Sensitive and specific detection of Ewing sarcoma minimal residual disease in ovarian and testicular tissues Ewing sarcoma (EWS) is a common pediatric solid tumor with high metastatic potential. Due to toxic effects of treatments on reproductive functions, the cryopreservation of ovarian tissue (OT) or testicular tissue (TT) is recommended to preserve fertility. However, the risk to reintroduce residual metastatic tumor cells should be evaluated before fertility restoration. Our goal was to validate a sensitive and specific approach for EWS minimal residual disease (MRD) detection in frozen germinal tissues. Thawed OT (n=12) and TT (n=14) were contaminated with tumor RD-ES cells (10, 100 and 1000 cells) and EWS-FLI1 tumor-specific transcript was quantified with RT-qPCR. All contaminated samples were found to be positive, with the strong correlation between RD-ES cell numbers and EWS-FLI1 levels in OT (r=0.93) and TT (r=0.96) (P<0.001). No transcript was detected in uncontaminated control samples. The invasive potential of Ewing cells was evaluated using co-culture techniques. After co-culturing, tumor cells were detected in OT/TT with histology, FISH and RT-qPCR. In addition, 4 OT and 4 TT samples from children with metastatic EWS were tested, and no MRD was found using RT-qPCR and histology. We demonstrated the high sensitivity and specificity of RT-qPCR to detect EWS MRD in OT/TT samples.