Serous fluid: Metastatic sarcomas, melanoma, and other non-epithelial neoplasms

Rhabdomyosarcoma

Rhabdomyosarcoma is the most common tumor of adolescents and young adults and is rare in patients older than 40 years of age. There are four major histological subtypes: embryonal (most common), alveolar, sclerosing/ spindle cell, and pleomorphic. The most common sites of metastasis are the lungs, heart, and lymph nodes. Involvement of serous membranes as well as intraperitoneal spread can occur. Rhabdomyosarcomas can also occur in the setting of a sarcomatous transformation in patients with germ cell tumors or malignant mixed Müllerian tumors (MMMT) of the ovary or endometrium.²¹ The presence of a moderate or massive effusion is an unfavorable prognostic factor in children with rhabdomyosarcoma.²² Cytologic material from pleural, peritoneal, and pericardial fluids may be diagnostic of metastasis [Figure 4], but usually require the help of ancillary studies.^23-25 By immunohistochemistry they show nuclear immunoreactivity for myogenin and MyoD1 with cytoplasmic immunoreactivity for desmin, as well as myoglobin in more differentiated cells. These stains may only be focal in myogenic tumor cells.

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Figure 4:

Pleural fluid samples from different patients with metastatic rhabdomyosarcoma. Tumor cells are typically characterized as small round cells with minimal cytoplasm (a). Tumor cells can sometimes have more moderate cytoplasm and nuclear pleomorphism (b). Tumor cells can form clusters (c). Multinucleated giant cells as shown in the center of the cell-block may be seen with alveolar rhabdomyosarcoma (d). [a. Smear, Pap stain, 60x; b. Smear, DQ stain, 40x; c. Smear, DQ stain, 40x; d. cell-block, H&E stain, 40x]

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Embryonal rhabdomyosarcoma accounts for nearly 80% of all rhabdomyosarcomas. The most common site of occurrence is the head and neck, followed by genitourinary (e.g. sarcoma botryoides) and paratesticular locations, as well as the extremities. Metastases to the body fluids have been reported.²⁶ This sarcoma shows aggregates of small cells, which are about 2–5 times the size of mature lymphocytes. Malignant samples may also contain scattered large isolated cells with conspicuous perinuclear clearing. Their nuclei are typically enlarged, hyperchromatic, and pleomorphic. The cytoplasm of tumor cells is somewhat bubbly with indistinct borders. In Diff-Quik (DQ)-stained smears, the cells appear poorly differentiated and blast-like. Occasionally, well-differentiated rhabdomyoblasts may be seen as large, round, bizarre or strap cells with cross striations. These cross striations are specific for rhabdomyosarcoma, but are present only in 30% of cases.

Alveolar rhabdomyosarcoma (ARMS) is the second most common subtype seen in childhood. These tumors have either PAX3-FOXO1 or a PAX7-FOXO1 gene fusion. They commonly involve the deep soft tissue of the extremities, head and neck, paraspinal region, and perineum. On Papanicolaou (Pap) stain, cytology samples may be cellular with a mixture of reactive mesothelial cells and singly scattered, rare, bizarre, dyshesive, pleomorphic, giant tumor cells. Rhabdomyoblasts and multinucleated tumor giant cells with peripheral wreath-like nuclei, when present, are helpful diagnostic clues. Mitotic figures are easily identified. The cytoplasm of these tumor cells is eosinophilic, moderate in amount, and sometimes vacuolated. Due to their cytomorphologic variability in effusions ARMS can mimic reactive mesothelial cells and adenocarcinoma cells.²⁷

Desmoplastic small round cell tumor (DSCRT)

DSRCT typically involves the abdominal cavity, typically in young males, and has an aggressive course. Patients may manifest with numerous serosal implants. DSRCT of the pleura may rarely occur and present with a pleural effusion.²⁸ This tumor is characterized by nests of proliferating small blue cells in a dense desmoplastic stroma. Effusion samples either show singly scattered tumor cells [Figure 5] or cohesive cell clusters [Figure 6] with similar cytologic features to other SRBCTs.^29-30 The tumor cells tend to be uniform, have round to oval or even reniform nuclei, fine chromatin and small nucleoli. Some tumors can have clear cytoplasm and larger cells with notable anisonucleosis. There may be nuclear molding, mitoses, and apoptotic cells. Immunohistochemically, these tumors demonstrate immunoreactivity with epithelial (cytokeratin, EMA), neural (NSE, GFAP), and muscle immunomarkers (paranuclear and dot-like desmin immunostaining). WT-1 (polyclonal C-terminus antibody) is also immunoreactive in around 90% of these tumors. DSRCT has been shown to have a novel translocation t(11;22) (p13;q12), which is a chimeric fusion product of the EWSR1-WT1 gene.

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Figure 5:

Desmoplastic small round cell tumor (DSRCT) metastatic to pleural fluid. Hypercellular sample with numerous isolated tumor cells are shown with high N:C ratios, round nuclei and finely granular chromatin. [a. Cytospin, Pap stain, 40x]

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Figure 6:

Desmoplastic small round cell tumor (DSRCT) involving peritoneal fluid. Tumor cells are shown forming a cohesive cluster (a). DSRCT tumor cells showing nuclear immunoreactivity for WT1 (arrows in b). [(a. Pap stain, 100x; b. WT-1 immunostain, 100x] (From Cytojournal 2005, 2:6. Courtesy of Granja NM et al.⁴²)

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Ewing sarcoma

The Ewing sarcoma family are sarcomas of neuroectodermal differentiation. They include Ewing sarcoma (most undifferentiated), peripheral primitive neuroectodermal tumor (PNET) and Askin tumor of the chest wall. They are the second most common sarcoma of bone (after osteosarcoma) and soft tissue (after rhabdomyosarcoma) in children and young adults. Visceral involvement can also occur. Primary extraskeletal Ewing sarcoma of the pleura is rare.³¹ Approximately 25% of afflicted patients may present with metastases at the time of diagnosis and spread is preferentially to the lungs, bone marrow, and lymph nodes.

