An in vitro model for purging of tumour cells from ovarian tissue

C.P. Schröder1, H. Timmer-Bosscha1, J.G. Wijchman2, L.F.M.H. de Leij3, H. Hollema3, M.J. Heineman2 and E.G.E. de Vries1,4

Department of 1 Medical Oncology, 2 Obstetrics and Gynaecology, and 3 Pathology and Laboratory Medicine, University Hospital, Groningen, The Netherlands

4 To whom correspondence should be addressed at: Department of Medical Oncology, University Hospital Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands. e-mail: e.g.e.de.vries{at}int.azg.nl


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Cryopreservation and autografting of ovarian tissue may preserve fertility after cancer treatment, but could be hampered by tumour cell contamination. Epithelial tumour cell lysis can be obtained with cytotoxic T cell retargeting through the bispecific antibody BIS-1, with combined affinity for the T-cell receptor and epithelial glycoprotein-2 (EGP-2). Our aim was to study the concept of tumour cell purging in the setting of a suspension of ovarian tissue. METHODS: Human ovarian tissue was brought into suspension by mechanical and enzymatic treatment. Cells of the MCF-7 breast cancer cell line and activated human lymphocytes were co- incubated for 4 h with or without BIS-1 and with or without ovarian suspension. After incubation, MCF-7 cell death and cell growth were evaluated by fluorescent cell detection and MTT assay, respectively. Ovarian tissue morphology was evaluated immunohistochemically. RESULTS: MCF-7 cell death and cell growth inhibition increased with increasing ratios of lymphocytes to MCF-7 cells. BIS-1 addition gave further augmentation, with a maximum depletion of growing MCF-7 cells of 89% (SD 11%) versus without BIS-1, 23% (SD 15%; P < 0.001). Follicles remained morphologically intact. CONCLUSIONS: Purging of added epithelial tumour cells from ovarian tissue with BIS-1 is possible in vitro. Morphologically, follicles remain intact after this procedure. This method may contribute to safer replacement of ovarian tissue in female cancer survivors.

Key words: cancer/fertility/ovary/tumour cell lysis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In females, chemotherapy and/or radiotherapy for the treatment of cancer can cause a reduction of the follicle population within the ovaries, which can lead to a premature menopause (Bokemeyer et al., 1994Go; Bonadonna and Valagussa, 1995Go; Cobleigh et al., 1995Go; Swerdlow et al., 1996Go; Hensley and Reichmen, 1998Go; Burstein and Winer, 2000Go). The cryopreservation of ovarian tissue obtained before cancer therapy is a promising new method for conserving the fertility of these cancer patients (Aubard et al., 2000Go). In animal studies, the transplantation of frozen–thawed ovarian autografts has led to a resumption of endocrine function and the restoration of fertility (Candy et al., 1994Go, 1995; Gosden et al., 1994Go; Harp et al., 1994Go; Baird et al., 1999Go; Dobson, 1999Go; Oktay et al., 2000Go; Wang et al., 2002Go). The successful re-transplantation of cryopreserved ovarian tissue has been described in a patient with Hodgkin’s lymphoma (after high-dose chemotherapy; Radford et al., 2001Go). Although safe transplantation of ovarian tissue from lymphoma patients has been reported in SCID mice (Kim et al., 2001bGo), the possibility of reintroducing tumour cells in cancer patients by autografting of ovarian tissue cannot be excluded (Shaw et al., 1996Go; Meirow, 1999Go).

