Cryopreservation of ovarian tissue for fertility preservation: no evidence of malignant cell contamination in ovarian tissue from patients with breast cancer
2011; Elsevier BV; Volume: 95; Issue: 6 Linguagem: Inglês
10.1016/j.fertnstert.2010.12.019
ISSN1556-5653
AutoresMikkel Rosendahl, Vera Timmermans Wielenga, Lotte Nedergaard, Stine Gry Kristensen, Erik Ernst, Per Emil Rasmussen, Michael K. Anderson, Kirsten Tryde Schmidt, Claus Yding Andersen,
Tópico(s)Pluripotent Stem Cells Research
ResumoCryopreserved ovarian cortical biopsies from 51 patients with breast cancer were examined by histologic and immunohistochemical analysis and showed no sign of metastases. Autotransplantation of ovarian cortex to patients with low-stage breast cancer disease appears safe, but confirmatory studies are required, including xenotransplantation studies. Cryopreserved ovarian cortical biopsies from 51 patients with breast cancer were examined by histologic and immunohistochemical analysis and showed no sign of metastases. Autotransplantation of ovarian cortex to patients with low-stage breast cancer disease appears safe, but confirmatory studies are required, including xenotransplantation studies. Breast cancer accounts for 27% of all cancers in the female population in the Nordic countries, corresponding to a 9% lifetime risk of developing this condition. Approximately 25% of the cases, or 40 cases per 100,000 women, occur in premenopausal women (1NORDCAN. Association of the Nordic Cancer Registries 2008. Available at: http://www-dep.iarc.fr/nordcan.htm. Last accessed January 7, 2011.Google Scholar). Because of improving treatment regimes, long-term survival after early-stage breast cancer is increasing (2Early Breast Cancer Trialists' GroupEffects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials.Lancet. 2005; 365: 1687-1717Abstract Full Text Full Text PDF PubMed Scopus (6347) Google Scholar). However, an important long-term side effect to chemotherapy is the potential toxic effect on the ovaries, which for some women results in premature ovarian failure, including amenorrhea and infertility (3Petrek J.A. Naughton M.J. Case L.D. Paskett E.D. Naftalis E.Z. Singletary S.E. et al.Incidence, time course, and determinants of menstrual bleeding after breast cancer treatment: a prospective study.J Clin Oncol. 2006; 24: 1045-1051Crossref PubMed Scopus (297) Google Scholar).Cryopreservation of ovarian tissue is a method to preserve fertility with many advantages; it can be performed on short notice, does not require a male partner, and results in the storage of a large pool of primordial follicles. The method is also applicable in prepubertal girls. To date, transplantation of ovarian tissue has resulted in the birth of at least eleven children (4Donnez J. Dolmans M.M. Demylle D. Jadoul P. Pirard C. Squifflet J. et al.Livebirth after orthotopic transplantation of cryopreserved ovarian tissue.Lancet. 2004; 364: 1405-1410Abstract Full Text Full Text PDF PubMed Scopus (1341) Google Scholar, 5Meirow D. Levron J. Eldar-Geva T. Hardan I. Fridman E. Zalel Y. et al.Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy.N Engl J Med. 2005; 353: 318-321Crossref PubMed Scopus (752) Google Scholar, 6Demeestere I. Simon P. Emiliani S. Delbaere A. Englert Y. Fertility preservation: successful transplantation of cryopreserved ovarian tissue in a young patient previously treated for Hodgkin's disease.Oncologist. 2007; 12: 1437-1442Crossref PubMed Scopus (332) Google Scholar, 7Andersen C.Y. Rosendahl M. Byskov A.G. Loft A. Ottosen C. Dueholm M. et al.Two successful pregnancies following autotransplantation of frozen/thawed ovarian tissue.Hum Reprod. 2008; 23: 2266-2272Crossref PubMed Scopus (394) Google Scholar, 8Ernst E. Bergholdt S. Jorgensen J.S. Andersen C.Y. The first woman to give birth to two children following transplantation of frozen/thawed ovarian tissue.Hum Reprod. 2010; 25: 1280-1281Crossref PubMed Scopus (158) Google Scholar, 9Sanchez-Serrano M. Crespo J. Mirabet V. Cobo A.C. Escriba M.J. Simon C. et al.Twins born after transplantation of ovarian cortical tissue and oocyte vitrification.Fertil Steril. 2010; 93 (268–3)Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 10Demeestere I. Simon P. Moffa F. Delbaere A. Englert Y. Birth of a second healthy girl more than 3 years after cryopreserved ovarian graft.Hum Reprod. 2010; 25: 1590-1591Crossref PubMed Scopus (59) Google Scholar, 11Silber S.J. DeRosa M. Pineda J. Lenahan K. Grenia D. Gorman K. et al.A series of monozygotic twins discordant for ovarian failure: ovary transplantation (cortical versus microvascular) and cryopreservation.Hum Reprod. 