Metastases involving pleural effusions are infrequent and found in less than 10% of cases.³² A malignant effusion may be the primary presentation in some patients.³³ Effusions involved by Ewing sarcoma may show loose groups of tumor cells or many solitary cells. These cells have a high nuclear-cytoplasmic ratio characterized by oval nuclei with scant, ill-defined cytoplasm [Figure 7]. Their chromatin is finely granular, and nucleoli are small and indistinct. Apoptosis and necrosis may be evident.

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Figure 7:

Ewing sarcoma metastatic to pleural fluid. This loose cluster is comprised of small round tumor cells with minimal cytoplasm, round to oval nuclei, finely granular chromatin and distinct nucleoli. [a. Smear, Pap stain, 60x]

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The tumor cells contain intracytoplasmic glycogen instead of mucin, which can be demonstrated with a positive periodic acid-Schiff (PAS) stain. The typical tigroid background due to smeared glycogen seen on stained slides is missing in effusions. These tumors can be differentiated from other SRBCTs using ancillary studies. There is often strong and diffuse membranous immunoexpression of CD99 (antibody to MIC2) and diffuse NKX2.2 staining. However, CD99 is also immunoexpressed in most of the other SRBCTs except for neuroblastoma and so may be relatively non-specific. Almost 25% of cases may express keratin and rarely there may be desmin immunoreactivity. Most tumors demonstrate t(11;22) EWSR1-FLI1 (85%) or t(21;22) EWSR1-ERG (10%) gene fusions. Detecting transcripts may be necessary to clinch the diagnosis.

Neuroblastoma

Neuroblastoma is the most common extracranial solid tumor encountered in children. Neuroblastoma and related tumors are derived from primordial neural crest cells that migrate from the mantle layer of the spinal cord and populate the primordia of the sympathetic ganglia and adrenal medulla. The age of presentation ranges from 2 months to 5 years, with a peak incidence at 18 months. The distribution of neuroblastomas follows the sympathetic chain and can arise anywhere from the base of skull to pelvis, including the adrenal medulla and dorsal root ganglia. About 90% of neuroblastomas are associated with elevated levels of catecholamines. Measurement of these metabolites in the urine is useful in monitoring the course of disease. The incidence of pulmonary involvement at the time of diagnosis is low, averaging about 4% in children over 1 year old with stage IV neuroblastoma.

In a report by Cowie et al, the incidence of pleural disease or pleural effusion in a group of 1,245 patients with neuroblastoma was 0.7%.³⁴ Pleural effusions usually signify a neuroblastoma with unfavorable biologic features and high-risk disease.³⁵ Cytologically, neuroblastomas are composed of small blue tumor cells [Figure 8]. These cells may form Homer-Wright rosettes, that can rarely be observed in effusions. The cytoplasm of neuroblastoma cells is scant and their nuclei have salt and pepper-type chromatin. Ganglion cells seen with maturation are unlikely to be encountered among metastatic tumor cells to body fluids. Neuroblastoma cells are variably immunoreactive for synaptophysin and chromogranin, and may also immunostain with GFAP, GATA3, tyrosine hydroxylase, and PHOX2B. A helpful immunopanel to distinguish neuroblastoma from other small blue cell tumors includes neuron specific enolase (NSE) and protein gene product 9.5 (PGP 9.5). However, these immunostains are relatively non-specific. Flow cytometry has proven to be helpful, for the detection of CD56+ neuroblastoma cells.³⁶ Several cytogenetic abnormalities have been reported, some of which are associated with a poor prognosis (e.g. ATRX, MYCN, and ALK amplifications).

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Figure 8:

Neuroblastoma metastatic to peritoneal fluid. Tumor cells shown forming a cohesive cluster (a). Hypercellular bloody fluid sample with many small round blue tumor cells (b). [a. Smear, DQ stain, 60x; b. Cell-block, H&E stain, 40x]

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Wilms tumor

Wilms tumor (WT) or nephroblastoma, a pediatric renal malignancy, is the most common solid tumor in children. Relapse usually occurs within 4 years of initial presentation, and the most common site of metastasis is the lung. Pleural effusion at the time of presentation is rare and is not associated with an adverse prognosis.³⁷ Occasionally, a Wilms tumor can rupture spilling tumor cells into the abdominal cavity. Histologically, WT is heterogeneous with epithelial, stromal, and blastemal (undifferentiated) components. When there is a predominance of one of these components, this will make the diagnosis challenging, especially in a metastatic setting. The cytology of WT in effusions has been rarely reported in the literature.³⁸ Tumor cells present mostly as cellular aggregates [Figure 9]. Blastema consists of small blue tumor cells. More complex cystic and tubular structures may be noted when epithelial components are present. Immunohistochemistry is helpful to distinguish these tumors from other SRBCTs. Strong immunoreactivity for WT-1 is typically demonstrated in epithelial and blastemal components, but not stromal components. Well-differentiated blastemal components may exhibit cytokeratin immunoreactivity. Molecular aberration of chromosome 11p, where the WT-1 gene is located, may be useful in establishing the diagnosis.

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Figure 9:

Wilms tumor metastatic to peritoneal fluid. Tumor cells are shown with variable N:C ratios, round nuclei and uniform chromatin. [Smear, DQ stain, 60x]

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