Risk of transferring malignant cells with transplanted frozen-thawed ovarian tissue

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1 Risk of transferring malignant cells with transplanted frozen-thawed ovarian tissue Marie-Madeleine Dolmans, M.D., Ph.D., a Valerie Luyckx, M.D., a Jacques Donnez, M.D., Ph.D., b Claus Yding Andersen, D.M.Sc., c and Tine Greve, M.D. c a P^ole de Recherche en Gynecologie, Institut de Recherche Experimentale et Clinique, Universite Catholique de Louvain, Cliniques Universitaires Saint Luc; b Societe de Recherche pour l'infertilite, Brussels, Belgium; and c Laboratory of Reproductive Biology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark Ovarian tissue cryopreservation and transplantation is a real option to preserve and restore fertility in young cancer patients. However, there is a concern regarding the possible presence of malignant cells in the ovarian tissue, which could lead to recurrence of the primary disease after reimplantation. A review of the existing literature was done to evaluate the risk of transplanting malignant cells in case of the main malignant indications for ovarian tissue cryopreservation. For ovarian tissue from patients with hematologic malignancies, it is of paramount importance to identify minimal residual disease before ovarian tissue transplantation. Indeed, these pathologies, reviewed here in detail, are considered to be most at risk of ovarian metastasis. (Fertil Steril Ò 2013;99: Ó2013 by American Society for Reproductive Medicine.) Key Words: Ovarian tissue cryopreservation, ovarian tissue transplantation, cancer, risk, malignant cells Discuss: You can discuss this article with its authors and with other ASRM members at fertstertforum.com/dolmansmm-risk-malignant-cells-ovarian-tissue-transplantation/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. Cryopreservation of ovarian tissue is the main option available to preserve fertility in women who require cancer treatment but cannot delay the start of chemotherapy and in prepubertal patients. In our department, hematologic malignancies are the most common indication for ovarian tissue cryopreservation, representing 37.5% of all indications (1). In this category, Hodgkin lymphoma (HL) is the most frequent, followed by leukemia and then non-hodgkin lymphoma (NHL). According to the current SEER data (NCI, USA, Surveillance, Epidemiology, and End Results), the most common hematologic cancer in females under the age of 20 years is leukemia, followed by HL and then NHL. Reversing treatment-related premature ovarian failure with autotransplantation of frozen-thawed ovarian tissue is now becoming a reality, with 24 live births reported so far using this technique (2). It is expected that in the near future, more and more cancer patients cured of their disease will request reimplantation Received February 7, 2013; revised and accepted March 13, 2013; published online March 29, M.-M.D. has nothing to disclose. V.L. has nothing to disclose. J.D. is a board member of Preglem and has received payment for lectures from Serono, MSD, Organon, and Ferring. C.Y.A. has nothing to disclose. T.G. has nothing to disclose. Supported by grants from Fonds National de la Recherche Scientifique de Belgique, Fonds Speciaux de Recherche, Fondation Saint-Luc, Foundation Against Cancer, and donations from Mr. Pietro Ferrero, Baron Frere, and Viscount Philippe de Spoelberch (M.-M.D. and V.L.) and the Danish Cancer Society (T.G.). Reprint requests: Marie-Madeleine Dolmans, M.D., Ph.D., P^ole de Recherche en Gynecologie, IREC Institut de Recherche Experimentale et Clinique, Universite Catholique de Louvain, Avenue Mounier, 52, B152.02, B-1200 Brussels, Belgium ( marie-madeleine.dolmans@uclouvain.be). Fertility and Sterility Vol. 99, No. 6, May /$36.00 Copyright 2013 American Society for Reproductive Medicine, Published by Elsevier Inc. of their cryopreserved ovarian tissue. However, there is concern regarding the possible presence of malignant cells in the ovarian tissue, which could lead to recurrence of the primary disease after reimplantation (3). To increase the safety of ovarian tissue transplantation, identification of tumor involvement in the ovaries and detection of the presence of cancer cells in ovarian tissue is of paramount importance (3). The aim of this review is to examine all available evidence of the risk of transplanting malignant cells with frozen-thawed ovarian tissue from cancer patients. The risks are evaluated for each of the main indications for ovarian tissue cryopreservation. LEUKEMIA In leukemia, malignant cells may be present in the bloodstream, and therefore also in the ovaries, so there may be a risk of transferring diseased cells with the ovarian tissue. A study by 1514 VOL. 99 NO. 6 / MAY 2013

2 Fertility and Sterility Jahnukainen et al. (5) showed that transplantation of testicular cells from leukemic donor rats transmits acute leukemia to healthy recipients. For ovarian tissue from hematologic cancer patients, it is of great importance to identify minimal residual disease (MRD) before ovarian tissue transplantation (3, 6). Meirow et al. (3) were the first to detect chronic myeloid leukemia (CML) cells in frozen-thawed ovarian tissue from a patient with the disease by real-time quantitative polymerase chain reaction (RT-qPCR), which proved to be positive for BCR-ABL transcripts, and therefore excluded transplantation of the stored ovarian tissue. A recent retrospective analysis of 5,571 autopsy findings of female patients under the age of 40 years in Japan found, by histology, leukemic involvement of the ovaries in 8.4% of leukemia patients (7). Very little has been published on the incidence of