Purging, or clearing of minor quantities of tumour cells has been described in the haematopoietic stem cell transplantation setting (Champlin, 1996Go; Kvalheim et al., 1996Go), but not for solid (tumour) tissue. Instead of ovarian cortex, a suspension of primordial or primary follicles may be autografted in plasma clots. This approach has restored estrogenic activity and fertility in a mouse model (Carroll et al., 1990Go; Newton, 1998Go). Possibly, making a suspension of follicles may allow purging of unwanted cell types in a similar way as for haematopoietic stem cells. A purging method, developed for haematopoietic stem cells by our institution, resulted in >99.9% tumour cell death without affecting colony formation by the stem cells (Schröder et al., 2000Go). This method makes use of the bispecific antibody BIS-1, directed against the epithelial-related membrane antigen epithelial glycoprotein-2 [EGP-2, widely expressed on breast and ovarian carcinomas (De Leij et al., 1998Go; Kroesen et al., 1998Go)], and CD3 on T lymphocytes. BIS-1 creates functional cross-linking of T cells and tumour cells, allowing the delivery of a tumour cell-specific lethal hit inducing specific epithelial tumour cell death in vitro and in vivo (Kroesen et al., 1993Go, 1994). This study was conducted to evaluate the concept of purging in the setting of a suspension of ovarian tissue.

Therefore, tumour cell death and morphological follicle survival were studied in an in vitro model in which activated lymphocytes and BIS-1 were added to the breast cancer cell line MCF-7, in the presence or absence of a suspension of human frozen–thawed ovarian tissue. Two interactions were studied: (i) the effect of the purging process on the follicles; and (ii) the effect of the presence of an ovarian tissue suspension on the lymphocyte–tumour interaction, to determine whether this would reduce lymphocyte activity.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ovarian tissue
Freezing procedure. Human ovarian tissue, obtained with laparoscopy, was frozen from eligible patients from 1998 onwards. The freezing procedure of ovarian tissue for a possible transplantation was considered part of the regular patient care by the Medical Ethical Committee of our institution; the use of ovarian tissue for the in vitro purging procedure (as described below), in case of the death of the patient prior to possible transplantation, was approved by the Medical Ethical Committee. All patients gave informed consent. The freezing and thawing procedure was performed as described by Gosden’s group (Oktay et al., 2000Go). Briefly, after collection in sterile, buffered medium, the ovary was cut into two parts under sterile conditions. One part was fixed in buffered formalin and embedded in paraffin, after which sections for standard haematoxylin–eosin (HE) staining were cut; the other part was used for preparation of the ovarian cortex. Pieces of the cortex of ~x 3 mm, 1 mm thickness were incubated for 30 min in Leibovitz L15 medium (Life Technologies, Paisley, UK) containing 10% autologous patient serum and cryoprotecting agents [1.5 mol/l dimethylsulfoxide (DMSO) and 0.1 mol/l sucrose]. Thereafter, they were cooled to –140°C in a programmable freezer (Planer Kryo 10, series II; cooled at –2°C/min to –9°C, seeded manually at –9°C; cooled at –0.3°C/min to –40°C; and cooled at –10°C/min to –140°C). Finally, the pieces were stored in 1.5 ml vials in liquid nitrogen.

Thawing and suspension procedure. Ovarian tissue was thawed in a water bath at 37°C for 2 min maximum, and washed in a diminishing sequence of DMSO in Leibovitz medium with 10% fetal calf serum (FCS; Life Technologies). In order to obtain a cell suspension suitable for tumour cell purging, ovarian tissue was treated further in two steps. First, the tissue was mechanically dissected into a suspension with 27-gauge needles under sterile conditions. Then, enzymatic treatment was performed in medium containing 10 U/ml DNase I (Sigma-Aldrich, Zwijndrecht, The Netherlands) and 300 U/ml collagenase IA (Sigma-Aldrich) for 2 h at 37°C. A combination of enzymatic treatment and microdissection has been used previously to suspend ovarian tissue (Oktay et al., 1997Go). The treatment appeared not to be toxic to tumour cells. The obtained cell suspension was transferred into RPMI medium (Life Technologies) with 10% FCS. Next, 100 µl of the cell suspension, was distributed into each well of a 96-well microtitre plate (Nunclon, Roskilde, Denmark). After overnight culture, the wells were inspected with an inverted phase-contrast microscope. In five independent experiments, the wells in which microscopically intact follicles were observed were counted. As the ovarian tissue suspension consisted of monocellular (stroma) cells and clumps of cells after enzymatic digestion, cell numbers were not standardized before purging. For wells in which follicles were present, the ovarian tissue suspension was carefully pipetted out and put into another 96-well plate in which chloromethyl fluorescein diacetate (CMFDA; Molecular Probes Europe BV, Leiden, The Netherlands)-labelled tumour cells were already present. These wells were used for purging experiments as described below.

