Dual-color, break-apart fluorescence in situ hybridization for EWS gene rearrangement distinguishes clear cell sarcoma of soft tissue from malignant melanoma

Abstract

Clear cell sarcoma of soft tissue (malignant melanoma of soft parts) is a soft tissue sarcoma with melanocytic differentiation that typically occurs in the tendons and aponeuroses of young adults. As demonstrated by cytogenetics and reverse-transcriptase polymerase chain reaction, between 70% and over 90% of clear cell sarcomas have a t(12;22) translocation, fusing the EWS and ATF1 genes on chromosomes 22q12 and 12q13, respectively. Identification of this translocation distinguishes clear cell sarcoma from histologic mimics, most importantly conventional malignant melanoma. We report our experience with a commercially available, dualcolor, break-apart fluorescence in situ hybridization (FISH) probe, which allows detection of EWS (22q12) gene rearrangement in formalin-fixed, paraffin-embedded tissues. Histologically and immunophenotypically wellcharacterized cases of clear cell sarcoma (n ¼ 10) and malignant melanoma (n ¼ 32) were evaluated with a 22q12 dual-color, break-apart probe (Vysis, Downer's Grove, IL, USA), which spans the known common breakpoints in the EWS gene on chromosome 22 (introns 7-10). Signals from tumor cell nuclei were counted under a fluorescence microscope and the presence of red-green break-apart signals was recorded. Of the clear cell sarcoma cases, seven of 10 showed evidence of an EWS gene rearrangement with a mean of 81.6% positive cells per sample (range: 60-95%). All cases of malignant melanoma (n ¼ 32) showed virtually absent break-apart signals in the EWS gene (less than 4% cells per case). FISH detects EWS gene rearrangement in a substantial proportion of clear cell sarcomas, with excellent specificity. Importantly, EWS FISH is negative in malignant melanoma, a clinically dissimilar tumor, which may closely mimic clear cell sarcoma histologically and immunohistochemically. As the studied probe can be utilized in routinely processed tissue, FISH provides an excellent alternative to reverse-transcriptase polymerase chain reaction in cases where fresh tissue is unavailable. Modern Pathology ( Clear cell sarcoma of soft tissue (malignant melanoma of soft parts) is a rare soft tissue sarcoma with melanocytic differentiation first described by Enzinger 1 in 1965. The tumor originates predominantly in the tendons, aponeuroses, and fascial structures of the extremities of young adults. [1][2][3][4][5] Clear cell sarcoma shares morphologic, immunohistochemical, ultrastructural, and molecular features with malignant melanoma. 2,[6][7][8][9][10] In contrast to malignant melanoma, a unique and recurrent t(12;22)(q13;q12) involving the EWS gene has been identified in 70% to over 90% of clear cell sarcomas by cytogenetics, reverse-transcriptase polymerase chain reaction (RT-PCR) looking for the resultant EWS-ATF1 fusion protein, and by fluorescence in situ hybridization (FISH). [11][12][13][14][15][16][17][18][19] Clear cell sarcoma is rare and the true incidence of this neoplasm is not well established; however, the increased utilization of molecular studies for the

Fluorescence in situ hybridization for MDM2 gene amplification as a diagnostic tool in lipomatous neoplasms

“…13 Blinded to the histologic diagnosis and using an Olympus IX-50 microscope (Olympus, Tokyo, Japan), cases were scored by counting a minimum of 40 nuclei per case under oil immersion at  100 magnification with a DAPI/green/red triple band pass filter. Only nuclei with at least two CEP12 signals were evaluated to minimize nuclear truncation artifact that occurs in paraffin-embedded 4-mm sections.…”

Histone deacetylase inhibitors induce growth arrest, apoptosis, and differentiation in clear cell sarcoma models

“…S1). 1 To investigate the gene transcriptional expression associated with possible induction of cartilaginous, skeletal muscle, adipose, and melanocytic differentiation, we performed quantitative real-time PCR analysis of the mRNA levels of SOX9, MYOD1, PPARG, and MITF. CDKN1A, known to be induced by histone deacetylase inhibitor treatment in other systems, was used as a positive control.…”

“…1). This translocation fuses the 5 ¶ portion of the Ewing sarcoma (EWSR1) oncogene on chromosome 22q with the 3 ¶ portion of the activating transcription factor 1 (ATF1) oncogene on chromosome 12q, resulting in the EWS-ATF1 chimeric fusion oncoprotein.…”

Detection and characterization of EWSR1/ATF1 and EWSR1/CREB1 chimeric transcripts in clear cell sarcoma (melanoma of soft parts)

“…Clear cell sarcomas, unlike melanoma, are genetically characterized by a recurrent translocation involving the EWSR1 gene located on chromosome 22q12. [11][12][13][14][15][16][17][18][19][20][21][22] The most common translocation partner is the activating transcription factor-1 (ATF1) gene on chromosome 12q13, of which four chimeric types have been described. [11][12][13]15 A second less common translocation partner is CREB1 located on chromosome 2q34, which results in a rearrangement t (2;22) that has been preferentially reported in clear cell sarcomas of the gastrointestinal tract and not in clear cell sarcomas of the soft tissue, until recently.…”