Table II.

Identification of EWS-FLI1 fusion in FFPE and CTCs in Ewing sarcoma patients.

A673 spiking (no. of cells)	NTC	0.15	0.5	1.5	5	15	50	150	500	Patient CTCs
PBMC (no. of cells)	2,000
∆∆Ct-1st	ND	33.821	33.224	30.651	30.364	28.16	26.707	24.18	22.884	ND
∆∆Ct-2nd	ND	34.292	33.346	30.531	30.276	28.058	26.454	24.206	22.937	ND

Reverse transcription-quantified PCR was performed after extracting RNA from the patient's CTC. The patient's ΔΔCt value was revealed to be undetermined as NTC. HeLa cells were the negative control. A673 cells were the positive control. EWS-FLI1 fusion was detected in FFPE of Ewing sarcoma, but not in the CTC. After spiking A673 Ewing sarcoma cells on normal PBMCs, the LOD for EWS-FLI1 fusion detection was determined. FFPE, formalin-fixed paraffin-embedded; CTCs, circulating tumor cells; NTC, no template control; PBMC, peripheral blood mononuclear cell; ND, not-detected (undetermined); PCR, polymerase chain reaction; LOD, limit of detection.