Effector and target cells; bispecific antibody
Target cells: fluorescent detection system. The MCF-7 breast cancer cell line was used as a EGP-2-positive tumour model. Cells were plated in microtitre plates (Nunclon) and cultured overnight for optimal adhesion. Cells were labelled with the fluorescent dye CMFDA for 30 min. The optimal CMFDA concentration was established using concentrations from 0.5 to 15 µmol/l.

Cell detection was adequate at 1.5 µmol/l, without signs of cytotoxicity, and therefore this CMFDA concentration was used in subsequent experiments.

Effector cells. Lymphocytes were obtained from peripheral blood of healthy volunteers by Lymphoprep (Nycomed Pharma AS, Oslo, Norway) isolation. They were washed and incubated in vitro with anti-CD3 antibody (at 0.5 µg of IgG/ml RPMI medium with 10% FCS) for 72 h, and then washed and subsequently cultured in the presence of recombinant human interleukin-2 (rh IL-2; Aldesleukin, Chiron, Amsterdam, The Netherlands) at 100 IU/ml RPMI medium with 10% FCS for 48 h. Thereafter, cells were washed, counted and resuspended in RPMI medium with 10% FCS. This sequence of adding activating agents, as described above, was shown earlier to induce T lymphocyte activation (Schröder et al., 2000Go). The activated lymphocytes were used for purging experiments.

BIS-1. The BIS-1-producing quadroma was produced in our department by fusion of the hybridomas RIV-9 and MOC-31, producing anti-CD3 (IgG3) and anti-EGP-2 (IgG1) antibodies, respectively (De Lau et al., 1989Go). Preparation and purification were as described earlier (De Leij et al., 1998Go). Briefly, BIS-1 was produced by means of a hollow fibre culture system (Endotronics, Minneapolis, MN). Purification of the hybrid antibodies (IgG3/IgG1) from parental-type antibodies, also produced by the quadroma, was performed by protein A column chromatography. BIS-1 F(ab')2 was then produced by means of digestion by pepsin followed by G150 Sephadex gel filtration, and added to a 0.9% sodium chloride solution to obtain a final concentration of 0.2 mg/ml.

Purging procedure
Tumour cell death, direct detection. Activated lymphocytes (effector cells) in the presence of 0.1 µg/ml BIS-1 were added to a 96-well plate (Nunclon), with 200, 500 or 1000 MCF-7 tumour cells (target cells) per well labelled with CMFDA, as described above. Ratios of effector to target cells were 100:1, 500:1 or 1000:1 (i.e. the addition of a 100-, 500- or 1000-fold more lymphocytes to the tumour cells), with an effector to target ratio of 0:1 (no lymphocytes added) as control. Co-incubation was performed in a total volume of 200 µl of RPMI medium with 10% FCS for 4 h at 37°C in a humidified, 5% CO2-containing atmosphere. The remaining tumour cells were counted directly by means of an inverted fluorescence microscope (Olympus IMT, Tokyo, Japan), at emission wavelength 516 nm, and excitation wavelength 492 nm. The results of the same effector to target ratios were taken together. Tumour cell death was assessed in five independent experiments.

The effect of the presence of the ovarian cortex suspension on tumour cell death efficiency was evaluated comparing tumour cell death as described above with tumour cell death in wells to which 100 µl of ovarian cortex suspension (prepared as described in ‘Thawing and suspension procedure’) was also added. Co-incubation was performed as described above. The prior fluorescent labelling of tumour cells allowed assessment of tumour cell death also in the presence of the ovarian cortex suspension. The effect of the addition of ovarian cortex suspension on tumour cell death efficiency was assessed with ovarian tissue of three patients.

