Cytologic Features of Clear Cell Sarcoma (Malignant Melanoma) of Soft Parts A Study of Fine-Needle Aspirates and Exfoliative Specimens

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1 Anatomic Pathology / CYTOLOGIC FEATURES OF CLEAR CELL SARCOMA Cytologic Features of Clear Cell Sarcoma (Malignant Melanoma) of Soft Parts A Study of Fine-Needle Aspirates and Exfoliative Specimens Andrew J. Creager, MD, 1 Martha B. Pitman, MD, 2 and Kim R. Geisinger, MD 3 Key Words: Cytology; Cytopathology; Fine-needle aspiration; Malignant melanoma of soft parts; Clear cell sarcoma; Pleural fluid; Ascites fluid; Cytogenetic; Immunohistochemistry; Electron microscopy Abstract We describe the cytologic features of clear cell sarcoma of soft tissue (CCS) in 11 fine-needle aspiration biopsy (FNAB) specimens and 6 exfoliative specimens from 11 patients. In 3 patients, FNAB was the initial method of tumor evaluation. In 6 of 11 cases, immunostaining with S-100 or HMB-45 was evaluated. Electron microscopic evaluation was performed in 1 case. Both the FNAB and exfoliative specimens varied in overall cellularity, although reproducible cytologic features were identified. A significant diagnostic pitfall, namely the potential of CCS to form microacinar structures mimicking adenocarcinoma, is described with particular reference to CCS metastatic to regional lymph nodes. A rare case of the granular cell variant of CCS is illustrated as well. Owing to the rarity of CCS, the diagnosis on cytologic smears is extremely difficult and is aided substantially by pertinent clinical data. The diagnosis can be made conclusively by FNAB in conjunction with immunocytochemical confirmation of HMB-45 or S-100 protein expression, cytogenetic demonstration of the t(12;22) translocation, or electron microscopic studies demonstrating melanosomes. Clear cell sarcoma of soft tissue (CCS), also designated malignant melanoma of soft parts, was first described by Enzinger 1 in 1965 and called clear cell sarcoma of tendons and aponeuroses. Although a rare tumor, CCS is a wellaccepted clinicopathologic entity with characteristic histologic features and an associated specific genetic t(12;22) chromosomal translocation involving the Ewing sarcoma and activating transcription factor 1 genes. 2 Historically, there has been controversy pertaining to the histogenesis of CCS. The demonstration of melanin pigment and melanosomes in CCS by light microscopy and electron microscopy, respectively, supports an origin from the neural crest. 3-9 CCS most commonly arises in the distal extremities of young adults, with a peak incidence in the third decade, and often is intimately associated with deep tendons and aponeuroses. 1,4,10-12 Clinically, CCS follows a slow yet progressive course characterized by frequent local recurrences with eventual lymph node metastases and distant visceral metastases. The pattern of metastases to lymph nodes is unusual for sarcomas and further supports a neural crest origin of CCS. Histologically distinctive, the tumor is composed of nests or fascicles of polygonal to fusiform cells separated by fibrous septa with occasional intervening tumor giant cells. The cells have pale, eosinophilic, or clear cytoplasm; vesicular nuclei, and either 1 large nucleolus or multiple small distinct nucleoli. Occasional cases demonstrate a variant with eosinophilic and distinctively granular cytoplasm. The presence of cytoplasmic melanin, expression of melanocytic markers by immunohistochemical analysis, demonstration of melanosomes ultrastructurally, and the presence of t(12;22) further support the diagnosis of CCS. American Society for Clinical Pathology Am J Clin Pathol 2002;117:

2 Creager et al / CYTOLOGIC FEATURES OF CLEAR CELL SARCOMA Table 1 Clinical Data Patient No./ Sex/Age (y) Cytologic Specimen Primary Site Recurrence Site Pertinent Ancillary Studies 1/F/53 FNAB, left thigh and lung; Left knee, soft tissue Thigh, lung ND pleural fluid 2/F/48 FNAB, lung Left knee, soft tissue Lung ND 3/M/35 * FNAB, right wrist Right wrist, soft tissue Axillary lymph nodes, lung S-100, HMB-45 positive 4/F/58 FNAB, right axillary lymph node Right axilla, soft tissue Axillary lymph nodes S-100, NSE positive; HMB-45 negative 5/F/15 FNAB, left thigh; pleural fluid Left thigh Thigh, lung S-100, HMB-45, and NSE positive 6/F/56 * FNAB, right calf Right calf S-100 positive; HMB-45 negative 