Detection of fusion gene in cell-free DNA of a gastric synovial sarcoma

Figure 1 Identification of the SYT-SSX2 fusion transcript. A: Structure of the SYT-SSX2 fusion transcript. Exon 10 of the SYT gene and exon 6 of the SSX2 gene are fused together in this transcript. The sequencing of exon is shown. Forward and reverse primers were designed in exon 10 of the SYT gene and exon 6 of the SSX2 gene, respectively; B: The RT-PCR product, including the SYT-SSX2 fusion site, was obtained from tumor tissue samples only. This analysis was performed more than three times, and a representative result of an electrophoresis is shown; C: The result of direct sequencing of the PCR product is shown. Exon 10 of the SYT gene is fused to exon 6 of the SSX2 gene.

Figure 2 Detection of the SYT-SSX2 fusion sequence. A: The intronic primer settings in the SYT and SSX2 genes are shown; B: Genomic PCR products were obtained from tumor tissue samples only. This analysis was performed more than two times, and a representative result of an electrophoresis is shown; C: The intronic breakpoint was confirmed by direct sequencing.

Figure 3 Intronic breakpoint and structure of the fusion sequence with specific probe and primer sets. A: The arrowheads indicate the position and sequence of the intronic breakpoint of the SYT and SSX2 genes; B: The structure and sequence of the FAM-labeled probe and primer sets specific to the intronic breakpoint of the SYT-SSX fusion sequence.

Figure 4 Detection of the fusion gene sequence in cell-free DNA using specific probe and primer sets. A: The range of reproducibility of rt-PCR with the specific probe and primer sets was confirmed. Diluted serial tumor gDNA of 1-0.063 ng was used. The dotted line indicates an approximate curve (R² = 0.9742, P = 0.0018); B: cfDNA samples of a patient and 10 healthy volunteers were analyzed using rt-PCR. rt-PCR was performed in duplicate, and mean Ct values are indicated. Standard deviation was calculated from duplicate samples. cfDNA: Cell-free DNA.