Small round cell malignant tumors (SRCMT) in adults comprise a heterogeneous group of neoplasms that share a common morphologic appearance but are treated with divergent therapeutic agents. Based on cytomorphology alone, a specific diagnosis is extremely difficult to render with certainty. The advances in immunohistochemistry and molecular biology have facilitated the diagnosis not only in large tumor samples but also in small cytologic preparations.

In adult soft tissue tumors, the differential diagnosis of SRCMT includes Non-Hodgkin lymphoma, extraskeletal Ewing’s/PNET, round cell liposarcoma, alveolar rhabdomyosarcoma, mesenchymal chondrosarcoma, desmoplastic round cell tumor, poorly differentiated synovial sarcoma, metastatic small cell carcinomas or other neuroendocrine tumors, and germ cell tumors (eg, embryonal carcinoma, immature teratomas). Of these entities, lymphoma and sarcomas comprise the majority.  In bone, the small cell variant of osteosarcoma, Ewing’s sarcoma, metastatic small cell carcinoma, and lymphoma are among the most common possibilities. [TABLE 1]

Table 1.  Differential Diagnosis of Small Round Malignant Tumors in Adults

Non-Hodgkin Lymphoma

Ewing’s sarcoma/Primitive neuroectodermal tumor

Other sarcomas (eg, round cell liposarcoma, alveolar rhabdomyosarcoma, mesenchymal chondrosarcoma, small cell variant of osteosarcoma, CIC-DUX sarcoma and poorly differentiated synovial sarcoma) 

Carcinoma (eg, small cell neuroendocrine carcinoma of the lung or extrapulmonary sites)

Germ cell tumors (eg, embryonal carcinoma, immature teratomas with prominent neuroectodermal component)

The finding of SMRCT of bone, as in cytologic findings of any bone tumor, needs to be strongly correlated with the imaging studies. Osteoid matrix production favors osteogenic sarcoma over lymphoma and Ewing’s, and this can be difficult to identify in small biopsy samples or aspirates, whereas radiological imaging usually is helpful to look for evidence of matrix-production.  While the same advises apply to SMRCT in soft tissue, the radiographic information is often less characteristic.  A combination of the radiological findings, age, gender, and extent of disease can usually help in narrowing the differential diagnosis prior to morphological review.

On fine needle aspiration biopsy (FNAB), Ewing’s sarcoma can be established if sufficient material is obtained for ancillary studies. The smears are markedly cellular and composed of mostly individually dispersed cells with possible clustering that is usually due to hypercellularity. [Figures 1 and 2]  There is usually a light and dark pattern seen at low power, which is thought to be attributed to some cells undergoing apoptosis.  The tumor cells are generally uniform with monomorphic round nuclei and evenly distributed chromatin with inconspicuous nucleoli.[Figure 3] A necrotic background accompanied by increased karyorrhectic debris can also be seen.[Figures 3-4]  Nuclear membranes are smooth and the nuclear-to-cytoplasmic ratio is extremely high.(2)  Cytoplasmic vacuoles and Homer Wright rosettes are rarely appreciated, but when present, they are highly suggestive(4) (see figures 4-5). In addition, since Ewing’s Sarcoma is a glycogenated tumor, a tigroid background can be appreciated between the cells on the air dried, Diff-Quik stained smears.  In addition, PAS can also be used to highlight the tumor cells given the presence of glycogen.  While typical Ewing’s sarcoma shows little variation among tumor cells, it exists several atypical variants where larger cell size, prominent nucleoli, mild pleomorphism and slight spindling can be accepted(1).

These features differ from usual large cell lymphomas which tend to show more complex nuclear membranes, vesicular to coarse chromatin with small nucleoli. The background is almost always necrotic. Lymphoglandular bodies are commonly seen, and their presence is always indicative of lymphoid malignancies (5). Alveolar Rhabdomyosarcoma (ARMS) is characterized by the presence of monomorphic population of small round cells similar to other tumors in this category, however with distinct cytologic features including general absence of cytoplasmic content, nuclear membrane irregularity, hyperchromasia, absence of nucleoli, and the occasional presence of rhabdomyoblastic differentiation (strap cells).  In addition, ARMS is characterized by a rearrangement of the FKHR (FOXO1) gene with either PAX3 or PAX7.

Subset of undifferentiated small blue round cell sarcoma with either CIC-DUX4, t (4;19) (q35;q13) or CIC-DUX4L, t(10;19)(q26;q13) fusions are also in differential diagnosis of Ewing’s sarcoma. These sarcomas occur over a wide age range but predominantly are seen in young adults. The tumors have histomorphology including round or spindle high-grade nuclear features (vesicular chromatin, moderate nuclear pleomorphism, and prominent nucleoli), lobular growth pattern, geographic necrosis, round cell cytomorphology, patchy clear cell areas, and distinctive foci of myxoid change. These tumors demonstrated clinical behavior at least as aggressive or more aggressive as Ewing sarcomas.  Immunohistochemistry for CD99 tends to be less diffuse in CIC-DUX sarcomas, however FLI1 and ERG are usually positive. This poses significant pitfall in diagnosis, particularly in small biopsies and cytology specimens.  Recent studies indicate that DUX4 immunohistochemistry is sensitive and specific marker for CIC-DUX4 fusion.  

Cytogenetics and immunohistochemistry performed on cell blocks or concurrent core biopsy specimens can help confirming the diagnosis of Ewing’s Sarcoma.  Over 90% of Ewing’s sarcomas express CD99 (O13), and 70-90% express Fli-1 (see figures 8-9). The cautious interpretation of the two latter stains is advised as CD99 is known to lack specificity and Fli-1 sensitivity. Neuroendocrine and neuroectodermal markers (S100, CD57, Synaptophysin and NSE) are variably expressed when neuroectodermal differentiation is present and their inclusion in the immunopanel can be of significant help. Cytokeratin expression (AE1/3) is not uncommon, as roughly 10-20% of cases demonstrate focal expression. However, strong and diffuse expression is very unusual. Ewing’s sarcoma should be negative for CD45 (lymphoid marker), S100, and desmin or other muscle markers. Molecular confirmation of the fusion transcripts EWS/FLI-1, ERG or other rare variants can further confirm the diagnosis and can be performed either by FISH or PCR.  Some laboratories use a chromosome 22 break apart FISH probe, which does not tell you the specific translocation in Ewing’s sarcoma, but can help to support a EWS rearrangement in a case that morphologically and immunophenotypically is consistent with Ewing’s sarcoma. 

In regard to the diagnosis made for this case, Immunohistochemistry as provided and detailed above is supportive of the diagnosis. Additionally, the diagnosis was further confirmed by RT-PCR amplification studies for chromosomal translocation.  A formalin fixed, paraffin embedded block was used for the test. The RT-PCR studies showed a fusion transcript between the EWS and FLI1 genes, corresponding to a type 2 fusion transcript. This was confirmed by sequencing of the PCR product. Thus, the molecular studies showed evidence of a t(11;22)(q24;q12) chromosomal translocation which can be observed in the Ewing Sarcoma family of peripheral/primitive neuroectodermal tumors (pPNETS).