7/F/56 * FNAB, right calf and right knee Right calf Right knee HMB-45, S-100 positive 8/F/23 FNAB, left forearm; pelvic fluid Left forearm Axillary and supraclavicular ND lymph nodes; limb stump 9/F/54 FNAB, left foot Left foot Thigh, pelvic fluid ND 10/M/26 FNAB, right foot Right foot Inguinal lymph nodes and ND soft tissue 11/M/43 Pleural fluid Lung Lung ND FNAB, fine-needle aspiration biopsy; ND, not done; NSE, neuron specific enolase. * FNAB was the initial diagnostic modality. While several large series have described the histopathologic features of CCS, 1,4,10-12 there is only 1 large series (9 patients) describing the cytologic features of CCS. 13 A handful of smaller series, the largest of which describes 3 patients, also are present in the literature We describe the cytologic features of 11 patients with CCS, using both fine-needle aspiration biopsy (FNAB) and exfoliative specimens. To our knowledge, the present series represents the largest cytologic series of CCS; it includes primary, metastatic, and recurrent tumors. The differential diagnosis and cytologic features of this unusual soft tissue neoplasm are defined with emphasis on the contribution of ancillary studies and clinical data to the diagnosis. Materials and Methods The case files from Wake Forest University Baptist Medical Center, Winston-Salem, NC, and the Massachusetts General Hospital, Boston, were searched for cytology specimens in which the diagnosis of CCS or malignant melanoma of soft parts was made on FNAB, body fluid, or a subsequent histologic specimen. The cytologic features of 17 specimens obtained from 11 patients with CCS were reviewed. Specimen representation included 12 FNAB specimens (2 primary tumor, 7 local recurrences, and 3 metastases) and 5 exfoliative specimens (3 pleural fluids and 2 pelvic fluids). Histologic preparations of the primary tumor and/or metastases were available for review in all but 2 cases. Clinical information, including presenting symptoms, treatment modalities, and pertinent clinical follow-up data, were obtained from the patients charts. Direct smears of exfoliative specimens were immediately fixed in 95% ethanol and stained using the Papanicolaou technique. Remaining material from exfoliative specimens was rinsed into saline and centrifuged. Cytocentrifuged preparations or paraffin cell blocks were prepared. Palpable tumors were aspirated by a cytopathologist or a surgeon using 22- to 25-gauge needles attached to a 10- or 20-mL syringe using an aluminum syringe holder. Deep soft tissue metastases and visceral metastases were aspirated by a radiologist using ultrasound guidance. FNAB smears were fixed in 95% ethanol and stained using the Papanicolaou technique and/or air dried and stained using the rapid Romanowsky method. The remainder of the aspirate material was rinsed into saline and centrifuged. Cytocentrifuged preparations or paraffin cell-blocks were prepared. Immunohistochemical studies were performed on cell block material from patients 3 and 6 using the avidin-biotinperoxidase complex method described previously. 25 The primary antibodies included vimentin (BioGenex, San Ramon, CA), cytokeratin AE1/AE3 (DAKO, Carpinteria, CA), leukocyte common antigen (DAKO), epithelial membrane antigen (EMA; DAKO), O13 (CD99; Signet, Dedham, MA), muscle specific actin (Enzo, Farmingdale, NY), S-100 (DAKO), and HMB-45 (DAKO). The specificity of the immunoreactions was verified by staining known positive and negative control tissue sections. Results Clinical Data The clinical findings, cytologic features, histologic confirmation, and pertinent immunohistochemical results for the 11 patients are summarized in Table 1. The most 218 Am J Clin Pathol 2002;117: American Society for Clinical Pathology

