Pediatric Pathology

Ewing Sarcoma

Paul Jedlicka
The Children's Hospital
University of Colorado Denver
Aurora CO


Clinical History
A previously healthy 2-year old female presented with a 1-month history of progressive bilateral lower extremity weakness and a neurogenic bladder. Imaging revealed an epidural tumor, which was biopsied and debulked to relieve the symptoms of spinal cord compression. Metastatic work-up was negative. She underwent chemotherapy and radiotherapy with good response and is currently in disease remission 4 years later.


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Figure 1
Touch preparation performed on tumor biopsy tissue, showing discohesive population of relatively uniform, small to medium-sized cells with high nuclear/cytoplasmic ratio and apoptotic activity.
Figure 2
Low-power histomorphologic features of tumor, showing a dense, small blue cell population with patchy necrosis, infiltrating fibrocollagenous tissue.
Figure 3
Intermediate-power histomorphologic features of tumor, showing a relatively uniform cell population with clear cytoplasm.

Figure 4
High-power histomorphologic features of tumor, showing cells with fine chromatin, inconspicuous nuclei, amphophilic to clear cytoplasm and scattered apoptotic activity.
Figure 5
CD99 immunohistochemical staining of tumor, showing uniform membranous staining of tumor cells in well-preserved areas.

Figure 6
Higher-power view of tumor CD99 immunostaining.
Figure 7
Ultrastructural features of tumor, showing cells with high nuclear-cytoplasmic ratio, and organelle-poor cytoplasm containing abundant glycogen in the polyparticulate form.


Pathologic Features
The biopsy consisted of multiple pieces of soft tissue. Touch preparation revealed a discohesive population of relatively uniform small to medium-sized cells with high nuclear/cytoplasmic ratio and apoptotic activity. Histologic examination showed a dense population of malignant small blue cells with clear to amphophilic cytoplasm, punctate chromatin, small nucleoli, and scattered apoptotic and mitotic activity. Immunophenotyping revealed the tumor cells to be strongly and diffusely positive for CD99 in a crisp membranous pattern, and immunongative for pancytokeratin, synaptophysin, neuron-specific enolase, desmin, myogenin, CD45 and TdT. Electron microscopic examination showed poorly differentiated cells containing abundant cytoplasmic glycogen in the polyparticulate form. Fluorescence in-situ hybridization studies revealed rearrangement of the EWSR locus, and molecular diagnostic studies demonstrated the presence of the EWS/Fli1 oncogenic fusion transcript. Together, these findings unequivocally established the diagnosis of Ewing Sarcoma/ Peripheral Primitive Neuroectodermal Tumor (PNET).

Discussion
Ewing Sarcoma is an enigmatic malignancy. First described by James Ewing in 1921 as an endothelioma of bone, the histogenesis of this aggressive, poorly differentiated tumor remains uncertain to this day. Ewing Sarcoma is a tumor of bone and soft tissue, and less commonly viscera. The current definition encompasses the entities of Ewing Sarcoma, Peripheral Primitive Neuroectodermal Tumor, Peripheral Neuroepithelioma and Askin Tumor, all of which share the same EWS/Ets oncogenic fusions and similar biologic behavior. Ewing Sarcoma represents the second most common bone and soft tissue malignancy in adolescents and young adults, peaking in incidence at about 5 per million in this age group. It is slightly more common in males, more common in Caucasians and rarely arises in individuals of African ancestry. As with other bone and soft tissue malignancies, Ewing Sarcoma presents with pain and/or a mass, and a destructive/infiltrative lesion on imaging. The most common bony sites are the long bones of the extremities, pelvis, chest wall and spine. Lesions of long bones typically involve the diaphysis.

Pathologic examination is key to diagnosis. Well-preserved biopsy specimens show characteristic histomorphology and ultrastructure, as exemplified in the above case, including relatively uniform, malignant, small to medium-sized cells with little amphophilic to clear cytoplasm, punctate chromatin, inconspicuous nuclei and abundant cytoplasmic glycogen in the polyparticulate form. Immunophenotyping helps to support the diagnosis. Well-preserved specimens are positive for CD99 in a diffuse membranous pattern. A number of other tumors may show varying degrees of CD99 immunopositivity, but in an experienced laboratory, diffuse CD99 positivity in a membranous pattern is highly suggestive of the diagnosis of Ewing Sarcoma in the right morphologic context. The tumor may also be variably positive for cytokeratin, synaptophysin and neuron-specific enolase. In pediatric patients, the main differential diagnosis includes leukemia/lymphoma, poorly differentiated rhabdomyosarcoma, and, depending on site, undifferentiated neuroblastoma; these diagnoses are excluded by CD45 and TdT negativity, myogenin and desmin negativity, and CD99 positivity, respectively. Occasional Ewing Sarcomas may show divergent skeletal muscle differentiation, and one must rely on cytogenetic/molecular studies (presence of EWS/Ets fusion) to arrive at the correct diagnosis. For bone lesions, absence of malignant osteoid further excludes small cell osteosarcoma; similarly, absence of a cartilaginous component helps exclude mesenchymal chondrosarcoma. On small biopsies, additional diagnostic considerations may include poorly differentiated synovial sarcoma (SS) and desmoplastic small round cell tumor (DSRCT); these can be differentiated by immunophenotyping (desmin positivity and CD99 negativity in DSRCT) and cytogenetic/molecular studies (X;18 translocation in SS and EWS/WT1 oncogenic fusion in DSRCT). Ultimately, if cytogenetic and/or molecular studies are available, the diagnosis of Ewing Sarcoma can be confirmed, and alternative diagnoses excluded, by the demonstration of an EWS/Ets oncogenic fusion. When available, ultrastructural studies can also be very helpful in navigating the differential diagnosis of Ewing Sarcoma. Occasionally one encounters Ewing-like tumors that lack the characteristic immunophenotype and (known) EWS/Ets fusions; these are diagnosed as unclassified/undifferentiated small round cell malignancies/sarcomas. This scenario underscores the importance of allocating fresh biopsy tissue for additional (cytogenetic, molecular and ultrastructural), studies whenever possible, as this may result in the identification of novel diagnostic features in such cases. In the not-so-distant future, such tumors may also be further subclassified by gene expression profiling.