ovarian metastasis in CML. In case of acute lymphoblastic leukemia (ALL), ovarian metastases have been found in up to 30% of patients at autopsy, but are rarely detected clinically (8). In recent studies, molecular biology was used to evaluate the presence of leukemic cells in cryopreserved ovarian tissue from patients with CML, acute myeloid leukemia (AML), and ALL, leukemia types that are the most frequent indications for cryopreservation of ovarian tissue (1, 9 12). Ovarian Tissue Histology In the above-mentioned studies, histology and immunohistochemistry failed to detect malignant cells in fresh or frozen ovarian tissue from any leukemia patients. Indeed, samples were analyzed by experienced pathologists, and no malignant cells were found in ovarian tissue of patients with any form of the disease (ALL, CML, or AML). Moreover, ovarian follicles were present in all the samples. Nevertheless, we should stress that since these publications, malignant cells have been detected by histology/immunohistochemistry in three cases of ALL (1). Polymerase Chain Reaction Analysis Determination of BCR-ABL fusion gene transcripts. The Philadelphia chromosome is pathognomonic for CML. It is the result of reciprocal translocation t(9;22), which brings the ABL gene from chromosome 9 together with the BCR gene from chromosome 22, creating a fusion gene known as BCR-ABL. Tumor-specific breakpoint cluster region/ proto-oncogene tyrosine protein kinase ABL1 (BCR ABL1) transcripts can be detected with use of RT-qPCR (3, 13). Molecular detection of leukemic cells in ovarian tissue can always be carried out for CML, because the presence of the BCR-ABL fusion gene is characteristic of the disease. Other specific translocations [e.g., t(1;19)] can be present in ALL disease. In MRD studies, PCR analysis at the time of diagnosis was performed on blood and bone marrow, and the same markers were used on frozen-thawed ovarian tissue. Detection of clonal immunoglobulin and T-cell receptor gene recombinations. MRD in case of ALL is evaluated by RT-qPCR analysis of leukemia-specific junctional regions of rearranged immunoglobulin (Ig) genes and T-cell receptor (TCR) genes, which can be considered as DNA fingerprints of leukemic cells (14). For ALL, genetic markers are not always detected. Recurrent chromosome translocations are reported in 38% of ALL patients (15, 16). If no chromosomal abnormalities are evidenced in blood or bone marrow of ALL patients, specific Ig gene rearrangements can often be tested, even if the sensitivity of these tests is lower. Nevertheless, some ALL cases do not display any markers at all, and so far there are no available molecular methods to evaluate the risk of contamination by malignant cells. PCR products obtained from Ig and TCR gene rearrangements were thus analyzed by gene scanning, as described by Van Dongen et al. (17). The present review reports both the Belgian and Danish experience in the field of ovarian tissue reimplantation in leukemia patients. THE BELGIAN EXPERIENCE (DOLMANS ET AL., BLOOD 2010) (9) Is there a risk of malignant cell contamination when ovarian tissue is retrieved from patients with leukemia in the active phase? CML and ALL In vitro study. Among CML patients (n ¼ 6), two were found to be positive for the BCR-ABL leukemic marker in their ovarian tissue (patients 1 and 3; Table 1). All CML patients received hydroxycarbamide with or without imatinib before ovarian tissue cryopreservation. Cryopreservation was carried out as bone marrow transplantation (BMT) was scheduled in these patients. Amplification curve results for the BCR-ABL fusion gene showed a positive signal in the bone marrow of diseased patients at diagnosis and also in their frozenthawed ovarian tissue (Table 1). Among ALL patients (n ¼ 12), two were excluded from PCR analysis because no molecular markers were available. Of the remaining ten, seven showed positive molecular markers in their cryopreserved ovarian tissue. One was positive for the BCR-ABL fusion gene, the second for t(1;19)(q23;p23.3), and the other five for Ig and/or TCRg rearrangement genes (Table 1). Among the seven patients who were positive for ALL markers in their ovarian tissue, four had not received any chemotherapy before ovarian tissue cryopreservation (patients 8, 11, 13, and 14) and three had already undergone one regimen of chemotherapy (patients 7, 10, and 18). In conclusion, using disease-specific PCR techniques, the authors found contamination of ovarian tissue in 33% and 70% of CML and ALL patients, respectively (9). In case of CML with indications for BMT, hematologists and oncologists have time to send their patients to the gynecologist for fertility preservation options such as ovarian tissue cryopreservation. In Dolmans et al.'s series (9), all CML patients underwent ovarian tissue cryopreservation before sterilizing chemotherapy, but after receiving hydroxycarbamide with or without imatinib. Nevertheless, we should stress that even after a minimum of 6 months of this treatment, PCR results showed that ovarian tissue was still positive (patients 1 and 3). VOL. 99 NO. 6 / MAY

