Contamination of Ovarian Tissue by RT-PCR in Participants With Solid Tumors

Fertility Preservation, Minimal Residual Disease

Polymerase Chain Reaction, fertility preservation

For prepubertal patients, cryopreservation of ovarian tissue is the only option available to preserve their fertility before cancer treatment. But ovarian autograft raises the issue of the risk of reintroduction of potentially malignant cells. The aim of our study is to develop a specific and sensitive method for residual disease detection in the ovarian tissue from patients treated for a solid tumor during infancy, whose fertility may have been compromised by treatments and who benefited of ovarian tissue cryopreservation.

Some solid tumors have high risk of metastatic localization including in ovaries. There is concern over the possible presence of malignant cells in ovarian tissue that could cause a recurrence of the primary disease after reimplantation. Thus, the possibility of ovarian tissue involvement needs to be evaluated with sensitive molecular methods. Those techniques are now available for leukaemia but histology is still the only way for solid tumors. Based on our experience in detection by RT-PCR of minimal residual disease (MRD) in neuroblastoma since 1994 \[Tchirkov et al. 2003\] and in ovarian tissue cryopreservation since 1995 \[Schubert et al. 2005, Chambon in press\], we want to develop a specific and sensitive method for residual disease detection by RT-PCR in order to evaluate the tumor contamination of ovarian harvested tissue. Study population: We chose 3 models of pediatric solid tumors with high risk of metastases and which often require sterilizing treatments (chemo and/or radiotherapy): neuroblastoma, Ewing tumor and alveolar rhabdosarcoma. We will use four tumor cells lines: IMR32 and SK-NSH for neuroblastoma; RD-ES for Ewing tumor; RH-30 for rhabdosarcoma. We plan to use 20 fragments per line. Study duration: 12 months Study design: Ovarian tissue without known malignancy but with a condition warranting laparoscopy (benign cysts) will be harvested during kystectomy (perikystic tissue). The harvested ovarian cortex will be cut: one half will be frozen according to our routine protocol \[Schubert et al. 2005\] and the other half will be contaminated with tumor cells lines. Then, detection of the specific transcript will be done by RT-PCR in fresh tissue and after freeze/thaw. Total RNA will be extracted with the TRI-reagent and qRT-PCR will be performed using the TaqMan technology. Primary endpoint: to reach a sensitivity about 1/106 cells.

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