2008; 23: 1531-1537Crossref PubMed Scopus (216) Google Scholar, 12Roux C. Amiot C. Agnani G. Aubard Y. Rohrlich P.S. Piver P. Live birth after ovarian tissue autograft in a patient with sickle cell disease treated by allogeneic bone marrow transplantation.Fertil Steril. 2010; 93: 2413-2419Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar).The safety of the procedure, however, is an important issue. The tissue is removed before the patients receive chemotherapy; therefore, any malignant cells in the ovarian tissue will not have been targeted by the anticancer therapy.Malignant cells in the ovarian cortex can thus occur in the frozen tissue and possibly, if transplanted, cause a relapse of the cancer. In retrospective studies, ovarian metastases account for 13.6%–15% of ovarian malignancies (13Lee S.J. Bae J.H. Lee A.W. Tong S.Y. Park Y.G. Park J.S. Clinical characteristics of metastatic tumors to the ovaries.J Korean Med Sci. 2009; 24: 114-119Crossref PubMed Scopus (44) Google Scholar, 14de Waal Y.R. Thomas C.M. Oei A.L. Sweep F.C. Massuger L.F. Secondary ovarian malignancies: frequency, origin, and characteristics.Int J Gynecol Cancer. 2009; 19: 1160-1165Crossref PubMed Scopus (80) Google Scholar). Most of the secondary ovarian malignancies (28%–66%) derive from the gastrointestinal tract, breast cancer (6.7%–28%), and endometrial cancer (10%–22%) (13Lee S.J. Bae J.H. Lee A.W. Tong S.Y. Park Y.G. Park J.S. Clinical characteristics of metastatic tumors to the ovaries.J Korean Med Sci. 2009; 24: 114-119Crossref PubMed Scopus (44) Google Scholar, 14de Waal Y.R. Thomas C.M. Oei A.L. Sweep F.C. Massuger L.F. Secondary ovarian malignancies: frequency, origin, and characteristics.Int J Gynecol Cancer. 2009; 19: 1160-1165Crossref PubMed Scopus (80) Google Scholar, 15Turan T. Aykan B. Koc S. Boran N. Tulunay G. Karacay O. et al.Analysis of metastatic ovarian tumors from extragenital primary sites.Tumori. 2006; 92: 491-495PubMed Google Scholar). These cases, however, were mostly advanced, whereas patients who are being considered for ovarian tissue cryopreservation usually are in the earlier stages of disease. The incidence of ovarian metastases of micrometastases in patients with low-stage breast cancer is unknown.In this article, we aim to systematically search for residual malignant cells in the ovarian cortex from Danish women with breast cancer who had the ovarian cortex cryopreserved for fertility preservation.The Danish Fertility Preservation Program was initiated in 1999. Since then, more than 450 patients have had ovarian tissue cryopreserved for clinical purposes. Since 2003, 19 autotransplantations have been performed in 13 women, resulting in seven pregnancies, of which three resulted in the birth of healthy babies (7Andersen C.Y. Rosendahl M. Byskov A.G. Loft A. Ottosen C. Dueholm M. et al.Two successful pregnancies following autotransplantation of frozen/thawed ovarian tissue.Hum Reprod. 2008; 23: 2266-2272Crossref PubMed Scopus (394) Google Scholar, 8Ernst E. Bergholdt S. Jorgensen J.S. Andersen C.Y. The first woman to give birth to two children following transplantation of frozen/thawed ovarian tissue.Hum Reprod. 2010; 25: 1280-1281Crossref PubMed Scopus (158) Google Scholar, 16Greve T. Ernst E. Markholt S. Schmidt K.T. Andersen C.Y. Legal termination of a pregnancy resulting from transplanted cryopreserved ovarian tissue.Acta Obstet Gynecol Scand. 2010; 89: 1589-1591Crossref PubMed Scopus (8) Google Scholar).At the time of inclusion, 83 patients with breast cancer had ovarian tissue cryopreserved in the Danish Fertility Preservation Program. Six of the patients died, and the remaining 77 patients received a letter with written information. After additional oral information, 51 patients gave written consent to participate in the study. The consent gave permission to thaw one or two pieces of ovarian cortex for evaluating residual malignant disease. The study was approved by the Danish Data Protection Agency and the Danish National Committee on Biomedical Research Ethics (H-A-2008-022), corresponding to a national institutional review board.One piece of ovarian cortex from each of the 51 participating patients was thawed for detection of malignant cells. The detailed protocol for removal, transport, and freezing of the ovarian tissue has been described previously (17Schmidt K.L. Ernst E. Byskov A.G. Nyboe A.A. Yding A.C. Survival of primordial follicles following prolonged transportation of ovarian tissue prior to cryopreservation.Hum Reprod. 