Ewing sarcoma/Primitive neuroectodermal tumor family (EWS/PNET) is a family of sarcomas characterized by an EWS break-apart to form a number of unique, non-random translocations [17]. These tumors differ in their extent of cellular differentiation. At one end of the spectrum are nearly undifferentiated small round cell malignancies, classical Ewing sarcoma. At the other end of the spectrum tumors show neuronal differentiation [18], and are termed peripheral neuroectodermal tumor [19].

Most of the tumors are located in bones, and only 10–20% are located in soft tissues [17, 19]. The most common locations are long bones of lower extremities, followed by pelvic bones and ribs [19, 20].

Although the incidence rate is low, Ewing sarcoma is the second most common sarcoma of bones in children and adolescents [17, 19]. Males are more commonly affected than females.

Tumor and pain are the leading symptoms, often accompanied by systemic symptoms such as fever and increased sedimentation rate, anaemia, and leukocytosis.

Radiography shows most lesions in the diaphysis, and they are osteolytic, intramedullary (symmetric or eccentric) with cortical destruction [21]. A reactive, onion-skin-like new bone formation is characteristic [17], and a soft tissue mass is often present.

Smears are usually very cellular, even in primary bone lesions, due to extra-skeletal tumor extension, which facilitates FNA. Numerous dissociated cells and naked nuclei predominate, and the number of cell groups is variable, but they are seldom numerous.

Cell groups are in the form of chance formations (Fig. 3.13) , tight cell groups (Fig. 3.14), and sometimes rosettes . (Fig. 3.15) Groups and tissue fragments do not have stroma, and cells are sometimes arranged around capillaries. Isolated stromal or capillary fragments can be present. Rosettes are seen in approximately 25% of cases and have no neutropil [22].

In their most characteristic form the tumor population is monomorphous (Figs. 3.13 and 3.16) or with mild anisonucleosis, which is seen in 70–80% of cases. Only 6% of cases analyzed at the Institute of Oncology, Ljubljana had severe anisonucleosis [23].

Nuclei are round and sometimes oval, centrally located. In some cells, nuclei can be eccentric (plasmacytoid cells) (Fig. 3.17) . Oval nuclei seldom predominate. Nucleoli are single or multiple, usually small and indistinct. In many smears nucleoli may be absent. Mitotic figures are not frequent. Pyknotic nuclei are often present and they can be numerous. As a result, many authors describe the smears as containing dark and light cells [18, 24].

Cytoplasm is mostly scant and poorly demarcated, sometimes moderate, plasmacytoid, or with unipolar tail-like extensions. Cells with dark, pyknotic nuclei have scant cytoplasm, while light cells have moderate amount of cytoplasm. Vacuoles that contain glycogen can be present in well-preserved cells . (Fig. 3.18).

The background composition can be variable: eosinophilic matrix in a lace-like pattern is the most common (Fig. 3.16). There can also be necrosis with macrophages and neutrophils, calcifications, lymphoglandular bodies, or some myxoid material (Fig. 3.19).

CD99 is positive in 90% of EWS/PNET ; however it is not specific (Fig. 3.20). Twenty to thirty percent of cases are positive for cytokeratin, diffusely or focally [19, 25]. Neuroendocrine markers are absent in classical Ewing sarcoma but positive in PNET. Chromogranin, synaptophysin, and Leu-7 have been reported [2]. CD56 may be present focally [9] and can therefore be negative in FNA samples. NB-84 has been reported positive in 30% of EWS/PNET [13]. Fourteen percent of EWS/PNET in our series were positive for desmin [10]. WT1 is not expressed in EWS/PNET [26].

Approximately 85% of EWS/PNET possess t(11;22)(q24;q12) translocation while others can harbor one of the nine alternate translocations [19]. The presence of translocations can be demonstrated by karyotyping or by demonstrating fusion products using reverse transcription polymerase chain reaction (RT-PCR) [27]. Fluorescence in situ hybridization (FISH) can be used to demonstrate a split EWSR1; we have to consider, however, that several other sarcomas have EWSR1 rearrangement [19]. Therefore, EWS/PNET cannot be differentiated from desmoplastic small round cell tumor or from extra-skeletal myxoid chondrosarcoma using FISH.

Differential diagnoses vary somewhat with EWS/PNET located in bones, as opposed to soft tissue locations.

Small cell osteogenic sarcoma is a rare tumor and there is very little experience with it in cytopathology. It cannot be differentiated from EWS/PNET on the basis of morphology. Immunocytochemistry is not very helpful, because both tumors express CD99. FLI1 may be of some help because it is negative in small cell osteosarcoma and positive in EWS/PNET. Molecular biology is necessary for demonstrating the presence or absence of the typical translocation found in EWS/PNET [28].

