title: Minimal residual disease detection of leukemic cells in ovarian cortex by eight-color flow cytometry.
abstract: OBJECTIVE
  How can leukemic cells be detected in cryopreserved ovarian tissue?
  
  
  CONCLUSIONS
  Multicolor flow cytometry (FCM) is useful to evaluate the presence of viable leukemic cells in the ovarian cortex with a high specificity and a robust sensitivity.
  
  
  BACKGROUND
  Storing ovarian tissue is an option to preserve fertility before gonadotoxic radiotherapy or chemotherapy treatments. However, transplantation of cryopreserved ovarian cortex to women cured of leukemia is currently not possible due to the risk of cancer re-seeding.
  
  
  METHODS
  We developed an automated ovarian cortex dissociation technique and we used eight-color FCM to identify leukemic cells with a series of dilutions added to ovarian single cell suspensions obtained from healthy cortex.
  
  
  METHODS
  Healthy ovarian cortex originated from women between 23 and 39 years of age undergoing laparoscopic ovarian drilling for polycystic ovary syndrome. Blood or bone marrow cells were collected in acute lymphoblastic leukemia (ALL) patients at diagnosis.
  
  
  RESULTS
  The tissue dissociation technique yield was 1.83 ± 1.49 × 10(6) viable nucleated cells per 100 mg of ovarian cortex. No cell exhibiting a leukemic phenotype was present in the normal ovarian cortex. Added leukemic cells were detected using their leukemia-associated phenotype up to a dilution of 10(-4). When specific gene rearrangements were present, they were detected by real-time quantitative PCR at the same dilution. The ovarian cortex from two leukemia patients was then used, respectively, as positive and negative controls.
  
  
  CONCLUSIONS
  Making available minimal residual disease (MRD) detection techniques (multicolor FCM, PCR and xenograft), that can be used either alone or together, is essential to add a fail-safe oncological dimension to pre-autograft monitoring.
  
  
  CONCLUSIONS
  This approach can be performed on fresh ovarian tissue during cryopreservation or on frozen/thawed tissue before reimplantation and it is currently the only available technique in cases of ALL where no molecular markers are identified. This new perspective should lead to studies on ovarian tissue from leukemia patients, for whom the presence of MRD should be established before autograft.
  The study was supported by the BioMedicine Agency, the Committee of the League against Cancer, the Besançon University Hospital, DGOS/INSERM/INCa and the regional Council of Franche-Comté. There were no conflicts of interest to declare.
authors: INSERM UMR1098, 1 Bd A Fleming, Besançon Cedex F-25020, France. clotilde.amiot@univ-fcomte.fr
authors: Clotilde
authors: C
authors: Amiot
authors: Fanny
authors: F
authors: Angelot-Delettre
authors: Tristan
authors: T
authors: Zver
authors: Magalie
authors: M
authors: Alvergnas-Vieille
authors: Philippe
authors: P
authors: Saas
authors: Francine
authors: F
authors: Garnache-Ottou
authors: Christophe
authors: C
authors: Roux
mesh_terms: D000328:Adult; D015925:Cryopreservation; D005260:Female; D059247:Fertility Preservation; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007938:Leukemia / Q000473:pathology*; D018365:Neoplasm, Residual / Q000473:pathology*; D010053:Ovary / Q000473:pathology* / Q000637:transplantation; D014182:Transplantation, Autologous
keywords: cryopreservation; flow cytometry; leukemia; minimal residual disease; ovarian tissue