A multiplex real-time pcr assay for the detection of gene fusions observed in solid tumors

Specific gene fusions observed in solid tumors are extremely useful diagnostic markers. We report the development of a method based on real-time PCR which enables the detection upon identical PCR conditions of the different fusions specifically observed in Ewing tumors (ET), alveolar rhabdomyosarcoma (ARMS), synovial sarcoma (SS), small round cell desmoplastic tumors (SRCDT), extraskeletal myxoid chondrosarcoma, malignant melanoma of soft parts, congenital fibrosarcoma, and anaplastic large cell lymphoma. A simple assay, based on multiplexing of primers and probes, is described for the routine genetic diagnosis of small round cell tumors of children. It enables the detection of the five EWS-ETS, the two PAX-FKHR, the three SYT-SSX, and the EWS-WT1 fusions of ET, ARMS, SS, and SRCDT, respectively. The sensitivity of this test is high enough to detect all fusions, including the large EWS-FLI-1 transcripts, with the equivalent of 100 tumor cells as a starting material. This multiplex fluorescent analysis of chromosome translocations (MFACT) was validated in comparison with conventional RT-PCR on a series of 79 tumors. A major advantage of this method is that it completely abolishes the manipulation of PCR-products. It, therefore, considerably lowers the risk of cross-contamination linked to carry-over of RT-PCR products. It also constitutes an important step toward the complete automation of the detection of cancer-specific gene fusions.