Sensitive and Specific Detection of Ewing Sarcoma Minimal Residual Disease in Ovarian and Testicular Tissues in an In Vitro Model

Abstract

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 of reintroducing 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 a 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, four OT and four 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. Clinical trial: NCT 02400970.

Testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys: A 6 year experience from a Swiss multi-center network

“…In patients with malignant disease, examination by molecular techniques of the tissue is mandatory to exclude reintroduction of malignant cells, whereas in non-malignant diseases like hemoglobinopathies there is no restriction for transplantation of the tissue. However, methods for detection of minimal residual disease as multicolor flow cytometry, RT-qPCR, next-generation sequencing and xenograft of tissue in immunodeficient mice are still in development in this context, mainly in ovarian tissue preservation, and validated strategies to ensure the complete safety are still lacking ( 23 – 25 ). A birth via autografting has been achieved in non-human primates, indicating the potential feasibility of this technique in humans ( 6 ).…”

Minimal Infiltrative Disease Identification in Cryopreserved Ovarian Tissue of Girls with Cancer for Future Use: A Systematic Review

“…The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/cancers15174199/s1, Table S1: GRADE criteria adapted for the systematic review for detection of minimal infiltrative disease in cryopreserved pediatric ovarian tissue; Table S2: Evidence tables of articles included in the systematic review [15,16,[24][25][26][27][28][29][30][31][32][33][34][35][36][37][38].…”

Whole-genome sequencing facilitates patient-specific quantitative PCR-based minimal residual disease monitoring in acute lymphoblastic leukaemia, neuroblastoma and Ewing sarcoma

“…The primers and probes used for RT-PCR analysis are listed in Supplementary Table 3. HR-NB and EWS mRNA transcripts were analysed as described previously [34,35]. PCR was performed in 25 μL reactions consisting of 12.5 μL Taqman Universal PCR Master Mix (2×, Applied Biosystems), 0.25 μL of 10 μM forward and reverse primers (100 nM), 0.125 μL of 20 μM probe (100 nM) and 5 μL of complementary DNA generated from 100 ng of total RNA.…”

“…Unlike ALL, where Ig/ TCR rearrangements are readily identified in most patients [17,18], developing patient-specific, DNA-based quantitative PCR MRD (qPCR-MRD) assays in HR-NB and EWS has been limited by the absence of readily identifiable, tumour-specific targets. Instead, MRD detection in these cancers has focussed on the detection of tumour-specific messenger RNA (mRNA) using qRT-PCR of NBspecific RNA markers, such as TH and PHOX2B, or EWSR1 gene fusion transcripts with the ETS family or FLI1 gene for EWS-specific markers [33][34][35][36].…”