Poorly differentiated neuroblastoma lacks typical Homer-Wright rosettes and neuropil and is therefore morphologically very similar to EWS/PNET. Dot-like positivity for CD99 has been reported in 30% of neuroblastomas in cytology [10]. Even though it is not a membranous positivity characteristic of EWS/PNET, it represents an additional difficulty. Klijanienko et al. have reported mistaking two of the 50 EWS/PNET cases for neuroblastoma [27] that were located in the mediastinum and intra-abdominally. We have misdiagnosed a bone metastasis of neuroblastoma for EWS/PNET.

EWS/PNET simulates alveolar rhabdomyosarcoma especially when cells have a plasmacitoid or tadpole appearance [5]. Desmin positivity in EWS/PNET in almost 14% of cases is an additional drawback, as is the fact that MyoD1 and myogenin are difficult to demonstrate in cytology.

Poorly differentiated synovial sarcomas are composed of undifferentiated small cells with hyperchromatic nuclei and scant cytoplasm. Åkerman et al. reported predominantly dissociated tumor cells that vary in size and shape from round and ovoid to plasmacytoid, and therefore resemble EWS/PNET [28]. Since synovial sarcoma is CD99 positive in approximately one-third of cases and 60% are CK positive, morphology and immunocytochemistry are not enough for a definitive cytological diagnosis [29].

Non-Hodgkin lymphoma may appear as primary tumor in bones, less common in soft tissues. Morphologically it can resemble EWS/PNET, but positivity for lymphatic antigens in immunocytochemistry or flow cytometry usually solves the problem. A broader pattern of B and T lymphocytic markers should be used because lymphocytic common antigen (LCA) is sometimes negative in high-grade lymphomas.

Desmoplastic small round cell tumors may be morphologically and immunocytochemically similar to EWS/PNET. However, this tumor is rare in young children and located mostly on serosal surfaces of the abdominal cavity.

Mesenchymal chondrosarcoma is a very rare tumor in teenagers and young adults. It comes into differential diagnosis with EWS/PNET in cases in which chondroid matrix is not present in the smear.

There are few reports describing series of EWS/PNET in FNA smears including data on accuracy of primary tumor diagnosis. Fröstad et al. [30] analysed 24 cases over a 10-year period, using ICC as the ancillary technique. The authors report a correct diagnosis of EWS/PNET in 22 cases; in one case neuroblastoma was suggested together with EWS/PNET. The misdiagnosed case proved on histology to be small cell osteosarcoma. Klijanienko et al. analysed 50 cases of EWS/PNET aspirated during a 15-year period between 1994 and 2009 [27]. They used ICC and molecular diagnostics as ancillary techniques. The correct diagnosis was given on morphology alone in 46 cases. Two were misdiagnosed as neuroblastoma and two others as RMS and nephroblastoma. EWS/PNET fusion transcript was not detected in six cases, including four with correct morphological FNA diagnosis. The results of our analysis at the Institute of Oncology in Ljubljana are not as good, probably because the EWS/PNET cases dated from 1972–2002 [23]. ICC was not in use during the first 15 years of the selected 30-year period and a limited number of antibodies were used in the 1990s. Comparison of diagnostic accuracy between the two 15-year periods showed an increase from 58 to 71%.

Since EWS/PNET is the second-most common bone tumor in children, while other primary bone tumor or metastases of round cell tumors to bones are almost curiosities in daily cytopathological practice, it is easy to fall into this trap. In addition, many small round cell tumors share CD99 positivity as well as positivity to other antigens. Therefore a broad spectrum of antibodies should be used, as well as, if possible, molecular diagnostics, in order to arrive at the right diagnosis.

Desmoplastic small round cell tumor (DSRCT) is a member of the extended EWS family of tumors in which the fusion partner of EWS gene is WT1 [31]. In the WHO classification of tumors of soft tissue and bone it is classified within the group of sarcomas with uncertain differentiation [32].

DSRCT is an uncommon tumor in children and incidence data are not available. DSRCT has a peak incidence in the third decade. In the series reported by Dehner, which included children up to 18 years, the majority of cases occurred in the second decade [30]. The most common location is in the abdominal cavity, where there is a widespread serosal growth (retroperitoneum, mesentery, omentum, pelvis). Less common locations are in the thoracic cavity, paratesticular tissue, liver, and ovary.

When all ages are considered, there is a male predominance. Among the 19 childhood DSRCT reported by Dehner there was no sex predominance [30].

Symptoms accompanying abdominal tumors are abdominal distention, pain, palpable mass, and acute abdomen.

Imaging techniques show tumor location and extent of disease.

Smears are moderately or highly cellular with many cell groups, tissue fragments, dissociated cells, and naked nuclei. The number of dissociated cells varies, while cell groups are usually abundant (Figs. 3.21 and 3.22).