title: Evaluation of minimal disseminated disease in cryopreserved ovarian tissue from bone and soft tissue sarcoma patients.
abstract: What is the risk of finding malignant cells in cryopreserved ovarian tissue from sarcoma patients?
  
  
  
  Minimal disseminated disease (MDD) was not detected in frozen-thawed ovarian tissue from 26 patients by any of the sensitive methods applied.
  
  
  
  In case of leukemia, the risk of malignant cell transmission through the graft is well known and widely documented. However, for bone cancer, like Ewing sarcoma or osteosarcoma, only a small number of case reports, have been published. These cancers often affect prepubertal girls, in whom ovarian tissue cryopreservation and transplantation is the only option to preserve fertility.
  
  
  
  The presence of malignant cells in cryopreserved ovarian tissue from patients with bone/soft tissue sarcoma was investigated with disease-specific markers for each patient, using immunohistochemistry (IHC), FISH and real-time quantitative RT-PCR (qPCR), with the original tumor serving as a positive control.
  
  
  
  Forty-eight sarcoma patients were enrolled in the study, 12 of whom subsequently died. In each case, tissue from the primary tumor was investigated in order to identify markers (immunohistochemical and/or molecular) to analyze the ovarian tissue case by case. Ovarian tissue from osteosarcoma (n = 15), liposarcoma (n = 1) and undifferentiated sarcoma (n = 5) patients could not be evaluated, as no specific markers were detected by FISH or sensitive IHC in any of their primary tumoral tissue. One patient with Li-Fraumeni syndrome was also excluded from the study. IHC analyses were therefore performed on ovarian tissue from 26 patients and qPCR on 19. The primary tumors involved were Ewing sarcoma family of tumors (n = 14), rhabdomyosarcoma (n = 7), synovial sarcoma (n = 2), clear cell sarcoma (n = 2) and a malignant peripheral nerve sheath tumor (n = 1).
  
  
  
  MDD was not detected in any of the 26 analyzed samples using sensitive techniques in this largest reported series, even from patients who subsequently died and/or those who presented with metastasis (11/26), hence the most aggressive forms of bone cancer. Indeed, anti-CD99 IHC and PCR performed on patients presenting with Ewing sarcoma family of tumors (n = 14) was negative in all cases. In patients with soft tissue sarcoma (n = 12) primitive tumor markers were detected by IHC and were negative in ovarian tissue. PCR could only be performed in 6/12 of these patients, again proving negative.
  
  
  
  Cryopreserved ovarian fragments to be transplanted cannot be tested, so this analysis of malignant cells cannot guarantee that all cryopreserved fragments will not contain any disseminated disease. Moreover, molecular markers are not readily available for all types of tumors.
  
  
  
  These results are reassuring regarding the risk of malignant cells in the ovary for transplantation, as the study involves a large series including different types of sarcomas. We believe this will help clinicians in their patient counseling for fertility preservation and restoration.
  
  
  
  This work was supported by the Fonds National de la Recherche Scientifique de Belgique-FNRS under Grants Nos 7.4578.14 (Télévie to MS) and 5/4/150/5 to MMD. The authors declare no competing financial interests.
authors: Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain (UCL), Brussels, Belgium Gynecology Department, Cliniques Universitaires Saint-Luc, Brussels, Belgium marie-madeleine.dolmans@uclouvain.be.
authors: M M
authors: MM
authors: Dolmans
authors: Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain (UCL), Brussels, Belgium.
authors: Y
authors: Y
authors: Iwahara
authors: Society for Research into Infertility (SRI), Brussels, Belgium.
authors: J
authors: J
authors: Donnez
authors: Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain (UCL), Brussels, Belgium.
authors: M
authors: M
authors: Soares
authors: Department of Clinical Biology, Université Catholique de Louvain, Brussels, Belgium.
authors: J L
authors: JL
authors: Vaerman
authors: Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain (UCL), Brussels, Belgium.
authors: C A
authors: CA
authors: Amorim
authors: Center for Human Genetics, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium.
authors: H
authors: H
authors: Poirel
mesh_terms: D000293:Adolescent; D000328:Adult; D001859:Bone Neoplasms / Q000473:pathology*; D002648:Child; D015925:Cryopreservation; D005260:Female; D059247:Fertility Preservation / Q000379:methods*; D006801:Humans; D010053:Ovary / Q000473:pathology*; D012509:Sarcoma / Q000556:secondary*; D055815:Young Adult
keywords: Ewing sarcoma; fertility preservation; malignant cell detection; minimal disseminated disease; molecular markers; ovarian metastasis; ovarian tissue cryopreservation; rhabdomyosarcoma