| Abstract|| |
Clear cell sarcoma of soft parts is a rare tumor in children and it requires a high index of suspicion for accurate diagnosis. Early diagnosis leads to radical surgical excision and limits the aggressive behavior of this tumor. We report a case of a 12-year-old boy with a recurrent soft-tissue tumor in the scalp, misdiagnosed on three occasions as epitheloid sarcoma owing to the poorly differentiated appearance of cells. In spite of focal S-100 expression, this tumor was not recognized as a tumor of melanocytic origin till melanosomes were demonstrated on electron microscopy (EM). Detection of melanosomes on electron microscopy helped in clinching the histology diagnosis, reiterating the definite role of EM in diagnosing these tumors. Failure to accurately diagnose this tumor resulted in institution of preoperative chemotherapy, delayed surgical excision, tumor progression and death of patient within a year and half of presentation.
Keywords: Clear cell sarcoma of soft tissue, electron microscopy, pediatric
|How to cite this article:|
Shirazi N, Kadam V, Deodhar K, Shet T. Ultrastructure in resolving a diagnosis of poorly differentiated clear cell sarcoma of soft parts in an adolescent male. Indian J Pathol Microbiol 2008;51:280-3
|How to cite this URL:|
Shirazi N, Kadam V, Deodhar K, Shet T. Ultrastructure in resolving a diagnosis of poorly differentiated clear cell sarcoma of soft parts in an adolescent male. Indian J Pathol Microbiol [serial online] 2008 [cited 2019 Oct 21];51:280-3. Available from: http://www.ijpmonline.org/text.asp?2008/51/2/280/41696
| Introduction|| |
Clear cell sarcoma of soft parts (CCSSP) is an extremely rare tumor in children and adolescents, affecting 0.8% to 2.2% of the pediatric population. , This is an aggressive tumor which requires early surgical intervention to prevent mortality associated with the tumor.
This tumor affects young adults between the ages of 20 years and 40 years, but it can occur at any age.  It usually occurs in the soft tissues of extremities and only 0.8% occur in the head and neck region.  A typical CCSSP on histology shows the classical clear cell pattern with prominent nucleoli, but eosinophilic variants that mimic other epithelioid sarcomas of soft tissues are known. ,
In an odd clinical setting or at an odd site or in poorly differentiated tumors, CCSSP is frequently misdiagnosed, resulting in delayed or incomplete surgery with frustrating recurrences.
We discuss a CCSSP that occurred in a boy, which was missed histologically on several occasions. Detection of melanosomes on electron microscopy (EM), however, helped in clinching the diagnosis, reiterating the definite role of EM in diagnosing these tumors.
| Case History|| |
A 12-year-old boy presented in early 2004 with a scalp swelling involving the right temporal soft tissues since 7 to 8 months. He was operated at an outside hospital and labeled as a case of high-grade sarcoma. No further treatment was given and the patient had a recurrence in 2 months' time. Following this, the patient was referred to our institute for further management. As the histology review of the slide submitted was an epithelioid sarcoma, the recurrent tumor was widely excised along with reconstruction of the defect. The histopathological diagnosis from this recurrence was a poorly differentiated sarcoma involving the skull bones. Within 4 months of surgery, the patient developed yet another recurrence. This time, the formalin-fixed material was submitted for electron microscopy. Reconsidering the aggressive clinical behavior, a decision to treat the patient with a rhabdomyosarcoma type of chemotherapy was taken. The chemotherapy regimen used was the RCT II protocol, which includes a sandwich treatment with VIE/VAC/VIE (V = Vincristine; A = Adriamycin; C = Cyclophosphamide; I = Ifosfamide; E = Etoposide).
After two cycles of the above chemotherapy, the patient developed severe headache and vomiting. A CT scan of the brain revealed a third recurrence with intracranial extension. The patient received palliative external radiotherapy to the brain (30 Gy with 9 Gy boost), followed by chemotherapy. While on chemotherapy, the patient developed pleural effusion, extensive lung metastases and died within a year and half of presentation.
The excision specimen from the first recurrence showed a large fleshy tumor measuring 19 cm × 11 cm × 3 cm and involving the underlying temporal bone. The specimen was fixed in neutral buffered formalin and routinely processed.