3 VIEWS AND REVIEWS TABLE 1 Patients and pathology characteristics with PCR markers tested on frozen-thawed ovarian tissue. Gonadotoxic chemotherapy before Patient no. Age at OTC Pathology OTC Molecular markers PCR results in ovarian tissue 1 31 CML 0 t(9;22)(q34;q11.2); BCR-ABL Pos CML 0 t(9;22)(q34;q11,2); BCR-ABL Neg CML 0 t(9;22)(q34;q11.2); BCR-ABL Pos CML 0 t(9;22)(q34;q11,2); BCR-ABL Neg CML 0 t(9;22)(q34;q11,2); BCR-ABL Neg CML 0 t(9;22)(q34;q11,2); BCR-ABL Neg ALL 1 (1, 8) a t(9;22)(q34;q11.2); BCR-ABL Pos ALL 0 t(1;19)(q23;p13.3); E2A-PBX1 Pos ALL 2 (2, 3, 4, 5, 6) Ig and TCRg rearrangement genes Neg ALL 1 (1, 2, 3, 4, 7, 8) Ig and TCRg rearrangement genes Pos ALL 0 Ig rearrangement genes Pos ALL 1 (1, 2) No molecular marker found NA ALL 0 Ig rearrangement genes Pos ALL 0 Ig rearrangement genes Pos ALL 1 (1, 2) Ig rearrangement genes Neg ALL 1 (1, 2) Ig and TCRg rearrangement genes Neg ALL 1 (1, 2) No molecular marker found NA ALL 1 (1, 2) TCRg rearrangement genes Pos. Note: Table showing the number of chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) patients, their age at the time of ovarian tissue cryopreservation (OTC), and the type of gonadotoxic chemotherapy received before OTC (CML patients received hydroxycarbamide with or without imatinib). Molecular markers present in blood or bone marrow at the time of diagnosis were tested by polymerase chain reaction (PCR) on frozen-thawed ovarian tissue. Adapted from Dolmans et al. (9). Ig¼ immunoglobulin; NA ¼ not applicable; TCR ¼ T-cell receptor. a In parentheses: 1 ¼ methotrexate; 2 ¼ cortisone; 3 ¼ cyclophosphamide; 4 ¼ vincristine; 5 ¼ doxorubicin; 6 ¼ cytosine arabinoside; 7 ¼ daunorubicin; 8 ¼ asparaginase. However, in case of ALL, gonadotoxic chemotherapy is often initiated before fertility preservation options can be proposed because of the urgency of treatment. It is thus important to establish whether previous chemotherapy can help to eliminate malignant cells from ovarian tissue. In this study (9), among the ten ALL patients with available molecular markers, four who underwent ovarian tissue cryopreservation before receiving any chemotherapy had positive PCR results for the presence of leukemic markers in their ovarian tissue (patients 8, 11, 13, and 14). The remaining six ALL patients with available molecular markers had already received some chemotherapy before ovarian tissue cryopreservation. Despite these first regimens of chemotherapy, three patients still showed positive ovarian tissue (patients 7, 10, and 18). Moreover, one of these patients (patient 10) had already received a full cycle of six chemotherapeutic agents (cortisone, methotrexate, cyclophosphamide, vincristine, daunorubicin, and asparaginase). One cycle of chemotherapy is therefore insufficient to completely eliminate all leukemic cells. In vivo study. For each patient, frozen-thawed ovarian tissue was xenografted to a SCID mouse for 6 months. The authors found evidence that ovarian contamination by leukemic cells is possible in ALL as well as CML and that human ovarian tissue positive for ALL markers by PCR can transmit the disease to mice (9). The xenografting experiments demonstrated the viability and malignant potential of leukemic cells present in frozen ovarian tissue (Fig. 1). Moreover, among the two ALL patients in whom no molecular markers were found, xenografting led to leukemic invasion in the mouse in one case (9). From a clinical point of view, knowing that patients in remission can present with negative molecular results in blood and/or bone marrow and still relapse, we must bear in mind that there is a potential risk of disease recurrence in case of ovarian tissue reimplantation, even if molecular tests are negative in ovarian tissue. THE DANISH EXPERIENCE (GREVE ET AL., BLOOD, 2012) (12) Is the risk diminished if ovarian tissue to be cryopreserved is taken from patients in complete remission? FIGURE 1 Histologic analysis of an ovarian fragment recovered from a mouse after 6 months' grafting (acute lymphoblastic leukemia patients). A human follicle is encircled by a large number of lymphocytes. Normal ovarian stroma is no longer present VOL. 99 NO. 6 / MAY 2013

4 Fertility and Sterility ALL and AML In vitro study. Although viable malignant cells have been evidenced in ovarian tissue retrieved from women/girls in the active phase of acute leukemia (9), this has not been demonstrated in patients who received chemotherapy before retrieval of ovarian tissue (12). At the time of diagnosis, a patient with leukemia has a huge presence of circulating malignant cells. However, the first round or first few rounds of chemotherapy (induction therapy) eliminate most of these malignant cells from the blood stream and bone marrow. The patient enters morphologic remission characterized by a normal blood count and normal bone marrow smear. In a number of patients, however, malignant cells persist, even if they are undetectable, and continued chemotherapy is needed (consolidation therapy) to stop the patient from relapsing. A subgroup of patients requires allogeneic hematopoietic stem cell transplantation (HSCT) to be cured. This treatment is gonadotoxic and has a very high risk of inducing sterility (18, 19). Among others, the Danish team evaluated the risk of malignant cell contamination of cryopreserved ovarian cortex from patients with leukemia, and they found that even though patients were in complete remission, traces of malignant cell contamination could be detected by RT-qPCR in two of four patients with a known molecular marker (10, 12). Recently, Jahnukainen et al. used RT-qPCR to examine cryopreserved ovarian tissue originating from girls who had died from leukemia (20). Some of the tissue had been collected in the active phase and some from patients in complete remission. In two of the ten patients in complete morphologic bone marrow remission, traces of malignant cells were still detected in ovarian cortex, albeit at low levels. RT-qPCR of the ovarian cortex from all patients in the active phase demonstrated the presence of malignant cells. With use of very sensitive RT-qPCR techniques, it is possible to identify low levels of contamination by malignant cells in ovarian cortical samples frozen for fertility preservation, even from patients in