3 Anatomic Pathology / ORIGINAL ARTICLE Image 1 Cell block from granular cell variant of clear cell sarcoma. There is abundant eosinophilic and distinctly granular cytoplasm. Nuclei are eccentrically placed, and nuclear/cytoplasmic ratios are low (H&E 400). Image 2 Clear cell sarcoma demonstrating both cohesive cellular groups and abundant epithelioid single cells with retained cytoplasm. This pattern, especially when obtained from a lymph node metastasis, mimics metastatic carcinoma (rapid Romanowsky, 100). common initial manifestation was a slowly enlarging, painless mass. Three patients initially were evaluated by FNAB. After FNAB, these patients underwent axillary lymph node dissection, excision followed by limb amputation, or excision alone. Of these 3 patients, none was given a diagnosis of CCS on FNAB and no ancillary studies besides immunocytochemical analysis were performed on these specimens (Table 1). The tumor in patient 3 originally was diagnosed by FNAB as spindle cell sarcoma, not otherwise specified, after immunohistochemical stains revealed positive staining for vimentin and negative immunoreactivity for cytokeratins and leukocyte common antigen. The diagnosis of CCS was made on a subsequent right axillary lymph node excision with immunohistochemical confirmation of S-100 and HMB-45 expression (Table 1). The original FNAB material obtained from patient 6 demonstrated cells with distinctly granular eosinophilic cytoplasm, small eccentrically placed nuclei, and low nuclear/cytoplasmic (N/C) ratios Image 1. The cells were strongly positive for S-100 protein and negative for cytokeratins, EMA, CD99 (O13), and muscle specific actin. The differential diagnosis included a peripheral nerve sheath tumor vs a granular cell tumor. A subsequent excision of this tumor was diagnosed as CCS with granular features. Patient 7 originally was evaluated by FNAB, and a diagnosis of small cell neoplasm, not otherwise specified, was made. No ancillary studies were performed on the FNAB specimen. A subsequent tumor excision was diagnosed as CCS after histologic examination and positive immunoreactivity for S-100 and HMB-45. The tumors in 8 patients originally were diagnosed by conventional excisional or incisional biopsies followed by wide excision of the biopsy site or limb amputation. Of these 8 patients, local recurrence or distant metastasis was confirmed cytologically by FNAB or exfoliative cytologic sampling. In 1 patient, the initial incisional and subsequent excisional biopsy specimens were diagnosed as malignant peripheral nerve sheath tumor. The diagnosis of CCS was not made until a subsequent axillary lymph node recurrence was aspirated and the previous surgical pathology cases were reevaluated. Lymph nodes were evaluated for metastases in 4 patients. Two patients underwent axillary lymph node dissection, and 2 patients underwent lymph node FNAB. Three of these patients had lymph node metastases. Five patients developed pulmonary metastases, and 4 of these metastases were evaluated by FNAB, exfoliative cytologic sampling, or both. Three patients are known to have died of disease (mean survival, 23 months). Survival data are unknown or not available for 7 patients, and at last followup, 1 patient was alive with metastatic disease. Pathologic Findings The sources of the specimens are given in Table 1. FNAB specimens varied from very cellular to paucicellular. Cellular samples consisted of cohesive groups of cells and abundant single tumor cells with retained cytoplasm Image 2. The American Society for Clinical Pathology Am J Clin Pathol 2002;117:

4 Creager et al / CYTOLOGIC FEATURES OF CLEAR CELL SARCOMA Image 3 Clear cell sarcoma demonstrating epithelioid and round to polygonal cells with eccentric nuclei, increased nuclear/cytoplasmic ratios, and a large prominent nucleolus or several less prominent nucleoli. Globular metachromatic cytoplasmic material is observed in some cells, whereas others contain homogeneous basophilic cytoplasm (rapid Romanowsky, 400). cohesive groups demonstrated cytomorphologic features ranging from epithelioid, round to polygonal cells with high N/C ratios suggesting the diagnosis of carcinoma to crowded fusiform and spindled cells suggesting the diagnosis of sarcoma Image 3. The cohesive groups of some cases demonstrated 3-dimensional clustering and pseudoacinar structures mimicking adenocarcinoma Image 4. Dispersed malignant single cells had a moderate amount of cytoplasm, which in the majority of cases was transparent and basophilic and frequently contained large, globular, metachromatic material on rapid Romanowsky stain (Image 3). A single case demonstrated eosinophilic granular cytoplasm (Image 1). Most cells had round to kidney bean shaped, eccentrically placed nuclei with moderate pleomorphism, finely granular chromatin, and 1 prominent centrally placed nucleolus or, less commonly, 2 or 3 less prominent nucleoli Image 5. Occasional multinucleated tumor giant cells and mirror-image binucleated cells