2003; 18: 2654-2659Crossref PubMed Scopus (120) Google Scholar, 18Rosendahl M. Schmidt K.T.L. Ernst E. Rasmussen P.E. Loft A. Byskov A.G. et al.Cryopreservation of ovarian tissue for a decade in Denmark-a view of the technique.Reprod Biomed Online. 2011; 22: 162-171Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The tissue was thawed for clinical transplantation as described previously (19Rosendahl M. Andersen M.T. Ralfkiaer E. Kjeldsen L. Andersen M.K. Andersen C.Y. Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia.Fertil Steril. 2010; 94: 2186-2190Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 20Tryde Schmidt K.L. Yding A.C. Starup J. Loft A. Byskov A.G. Nyboe A.A. Orthotopic autotransplantation of cryopreserved ovarian tissue to a woman cured of cancer—follicular growth, steroid production and oocyte retrieval.Reprod Biomed Online. 2004; 8: 448-453Abstract Full Text PDF PubMed Scopus (85) Google Scholar). The frozen-thawed material was embedded in paraffin, and 4-μm sections were stained with hematoxylin and eosin (H&E)—the keratin markers CK7, CK-aecam, CA125, and WT1—as markers for ovarian epithelium. Antigens were retrieved with Dako Target Retrieval Solutions High pH for 20 minutes at 97°C (Code K 8002) using the Dako pretreatment link. After blocking endogenous peroxidase activity with Dako EnVision Flex+ (Dako K 8002) for 5 minutes, tissue sections were incubated with primary antibody and diluted in Dako antibody diluent (Dako K 8006). The sections were counterstained with hematoxylin. Antibodies: CA 125, Clone OV185: Novocastra NCL-CA125, diluted 1:200. WT-1, cloneWT49: Novocastra NCL-WT1-562, diluted 1:10. CK-AECAM, clone AE1/AE3+5DT: diluted 1:400. CK7, clone OV-TL 12/30, Dako M7018; diluted 1:400.To validate the method, a piece of ovarian tissue with known involvement of breast cancer was used as a positive control. The patient, a 32-year-old woman with disseminated invasive ductal carcinoma, had ovarian tissue removed outside the clinical program. After oophorectomy, dissection of the ovary showed macroscopic suspect areas, and histologic analysis confirmed metastasis from ductal carcinoma.The median age of the patients at the time of removal and cryopreservation of the ovarian tissue was 31.0 years (range, 23.7–39.8 years). Tumor types were:Ductal carcinoma, 93% (n = 43); mucinous tumor, 4% (n = 2); medullary carcinoma, 2% (n = 1); unknown or unclassified, 10% (n = 5). Median tumor size was 18 mm (range, 5–75 mm; n = 47).Vascular invasion of the tumor was recorded in 25% (n = 12) of the cases of 48 patients (94%) when the information was available. The malignancy grade was available in 43 cases (84%): grade I, 14% (n = 6); grade II, 35% (n = 15); grade III, 51% (n = 22). Local lymph node involvement was recorded in 44% (n = 21) of 44 cases when the information was available. None of the patients had distant metastases.All patients had one piece of ovarian cortex examined. Morphologically normal primordial follicles were seen in all specimens. None of the specimens showed immunohistochemical or histologic signs of malignant cell infiltration. Figure 1 shows a specimen of the frozen-thawed ovarian cortex stained with H&E, and marked with ovarian markers CA125 and WT1 and the keratin marker CK7. The result is normal ovarian cortical morphology and no indication of malignant infiltration. Figure 2 shows samples of the positive control. The result is ovarian stroma with invasive ductal carcinoma, which is consistent with the patient's history.Figure 2Positive control. Ovarian cortex from a 32-year-old patient with invasive ductal carcinoma. Ovarian cortex shows metastatic infiltration. (A) H&E. (B) WT-1. (C) CK-7. Magnification, ×10.View Large Image Figure ViewerDownload Hi-res image Download (PPT)At initial presentation, approximately 3%–10% of patients with breast cancer will have distant metastases (21Ernst M.F. van de Poll-Franse L.V. Roukema J.A. Coebergh J.W. van Gestel C.M. Vreugdenhil G. et al.Trends in the prognosis of patients with primary metastatic breast cancer diagnosed between 1975 and 2002.Breast. 2007; 16: 344-351Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Based on findings at autopsies and prophylactic and therapeutic oophorectomy, the incidence of ovarian metastases from breast cancer has been found to range from 13%–38% (14de Waal Y.R. Thomas C.M. Oei A.L. Sweep F.C. Massuger L.F. Secondary ovarian malignancies: frequency, origin, and characteristics.Int J Gynecol Cancer. 2009; 19: 1160-1165Crossref PubMed Scopus (80) Google Scholar, 15Turan T. Aykan B. Koc S. Boran N. Tulunay G. Karacay O. et al.Analysis of metastatic ovarian tumors from extragenital primary sites.Tumori. 