complete remission. However, to what extent this translates into a real risk of introducing viable malignant cells into patients undergoing transplantation is currently not known with any certainty. In vivo study. The Belgian and Danish teams xenotransplanted tissue from patients with leukemia and found that no mice developed the disease with tissue from patients in complete remission, even though the tissue was PCR-positive (9, 12). In Denmark, Greve et al. transplanted ovarian cortex from 21 patients with ALL or AML to immunodeficient mice, and none of the mice developed leukemia. Seventeen of the 21 were in complete remission when the ovarian cortex was collected and cryopreserved (12). No mice developed leukemia after grafting ovarian tissue collected from patients who had received chemotherapy, indicating that no or too few copies of malignant cells were present to transmit the disease to mice. What the implication of this result is for humans is less certain, because mice models might underestimate the real risk due to a lack of speciesspecific growth factors, or overestimate the risk because of a lack of protection from the immune system of the mouse. CML Before approval of the first-generation tyrosine kinase inhibitor imatinib, standard treatment for children and young adults diagnosed with CML was HSCT, because it was the only treatment that could cure patients (21, 22). Because HSCT runs a very high risk of causing infertility (19, 20), the girls were offered cryopreservation of their ovarian tissue. New and possibly lifelong treatment with imatinib can remove malignant cells, so they cannot be detected by either blood count (complete hematologic response), cytogenetic detection of the Philadelphia chromosome (complete cytogenetic response), or RT-qPCR (complete molecular remission). If patients stop treatment with imatinib, some will relapse (21 23). While imatinib probably does not pose a threat to fertility (22), it is teratogenic (24). Patients treated only with tyrosine kinase inhibitors should not be offered cryopreservation of ovarian tissue, because the treatment probably does not cause ovarian insuffiency. Only patients with CML who cannot be brought into complete remission (chronic phase with detectable disease in blood and bone marrow) need to undergo HSCT and are thus candidates for ovarian tissue cryopreservation. As previously stated, BCR-ABL transcripts have been found by RT-qPCR, albeit at low levels, in ovarian cortex from CML patients (3, 9, 12, 25). Dolmans et al. and Greve et al. performed long-term xenotransplantation to immunodeficient mice, but ovarian cortex from CML patients with positive RT-qPCR failed to produce disease in the animals. The reason could have been that the cells were not viable or that the cells could not proliferate in mice (9, 12, 26). LESSONS AND QUESTIONS So far, three different teams (3, 9, 12) have demonstrated by PCR that ovarian tissue from leukemia patients is positive for malignant cells in >50% of cases. Greve et al. reported that ovaries from leukemia patients in complete remission do not appear to contain viable malignant cells, in contrast to ovarian tissue retrieved before chemotherapy (12). In an editorial recently published in Blood (26),Dolmans suggested that differences between the results of the Danish and Belgian teams may be explained, at least in part, by the fact that in Greve et al.'s study, almost all the women were in complete remission at the time of ovarian biopsy and cryopreservation (12). Nine patients in the Danish study died. Those patients probably presented with a more aggressive form of the disease and were therefore most at risk of carrying malignant cells in their ovarian tissue. Unfortunately, evaluation of ovarian tissue from the deceased women was not authorized by the local ethics committee, according to the protocol used. It would have been extremely valuable to confirm the presence or absence of malignant cells in the ovarian tissue of these patients, because they were in complete remission at the time of the cryopreservation procedure, but subsequently suffered disease recurrence. Even if some of the results of the Danish team are reassuring and demonstrate that ovarian tissue taken from leukemia patients in complete remission after chemotherapy does not induce tumors in a xenograft model, the presence of positive PCR (in four of seven patients) leads us to interpret these results with caution. VOL. 99 NO. 6 / MAY

5 VIEWS AND REVIEWS In conclusion, we believe that the risk of finding viable malignant cells in cryopreserved ovarian tissue from leukemia patients in complete remission is low. However, it cannot be completely excluded. The study by Greve et al. should be confirmed by other teams before transplantation can be safely performed in these patients. Alternative methods, such as in vitro maturation and isolated follicle transplantation, should be evaluated for fertility preservation in leukemia patients. Because in vitro growth of ovarian cortex is still in its infancy, it is not possible to exclude the possibility that in vitro growth of follicles and in vitro maturation of oocytes could be compromised by exposure of resting primordial follicles to chemotherapy. Nevertheless, the risk of viable malignant cells being present in ovarian cortex retrieved before chemotherapy should be balanced against the theoretical risk of compromised in vitro growth. Furthermore, each type of leukemia may represent a different risk scenario depending on how induction is performed, how far in remission the patient is, and other factors, such as the number of viable malignant cells that can cause relapse. LYMPHOMA HL and NHL are frequent indications