Highly Multiplexed Detection of Translocation Fusion Transcripts without Amplification Using the NanoString Platform
A Luina Contreras, S Jackson, M Ladanyi. Memorial Sloan-Kettering Cancer Center, New York, NY; NanoString Technologies, Inc, Seattle, WA
Background: Translocation-associated gene fusions are useful diagnostic markers. Current methods of detection include fluorescent in-situ hybridization (FISH) and reverse transcriptase PCR (RT-PCR). Here, we describe the development of an assay, based on the NanoString platform, for detecting sarcoma translocations using color-coded probe pairs without any PCR amplification step.
Design: We designed a multiplex probe library for EWS-FLI1; EWS-WT1; SYT-SSX1 and SYT-SSX2; and PAX3-FKHR and PAX7-FKHR, based on paired oligonucleotide probes. RNA molecules are immobilized using a 50 nt capture probe, corresponding to the portion immediately 5' to the chimeric transcript fusion junction. The second probe, the reporter probe, is a 50 nt probe hybridizing to the portion immediately 3' to the chimeric transcript fusion point. The reporter probe is coupled to a tag containing a fluorescent barcode that provides the detection signal and identifies the transcript. We analyzed 42 sarcomas: 10 Ewing sarcomas (6 EWS-FLI1 type-1 and 4 type-2); 12 desmoplastic round blue cell tumors (all EWS-WT1), 17 synovial sarcomas (11 SYT-SSX1 and 6 SYT-SSX2) and 3 alveolar rhabdomyosarcomas (2 PAX3-FKHR, 1 PAX7-FKHR). All cases were corroborated by RT-PCR. Twenty-two cases were fresh frozen tissue (FFT) and 20 formalin-fixed paraffin embedded (FFPE). Sensitivity controls consisted of serial dilutions (50%, 25%, 10%, 5%, 1% and 0.5%) of 6 sarcoma cell lines bearing these translocations.
Results: This NanoString assay detected the correct fusion transcript in all 39 tumor samples with satisfactory results and in 6/6 sarcoma cell lines. One of 22 FFT (5%) and 2 of the 20 FFPE samples (10%) gave uninterpretable results due to poor RNA quality. There were no false positives. The technical detection sensitivity ranged from 5-10% tumor in 90-95% normal. The standard assay design cannot discriminate between fusion transcripts with the same 3' fusion partner (e.g. PAX3-FKHR and PAX7-FKHR).
Conclusions: The NanoString Platform is well suited to the multiplex detection of cancer translocation fusion transcripts, requiring no amplification and minimal technical hands-on time. The assay is tolerant of partially degraded RNA and its technical detection sensitivity is suitable for routine diagnostic testing. This is a promising platform for broad screening of cancer translocation fusion transcripts in clinical laboratories.
Category: Pan-genomic/Pan-proteomic Approaches to Diseases
Tuesday, March 23, 2010 8:00 AM
Platform Session: Section H 1, Tuesday Morning