2006; 92: 491-495PubMed Google Scholar, 22Perrotin F. Marret H. Bouquin R. Fischer-Perrotin N. Lansac J. Body G. Incidence, diagnosis and prognosis of ovarian metastasis in breast cancer.Gynecol Obstet Fertil. 2001; 29: 308-315Crossref PubMed Scopus (33) Google Scholar). Nevertheless, these are often advanced cases in contrast to the patients for whom cryopreservation of ovarian tissue is an option. These women usually have early-stage disease, in which the risk of ovarian involvement is presumed to be low.In this study, we have examined one piece of ovarian cortex from 51 patients. The negative findings in the present study do not, however, rule out metastases in the ovarian tissue, and an increased number of tissue fragments would increase the validity of the result.Sánchez-Serrano et al. did not find evidence of malignant cell contamination in a series of 100 cortical biopsies from 63 patients with breast cancer and in six entire cortex from patients with breast cancer (23Sánchez-Serrano M. Novella-Maestre E. Rosello-Sastre E. Camarasa N. Teruel J. Pellicer A. Malignant cells are not found in ovarian cortex from breast cancer patients undergoing ovarian cortex cryopreservation.Hum Reprod. 2009; 24: 2238-2243Crossref PubMed Scopus (75) Google Scholar). In a study from Israel, the investigators did not find evidence of malignant cells in the ovarian cortex from 13 patients with breast cancer by using H&E staining and light microscopy (24Azem F. Hasson J. Ben-Yosef D. Kossoy N. Cohen T. Almog B. et al.Histologic evaluation of fresh human ovarian tissue before cryopreservation.Int J Gynecol Pathol. 2010; 29: 19-23Crossref PubMed Scopus (36) Google Scholar).More than 160 ovarian cortical biopsies, and six entire cortex biopsies, from 133 patients with breast cancer, who had the tissue removed for cryopreservation, have now been examined without evidence of malignant cell contamination. However, a biopsy may not be representative of the entire ovarian cortex, and hence malignant cell infiltration may be overlooked. Furthermore, histologic and immunohistochemical analysis may not be sufficiently sensitive to detect infiltration with a small number of cells. Therefore, there is still a potential risk involved in autotransplantation of the ovarian cortex from these patients. The reverse transcriptase polymerase chain reaction (RT-PCR) assay is able to detect even a minimal quantity of mRNA of a given protein. In 2010, our group applied RT-PCR in the ovarian cortex from patients with leukemia, and we were able to detect disease specific transcripts in 6 of 8 patients (19Rosendahl M. Andersen M.T. Ralfkiaer E. Kjeldsen L. Andersen M.K. Andersen C.Y. Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia.Fertil Steril. 2010; 94: 2186-2190Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). RT-PCR may also be able to detect breast cancer–derived cells in ovarian tissue. Detection of genes coding for the breast cancer proteins, mammaglobin genes MGB1 and –2, may increase the sensitivity in sentinel node examination in patients with breast cancer (25Ouellette R.J. Richard D. Maicas E. RT-PCR for mammaglobin genes, MGB1 and MGB2, identifies breast cancer micrometastases in sentinel lymph nodes.Am J Clin Pathol. 2004; 121: 637-643Crossref PubMed Scopus (50) Google Scholar), although this is not standard (26Douglas-Jones A.G. Woods V. Molecular assessment of sentinel lymph node in breast cancer management.Histopathology. 2009; 55: 107-113Crossref PubMed Scopus (20) Google Scholar). This method may also prove useful in detecting malignant cells in a frozen-thawed ovarian cortex.Nonetheless, the detection of malignant cells in the ovarian cortex does not equal viability. Furthermore, it is unknown how many viable cells are required to reintroduce the malignancy.When approaching the safety of autotransplanting the cryopreserved ovarian cortex from women with breast cancer, one method with higher certainty would be to transplant the cortex to immune-compromised mice. This method was recently validated using the ovarian cortex from women with leukemia when the mice showed evidence of reactivation of the leukemia after transplantation with the contaminated ovarian cortex (27Dolmans M.M. Marinescu C. Saussoy P. van L.A. Amorim C. Donnez J. Reimplantation of cryopreserved ovarian tissue from patients with acute lymphoblastic leukemia is potentially unsafe.Blood. 