for ovarian tissue cryopreservation (1, 27). However, because Shaw et al. (28) reported that ovarian tissue collected from AKR mice with lymphoma could transfer the disease to healthy recipient animals, caution is required. Hodgkin Lymphoma Several studies (Table 2) have tested the safety of grafting cryopreserved human ovarian tissue from HL patients, and they suggest that ovarian tissue transplantation may be considered safe in this case (3, 29 31). One case report showed ovarian involvement in stage III HL (32) and, in another study (7), the ovaries were found to be affected by HL in 4.3% of autopsies. Fifteen autotransplantations have been reported in HL patients and none of them experienced disease recurrence after their ovarian transplantation (some cases are described in more than one paper) (33 44). The maximum follow-up after autotransplantation in a patient with HL is now >8years(45, 46). Non-Hodgkin Lymphoma Six autotransplantations of frozen-thawed ovarian tissue from NHL patients have been reported, and they failed to demonstrate any signs of disease recurrence (Table 2) (41, 45 51). Although Kyono et al. (7) reported a 9.8% incidence of ovarian involvement in NHL, histology did not reveal the presence of malignant cells in ovarian cortex in a number of studies (3, 30, 52). Kim et al. (30) did not find evidence of any metastasis after xenografting tissue to SCID mice for 16 weeks (n ¼ 13 patients with HL, 5 with NHL). However, the Belgium team detected malignant cells by histologic evaluation in two patients (6%) out of 32 with NHL (Fig. 2). NHL cells were found by immunohistochemistry in the medulla (1/32) and in the cortex (1/32; Fig. 2). Although the risk is low, it nevertheless exists and therefore warrants further investigation (1). GYNECOLOGIC CANCER Breast Cancer Breast cancer is the most common malignancy in women. Studies estimate that there were around 230,000 new invasive breast cancer cases in the United States female population in 2011, and 5% were in women under the age of 40 years at the time of diagnosis (53). The incidence of ovarian metastasis in breast cancer patients can vary from 13.2% to 37.8% (7, 54), but is more commonly observed in case of advanced-stage breast cancer (55, 56). Sanchez et al. (57) and Rosendhal et al. (58) analyzed frozen-thawed ovarian cortical biopsies from breast cancer patients. Neither study revealed any evidence of malignant cell infiltration of the cryopreserved ovarian tissue by histology or immunohistochemistry. Autotransplantation of frozen-thawed ovarian fragments appears to be safe in patients with low-stage breast cancers, as demonstrated in the series of Donnez et al. (2, 45). In advanced-stage breast cancers, however, the risk of ovarian metastases cannot be excluded. One patient suffered a local relapse at the site of the original breast tumor 1 year after autotransplantation of ovarian cortex, but there was no sign of metastatic activity in the ovarian graft (11, 58). In conclusion, although these studies are so far reassuring, they nevertheless demonstrate that further procedures, such as PCR and long-term xenografting, are necessary to prove the safety of frozen-thawed ovarian tissue transplantation in women with breast cancer. TABLE 2 Experimental studies on the risk of transmitting malignant cells in case of lymphoma. n Histology IHC Xenografting Autografting (patients) HL Meirow et al. (29) 7 Neg Kim et al. (30) 13 Neg 16 wk (no relapse) Seshadri et al. (31) 26 Neg Neg Meirow et al. (3) 33 Neg Neg (n ¼ 1) 1 (no relapse) NHL Meirow et al. (3) 14 Neg 2 (no relapse) Kim et al. (30) 5 Neg 16 wk (no relapse) Note: Experiments are divided into two groups, depending on whether ovarian tissue from Hodgkin lymphoma (HL) or non-hodgkin lymphoma (NHL) patients was used. All studies used histology, and some were completed by the addition of either immunohistochemistry or xenografting experiments. One study reported autografting results VOL. 99 NO. 6 / MAY 2013

6 Fertility and Sterility FIGURE 2 Ovarian fragments from a non-hodgkin lymphoma (NHL) patient. (A) Immunohistochemistry anti-cd20 (against NHL cells) is negative in the cortex (blue staining) and positive in the medulla (brown staining). Magnification of the white rectangle is represented in (B) and magnification of the black rectangle in (C). (B) Cells with an enlarged nucleus and patchy chromatin are disseminated in the tissue and stain strongly for anti-cd20. (C) Piece of cancer cell-free cortex. Follicles present in the cortex appear healthy. (D) Another fragment from the same patient shows massive invasion by NHL cells in the cortex. Cervical Carcinoma Reports on ovarian tissue autotransplantation after cervical carcinoma have been published by Kim et al. (59 61). Three patients with cervical cancer had their tissue heterotopically transplanted, the first of whom suffered a pelvic relapse and died soon after transplantation (61). Schmidt et al. (42) reported autotransplantation of cryopreserved ovarian tissue in a woman with cervical cancer, and Donnez et al. (2) also performed reimplantation in a patient cured of squamous cell cervical carcinoma (follow-up 1 year). No sign of relapse has so far been noted in either of these two clinical cases. It is well known that ovarian involvement is more frequent in adenocarcinoma than in squamous cell carcinoma. Ovarian metastasis has been reported in 0.7% 2.5% of patients with squamous cell carcinoma and in 0 6.8% of patients with adenocarcinoma of the cervix (62, 63). Endometrial Carcinoma According to histologic studies, the risk of ovarian metastasis with endometrial cancer ranges from 1.9% (FIGO stage I tumors) (64) to 41.7% (FIGO stage I III