were present in some cases. Although intranuclear cytoplasmic pseudoinclusions were identified in most cases, melanin pigment was not identified in any case. Mitotic figures were readily identifiable. Abundant necrosis was present in 2 cases and in 1 case was attributed to previous chemotherapy. Exfoliative specimens tended to have a similar cytomorphologic presentation, although overall the samples were less cellular and had fewer cohesive groups. The cells were readily identifiable as malignant based on the high N/C ratios, mitotic activity, and the prominent nucleoli. The peripheral placement of the nucleus and the intranuclear pseudoinclusions were both less frequent in exfoliative specimens. Exfoliative specimens demonstrated a higher degree of degenerative changes than the aspirates. Tumor cells were easily distinguished from mesothelial cells when the latter were present Image 6. Immunohistochemical analysis was performed on either the FNAB material or the permanent paraffinembedded tissue sections in most cases, although for some A B Image 4 A, Microacinar structures with malignant cytologic features mimicking adenocarcinoma (rapid Romanowsky, 400). B, Paraffin-embedded section demonstrating histologic correlate of the microacinar-like structures observed on direct smears (H&E, 400). 220 Am J Clin Pathol 2002;117: American Society for Clinical Pathology

5 Anatomic Pathology / ORIGINAL ARTICLE Image 5 Loosely cohesive groups of epithelioid cells with high nuclear/cytoplasmic ratios, irregular nuclear contours, and a single prominent nucleolus or several smaller nucleoli are observed on this fine-needle aspiration biopsy specimen. In the center of the frame, a microacinar-like structure is present (Papanicolaou, 400). Image 6 A clear cell sarcoma cell present in a pleural fluid specimen. A malignant binucleate cell with eccentrically placed nuclei and prominent nucleoli is observed. Note the benign mesothelial cells in the background (Papanicolaou, 600). cases the results were not available (Table 1). All tumors that were stained for the presence of S-100 or HMB-45 proteins demonstrated positive immunostaining for at least one of these markers. No case that was examined for the presence of keratin antigens demonstrated positive immunostaining. Ultrastructural examination of a tissue fragment from 1 patient demonstrated the presence of melanosomes in various stages of development. Histologic sections of CCS were available for review in 9 of 11 cases, and the histomorphologic characteristics of CCS were represented in all cases. Discussion CCS has long been accepted as a distinct clinicopathologic entity with well-established histologic morphologic features. 1,4,10-12 CCS accounts for less than 1% of all soft tissue sarcomas and predominantly affects young adults. Specific recognition of CCS in cytologic specimens is difficult, due in part to the rarity of this tumor. Definitive diagnosis in the absence of ancillary studies is rarely achieved. Numerous large series have documented the histologic and clinical features of CCS. 1,4,10-12 Only a single large and several smaller series have been published describing the cytologic features of CCS Table 2. Based on our experience with the present series, CCS has many faithfully reproducible cytologic and clinical features that make its diagnosis possible. Unfortunately, there are also numerous diagnostic pitfalls that can lead even an experienced cytologist astray from the correct diagnosis. In our review of the literature, a total of 23 FNAB specimens, 13,16-20,22,23 exfoliative specimens, and 1 intraoperative touch imprint specimen 21 have been described (Table 2). HMB- 45, S-100, and neuron specific enolase were used as ancillary studies in 21 FNAB specimens, 3 exfoliative specimens, and 1 intraoperative imprint specimen, respectively. Electron microscopy was performed in 7 previous cases ,20,21,23 In a clinicopathologic study, Deenik et al 12 reported the results of an additional 8 FNAB specimens in 1 primary lesion and 7 recurrences, although no cytologic features were reported. To our knowledge, cytogenetic confirmation of t(12;22) CCS has not been demonstrated from FNAB or exfoliative specimens. In the present study, cytogenetics was not performed on any FNAB case. While the cytologic features of the cases in this series were similar, overall cellularity varied among aspirate and exfoliative specimens. Both dispersed single cells with retained cytoplasm and cohesive clusters of cells were present in the smears. When the findings of our study were compared with those of previous studies, several features of CCS were common (Table 2). These findings include highly cellular smears containing cohesive groups and dispersed single cells. Dispersed round to polygonal cells with abundant cytoplasm, American Society for Clinical Pathology Am J Clin Pathol 2002;117:

6 Creager et al / CYTOLOGIC FEATURES OF CLEAR CELL SARCOMA Table 2 Summary of Cytologic Features of Clear Cell Sarcoma in Previously Reported Cases No. of Type (No.) Immunocytochemical Reference Patients of Specimens Stains Cytologic Features Caraway et al 13 9 FNAB (13) HMB-45 positive Dispersed, round to polygonal and occasionally fusiform cells with abundant cytoplasm; eccentric round nuclei with intranuclear cytoplasmic inclusions and 1 prominent nucleolus or 2-3 smaller nucleoli Nguyen et al 14 2 Exfoliative (2) S-100 positive Single cells or in variable sized clusters, marked cellular cohesiveness and molding; round and hyperchromatic nuclei with conspicuous nucleoli Keller et al 15 1 Exfoliative (1) S-100 negative; Single cells with abundant granular cytoplasm having round nuclei and HMB-45 positive prominent nucleoli Exfoliative (12) ND Findings similar to FNAB with the exception of decreased cellularity and absence of intranuclear cytoplasmic inclusions Schwartz and 2 FNAB (2) S-100 positive Discohesive polygonal to fusiform cells having clear cytoplasm, Zollars 16 eccentrically placed nuclei, and large nucleoli Maruyama et al 17 1 FNAB (1) S-100 and HMB-45 Cells with clear cytoplasm, hyperchromatic nuclei, and prominent positive nucleoli Garcia-Prats et al 18 1 FNAB (1) S-100 positive Large, noncohesive, spindle-shaped or polygonal cells with clear cytoplasm, an eccentric nucleus, and conspicuous nucleoli Almeida et al 19 3 FNAB (3) S-100 positive Highly cellular smears with dispersed relatively uniform cells, large eccentric polygonal or spindled nuclei with prominent nucleoli, and rare intranuclear cytoplasmic inclusions Husain and 1 FNAB (1) S-100 positive; Cellular smears with isolated pleomorphic cells with single or multiple Nguyen 20 HMB-45 negative hyperchromatic nuclei and prominent nucleoli; cytoplasmic melanin present Ishi et al 21 1 ITI (1) S-100 and NSE Clusters and individual polygonal to spindled cells with prominent positive nucleoli, rare multinucleate cells, and cytoplasmic melanin Hombal 22 1 FNAB (1) S-100 positive Cells with clear cytoplasm, hyperchromatic nuclei, and prominent nucleoli Kindblom 23 1 FNAB (1) ND Round to polygonal cells with central nuclei, large nucleoli FNAB, fine-needle aspiration biopsy; ITI, intraoperative touch imprint; ND, not done. eccentric nuclei, intranuclear cytoplasmic inclusions, and either 1 prominent nucleolus or 2 to 3 smaller nucleoli were frequently described. A potential cytologic pitfall to the diagnosis of CCS is its ability to mimic metastatic carcinoma by forming cellular aggregates. 13 Microacinar structures, identified in one of our cases, have not been described previously in CCS (Image 4). This specimen was aspirated from an axillary lymph node of a 56-year-old woman with a history of sarcoma in the ipsilateral arm. Despite this history, metastatic breast carcinoma could not be excluded on immediate interpretation of the smears because of the presence of microacinar structures, dispersed single cells with retained cytoplasm, and cohesive aggregates of cells. Final evaluation of both the cytologic material and previous histologic and immunohistochemical material made the diagnosis of CCS possible (Table 2). Exclusion of adenocarcinoma by absence of cytokeratin expression was confirmatory. The differential diagnosis in the clinical setting of a young adult with an extremity-based soft tissue mass and regional lymph node metastases includes synovial sarcoma, epithelioid sarcoma, and malignant melanoma. Synovial sarcoma, similar to CCS, arises in the deep soft tissues frequently adjacent to tendons and aponeuroses and can metastasize to regional lymph nodes. Synovial sarcoma demonstrates a very different cytologic spectrum depending on which subtype is present. The monophasic fibrous and biphasic subtypes yield moderate to highly cellular smears composed of a mostly individually dispersed, uniform population of ovoid to slightly spindled cells with ovoid hyperchromatic nuclei, high N/C ratios, and scant, tapering cytoplasm. 