2010; 116: 2908-2914Crossref PubMed Scopus (297) Google Scholar). We expect the method to be similarly sensitive in the case of breast cancer.In conclusion, this study has demonstrated that in patients with early-stage breast cancer, there is no histologic or immunohistochemical evidence of malignant cell contamination of the cryopreserved ovarian tissue. Nevertheless, until conclusive evidence on the safety of the autotransplantations in patients with breast cancer is obtained, the procedures should be performed only after thorough information on the patient, after a preoperative representative ovarian cortical biopsy, and under careful observation for signs of reactivation of the malignant condition. Breast cancer accounts for 27% of all cancers in the female population in the Nordic countries, corresponding to a 9% lifetime risk of developing this condition. Approximately 25% of the cases, or 40 cases per 100,000 women, occur in premenopausal women (1NORDCAN. Association of the Nordic Cancer Registries 2008. Available at: http://www-dep.iarc.fr/nordcan.htm. Last accessed January 7, 2011.Google Scholar). Because of improving treatment regimes, long-term survival after early-stage breast cancer is increasing (2Early Breast Cancer Trialists' GroupEffects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials.Lancet. 2005; 365: 1687-1717Abstract Full Text Full Text PDF PubMed Scopus (6347) Google Scholar). However, an important long-term side effect to chemotherapy is the potential toxic effect on the ovaries, which for some women results in premature ovarian failure, including amenorrhea and infertility (3Petrek J.A. Naughton M.J. Case L.D. Paskett E.D. Naftalis E.Z. Singletary S.E. et al.Incidence, time course, and determinants of menstrual bleeding after breast cancer treatment: a prospective study.J Clin Oncol. 2006; 24: 1045-1051Crossref PubMed Scopus (297) Google Scholar). Cryopreservation of ovarian tissue is a method to preserve fertility with many advantages; it can be performed on short notice, does not require a male partner, and results in the storage of a large pool of primordial follicles. The method is also applicable in prepubertal girls. To date, transplantation of ovarian tissue has resulted in the birth of at least eleven children (4Donnez J. Dolmans M.M. Demylle D. Jadoul P. Pirard C. Squifflet J. et al.Livebirth after orthotopic transplantation of cryopreserved ovarian tissue.Lancet. 2004; 364: 1405-1410Abstract Full Text Full Text PDF PubMed Scopus (1341) Google Scholar, 5Meirow D. Levron J. Eldar-Geva T. Hardan I. Fridman E. Zalel Y. et al.Pregnancy after transplantation of cryopreserved ovarian tissue in a patient with ovarian failure after chemotherapy.N Engl J Med. 2005; 353: 318-321Crossref PubMed Scopus (752) Google Scholar, 6Demeestere I. Simon P. Emiliani S. Delbaere A. Englert Y. Fertility preservation: successful transplantation of cryopreserved ovarian tissue in a young patient previously treated for Hodgkin's disease.Oncologist. 2007; 12: 1437-1442Crossref PubMed Scopus (332) Google Scholar, 7Andersen C.Y. Rosendahl M. Byskov A.G. Loft A. Ottosen C. Dueholm M. et al.Two successful pregnancies following autotransplantation of frozen/thawed ovarian tissue.Hum Reprod. 2008; 23: 2266-2272Crossref PubMed Scopus (394) Google Scholar, 8Ernst E. Bergholdt S. Jorgensen J.S. Andersen C.Y. The first woman to give birth to two children following transplantation of frozen/thawed ovarian tissue.Hum Reprod. 2010; 25: 1280-1281Crossref PubMed Scopus (158) Google Scholar, 9Sanchez-Serrano M. Crespo J. Mirabet V. Cobo A.C. Escriba M.J. Simon C. et al.Twins born after transplantation of ovarian cortical tissue and oocyte vitrification.Fertil Steril. 2010; 93 (268–3)Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 10Demeestere I. Simon P. Moffa F. Delbaere A. Englert Y. Birth of a second healthy girl more than 3 years after cryopreserved ovarian graft.Hum Reprod. 2010; 25: 1590-1591Crossref PubMed Scopus (59) Google Scholar, 11Silber S.J. DeRosa M. Pineda J. Lenahan K. Grenia D. Gorman K. et al.A series of monozygotic twins discordant for ovarian failure: ovary transplantation (cortical versus microvascular) and cryopreservation.Hum Reprod. 2008; 23: 1531-1537Crossref PubMed Scopus (216) Google Scholar, 12Roux C. Amiot C. Agnani G. Aubard Y. Rohrlich P.S. Piver P. Live birth after ovarian tissue autograft in a patient with sickle cell disease treated by allogeneic bone marrow transplantation.Fertil Steril. 