Molecular diagnosis of clear cell sarcoma: detection of EWS-ATF1 and MITF-M transcripts and histopathological and ultrastructural analysis of 12 cases

Abstract

Clear cell sarcoma (CCS), also known as melanoma of soft parts, is an uncommon deep soft tissue tumor presenting typically in the lower extremities of young adults. Previous cytogenetic studies have established the specificity of the recurrent t(12;22)(q13;q12), resulting in a EWS-ATF1 fusion, for CCS. The prevalence of the EWS-ATF1 fusion in CCS remains unclear, since most genetically confirmed CCS have been reported as isolated cytogenetic or molecular diagnostic case reports. We therefore studied histologically confirmed CCS from 12 patients for the presence of EWS-ATF1 by reverse-transcriptase polymerase chain reaction (RT-PCR), using RNA extracted from either frozen (four cases) or formalin-fixed paraffin-embedded (eight cases) material. All primary tumors were located in the deep soft tissues of the extremities. Histologically, 10 cases had a typical epithelioid nested appearance. Most or all cases showed immunostaining for HMB45 (12 of 12), S-100 protein (10 of 12), and MITF (12 of 12). Ultrastructural analysis showed melanosomes in six of seven cases. The presence of an EWS-ATF1 fusion transcript was identified by RT-PCR in 11 of 12 cases (91%), all of which showed the same fusion transcript structure, namely the previously described in-frame fusion of EWS exon 8 to ATF1 codon 65. RT-PCR analysis for the melanocyte-specific splice form of the MITF transcript was positive in all cases tested (4 of 4). These data confirm that EWS-ATF1 detection can be used as a highly sensitive diagnostic test for CCS and that CCS expresses the melanocyte-specific form of the MITF transcript, further supporting its genuine melanocytic differentiation.

Figure 1.

Microscopic appearance of nested epithelioid cells with clear cytoplasm and prominent nucleoli, separated by fibrous bands and showing scattered multinucleated giant cells (hematoxylin & eosin; ×10).

Figure 2.

Case CCS 6 showed a distinct microscopic appearance, with epithelioid cells, showing a higher degree of nuclear pleomorphism and anaplasia, arranged in an alveolar pattern of growth, due to a striking loss of cohesion (hematoxylin & eosin; ×20).

Figure 3.

Immunohistochemical study with D5 monoclonal antibody showing strong and diffuse nuclear labeling in the tumor cells and also in the multinucleated giant cells (×20).

Figure 4.

Ultrastructural appearance of case CCS 4 with few stage II melanosomes (arrows) and stage III pigmented melanosomes (arrowheads) (×29,400).

Figure 5.

Detection of EWS-ATF1 transcripts in CCS by RT-PCR using RNA extracted from frozen tissues. M1: Size marker (HaeIII digest of ΦX174); M2: 100-bp DNA ladder; ± , reverse transcriptase added/not added (the latter representing the “no RT” negative control).

Figure 6.

Detection of EWS-ATF1 transcripts in CCS by nested RT-PCR using RNA extracted from paraffin-embedded tissues. Top panel shows products of RT-PCR (first step); bottom panel shows products of nested PCR performed on products of first step. M: HaeIII digest of ΦX174. CCS + ctrl: RNA extracted from frozen tissue in CCS 1, used as positive control. CCS 11, a and b: different paraffin blocks used for RNA extraction. ±, reverse transcriptase added/not added (the latter representing the “no RT” negative control).

Figure 7.

Detection of melanocyte-specific (M) and consensus (C) MITF transcripts in CCS by RT-PCR. RNA from the SK-MEL-19 melanoma cell line provided a positive control. M1: Size marker (HaeIII digest of ΦX174); M2: 100-bp DNA ladder; ±, reverse transcriptase added/not added (the latter representing the “no RT” negative control).