The percentage tumour cell death was calculated as untreated tumour cells (control, effector to target ratio 0:1) minus the remaining tumour cells after treatment, divided by the control tumour cells, times 100%.

Tumour cell death, indirect detection. To evaluate longer term effects of the purging procedure on the growing potential of tumour cells, lymphocytes (effector cells) were co-incubated in a 96-well plate in the presence of 0.1 µg/ml BIS-1, with 2000 or 5000 MCF-7 tumour cells (target cells). Compared with the direct detection assay (see above), in this assay larger initial tumour cell numbers were necessary, in order to obtain an adequate formazan production in the MTT assay (see below) for measurement of tumour cell depletion (compared with the untreated control wells) after 5 days. The maximum total number of cells per well (due to culturing conditions in each well for 5 days) did not allow for higher effector to target ratios than used here, resulting in lower ratios than used in the direct detection assay.

Ratios of effector to target cells were 0:1, 10:1, 20:1 or 50:1, in a total volume of 200 µl of RPMI medium with 10% FCS (similar to that described in the experiment above). After 4 h of co-incubation at 37°C in a humidified, 5% CO2-containing atmosphere, the supernatant was removed. Cells were washed with fresh RPMI medium with 10% FCS and the supernatant was removed. Fresh medium (200 µl) was added and cells were cultured for 5 days (during which medium was refreshed one additional time) at 37°C in a humidified, 5% CO2-containing atmosphere. To establish tumour cell survival/growth after 5 days, the cellular reduction of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) by the mitochondrial dehydrogenase of viable cells to a blue formazan product was evaluated in a standard microculture tetrazolium assay (Carmichael et al., 1987Go). DMSO (100%, 200 µl) was used to dissolve the formazan crystals, and the plate was read in an enzyme-linked immunosorbent assay (ELISA) reader (Thermo Max, Molecular Devices, Sunnyvale, CA) at wavelength 490 nm. Comparisons were made with wells containing tumour cells and lymphocytes without BIS-1, tumour cells and BIS-1 without lymphocytes, or lymphocytes alone. In this experiment, no ovarian cortex suspension was added, as the described detection method does not allow discrimination between viable tumour cells or viable ovarian cells. Results of the same effector to target ratios were taken together. Depletion of growing tumour cells was assessed in four independent experiments.

The percentage depletion of growing cells was calculated as the number of untreated tumour cells (control) minus the number of remaining tumour cells after treatment, divided by the control number of tumour cells, times 100%. The background formazan production of lymphocytes was deducted.

Follicle morphology. Morphology and viability of follicles were assessed before freezing, after the freezing–thawing procedure of tissue, and before and after the purging procedure. Before and after the freezing–thawing procedure, pieces of tissue were fixed in buffered formalin, dehydrated through an alcohol series, and paraffin embedded. Before and after the purging procedure, ovarian cortex suspension was allowed to sediment onto glass slides, through a metal funnel-like device. Slides of all material were stained with standard Giemsa staining as well as the periodic acid–Schiff method (PAS) (Whitaker and Williams, 1994Go), Papanicolau method (PAP) (Whitaker and Williams, 1994Go) and the MOC31 antibody, directed against EGP-2, for tumour cell presence. Evaluation criteria for morphology on paraffin sections were eosinophilia of ooplasm, clumping of chromatin and wrinkling of the oocyte nuclear membrane as signs of atresia (Wright et al., 1999Go).