2010; 93: 2413-2419Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The safety of the procedure, however, is an important issue. The tissue is removed before the patients receive chemotherapy; therefore, any malignant cells in the ovarian tissue will not have been targeted by the anticancer therapy. Malignant cells in the ovarian cortex can thus occur in the frozen tissue and possibly, if transplanted, cause a relapse of the cancer. In retrospective studies, ovarian metastases account for 13.6%–15% of ovarian malignancies (13Lee S.J. Bae J.H. Lee A.W. Tong S.Y. Park Y.G. Park J.S. Clinical characteristics of metastatic tumors to the ovaries.J Korean Med Sci. 2009; 24: 114-119Crossref PubMed Scopus (44) Google Scholar, 14de Waal Y.R. Thomas C.M. Oei A.L. Sweep F.C. Massuger L.F. Secondary ovarian malignancies: frequency, origin, and characteristics.Int J Gynecol Cancer. 2009; 19: 1160-1165Crossref PubMed Scopus (80) Google Scholar). Most of the secondary ovarian malignancies (28%–66%) derive from the gastrointestinal tract, breast cancer (6.7%–28%), and endometrial cancer (10%–22%) (13Lee S.J. Bae J.H. Lee A.W. Tong S.Y. Park Y.G. Park J.S. Clinical characteristics of metastatic tumors to the ovaries.J Korean Med Sci. 2009; 24: 114-119Crossref PubMed Scopus (44) Google Scholar, 14de Waal Y.R. Thomas C.M. Oei A.L. Sweep F.C. Massuger L.F. Secondary ovarian malignancies: frequency, origin, and characteristics.Int J Gynecol Cancer. 2009; 19: 1160-1165Crossref PubMed Scopus (80) Google Scholar, 15Turan T. Aykan B. Koc S. Boran N. Tulunay G. Karacay O. et al.Analysis of metastatic ovarian tumors from extragenital primary sites.Tumori. 2006; 92: 491-495PubMed Google Scholar). These cases, however, were mostly advanced, whereas patients who are being considered for ovarian tissue cryopreservation usually are in the earlier stages of disease. The incidence of ovarian metastases of micrometastases in patients with low-stage breast cancer is unknown. In this article, we aim to systematically search for residual malignant cells in the ovarian cortex from Danish women with breast cancer who had the ovarian cortex cryopreserved for fertility preservation. The Danish Fertility Preservation Program was initiated in 1999. Since then, more than 450 patients have had ovarian tissue cryopreserved for clinical purposes. Since 2003, 19 autotransplantations have been performed in 13 women, resulting in seven pregnancies, of which three resulted in the birth of healthy babies (7Andersen C.Y. Rosendahl M. Byskov A.G. Loft A. Ottosen C. Dueholm M. et al.Two successful pregnancies following autotransplantation of frozen/thawed ovarian tissue.Hum Reprod. 2008; 23: 2266-2272Crossref PubMed Scopus (394) Google Scholar, 8Ernst E. Bergholdt S. Jorgensen J.S. Andersen C.Y. The first woman to give birth to two children following transplantation of frozen/thawed ovarian tissue.Hum Reprod. 2010; 25: 1280-1281Crossref PubMed Scopus (158) Google Scholar, 16Greve T. Ernst E. Markholt S. Schmidt K.T. Andersen C.Y. Legal termination of a pregnancy resulting from transplanted cryopreserved ovarian tissue.Acta Obstet Gynecol Scand. 2010; 89: 1589-1591Crossref PubMed Scopus (8) Google Scholar). At the time of inclusion, 83 patients with breast cancer had ovarian tissue cryopreserved in the Danish Fertility Preservation Program. Six of the patients died, and the remaining 77 patients received a letter with written information. After additional oral information, 51 patients gave written consent to participate in the study. The consent gave permission to thaw one or two pieces of ovarian cortex for evaluating residual malignant disease. The study was approved by the Danish Data Protection Agency and the Danish National Committee on Biomedical Research Ethics (H-A-2008-022), corresponding to a national institutional review board. One piece of ovarian cortex from each of the 51 participating patients was thawed for detection of malignant cells. The detailed protocol for removal, transport, and freezing of the ovarian tissue has been described previously (17Schmidt K.L. Ernst E. Byskov A.G. Nyboe A.A. Yding A.C. Survival of primordial follicles following prolonged transportation of ovarian tissue prior to cryopreservation.Hum Reprod. 2003; 18: 2654-2659Crossref PubMed Scopus (120) Google Scholar, 18Rosendahl M. Schmidt K.T.L. Ernst E. Rasmussen P.E. Loft A. Byskov A.G. et al.Cryopreservation of ovarian tissue for a decade in Denmark-a view of the technique.Reprod Biomed Online. 2011; 22: 162-171Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The tissue was thawed for clinical transplantation as described previously (19Rosendahl M. Andersen M.T. Ralfkiaer E. Kjeldsen L. Andersen M.K. Andersen C.Y. Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia.Fertil Steril. 