In prepubertal patients, cryopreservation of ovarian or testicular tissue is currently the only available method for fertility preservation prior to gonadotoxic cancer treatments. However, this approach carries the risk of reintroducing malignant cells upon autotransplantation, particularly in cases of metastatic cancers such as neuroblastoma and Ewing sarcoma. Therefore, it is crucial to employ highly sensitive techniques to detect minimal residual disease (MRD) in preserved gonadal tissues.

This study aims to identify the most effective detection method by comparing the sensitivity and specificity of reverse transcription quantitative PCR (RT-qPCR) and droplet digital PCR (ddPCR) in identifying MRD of neuroblastoma and Ewing sarcoma in ovarian and testicular tissues from patients treated for these malignancies during infancy.

Ovarian and testicular cryopreservation is the only option to preserve the fertility of children diagnosed with cancer. Cancer treatments such as chemotherapy and radiotherapy can lead to gonadal toxicity. However, there is a risk of reintroducing tumor cells when the germinal tissue is reused. This is known as residual disease. This risk is higher in the case of metastatic cancers such as neuroblastoma and Ewing sarcoma. These two solid tumors are associated with a high risk of relapse and disease progression after treatment. Neuroblastoma is the most common extracranial solid tumor in children. One characteristic of this neuroendocrine tumor is its high frequency of metastatic disease. Several cases of metastasis have been reported in ovarian and testicular tissues.

Regarding Ewing sarcoma, it is the second most common bone cancer in children. Approximately 27% of patients show evidence of metastasis at diagnosis; these circulating tumor cells can reach the germinal tissues.

Several detection methods are available for these two solid tumors, such as histopathological analysis with hematoxylin-eosin staining, and immunohistochemistry targeting cellular markers with fluorescent antibodies. However, the main limitation of these two methods is their low sensitivity for detecting only a few tumor cells. That is why RT-qPCR and ddPCR, which have high detection sensitivity, have been developed. These two molecular methods are currently used for the detection of residual disease in leukemia and lymphoma. However, no studies have investigated the detection of residual disease of neuroblastoma and Ewing sarcoma by ddPCR in ovarian and testicular tissues.

Therefore, the goal of this study is to validate the detection of residual disease of neuroblastoma and Ewing sarcoma by ddPCR in ovarian and testicular tissues. In addition, the second goal is to evaluate the specificity and sensitivity of residual disease detection for these two solid tumors in ovarian and testicular tissues using RT-qPCR and ddPCR.

First, an in vitro model has been developed to mimic the metastatic dissemination of neuroblastoma and Ewing sarcoma in germinal tissues. To do this, two tumor cell lines will be used: IMR-32 for neuroblastoma and RD-ES for Ewing sarcoma. Then, 10 ovarian tissues and 10 testicular tissues will be contaminated with increasing quantities of neuroblastoma tumor cells: 5, 10, 20, and 100 tumor cells. The same procedure will be applied in the case of Ewing sarcoma. Ovarian tissues are derived from women of any age diagnosed with a benign cyst requiring laparoscopy. Testicular tissues are derived from men of any age diagnosed with non-obstructive azoospermia. These ovarian and testicular models have been validated by our team through preliminary work. These models are as close as possible to prepubertal tissues with immature gametes. Next, the investigators will perform RNA extraction with TRIzol reagent, followed by reverse transcription to generate complementary DNA (cDNA) and RT-qPCR and ddPCR to detect the specific tumor genes: PHOX2B for neuroblastoma and EWSR1-FLI1 for Ewing sarcoma.

Residual disease detection will be conducted on ovarian and testicular tissues collected from prepubertal patients diagnosed and treated for neuroblastoma or Ewing sarcoma during infancy. These patients underwent germinal tissue cryopreservation as a fertility preservation strategy following their cancer diagnosis.

Total RNA will be extracted from the cryopreserved tissues using TRIzol reagent. Subsequently, reverse transcription will be performed. The presence or absence of tumor-specific transcripts will be assessed using both reverse transcription quantitative PCR (RT-qPCR) and droplet digital PCR (ddPCR). Specifically, the neuroblastoma-associated PHOX2B gene and the Ewing sarcoma-specific EWSR1-FLI1 fusion transcript will be targeted.

By comparing the sensitivity and specificity of RT-qPCR and ddPCR for detecting minimal residual disease in these tissues, this study aims to determine the most effective and reliable protocol for identifying residual tumor cells. The findings will support the safe future use of cryopreserved ovarian and testicular tissues in fertility restoration for pediatric cancer survivors.

Inclusion Criteria:

• Women of any age diagnosed with a benign cyst requiring laparoscopy may be included.
• Men of any age diagnosed with a non-obstructive azoospermia may be included.
• Prepubertal girls and boys diagnosed with neuroblastoma or Ewing sarcoma during infancy may be included.
• Capable of providing written informed consent to participate in the research study
• Affiliated with a social welfare service.
• For prepubertal patients, written informed consent to participate in the research study must be provided by their parents or legal guardians.

Exclusion Criteria:

• None

Residual disease detection will be conducted on ovarian and testicular tissues collected from prepubertal patients diagnosed and treated for neuroblastoma or Ewing sarcoma during infancy