Statistics
Tumour cell death, assessed by means of direct (fluorescent) detection or indirect (MTT) detection, was analysed by means of a two-sided Student’s t-test for independent samples. Also the effect of adding ovarian cortex suspension on tumour cell death was analysed by means of this test. All analyses were performed with the statistical software program SPSS. A P-value <0.05 was considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ovarian tissue
Ovarian tissue was used from three patients. The first two patients were 13 and 17 years old at the time of cryopreservation, and they suffered from acute lymphatic leukaemia and an ovarian germ cell tumour of the contralateral ovary, respectively. From the third patient, aged 35 years, cryopreservation of ovarian tissue was performed after prophylactic ovariectomy because of a BRCA1 mutation; this patient gave consent for cryopreservation of ovarian tissue for the purging procedure described here. In the ovarian tissue of the latter patient, no sign of tumour contamination was present.

Purging efficiency
Tumour cell death, direct detection. For each effector to target ratio, the proportion of tumour cells killed was the same, irrespective of the initial number of tumour cells (200, 500 or 1000). For instance, at an effector to target ratio of 1000:1 (i.e. 1000-fold more lymphocytes than tumour cells), tumour cell death for wells initially containing 200, 500 or 1000 tumour cells was 53 (n = 2), 46 ± 16 (n = 3) and 23 ± 7% (n = 3) without BIS-1 (P = NS); and 84 (n = 2), 79 ± 2 (n = 3) and 70 ± 14% (n = 3) with BIS-1 (P = NS). Tumour cell death was affected by BIS-1 (see below), but not significantly by the initial tumour cell count. Within each effector to target ratio (100:1, 500:1 and 1000:1, as well as the untreated control with effector to target ratio 0), we therefore pooled data for the three tumour cell concentrations (200, 500 or 1000 cells per well) before analysis. Figure 1A shows the percentage death of CMFDA-labelled tumour cells after lymphocyte-induced tumour cell death in the presence or absence of BIS-1 for 4 h. In the absence of BIS-1, increased tumour cell death is obtained with effector to target ratios of 500:1 and 1000:1 compared with the control with effector to target ratio 0, defined as 0% cell death. Adding BIS-1 to wells containing an effector to target ratio of 1000:1 further augmented this death (75.5% killed, SD 10.5, P = 0.002 compared with 40%, killed without BIS-1: SD 19). Tumour cell death is comparable with that in haematopoietic stem cell harvests (Schröder et al., 2000Go), with these effector to target ratios.




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Figure 1. Direct assessment of tumour cell death by fluorescent cell detection. The x-axis shows the effector (E) to target (T) ratios. The y-axis shows the percentage tumour cell death of CMFDA-labelled MCF-7 tumour cells after 4 h co-incubation with activated lymphocytes, relative to the death in untreated MCF-7 cells. An asterisk reflects a significant difference between the white and black bar; a plus sign reflects a significant difference from the untreated control sample (E/T ratio 0:1, defined as 0% death). (A) Effect of BIS-1. White bar, absence of BIS-1; black bar, presence of BIS-1. (B) Effect of ovarian cortex suspension in the presence of BIS-1. Black bar, absence of ovarian cortex suspension; striped bar, presence of ovarian cortex suspension.

 
The effect of the addition of ovarian tissue on tumour cell death efficiency is shown in Figure 1B. Tumour cell death in the presence of BIS-1 increases at effector to target ratios of 500:1 and 1000:1; adding ovarian tissue does not affect tumour cell death.

Tumour cell death, indirect detection. In Figure 2, the percentage depletion of growing MCF-7 tumour cells is shown, after a 4 h co-incubation with activated lymphocytes in the presence or absence of BIS-1 and subsequent culture for 5 days. Depletion of growing tumour cells is clearly increased after treatment with activated lymphocytes in the presence of BIS-1, compared with the absence of BIS-1. In this setting, a maximum tumour cell depletion of 89% is seen at effector to target ratio of 10:1 (SD 11%, P < 0.001 compared with depletion without BIS-1).



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Figure 2. Indirect assessment of tumour cell death by MTT assay. The x-axis shows the effector (E) to target (T) ratios. The y-axis shows the percentage depletion of growing MCF-7 tumour cells after 4 h co-incubation with activated lymphocytes, and subsequent culture for 5 days, compared with tumour cell depletion in untreated MCF-7 cells. White bar, absence of BIS-1, black bar: presence of BIS-1. An asterisk reflects a significant difference between the white and black bar; a plus sign reflects a significant difference from the untreated control sample (E/T ratio 0:1, defined as 0% depletion).

