[80] An RT-PCR Assay for FFPE Tissue Identifies a New FUS-DDIT3 Gene Fusion in Myxoid Liposarcoma

MP Powers, W-L Wang, VS Hernandez, KU Patel, AJF Lazar, DH Lopez-Terrada. Baylor College of Medicine and Texas Childrens Hospital, Houston, TX; M.D. Anderson Cancer Center, Houston, TX

Background: Myxoid Liposarcoma (MLS) is an adipocytic tumor with a myxoid background and shares a differential diagnosis with myxoma, myxofibrosarcoma, lipoblastoma, and extraskeletal myxoid chondrosarcoma. Two translocations, t(12;16)(q13;p11) and t(12;22)(q13;q12), are pathognomonic for myxoid liposarcoma resulting in at least 13 described alternative gene fusions between the FUS (16p11) or EWSR1 (22q12) genes with the DDIT3 gene (12q13). RT-PCR from formalin-fixed paraffin-embedded (FFPE) tissue can assist in the diagnosis.
Design: 21 FFPE archival samples of myxoid liposarcoma were obtained. Reverse transcription (RT)-PCR was attempted using previously published primer pairs and conditions. Multiple bioinformatics tools (Primer3, Primer-BLAST, BLAST, and PerlPrimer) were used to analyze the previously published primers and to design new, optimized RT-PCR primers for each of the known chimeric transcripts.
Results: Only a fraction of the MLS samples were amplified with previously published primers covering the most common, but not all, described breakpoints. Seven of the unamplified cases had confirmed DDIT3 rearrangements by fluorescence in-situ hybridization (FISH). Beta-actin amplified readily suggesting the RNA was intact. A first iteration of new primer design amplified 14 of 21 cases, including 5 of the 7 FISH-confirmed cases. Of the 7 negative cases, 5 were from or prior to 2003, had very weak actin amplification and/or had very low levels of RNA extracted. Bioinformatic analysis suggested multiple parameters that may have decreased primer efficiency. These same tools were used to optimize primer design, which resulted in more efficient amplification of the gene fusions in MLS. The first 6 cases tested all gave expected amplification. A larger than expected amplification product was seen in one sample using primers in exon 7 of FUS and exon 3 of DDIT3. Sequencing revealed a new, undescribed in-frame translocation between exon 9 of FUS and exon 3 of DDIT3.
Conclusions: Our results highlight the need for optimal assay and primer design when developing RT-PCR tests for FFPE tumor samples with a prominent acellular and myxoid stroma, such as the fusion transcripts characteristic of MLS. Additionally, we identified a new FUS-DDIT3 chimeric transcript, which needs to be considered in future assay development, diagnosis, and phenotypic characterization of myxoid liposarcoma.
Category: Bone & Soft Tissue

Monday, March 9, 2009 9:00 AM

Platform Session: Section E, Monday Morning