Utility of fluorescence in situ hybridization in sub- classifying unclassified high-grade sarcomas: A study of 40 cases using break-apart probes of EWSR1, FOXO1A, SS18 and DDIT3 genes

Abstract

Objectives: In an era of rapid advances in oncologic treatment, there is continuous emphasis for a definitive classification of undifferentiated sarcomas in order to select the most appropriate therapeutic regimens against these malignancies. EWSR1, FOXO1A, SS18 and DDIT3 gene break-aparts associated with chromosomal translocations are widely used as specific molecular markers in diagnosing the Ewing family of tumors, alveolar rhabdomyosarcoma, synovial sarcoma and myxoid liposarcoma, respectively. However, the utility of these markers for definitive categorization of previously unclassified high-grade sarcomas has not been studied.
Methods: We identified 40 sarcomas from 2003 to 2009, which on light microscopy exhibited a spindle cell or epithelioid morphology with marked nuclear atypia, high mitotic rate and necrosis. These sarcomas were high grade and poorly differentiated, and remained unclassified despite the use of immunohistochemical panels comprising neural, smooth muscle, skeletal muscle, melanocytic, epithelial, vascular and fibrohistiocytic markers.
Results: Thirty five cases were resection specimens, and the rest were needle biopsies. Fluorescence in situ hybridization (FISH) was applied to formalin fixed paraffin embedded tissue (FFEP) with break-apart probes for EWSR1, FOXO1A, SS18 and DDIT3 gene. In positive cases, RT-PCR was done to detect specific gene fusions associated with translocations. One of 40 cases was positive for DDIT3 gene break-apart, consistent with a dedifferentiated myxoid liposarcoma.
Conclusions: Subsequent reverse transcriptase (RT)-PCR demonstrated a FUS-DDIT3 fusion, supporting the FISH result. Another case was positive for the FOXO1A gene break-apart, consistent with alveolar rhabdomyosarcoma, even though RT-PCR failed to reveal specific gene fusion. Although with low frequency, FISH using a variety of probes can provide precise classification in a few otherwise unclassifiable high grade sarcomas. The findings also affirm the utility of FISH technology in FFPE tissues, including small biopsies.