1 Centre for Reproduction, Growth and Development, University of Leeds, Leeds, 2 Department of Medical Oncology, 3 Department of Histopathology, Christie Hospital NHS Trust, Manchester, 4 Department of Cancer Genetics, Paterson Institute for Cancer Research, Manchester, 5 Department of Reproductive Medicine, St. Mary's Hospital, Manchester and 6 Department of Endocrinology, Christie Hospital NHS Trust, Manchester, UK
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Abstract |
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Key words: cryopreservation/fertility/lymphoma/transplantation/xenografting
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Introduction |
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A new strategy, involving autotransplantation of ovarian cortical slices banked at low temperatures (Gosden et al., 1994), offers the possibility of reinstating fertility in women and children after sterilizing treatment. In addition, this approach may be helpful for women recovering fertility temporarily but who are expected to undergo therapy-related premature menopause. In either circumstance, the endocrine effects of autotransplanted ovarian tissue could be advantageous, and avoid the need for simultaneous hormone replacement therapy. Studies in sheep have demonstrated that frozenthawed and grafted ovarian tissue can restore ovulatory cycles (Baird et al., 1999
), and a single case report has provided proof of principle in humans (Oktay and Karlikaya, 2000
).
Before proceeding to clinical applications, it is vital to test whether ovarian tissue harvested from cancer patients harbours residual disease which could be transmitted by autotransplantation. This theoretical risk has been highlighted in a study of AKR strain mice susceptible to lymphoma (Shaw et al, 1996), where the disease was transmitted by grafts to healthy recipients. We have addressed the safety issue in humans by xenografting ovarian tissue from patients with lymphoma to immunodeficient mice.
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Materials and methods |
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Preparation of Positive Controls
For NHL controls, a disease-positive lymph node from a patient with recurrent follicular B-cell lymphoma was processed and cryopreserved using the same protocol.
For HL controls, the cell line L1236 derived from Hodgkin's disease tissue was cultured in RPMI 1640 medium (Sigma) supplemented with 10% heat inactivated fetal calf serum, 50 IU/ml penicillin, 50 µg/ml streptomycin and 4 mmol/l L-glutamine in a 5% CO2 atmosphere at 37°C. The total cultured tumour cells (45x106) were resuspended in 2500 µl of phosphate-buffered saline (PBS) and prepared into five aliquots (9x106 cells in 500 µl PBS) for inoculation.
Xenografting
Non-obese diabetic severe combined immunodeficient NOD/LtSzSCID female mice obtained from Jackson Laboratory, Bar Harbor, ME, USA were housed in sterile, positive pressure isolators with sterilized food and water. Prophylactic supplements of 2 mg/ml tetracycline were added to drinking water starting 2 days prior to surgery. At 12 weeks of age, 30 animals were anaesthetized by inhalation of an oxygen/halothane mixture. A 1 cm dorsolateral skin incision was made for s.c. inserting one or two pieces of human ovarian tissue. Tissue was anchored to muscle by suturing with non-absorbable suture (6.0 Prolene) and covered with 0.2 ml of Matrigel (10 mg/ml) (Collaborative Research, Bethesda, MD, USA) to stimulate adhesion of tumour cells. The skin incision was closed with Michel clips. Ovarian tissue from 13 patients with HL was grafted into 20 animals and ovarian tissue from 5 patients with NHL was grafted into 10 animals.
For NHL controls, 3 animals were grafted with lymph node tissue in the same manner. For HL controls, viable cells (9x106) from cultured cell line L1236 mixed with 0.2 ml Matrigel were inoculated s.c. into the flanks of 5 animals with a 26 gauge needle. Post-operatively, the animals were monitored daily and any becoming cachectic were euthanized and autopsied. At 16 weeks post-surgery the animals were killed by CO2 inhalation. During a careful autopsy, the graft was removed for study, and specimens were taken from liver, spleen, sternum, para-aortic lymph nodes and thymus.
Histology and immunohistochemistry
Tissue from the graft sites and host organs was fixed in 4% paraformaldehyde and processed as 4 µm wax sections stained with haematoxylin and eosin. Selected tissues were stained immunohistochemically using the streptavidin ABC/HRP method (DAKO Ltd., Ely, UK) with the following antibodies: anti-CD3 for T-cell expression; anti-CD79a and anti-CD20/L26 for B-cell expression; anti-CD30/Ber-H2 (DAKO Ltd) and anti-CD15/Leu M1 (Becton Dickinson, Oxford, UK) for monocyte expression (particularly in HL).
