title: Highly sensitive assessment of neuroblastoma minimal residual disease in ovarian tissue using RT-qPCR-A strategy for improving the safety of fertility restoration.
abstract: Ovarian tissue cryopreservation (OTC) is the only option available to preserve fertility in prepubertal females with neuroblastoma (NB), a childhood solid tumor that can spread to the ovaries, with a risk of reintroducing malignant cells after an ovarian graft.
  
  
  
  We set out to determine whether the analysis of TH (tyrosine hydroxylase), PHOX2B (paired-like homeobox 2b), and DCX (doublecortin) transcripts using quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) could be used to detect NB contamination in ovarian tissue. Analyses were performed on benign ovarian tissue from 20 healthy women between November 2014 and September 2015 at the University Hospital of Clermont-Ferrand. Pericystic benign ovarian tissues were collected and contaminated with increasing numbers of human NB cells (cell lines IMR-32 and SK-N-SH) before detection using RT-qPCR.
  
  
  
  TH and DCX transcripts were detected in uncontaminated ovarian tissue from all the donors, hampering the detection of small numbers of tumor cells. By contrast, PHOX2B was not detected in any uncontaminated ovarian fragment. PHOX2B levels were significantly increased from 10 NB cells. Our study is the first to evaluate minimal residual disease detection using NB mRNAs in human ovarian tissue. Only PHOX2B was a reliable marker of NB cells contaminating ovarian tissue.
  
  
  
  These results are encouraging and offer hope in the near future for grafting ovarian tissue in women who survive cancer, whose fertility has been jeopardized by treatment, and who could benefit from OTC without oncological risk.
authors.affiliation: CHU Clermont-Ferrand, Service d'Hématologie et d'Oncologie Pédiatrique, Clermont-Ferrand, France.|INSERM-CIC 1405, Unite CRECHE, Clermont-Ferrand, France.
authors.forename: Victoria
authors.initials: V
authors.lastname: Grèze
authors.affiliation: Faculté de Médecine, Université d'Auvergne, Université Clermont 1, Clermont-Ferrand, France.|CHU Clermont-Ferrand, Biologie et médecine de la reproduction, AMP-CECOS, Clermont-Ferrand, France.|Laboratoire Génétique Reproduction et Développement, CNRS 6293-INSERM 1103, Université Clermont 1, UFR Médecine, Clermont-Ferrand, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Brugnon
authors.affiliation: CHU Clermont-Ferrand, Service d'Hématologie et d'Oncologie Pédiatrique, Clermont-Ferrand, France.|INSERM-CIC 1405, Unite CRECHE, Clermont-Ferrand, France.
authors.forename: Fanny
authors.initials: F
authors.lastname: Chambon
authors.affiliation: CHU Clermont-Ferrand, Centre de Biothérapie d'Auvergne, Hôpital Estaing, Clermont-Ferrand, France.
authors.forename: Pascale
authors.initials: P
authors.lastname: Halle
authors.affiliation: Faculté de Médecine, Université d'Auvergne, Université Clermont 1, Clermont-Ferrand, France.|CHU Clermont-Ferrand, Biologie et médecine de la reproduction, AMP-CECOS, Clermont-Ferrand, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Canis
authors.affiliation: INSERM-CIC 1431, Besançon, France.|Faculté de Médecine et de Pharmacie, Université de Franche-Comté, Besançon, France.|CHU Besançon, Service de Génétique Biologique,-Histologie, Biologie du Développement et de la Reproduction, Cryobiologie-CECOS, Besançon, France.
authors.forename: Clotilde
authors.initials: C
authors.lastname: Amiot
authors.affiliation: Faculté de Médecine, Université d'Auvergne, Université Clermont 1, Clermont-Ferrand, France.|CHU Clermont-Ferrand, Biologie et médecine de la reproduction, AMP-CECOS, Clermont-Ferrand, France.
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Grémeau
authors.affiliation: CHU Clermont-Ferrand, Unité de Biostatistiques, Délégation à la Recherche Clinique et l'Innovation, Clermont-Ferrand, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Pereira
authors.affiliation: Pediatric Oncology Unit, Hospital Universitari i Politècnic La Fe, Avda Fernando Abril Martorell, Valencia, Spain.
authors.forename: Yania
authors.initials: Y
authors.lastname: Yáñez Peralta
authors.affiliation: Faculté de Médecine, Université d'Auvergne, Université Clermont 1, Clermont-Ferrand, France.|CHU Clermont-Ferrand et Centre de Lutte contre le Cancer Jean Perrin, Clermont-Ferrand, France.|CHU Clermont-Ferrand, Service de Cytogénétique Médicale, Clermont-Ferrand, France.
authors.forename: Andrei
authors.initials: A
authors.lastname: Tchirkov
authors.affiliation: CHU Clermont-Ferrand, Service d'Hématologie et d'Oncologie Pédiatrique, Clermont-Ferrand, France.|INSERM-CIC 1405, Unite CRECHE, Clermont-Ferrand, France.|Faculté de Médecine, Université d'Auvergne, Université Clermont 1, Clermont-Ferrand, France.|CHU Clermont-Ferrand, Centre de Biothérapie d'Auvergne, Hôpital Estaing, Clermont-Ferrand, France.
authors.forename: Justyna
authors.initials: J
authors.lastname: Kanold
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics; D045744:Cell Line, Tumor; D002648:Child; D002675:Child, Preschool; D015925:Cryopreservation; D000089622:Doublecortin Domain Proteins; D000089662:Doublecortin Protein; D005260:Female; D005298:Fertility; D059247:Fertility Preservation / Q000379:methods*; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics; D009364:Neoplasm Recurrence, Local / Q000517:prevention & control*; D018365:Neoplasm, Residual / Q000235:genetics*; D009447:Neuroblastoma / Q000235:genetics* / Q000473:pathology*; D009479:Neuropeptides / Q000235:genetics; D010053:Ovary / Q000166:cytology; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics*; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics
chemical_list: D014408:Biomarkers, Tumor; C000714328:DCX protein, human; D000089622:Doublecortin Domain Proteins; D000089662:Doublecortin Protein; D018398:Homeodomain Proteins; D008869:Microtubule-Associated Proteins; C109220:NBPhox protein; D009479:Neuropeptides; D012333:RNA, Messenger; D014157:Transcription Factors; D014446:Tyrosine 3-Monooxygenase
keywords: RT-qPCR; fertility preservation; minimal residual disease; neuroblastoma; ovarian tissue