On microscopy, the tumor showed a prominent nodular appearance with large areas of necrosis [Figure 1]. The nodules were composed of tumor cells with a fair amount of cytoplasm. The cytoplasm of most cells was eosinophilic [Figure 2], but few areas showed tumor cells with a clear cytoplasm [Figure 3]. The nuclei in few cells showed prominent nucleoli. The tumor showed brisk with few atypical mitosis.
Immunohistochemistry was performed on the routinely processed paraffin section by the Avidin Biotin Complex method using the following panel of antibodies from DAKO Corporation: Cytokeratin (monoclonal MNF116; 1:100), epithelial membrane antigen (monoclonal E29; 1:50), Desmin (monoclonal D33; 1:400), Myoglobin (polyclonal; 1:500), Mic2 (monoclonal 12E7; 1:50), Vimentin (monoclonal V9; 1:50), neuron-specific enolase (monoclonal BBS/NC/VI- H14; 1:50), CD117 (polyclonal; 1:100), Chromogranin (monoclonal; 1:100), Synaptophysin (monoclonal; 1:50), HMB45 (monoclonal; 1:50) and CD117/c kit (polyclonal; 1:100).
On immunohistochemistry, the tumor cells were strongly positive for vimentin and showed focal expression of S-100 protein [Figure 4]. All other markers like HMB45, c kit, CK, EMA; and all other markers enumerated above were negative.
Electron microscopy was done on the buffered formalin-fixed tissue, refixed in the universal fixative (combination of formaldehyde and glutaraldehyde). Tumor was then secondarily fixed in 1% osmium tetra oxide for 2 hours, dehydrated through serial dilutions of ethanol, transferred to propylene oxide and embedded in epoxy resin. The ultra-thin sections were stained with uranyl acetate and lead acetate and examined under a Zeiss electron 109 microscope.
Ultrastructure revealed tumor cells with abundant clear cytoplasm, which was poorly preserved due to the prior formalin fixation. Tumor cells showed unevenly distributed chromatin with peripheral condensation and nucleoli in few cells. The cytoplasm was abundant and contained membrane-bound vesicles and few swollen mitochondria. Glycogen could not be appreciated due to suboptimal cytoplasmic preservation. Some of the tumor cells, however, showed type III or pigmented melanosomes [Figure 5]. Based on this ultrastructure finding, all previous histology was reviewed and confirmed as a poorly differentiated variant of clear cell sarcoma.
| Discussion|| |
The histogenetic origin of clear cell sarcoma of soft parts (CCSSP) is still debated. Clear cell sarcoma of soft parts (CCSSP) was called "malignant melanoma of soft parts" in earlier days because it expressed melanocytic markers like S-100 and HMB45 and showed premelanosomes and melanosomes on ultrastructural examination. , CCSSP is however more deeply located, intimately associated with tendons or aponeurosis; and it clinically behaves more like a high-grade sarcoma rather than a melanoma. In his textbook, Enzinger lists CCSSP under the category of "nerve sheath sarcomas."  On ultrastructural examination, some authors have demonstrated schwannian differentiation in CCSSP.  The detection of a specific translocation t (12; 22) (q13; q12.2-12.3) or the EWS-ATF1 gene fusion in CCSSP has established this tumor as a definite sarcoma distinct from the usual malignant melanoma or nerve sheath tumors. 
An accurate diagnosis of CCSSP based on conventional morphology alone is difficult. While a typical CCSSP shows a classical pattern of variably sized nests of plump or spindle cells with clear cytoplasm separated by fibrous stroma. Histological variants with a substantial proportion of epitheloid cells, moderate-to-marked nuclear pleomorphism, predominantly diffuse growth, or a microcystic pattern are recorded.  Prominent nucleolus is an important diagnostic feature of CCSSP, but nuclei of some tumor cells showing finely granular chromatin with vesicular nuclei and nucleoli may be infrequent.  Though melanosomes are seen in all tumors on ultrastructural examination, few tumors even on histology show melanin pigment.  Melanin pigment is demonstrated in 50% to 75% of cases with use of special stains like Masson's Fontana or a Warthin Starry stain. 