tumors) (65). COLORECTAL CANCER Dittrich et al. (66) described a patient with anal cancer (stage not mentioned) who underwent ovarian tissue transplantation, in whom no malignant cells were detected in grafts. However, metastases from colorectal cancer to the ovaries have been established in many autopsy studies. Kyono et al. reported a frequency varying from 16.7% to 31.1%, depending on the patient's age (7). CENTRAL NERVOUS SYSTEM TUMORS Sonmezer and Oktay (4) classified neuroblastoma together with leukemia in the high-risk category for ovarian involvement. Primitive neurectodermal tumors (PNETs), including medulloblastoma (the most common) and neuroblastoma, generally occur in children and are classified into two types: peripheral PNET (Ewing sarcoma) and central nervous system PNET. Donnez et al. (67) performed reimplantation of frozenthawed ovarian tissue in a patient cured of a metastatic neurectodermal tumor of the orbit, who had her tissue cryopreserved before BMT. Histologic evaluation of an ovarian sample before transplantation did not reveal ovarian metastasis, and the patient has now delivered her third child. The Danish team performed transplantation of cryopreserved ovarian tissue in a patient treated for Ewing sarcoma (42, 68), who subsequently delivered a healthy baby twice. Other studies reporting histologic analyses of ovarian tissue from Ewing sarcoma patients did not find any sign of metastases (52, 69). Abir et al. (70) analyzed ovarian samples from eight patients with Ewing sarcoma by histology, VOL. 99 NO. 6 / MAY

7 VIEWS AND REVIEWS TABLE 3 Risk of ovarian metastasis according to cancer type. High risk Moderate risk Low risk Leukemia Breast cancer Breast cancer Stage IV Stage I II Infiltrating lobular subtype Infiltrating ductal subtype Neuroblastoma Colon cancer Squamous cell carcinoma of the cervix Burkitt lymphoma Adenocarcinoma of the Hodgkin's lymphoma cervix Non-Hodgkin lymphoma Osteogenic carcinoma Ewing sarcoma Nongenital rhabdomyosarcoma Wilms tumor Note: Adapted from Sonmezer and Oktay (4) and modified according to the recent literature: Ewing sarcoma and NHL were recategorized from low to moderate risk. immunohistochemistry, and RT-PCR. There was no evidence of the disease in ovaries from pathologic studies (n ¼ 7), but in one patient out of five, RT-PCR proved positive for the Ewing sarcoma translocation. Poirot et al. (69) found no sign of ovarian metastases in their histologic studies of ovarian tissue from patients with Ewing sarcoma, medulloblastoma, or neuroblastoma. Nevertheless, case reports and autopsy data indicated that ovarian involvement does occur in case of neuroblastoma. SARCOMA Rosendahl et al. (71) xenotransplanted ovarian tissue from five patients with sarcoma and no tumor invasion was detected. Histologic analyses of ovarian tissue aiming to identify malignant cells in tissue from patients with bone sarcoma, osteosarcoma, and rhabdomyosarcoma are reassuring (52, 69). It should, however, be pointed out that some patients in these studies had already received chemotherapy before ovarian tissue cryopreservation. CONCLUSION Cryopreservation of ovarian tissue followed by orthotopic reimplantation is the option of choice to restore fertility in women requiring immediate chemotherapy and in prepubertal patients. This review analyzed the presence of malignant cells in ovarian tissue that could lead to recurrence of the primary disease after reimplantation. Sonmezer and Oktay (4) classified malignant diseases into three categories, representing low, intermediate, and high risk of ovarian involvement, which we slightly modified according to the recent literature, recategorizing Ewing sarcoma and NHL from low to moderate risk (Table 3). Among the different pathologies investigated, the highest risk of reimplanting malignant cells was found with leukemia. However, taking into account the Belgian and Danish data, the risk is likely to be very low if the ovarian cryopreservation procedure is carried out in women in complete remission. We strongly suggest that ovarian autotransplantation be discouraged when there is a risk of reimplanting malignant cells, especially in leukemia patients. Molecular markers are available in case of CML, but not always in case of ALL. In the other pathologies, the risk is low, but the authors stress the need to discuss it with patients before reimplantation. Acknowledgments: The authors thank Mira Hryniuk, B.A., for reviewing the English language of the manuscript and Anne Lepage, unit secretary, for her administrative assistance. REFERENCES 1. Dolmans MM, Jadoul P, Gilliaux S, Amorim AC, Luyckx V, Squifflet J, et al. A review of 15 years of ovarian tissue bank activities. J Assist Reprod Genet 2013; 2. Donnez J, Dolmans M-M, Pellicer A, Diaz-Garcia C, Sanchez Serrano M, Schmidt KT, et al. Restoration of ovarian activity and pregnancy after transplantation of cryopreserved ovarian tissue: a review of 60 cases of reimplantation. Fertil Steril 2013;99: Meirow D, Hardan I, Dor J, Fridman E, Elizur S, Ra'anani H, et al. Searching for evidence of disease and malignant cell contamination in ovarian tissue stored from hematologic cancer patients. Hum Reprod 2008;23: Sonmezer M, Oktay K. Fertility preservation in female patients [review]. Hum Reprod Update 2004;10: Jahnukainen K, Hou M, Petersen C, Setchell B, S oder O. Intratesticular transplantation of testicular cells from leukemic rats causes transmission of leukemia. Cancer Res 2001;61: Dolmans MM, Michaux N, Camboni A, Martinez-Madrid B, van Langendonckt A, Nottola S, et al. Evaluation of Liberase, a purified enzyme blend, for the isolation of human primordial and primary ovarian follicles. Hum Reprod 2006;21: Kyono K, Doshida M, Toya M, Sato Y, Akahira J, Sasano H. Potential indications for ovarian autotransplantation based on the analysis of 5571 autopsy finding of females under the age of 40 in Japan. Fertil Steril 2010;1: Slasky BS, Straub WH, Deutsch M. Acute lymphocytic leukemia of the ovary: the value of sonography. J Comput Tomogr 1982;6: Dolmans MM, Marinescu C, Saussoy P, van Langendonckt A, Amorim C, Donnez J. Reimplantation of cryopreserved ovarian tissue from patients with acute lymphoblastic leukemia is potentially unsafe. Blood 2010;116: Rosendahl M, Andersen MT, Ralfkiaer E, Kjeldsen L, Andersen MK, Andersen CY. Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia. Fertil Steril 2010;94: Rosendahl M, Greve T, Andersen CY. The safety of transplanting cryopreserved ovarian tissue in cancer patients: a review of the literature. J Assist Reprod Genet 2013;30: Greve T, Clasen-Linde E, Andersen MT, Andersen MK, Rosendahl M, et al. No signs of viable malignant cells in frozen-thawed ovarian cortex intended 1520 VOL. 99 NO. 6 / MAY 2013

8 Fertility and Sterility for fertility preservation from patients with leukaemia. Blood 2012;22: Gabert J, Beillard E, van der Velden VH, Bi W, Grimwade D, Pallisgaard N, et al. Standardization and quality control studies of real-time quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia a Europe Against Cancer program. Leukemia 2003;17: van der Velden VHJ, Cazzaniga G, Schrauder A, Hancock J, Bader P, Panzer- Grumayer ER, et al, European Study Group on MRD Detection in ALL. Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data. Leukemia 2007;21: Pui CH, Relling MV, Downing JR. Acute lymphoblastic leukemia. N Engl J Med 2004;350: Hoffman R, Furie B, Benz EJ, McGlave P, Silberstein LE, Shattil SJ. Hematology: basic principles and practice. 5th ed. Philadelphia: Elsevier; van Dongen JJ, Langerak AW, Br uggemann M, Evans PA, Hummel M, Lavender FL, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT Leukemia 2003;17: Jadoul P, Anckaert E, Dewandeleer A, Steffens M, Dolmans MM, Vermylen C, et al. Clinical and biologic evaluation of ovarian function in women treated by bone marrow transplantation for various indications during childhood or adolescence. Fertil Steril 2011;96: Borgmann-StaudtA, RendtorffR, Reinmuth S, Hohmann C, KeilT, SchusterFR, et al. Fertility after allogeneic haematopoietic stem cell transplantation in childhood and adolescence. Bone Marrow Transplant 2012;47: Jahnukainen K, Tinkanen H, Wikstr om A, Dunkel L, Saarinen-Pihkala UM, M akinen S, et al. Bone marrow remission status predicts leukemia contamination in ovarian biopsies collected for fertility preservation. Leukemia 2012; Andolina JR, Neudorf SM, Corey SJ. How I treat childhood CML. Blood 2012; 119: Goldman JM. How I treat chronic myeloid leukemia in the imatinib era. Blood 2007;110: Woodruff TK. Preserving fertility during cancer treatment. Nat Med 2009; 15: Pye SM, Cortes J, Ault P, Hatfield A, Kantarjian H, Pilot R, et al. The effects of imatinib on pregnancy outcome. Blood 2008;111: Courbiere B, Prebet T, Mozziconacci MJ, Metzler-Guillemain C, Saias- Magnan J, Gamerre M. Tumor cell contamination in ovarian tissue cryopreserved before gonadotoxic treatment: should we systematically exclude ovarian autograft in a cancer survivor? Bone Marrow Transplant 2010;45: Dolmans MM. Safety of ovarian autotransplantation. Blood 2012;120: Donnez J, Dolmans MM. Preservation of fertility in females with haematological malignancy. Br J Haematol 2011;154: Shaw JM, Bowles J, Koopman P, Wood EC, Trounson AO. Fresh and cryopreserved ovarian tissue samples from donors with lymphoma transmit the cancer to grafts recipients. Hum Reprod 1996;11: Meirow D, Ben Yehuda D, Prus D, et al. Ovarian tissue banking in patients with Hodgkin's disease: is it safe? Fertil Steril 1998;69: Kim SS, Radford J, Harris M, et al. Ovarian tissue harvested from lymphoma patients to preserve fertility may be safe for autotransplantation. Hum Reprod 2001;16: Seshadri T, Gook D, Lade S, et al. Lack of evidence of disease contamination in ovarian tissue harvested for cryopreservation from patients with Hodgkin lymphoma and analysis of factors predictive of oocyte yield. Br J Cancer 2006;94: Bittinger SE, Nazaretian SP, Good DA, Parmar C, Harrup RA, Stern CJ. Detection of Hodgkin lymphoma within ovarian tissue. Fertil Steril 2011;95: 803.e Radford JA, Lieberman BA, Brison DR, Smith AR, Critchlow JD, Russell SA, et al. Orthotopic reimplantation of cryopreserved ovarian cortical strips after high-dose chemotherapy for Hodgkin's lymphoma. Lancet 2001;357: Donnez J, Dolmans MM, Demylle D, Jadoul P, Pirard C, Squifflet J, et al. Livebirth after orthotopic transplantation of cryopreserved ovarian tissue. Lancet 2004;364: Schmidt KL, Andersen CY, Loft A, Byskov AG, Ernst E, Andersen AN. Followup of ovarian function post-chemotherapy following ovarian cryopreservation and transplantation. Hum Reprod 2005;20: Demeestere I, Simon P, Buxant F, Robin V, Fernandez SA, Centner J, et al. Ovarian function and spontaneous pregnancy after combined heterotopic and orthotopic cryopreserved ovarian tissue transplantation in a patient previously treated with bone marrow transplantation: case report. Hum Reprod 2006;21: Rosendahl M, Loft A, Byskov AG, Ziebe S, Schmidt KT, Andersen AN, et al. Biochemical pregnancy after fertilization of an oocyte aspirated from a heterotopic autotransplant of cryopreserved ovarian tissue: case report. Hum Reprod 2006;21: Demeestere 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: Donnez J, Squifflet J, van Eyck AS, Demylle D, Jadoul P, van Langendonckt A, Dolmans MM. Restoration of ovarian function in orthotopically transplanted cryopreserved