2010; 94: 2186-2190Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 20Tryde Schmidt K.L. Yding A.C. Starup J. Loft A. Byskov A.G. Nyboe A.A. Orthotopic autotransplantation of cryopreserved ovarian tissue to a woman cured of cancer—follicular growth, steroid production and oocyte retrieval.Reprod Biomed Online. 2004; 8: 448-453Abstract Full Text PDF PubMed Scopus (85) Google Scholar). The frozen-thawed material was embedded in paraffin, and 4-μm sections were stained with hematoxylin and eosin (H&E)—the keratin markers CK7, CK-aecam, CA125, and WT1—as markers for ovarian epithelium. Antigens were retrieved with Dako Target Retrieval Solutions High pH for 20 minutes at 97°C (Code K 8002) using the Dako pretreatment link. After blocking endogenous peroxidase activity with Dako EnVision Flex+ (Dako K 8002) for 5 minutes, tissue sections were incubated with primary antibody and diluted in Dako antibody diluent (Dako K 8006). The sections were counterstained with hematoxylin. Antibodies: CA 125, Clone OV185: Novocastra NCL-CA125, diluted 1:200. WT-1, cloneWT49: Novocastra NCL-WT1-562, diluted 1:10. CK-AECAM, clone AE1/AE3+5DT: diluted 1:400. CK7, clone OV-TL 12/30, Dako M7018; diluted 1:400. To validate the method, a piece of ovarian tissue with known involvement of breast cancer was used as a positive control. The patient, a 32-year-old woman with disseminated invasive ductal carcinoma, had ovarian tissue removed outside the clinical program. After oophorectomy, dissection of the ovary showed macroscopic suspect areas, and histologic analysis confirmed metastasis from ductal carcinoma. The median age of the patients at the time of removal and cryopreservation of the ovarian tissue was 31.0 years (range, 23.7–39.8 years). Tumor types were: Ductal carcinoma, 93% (n = 43); mucinous tumor, 4% (n = 2); medullary carcinoma, 2% (n = 1); unknown or unclassified, 10% (n = 5). Median tumor size was 18 mm (range, 5–75 mm; n = 47). Vascular invasion of the tumor was recorded in 25% (n = 12) of the cases of 48 patients (94%) when the information was available. The malignancy grade was available in 43 cases (84%): grade I, 14% (n = 6); grade II, 35% (n = 15); grade III, 51% (n = 22). Local lymph node involvement was recorded in 44% (n = 21) of 44 cases when the information was available. None of the patients had distant metastases. All patients had one piece of ovarian cortex examined. Morphologically normal primordial follicles were seen in all specimens. None of the specimens showed immunohistochemical or histologic signs of malignant cell infiltration. Figure 1 shows a specimen of the frozen-thawed ovarian cortex stained with H&E, and marked with ovarian markers CA125 and WT1 and the keratin marker CK7. The result is normal ovarian cortical morphology and no indication of malignant infiltration. Figure 2 shows samples of the positive control. The result is ovarian stroma with invasive ductal carcinoma, which is consistent with the patient's history. At initial presentation, approximately 3%–10% of patients with breast cancer will have distant metastases (21Ernst M.F. van de Poll-Franse L.V. Roukema J.A. Coebergh J.W. van Gestel C.M. Vreugdenhil G. et al.Trends in the prognosis of patients with primary metastatic breast cancer diagnosed between 1975 and 2002.Breast. 2007; 16: 344-351Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Based on findings at autopsies and prophylactic and therapeutic oophorectomy, the incidence of ovarian metastases from breast cancer has been found to range from 13%–38% (14de Waal Y.R. Thomas C.M. Oei A.L. Sweep F.C. Massuger L.F. Secondary ovarian malignancies: frequency, origin, and characteristics.Int J Gynecol Cancer. 2009; 19: 1160-1165Crossref PubMed Scopus (80) Google Scholar, 15Turan T. Aykan B. Koc S. Boran N. Tulunay G. Karacay O. et al.Analysis of metastatic ovarian tumors from extragenital primary sites.Tumori. 2006; 92: 491-495PubMed Google Scholar, 22Perrotin F. Marret H. Bouquin R. Fischer-Perrotin N. Lansac J. Body G. Incidence, diagnosis and prognosis of ovarian metastasis in breast cancer.Gynecol Obstet Fertil. 2001; 29: 308-315Crossref PubMed Scopus (33) Google Scholar). Nevertheless, these are often advanced cases in contrast to the patients for whom cryopreservation of ovarian tissue is an option. These women usually have early-stage disease, in which the risk of ovarian involvement is presumed to be low. In this study, we have examined one piece of ovarian cortex from 51 patients. The negative findings in the present study do not, however, rule out metastases in the ovarian tissue, and an increased number of tissue fragments would increase the validity of the result. Sánchez-Serrano et al. did not find evidence of malignant cell contamination in a series of 100 cortical biopsies from 63 patients with breast cancer and in six entire cortex from patients with breast cancer (23Sánchez-Serrano M. Novella-Maestre E. Rosello-Sastre E. Camarasa N. Teruel J. Pellicer A. Malignant cells are not found in ovarian cortex from breast cancer patients undergoing ovarian cortex cryopreservation.Hum Reprod. 