The case being discussed herein stresses on the diagnostic difficulties in clear cell sarcoma in young patients, especially in tumors with a poorly differentiated morphology. In the context of above variations in morphology, a high index of suspicion and ancillary methods are required for an accurate diagnosis. Immunohistochemistry (IHC) is useful in documenting the melanocytic differentiation and 95% of cases of CCSSP show S-100 positivity. However, a lower percentage (75-90%) of cases of CCSSP are HMB45 positive and interestingly 30% also express keratin. , This profile may overlap with other epithelioid soft-tissue sarcomas, leading to diagnostic problems as in our case.
The two ancillary methods that help in recognition of a CCSSP are electron microscopy (EM) and molecular methods demonstrating the t (12, 22) translocation. Melanosomes in various developmental stages are found in CCSSP.  On ultrastructural analysis, tumor cells also show continuous basal lamina, rudimentary cell junctions, abundant cytoplasm containing numerous mitochondria and aggregates of glycogen.
While EM aids in the recognition of CCSSP in cases that have overlapping immunohistochemistry findings or in those that are HMB45 negative, it does not help in differentiating a CCSSP from a melanoma. The t (12, 22) translocation seen in CCSSP is more diagnostic and is not present in a melanoma. One study compared both EM and molecular studies in CCSSP and found that ultrastructural analysis showed melanosomes in six (85%) of the seven cases, while EWS-ATF1 fusion transcripts were identified by real time polymerase chain reaction (RT PCR) in 91% of the cases.  RT-PCR analysis for the melanocyte-specific splice form of the MITF transcript was positive in all cases tested. These data confirm that EWS-ATF1 detection can be used as a highly sensitive diagnostic test for CCSSP and detection of the melanocyte-specific form of MITF transcript confirms the genuine melanocytic differentiation of this tumor. 
The molecular studies for CCSSP till recently required fresh tissue and this was the reason they could not be used in most cases such as ours. Non-availability of these tests as routine laboratories tests is also a problem. In such instances, EM examination of the formalin-fixed tumor tissue definitely helps in the diagnosis as demonstrated in this case report. In our case, EM, along with the clinical picture of a recurrent soft-tissue mass and morphology helped us to arrive at the correct diagnosis inspite of the absence of molecular confirmation of the translocation. Molecular advances have outstripped EM of all its past glory; but when fresh tissue is not available, EM continues to help to achieve correct diagnosis.
CCSSP is a rare tumor in pediatric patients and only 5 cases were recorded at the St. Jude's Children Hospital out of a total of 225 soft-tissue sarcomas (over 35 years) in children.  The median age at diagnosis was 15 years 3 months. Primary sites included the extremities ( n = 3), chest wall ( n = 1) and abdomen ( n = 1). Another larger study reported a 0.8% incidence, but no case was reported in the head and neck region. 
Obtaining a precise diagnosis of CCSSP is important, especially in children, because the treatment of non-rhabdomyosarcoma type of soft tissue sarcomas is primarily surgical and includes a wide spectrum of tumors with varying chemosensitivity.  Radical surgery is sometimes postponed in CCSSP until the tumor recurs and this impacts the survival negatively.  To this, we could add delays due to pathologic misdiagnosis. It is known that patients who develop local recurrence from a CCSSP frequently die of their disease. Early diagnosis and initial radical surgery are essential for a favorable outcome; and once regional lymph node metastasis or hematogenous dissemination occurs, the prognosis is dismal.  Tumor size, necrosis and nodal metastasis are documented as important prognostic factors.  Though CCSSP has poor prognosis, the 5-year survival rate in children is better than in adults and is 69% vs. 50% in adults. 
Recurrent or metastatic disease in CCSSP is predominantly managed by chemotherapy.  Radiotherapy has limited role and is advised only for residual disease. Amongst chemotherapy, both non-rhabdomyosarcoma and rhabdomyosarcoma protocols have been tried but they are not very effective.  The VAC type of chemotherapy administered to the patient we are discussing is a type of rhabdomyosarcoma protocol and was ineffective.
There is a report of dramatic complete pathologic response in a 40-year-old lady with interferon-alpha 2b therapy.  However, this needs to be confirmed in a larger series of patients.
To conclude, CCSSP is a distinct soft-tissue sarcoma occurring rarely in children and it often requires a high index of suspicion for odd histological features and ancillary methods for an accurate diagnosis. Accurate pathologic recognition could aid in the institution of prompt radical surgery and could delay or avoid recurrences.
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Source of Support: None, Conflict of Interest: None
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