Screening for human microsatellite DNA
DNA was prepared from 10 µm sections using procedures previously described (Varley et al., 1997). PCR using primers for human microsatellite DNA sequences were carried out using standard procedures incorporating 32P-dCTP, and the products were visualized by autoradiography after gel electrophoresis. The microsatellites analysed were D2S123, D3S1076, D6S292, D8S255, D9S162, D9S166 and D13S175.
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Results |
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Microscopic observations
Light microscopy showed no evidence of malignant cells in ovarian tissues prior to transplantation. All animals grafted with human ovarian tissues had evidence of spontaneous (murine) lymphoblastic lymphoma in their thymuses (Figure 1a,b), and in eight animals it had spread to one or more of the following: sternal marrow, spleen, liver and para-aortic lymph nodes. Immunohistochemistry of representative samples of these lesions showed that the lymphoma was T-cell derived (CD3+, CD79a, CD20, CD15, CD30).
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Discussion |
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This is the first reported in-vivo investigation of residual disease in human ovarian tissue using a xenograft. Grafting human cells into immunodeficient mice is a well-established method for studying haematological cancer (Greiner et al., 1995), and this model may help to inform clinical decisions about the risks of autotransplantation. The patients were deliberately selected for having high-risk disease and, in the three years following tissue harvesting, nine (50%) have died of lymphoma (HL 6; NHL 3). Thus, the study was based on worse-case scenarios in which the lymphomas were highly aggressive and likely to be disseminated at the time of tissue harvest, possibly including the ovaries. In fact, ovarian cortical strips were re-implanted into patients 11 and 13 (Table I), and both remain disease-free at the time of writing, one year later (Radford et al., 2001
).
None of the animals xenografted with human ovarian tissue from 13 patients with HL developed signs of the disease. Neither was there any microscopic evidence of residual disease in ovarian tissue prior to grafting, nor in any other tissues studied. Equally, the result with NHL was reassuring. There was no sign of human lymphoma in the animals xenografted with ovarian tissue from 5 NHL patients. In contrast, all 3 control mice grafted with human lymph node tissue from a patient with follicular lymphoma developed B-cell lymphoma of human origin, which was confirmed unequivocally by microsatellite DNA analysis. Our results give hope that ovaries from HL or NHL patients can be safely autotransplanted at the completion of treatment.
One of the most difficult parts of this study was selection of the specimen for the HL control, because the characteristic Hodgkin/Reed-Sternberg (H-RS) cells represent only a minor population in the tumour (<1%) (Kanzler et al., 1996). Therefore, propagation of malignant cells by engraftment or in-vitro culture is very difficult. Among 14 Hodgkin's disease derived cell lines, only a cell line L1236 has been proven by PCR for Ig gene rearrangement to derive from primary H-RS cells (Wolf et al., 1996
). Although it has been reported that SCID mice support growth of Hodgkin's disease derived cell lines, the frequency of transmission is only ~40% after 4 months observation (Kalle et al., 1992
). In our study, none of the 5 animals injected with L1236 cells developed the human disease. This may be due to a limited observation period (16 weeks) or a lower number of injected cells (9x106) compared with other studies (Kalle et al., 1992
; Kapp et al., 1994
).
This study introduces the NOD/LtSz-SCID mouse as a model for investigating the safety of ovarian transplantation, serving as a preclinical screen for disease. Whilst it has focused on lymphoma, the model could serve for other diseases where it is established that human tumour tissue survives and proliferates in vivo. The maximum period of study is, however, constrained by the short life span of the mice. It has been reported that over 80% of female NOD/LtSz-SCID mice have developed lethal thymic lymphomas by 20 weeks of age (Serreze et al., 1995), and this was consistent with our findings. Xenografting provides a functional test which is complementary to evidence from histopathology or PCR.
Our findings are of immediate relevance to women with lymphoma who are considering banking ovarian tissue for fertility conservation, as well as to their physicians who need to provide them with appropriate advice. We can now be more optimistic about the application of ovarian cryopreservation and transplantation in cancer patients, but these reassuring findings should not be interpreted as an absolute indication of safety. Hence, efforts to develop new techniques to detect residual disease need to be continued. In the long-term, it is hoped that oocytes in primordial follicles can be safely matured in vitro, since the zona pellucida provides a barrier to somatic cells. This technology is still at an early stage, even in animals (Eppig and O'Brien, 1996), yet it may be available for young females who will not require their stored tissue for a decade or more.
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Acknowledgements |
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Notes |
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8 To whom correspondence should be addressed at: University of Washington, Department of Obstetrics and Gynecology,4225 Roosevelt Way N.E., Suite 305, Seattle WA 98105, USA. E-mail: medssk{at}att.net
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References |
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Submitted on February 9, 2001; accepted on June 26, 2001.