The two most important prognostic indicators in the staging of Ewing Sarcoma are tumor size (greater or less than 8 cm) and presence or absence of metastatic disease. Additional prognostic information is conferred by fusion type: for tumors harboring the EWS/Fli1 fusion, the type 1 variant carries a better prognosis. More recently, additional molecular indicators of (poor) prognosis have been identified, including p53 mutation and p16/p14ARF deletion. Further, prognostically meaningful tumor "signatures" that predict disease outcome are emerging from global gene expression profiling studies. From the standpoint of treatment, the presence of absence of overt metastasis is the most important determinant, although all patients are presumed to harbor micrometastatic disease. Multi-agent chemotherapy has raised survival for patients without evidence of overt metastasis at the time of presentation from 10% to 50-60% at 5 years. However, survival for the 25% of patients with overt metastatic disease at presentation is less than 25% at 5 years; recurrent disease carries a similarly grim prognosis. Treatment of patients without overt metastatic disease consists of initial chemotherapy (for cytoreduction and eradication of any micrometastatic disease), followed by surgical resection, and then consolidation chemotherapy (to reduce the likelihood of recurrence). Treatment options for metastatic and recurrent disease include chemotherapy, surgery, radiation, biologically targeted therapy and bone marrow transplantation, but little progress has been made in improving survival in this group. Hence, there is a tremendous potential role for new therapies.

Biologically, Ewing Sarcoma is the canonical example of a malignancy driven by a fusion oncogene. Ewing Sarcomas harbor fusions between the amino terminus of the EWS gene on chromosome 22 and the carboxy terminus of one of five Ets family genes. The Ets gene is Fli1 in 85% of cases, Erg in 10% of cases, and Etv1, Etv4 or FEV in the remaining 5% of cases. EWS is a member of the TET family of proteins, which are ubiquitously expressed in all cells, and appear to be involved in transcription and/or RNA processing. Fli1 and the other Ets genes are tissue-specific transcription factors. In-frame fusion of EWS to an Ets factor in Ewing Sarcoma yields a highly expressed, non-physiologic, potent transcription factor, which dysregulates gene expression in the cell.

All experimental evidence points to EWS/Ets fusions being the key oncogenes in Ewing Sarcoma. Namely, EWS/Ets fusions are both necessary and sufficient, in the appropriate cellular context, to drive oncogenesis. Thus, the key to understanding Ewing Sarcoma pathogenesis centers on the biology of these fusions. A shortcoming of much early research on Ewing Sarcoma was the lack of experimental systems that closely model the disease. Recently, a number of advances have been made on this front. First, RNA interference technology has permitted the identification of bona fide cellular pathways mediating EWS/Ets oncogenicity, opening the way to the identification of new "druggable targets" for therapy. Second, two independent groups have shown that expression of EWS/Fli1 in bone marrow-derived mesenchymal stem cells is sufficient to induce Ewing Sarcoma-like tumors in a mouse xenograft model. In a related study, another group has shown that loss of EWS/Fli1 expression in Ewing Sarcoma results in change to a mesenchymal progenitor-type cell with multipotential differentiation capacity. Although not proof, these findings offer strong evidence in support of a mesenchymal stem cell origin for Ewing Sarcoma. This, in turn, has given rise to better experimental models, and finally opened the door to the possibility of an animal genetic model for the disease. The hope, of course, is that better understanding of Ewing Sarcoma pathogenesis through improved experimental systems will yield more effective therapies. To this end, identification of insulin-like growth factor signaling as essential for Ewing Sarcoma-genesis, has already led to biologically based therapies currently in Phase II clinical trials, with more likely to follow. The "holy grail" for a biologically targeted therapy remains blockade of the oncogenic EWS/Ets fusions themselves, a goal toward which, unfortunately, little progress has been made to date.

Bullet points:
  • Ewing Sarcoma is an aggressive malignancy affecting adolescents and young adults

  • Diagnosis is suggested by characteristic histomorphology, ultrastructure and immunophenotype, and confirmed by cytogenetic and/or molecular analysis

  • Metastatic and recurrent Ewing Sarcoma remains a disease with poor prognosis

  • EWS/Ets oncogenic fusions drive Ewing Sarcoma-genesis

  • Recent evidence supports a mesenchymal stem cell origin for Ewing Sarcoma

  • New model experimental systems offer hope for the discovery of more effective therapies

References
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