ovarian tissue: a pilot experience. Reprod Biomed Online 2008;16: Sanchez M, Novella-Maestre E, Teruel J, Ortiz E, Pellicer A. The Valencia programme for fertility preservation. Clin Transl Oncol 2008;10: Dolmans MM, Donnez J, Camboni A, Demylle D, Amorim C, van Langendonckt A, et al. IVF outcome in patients with orthotopically transplanted ovarian tissue. Hum Reprod 2009;24: Schmidt KT, Rosendahl M, Ernst E, Loft A, Andersen AN, Dueholm M, et al. Autotransplantation of cryopreserved ovarian tissue in 12 women with chemotherapy-induced premature ovarian failure: the Danish experience. Fertil Steril 2011;95: Andersen CY, Rosendahl M, Byskov AG, et al. Two successful pregnancies following autotransplantation of frozen/thawed ovarian tissue. Hum Reprod 2008;23: Dittrich R, Lotz L, Keck G, Hoffmann I, Mueller A, Beckmann MW, et al. Live birth after ovarian tissue autotransplantation following overnight transportation before cryopreservation. Fertil Steril 2012;97: Donnez J, Silber S, Andersen CY, Demeestere I, Piver P, Meirow D, et al. Children born after autotransplantation of cryopreserved ovarian tissue. a review of 13 live births. Ann Med 2011;43: Donnez J, Jadoul P, Pirard C, Hutchings G, Demylle D, Squifflet J, et al. Live birth after transplantation of frozen-thawed ovarian tissue after bilateral oophorectomy for benign disease. Fertil Steril 2012;98: Akar ME, Carrillo AJ, Jennell JL, Yalcinkaya TM. Roboticassisted laparoscopic ovarian tissue transplantation. Fertil Steril 2011;95: Meirow 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: Meirow D, Baum M, Yaron R, Levron J, Hardan I, Schiff E, et al. Ovarian tissue cryopreservation in hematologic malignancy: ten years' experience. Leuk Lymphoma 2007;48: Stern CJ, Toledo MG, Hale LG, Gook DA, Edgar DH. The first Australian experience of heterotopic grafting of cryopreserved ovarian tissue: evidence of establishment of normal ovarian function. Aust N Z J Obstet Gynaecol 2011; 51: Tryde Schmidt KL, Yding AC, Starup J, Loft A, Byskov AG, Nyboe AA. Orthotopic autotransplantation of cryopreserved ovarian tissue to a woman cured of cancer follicular growth, steroid production and oocyte retrieval. Reprod Biomed Online 2004;8: Azem 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: American Cancer Society. Breast Cancer Facts and Figures Available at: breastcancerfactsfigures/breast-cancer-facts-and-figures Last accessed April 17, VOL. 99 NO. 6 / MAY

9 VIEWS AND REVIEWS 54. Perrotin 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: Gagnon Y, T^etu B. Ovarian metastases of breast carcinoma. A clinicopathologic study of 59 cases. Cancer 1989;64: Li CI, Anderson BO, Daling JR, Moe RE. Trends in incidence rates of invasive lobular and ductal breast carcinoma. JAMA 2003;289: Sanchez-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: Rosendahl M, Timmermans Wielenga V, Nedergaard L, Kristensen SG, Ernst E, et al. Cryopreservation of ovarian tissue for fertility preservation: no evidence of malignant cell contamination in ovarian tissue from patients with breast cancer. Fertil Steril 2011;95: Kim SS, Hwang IT, Lee HC. Heterotopic autotransplantation of cryobanked human ovarian tissue as a strategy to restore ovarian function. Fertil Steril 2004;82: Kim SS, Lee WS, Chung MK, Lee HC, Lee HH, Hill D. Long-term ovarian function and fertility after heterotopic autotransplantation of cryobanked human ovarian tissue: 8-year experience in cancer patients. Fertil Steril 2009; 91: Kim SS. Asssessment of long term endocrine function after transplantation of frozen-thawed ovarian tissue to the heterotopic site: 10 year longitudinal follow-up study. J Assist Reprod Genet 2012;29: Morice P, Haie-Meder C, Pautier P, Lhomme C, Castaigne D. Ovarian metastasis on transposed ovary in patients treated for squamous cell carcinoma of the uterine cervix: report of two cases and surgical implications. Gynecol Oncol 2001;83: Nakanishi T, Wakai K, Ishikawa H, Nawa A, Suzuki Y, Nakamura S, et al. A comparison of ovarian metastasis between squamous cell carcinoma and adenocarcinoma of the uterine cervix. Gynecol Oncol 2001;82: Pan Z, Wang X, Zhang X, Chen X, Xie X. Retrospective analysis on coexisting ovarian cancer in 976 patients with clinical stage I endometrial carcinoma. J Obstet Gynaecol Res 2011;37: Dundar E, Tel N, Ozalp SS, Isiksoy S, Kabukcuoglu S, Bal C. The significance of local cellular immune response of women 50 years of age and younger with endometrial carcinoma. Eur J Gynaecol Oncol 2002;23: Dittrich R, Mueller A, Maltaris T, Hoffmann I, Magener A, Oppelt P, et al. Hormonal and histologic findings in human cryopreserved ovarian autografts. Fertil Steril 2009;91: Donnez J, Squifflet J, Jadoul P, Demylle D, Cheron AC, van Langendonckt A, et al. Pregnancy and live birth after autotransplantation of frozen-thawed ovarian tissue in a patient with metastatic disease undergoing chemotherapy and hematopoietic stem cell transplantation. Fertil Steril 2011;95: 1787.e Ernst E, Bergholdt S, Jørgensen JS, Andersen CY. The first woman to give birth to two children following transplantation of frozen/thawed ovarian tissue. Hum Reprod 2010;25: Poirot CJ, Martelli H, Genestie C, Golmard JL, Valteau-Couanet D, Helardot P, et al. Feasibility of ovarian tissue cryopreservation for prepubertal females with cancer. Pediatr Blood Cancer 2007;49: Abir R, Feinmesser M, Yaniv I, Fisch B, Cohen IJ, Ben-Haroush A, et al. Occasional involvement of the ovary in Ewing sarcoma. Hum Reprod 2010;25: Rosendahl M, Schmidt KT, Ernst E, Rasmussen PE, Loft A, Byskov AG, et al. Cryopreservation of ovarian tissue for a decade in Denmark an overview of the technique. Reprod Med Online 2011;22: VOL. 99 NO. 6 / MAY 2013