 
Follicle morphology
Effect of freezing. No differences were observed between the tissue that had been passed through the freezing procedure and fresh tissue that was directly embedded in paraffin, when scored with the morphological criteria described in Materials and methods.

Effect of thawing and suspension procedure. The effect of the mechanical and enzymatic suspension procedure after thawing was evaluated in the ovarian cortex suspension after overnight culture, as described above. The mean number of wells (in the 96-well plate) microscopically containing one or more follicles was 62 ± 30 (SD; n = 5 replicates, see also thawing and suspension procedure). In these wells, no fibroblast outgrowth or attachment of granulosa cells was observed. The wells that were not used for purging experiments were used for cytology slides. Intact follicles were detected in these suspensions, and a representative sample of frozen–thawed ovarian suspension is shown in Figure 3.



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Figure 3. Effect of the freezing–tthawing procedure on follicle morphology. Shown is a representative piece of frozen–thawed ovarian tissue, at 40 x 10 magnification, with PAS staining. Three intact follicles (1) are indicated. Scale bar = 60 µm.

 

Effect of purging. Morphological evaluation of the suspension including follicles, lymphocytes and tumour cells after the purging procedure, scored with the morphological criteria described above, revealed intact follicles remaining. A representative picture is shown in Figure 4. No MOC31-positive tumour cells were detected after the purging procedure.



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Figure 4. Effect of the purging procedure on follicle morphology. Shown is a representative part of frozen–thawed ovarian tissue, after the purging procedure including activated lymphocytes and BIS-1, at 40 x 10 magnification with PAP staining. One intact follicle (2) as well as lymphocytes (1) are indicated. Scale bar = 60 µm.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Improvement in anticancer therapies has resulted in more long-term survivors. This has increased the awareness of long-term effects, such as gonadal failure (Bokemeyer et al., 1994Go; Bonadonna and Valagussa, 1995Go; Cobleigh et al., 1995Go; Swerdlow et al., 1996Go; Hensley and Reichmen, 1998Go; Burstein and Winer, 2000Go). As there are only a few possibilities to limit the toxic effect of chemotherapy and radiotherapy on ovarian function (Abir et al., 1998Go; Morita et al., 2000Go), there is a growing need to study the possibilities of ovarian protection and preservation. Cryopreservation and autografting of ovarian tissue could be a potential method to maintain fertility, but concerns remain about possible disease transmission because of tumour cell contamination of the graft. This issue would be resolved if primordial follicle isolation and subsequent in vitro maturation were possible. However, this technique is very challenging and still in a developmental stage (Rutherford and Gosden, 1999Go; Kim et al., 2001aGo). Encouragingly, a two-step procedure with in vivo transplantation and in vitro culture was shown to result in live mice offspring (Liu et al., 2001Go), but this procedure has not been applied in the human setting so far. Alternatively, one of the procedures for autografting of cryopreserved ovarian tissue involves reinsertion of a follicle suspension in plasma clots (Carroll et al., 1990Go; Newton, 1998Go). We applied our purging procedure for haematopoietic stem cells (Schröder et al., 2000Go) to a suspension of isolated follicles. We used the same breast cancer cell model that was used in the previous study, but this purging concept may very well also be applicable to other tumour types, e.g. B-cell lymphoma, which is sensitive to immunological treatment with monoclonal antibody rituximab (Onrust et al., 1999Go), and for which a bispecific antibody was also developed in our institution (Withoff et al., 2000Go).