2009; 24: 2238-2243Crossref PubMed Scopus (75) Google Scholar). In a study from Israel, the investigators did not find evidence of malignant cells in the ovarian cortex from 13 patients with breast cancer by using H&E staining and light microscopy (24Azem F. Hasson J. Ben-Yosef D. Kossoy N. Cohen T. Almog B. et al.Histologic evaluation of fresh human ovarian tissue before cryopreservation.Int J Gynecol Pathol. 2010; 29: 19-23Crossref PubMed Scopus (36) Google Scholar). More than 160 ovarian cortical biopsies, and six entire cortex biopsies, from 133 patients with breast cancer, who had the tissue removed for cryopreservation, have now been examined without evidence of malignant cell contamination. However, a biopsy may not be representative of the entire ovarian cortex, and hence malignant cell infiltration may be overlooked. Furthermore, histologic and immunohistochemical analysis may not be sufficiently sensitive to detect infiltration with a small number of cells. Therefore, there is still a potential risk involved in autotransplantation of the ovarian cortex from these patients. The reverse transcriptase polymerase chain reaction (RT-PCR) assay is able to detect even a minimal quantity of mRNA of a given protein. In 2010, our group applied RT-PCR in the ovarian cortex from patients with leukemia, and we were able to detect disease specific transcripts in 6 of 8 patients (19Rosendahl M. Andersen M.T. Ralfkiaer E. Kjeldsen L. Andersen M.K. Andersen C.Y. Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia.Fertil Steril. 2010; 94: 2186-2190Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). RT-PCR may also be able to detect breast cancer–derived cells in ovarian tissue. Detection of genes coding for the breast cancer proteins, mammaglobin genes MGB1 and –2, may increase the sensitivity in sentinel node examination in patients with breast cancer (25Ouellette R.J. Richard D. Maicas E. RT-PCR for mammaglobin genes, MGB1 and MGB2, identifies breast cancer micrometastases in sentinel lymph nodes.Am J Clin Pathol. 2004; 121: 637-643Crossref PubMed Scopus (50) Google Scholar), although this is not standard (26Douglas-Jones A.G. Woods V. Molecular assessment of sentinel lymph node in breast cancer management.Histopathology. 2009; 55: 107-113Crossref PubMed Scopus (20) Google Scholar). This method may also prove useful in detecting malignant cells in a frozen-thawed ovarian cortex. Nonetheless, the detection of malignant cells in the ovarian cortex does not equal viability. Furthermore, it is unknown how many viable cells are required to reintroduce the malignancy. When approaching the safety of autotransplanting the cryopreserved ovarian cortex from women with breast cancer, one method with higher certainty would be to transplant the cortex to immune-compromised mice. This method was recently validated using the ovarian cortex from women with leukemia when the mice showed evidence of reactivation of the leukemia after transplantation with the contaminated ovarian cortex (27Dolmans M.M. Marinescu C. Saussoy P. van L.A. Amorim C. Donnez J. Reimplantation of cryopreserved ovarian tissue from patients with acute lymphoblastic leukemia is potentially unsafe.Blood. 2010; 116: 2908-2914Crossref PubMed Scopus (297) Google Scholar). We expect the method to be similarly sensitive in the case of breast cancer. In conclusion, this study has demonstrated that in patients with early-stage breast cancer, there is no histologic or immunohistochemical evidence of malignant cell contamination of the cryopreserved ovarian tissue. Nevertheless, until conclusive evidence on the safety of the autotransplantations in patients with breast cancer is obtained, the procedures should be performed only after thorough information on the patient, after a preoperative representative ovarian cortical biopsy, and under careful observation for signs of reactivation of the malignant condition. The authors thank Tiny Roed and Marjo Westerdahl for laboratory work, and the Danish Breast Cancer Cooperative Group for the patient and tumor characteristics information.
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