26,27 Small tumor cell aggregates may be present, and nucleoli are most commonly small and inconspicuous. While biphasic synovial sarcoma has a distinct epithelial component, it is well recognized that epithelioid cells are only rarely identifiable on cytologic smears and when present display abundant, minutely vacuolated cytoplasm Synovial sarcoma has a distinct immunohistochemical profile characterized by cytokeratin and EMA immunoreactivity and frequent CD99 (O13) expression. In addition, more than 90% of synovial sarcoma demonstrates a characteristic cytogenetic translocation, t(x;18)(p11;q11), creating the SYT-SSX fusion gene product. 29 Epithelioid sarcoma joins the differential diagnosis because of its predilection for young adults, its ability to metastasize to regional lymph nodes, and its cytomorphologic features. Epithelioid sarcoma is a rare tumor that usually occurs in young adults within the distal extremities, especially the hand. 30 FNAB specimens are moderately cellular and composed of discohesive and relatively uniform neoplastic cells exhibiting only mild nuclear pleomorphism. 27 Nuclei are round, eccentrically located, and 222 Am J Clin Pathol 2002;117: American Society for Clinical Pathology

7 Anatomic Pathology / ORIGINAL ARTICLE surrounded by slight to moderate amounts of dense cytoplasm. 27 Degenerating intracytoplasmic vacuoles may be present. Necrotic debris in the background is a common accompaniment to the neoplastic cells. Immunohistochemically, epithelioid sarcoma shows diffuse and strong positivity for cytokeratins and EMA. Often, CD34 is positive in epithelioid sarcoma, a finding that is extremely uncommon in carcinomas. 31 We are unaware of any consistent cytogenetic abnormality in epithelioid sarcoma cells. Distinguishing metastatic melanoma from CCS by aspiration cytology is difficult at best. The clinical data will probably be the best indicator in differentiating the two entities. CCS almost exclusively occurs in the extremities of young adults where it is most frequently deeply seated and associated with tendons and aponeuroses. Metastatic melanoma to the soft tissues is most commonly superficial and rarely involves tendons and aponeuroses. Immunocytochemically, the two tumors are similar. To our knowledge, t(12;22) has not been reported in malignant melanoma. The granular cell variant of CCS is exceedingly rare. 32 In the present study, only a single granular cell variant of CCS was observed. The differential diagnosis of this entity includes a malignant granular cell tumor or other peripheral nerve sheath tumors. 33,34 While both these tumors are known to express S-100 protein, to our knowledge, HMB-45 expression and the t(12;22) have not been described. Despite the tumor s name, clear cells are not always observed in CCS. Tumor cells can demonstrate a heterogeneous spectrum of cytoplasmic features when H&E-stained permanent sections are examined. These findings also are demonstrable on air-dried Romanowsky-stained cytologic preparations and consist of cells with clear, eosinophilic granular, homogeneous basophilic, pale, or lightly eosinophilic cytoplasm or any combination of the aforementioned cell types. Melanin pigment production visible at the light microscopic level was not present in any of our cases. We believe there are reproducible cytologic features of CCS, and we have detailed these. It must be pointed out, however, that in the present retrospective study when cytologic smears were used as the primary diagnostic modality, the diagnosis of CCS was not made in any instance. The clinical data and the support of immunohistochemical analysis, cytogenetics, and electron microscopy are invaluable adjuncts in the evaluation of these diagnostically difficult tumors. From the Department of Pathology, 1 Duke University Medical Center, Durham, NC; 2 Massachusetts General Hospital, Boston; 3 Wake Forest University Baptist Medical Center, Winston-Salem, NC. Address reprint requests to Dr Creager: Dept of Pathology, Duke University Medical Center, DUMC 3712, Durham, NC References 1. Enzinger FM. Clear cell sarcoma of tendons and aponeuroses: an analysis of 21 cases. 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