The cryopreservation and thawing of ovarian tissue in this study was performed according to protocols described by the group of Gosden (Oktay et al., 1997Go). In the recent literature, seeding temperatures down to –9°C have been described, indicating that there is no optimal or standardized protocol for ovary cryopreservation so far (Gook et al., 2001Go; Kim et al., 2001aGo; Newton and Illingworth, 2001Go; Radford et al., 2001Go). The integrity of frozen–thawed follicles after enzymatic isolation was confirmed by electron microscopy evaluation previously (Oktay et al., 1997Go). Our results, showing morphologically intact follicles after thawing by light microscopy, are in line with this, although subcellular damage induced by freezing–thawing cannot be excluded. We developed a fluorescent detection system to evaluate tumour cell depletion after the purging procedure. As our ultimate aim is to culture the suspension of ovarian tissue after the purging procedure, the Chromium51 release assay, commonly used to evaluate tumour cell depletion, was considered inadequate. With the fluorescent detection system, highly efficient tumour cell death by activated lymphocytes in the presence of BIS-1 was demonstrated. No (adverse) effect of the presence of ovarian tissue on tumour cell death was observed, and morphologically intact follicles were detected following the purging procedure. Therefore, this study supports the concept that solid tissue, rendered into suspension, can be purged in a similar way to haematopoietic stem cell material.

This study was performed to provide proof of principle, with morphological evaluation of the ovarian follicles. It is clear that future studies will have to address the important issue of the quantitative and functional survival of the follicles, according to studies performed by Hovatta et al. (Hovatta et al., 1997Go, 1999; Wright et al., 1999Go). Ideally, in vivo evaluation of follicle functionality and tumour cell outgrowth would also follow, for instance by reinserting the follicles in a plasma clot under the renal capsule of SCID mice. Recently, the reinsertion of ovarian tissue resulted in growing and hormonally responsive primordial human follicles (Van den Broecke et al., 2001Go). Furthermore, endogenous tumour cell infiltration of ovarian tissue instead of exogenously added tumour cells will have to be evaluated in this purging concept. As the enzymatic isolation of follicles most probably renders the endogenous tumour cells accessible for lymphocyte cell death, similar results to those with exogenous tumour cells are expected. It would be interesting to study whether (very) thin slices of ovarian tissue can also be purged in this way but, at present, no adequate in vitro model is available for the evaluation of the presence of residual tumour cells. To avoid potential non-specific lymphocyte activity directed against the ovarian tissue, the use of autologous patient lymphocytes will probably be preferred in a future patient-related setting, although no such activity was observed in this study. Previously, we already demonstrated that autologous patient serum does not affect purging efficiency (Schröder et al., 2000Go), but for practical reasons (available volume) FCS was used in the present study. In reference to the cell model chosen in this study, one might argue that restoring endocrine function and fertility is undesirable in breast cancer patients, because of possible hormonal growth stimulation of residual disease. However, there is no evidence so far that a pregnancy after breast cancer treatment increases the risk of poor prognosis (Burstein and Winer, 2000Go; Gelber et al., 2001Go). With regards to the relevance of this study with a breast cancer cell model, it should be noted that a considerable number of breast cancer patients are diagnosed in their childbearing years. In The Netherlands, this amounts to ±1000 patients per annum; ~10% of the women diagnosed yearly with breast cancer (Visser et al., 2000Go). Together with the trend towards postponed childbearing (Heck et al., 1997Go), preservation of fertility for these young cancer patients may become an issue of increasing importance.

In conclusion, this study provides a first step in the direction of purging cryopreserved ovarian tissue of tumour cells. This would imply that patients with an increased risk of tumour cell contamination of the ovary do not have to be excluded from gonadal cryopreservation beforehand. The safe replacement of ovarian tissue in female cancer survivors to restore their endocrine function and fertility would be a major step forward in the improvement of the quality of life for these women.


    Acknowledgements
 
We would like to thank O.Hovatta (Karolinska Institute, Huddinge, Sweden) for helpful comments. This study was supported by grant RUG 96-1277 of the Dutch Cancer Society, and by a grant from the Nijbakker-Morra foundation.


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 Discussion
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Submitted on August 22, 2003; accepted on March 10, 2004.