lucchesi C, et al. A new subtype of bone sarcoma defined by BCOR–CCNB3 gene fusion. Nat Genet. 2012;44(4):461–466.

P

A T H O L O G Y

31 (2014) 39–47

47

45. Linabery AM, Ross JA. Childhood and adolescent cancer survival in the US by race and ethnicity for the diagnostic period 1975–1999. Cancer. 2008;113(9):2575–2596. 46. Ludwig JA. Ewing sarcoma: historical perspectives, current state-of-the-art, and opportunities for targeted therapy in the future. Curr Opin Oncol. 2008;20(4):412–418. 47. Picci P, Bohling T, Bacci G, et al. Chemotherapy-induced tumor necrosis as a prognostic factor in localized Ewing0 s sarcoma of the extremities. J Clin Oncol. 1997;15(4):1553–1559. 48. Nakashima Y, Unni KK, Shives TC, Swee RG, Dahlin DC. Mesenchymal chondrosarcoma of bone and soft tissue. A review of 111 cases. Cancer. 1986;57(12):2444–2453. 49. Nakajima H, Sim FH, Bond JR, Unni KK. Small cell osteosarcoma of bone. Review of 72 cases. Cancer. 1997;79(11): 2095–2106. 50. Somers GR, Shago M, Zielenska M, Chan HS, Ngan BY. Primary subcutaneous primitive neuroectodermal tumor with aggressive behavior and an unusual karyotype: case report. Pediatr Dev Pathol. 2004;7(5):538–545. 51. Choi EY, Thomas DG, McHugh JB, et al. Undifferentiated small round cell sarcoma with t(4;19)(q35;q13.1) CIC–DUX4 fusion: a novel highly aggressive soft tissue tumor with distinctive histopathology. Am J Surg Pathol. 2013;37(9):1379–1386. 52. Graham C, Chilton-MacNeill S, Zielenska M, Somers GR. The CIC–DUX4 fusion transcript is present in a subgroup of pediatric primitive round cell sarcomas. Hum Pathol. 2012;43 (2):180–189. 53. Italiano A, Sung YS, Zhang L, et al. High prevalence of CIC fusion with double-homeobox (DUX4) transcription factors in EWSR1-negative undifferentiated small blue round cell sarcomas. Genes Chromosomes Cancer. 2012;51(3):207–218. 54. Kajtar B, Tornoczky T, Kalman E, Kuzsner J, Hogendoorn PC, Szuhai K. CD99-positive undifferentiated round cell sarcoma diagnosed on fine needle aspiration cytology, later found to harbour a CIC–DUX4 translocation: a recently described entity. Cytopathology. 2013. http://onlinelibrary.wiley.com/doi/ 10.1111/cyt.12079/abstract;jsessionid=0D364C52932E67456AA EFD5DBCE8C014.f02t03 [E-pub ahead of print]. 55. Kawamura-Saito M, Yamazaki Y, Kaneko K, et al. Fusion between CIC and DUX4 up-regulates PEA3 family genes in Ewing-like sarcomas with t(4;19)(q35;q13) translocation. Hum Mol Genet. 2006;15(13):2125–2137. 56. Machado I, Cruz J, Lavernia J, et al. Superficial EWSR1negative undifferentiated small round cell sarcoma with CIC/DUX4 gene fusion: a new variant of Ewing-like tumors with locoregional lymph node metastasis. Virchows Arch. 2013;463(6):837–842. 57. Rakheja D, Goldman S, Wilson KS, Lenarsky C, Weinthal J, Schultz RA. Translocation (4;19)(q35;q13.1)-associated primitive round cell sarcoma: report of a case and review of the literature. Pediatr Dev Pathol. 2008;11(3):239–244. 58. Richkind KE, Romansky SG, Finklestein JZ. t(4;19)(q35;q13.1): a recurrent change in primitive mesenchymal tumors? Cancer Genet Cytogenet. 1996;87(1):71–74. 59. Yoshimoto M, Graham C, Chilton-MacNeill S, et al. Detailed cytogenetic and array analysis of pediatric primitive sarcomas reveals a recurrent CIC–DUX4 fusion gene event. Cancer Genet Cytogenet. 2009;195(1):1–11. 60. Jung SC, Choi JA, Chung JH, Oh JH, Lee JW, Kang HS. Synovial sarcoma of primary bone origin: a rare case in a rare site with atypical features. Skeletal Radiol. 2007;36(1):67–71. 61. Lucas DR, Ryan JR, Zalupski MM, Gross ML, Ravindranath Y, Ortman B. Primary embryonal rhabdomyosarcoma of long bone. Case report and review of the literature. Am J Surg Pathol. 1996;20(2):239–244. 62. Murphy A, Stallings RL, Howard J, et al. Primary desmoplastic small round cell tumor of bone: report of a case with cytogenetic confirmation. Cancer Genet Cytogenet. 2005;156(2):167–171.