Acute leukemia is the most common childhood cancer. In 30–40% of the patients, leukemia carries a high-risk cytogenetic aberration, responds slowly to therapy, or relapses. To be cured, these patients require treatment with allogeneic hematopoietic stem cell transplantation. Permanent ovarian failure is observed in 70–90% of young patients following hematopoietic stem cell transplantation.1, 2 Rapid progress in the development of strategies to generate fertile gametes from cryopreserved ovarian tissue has opened possibilities to preserve fertility of these young girls and women. In adults, the cryopreservation of ovarian cortex before gonadotoxic chemotherapy and autotransplantation of thawed tissue back to the cured patient has been shown to re-establish the menstrual cycles and lead to the birth of healthy children.3 Cryopreservation of ovarian tissue from young, sexually immature girls has been proposed similarly to allow long-term storage of large numbers of primordial follicles for fertility preservation.4, 5 Although transplanted children and adolescents constitute the most important group of patients who would benefit from the novel ovarian autotransplantation techniques, concern has been expressed about the method possibly carrying a risk of leukemia relapse originating from residual leukemic cells in the ovarian tissue.6, 7, 8
In order to evaluate whether the bone marrow-remission status influences the minimal residual leukemia (MRD) in ovarian samples, individual Ig/TCR (immunoglobulin/T-cell receptor) gene rearrangements and translocation-specific fusion gene products were analyzed by quantitative real-time PCR (qPCR). MRD was determined in six ovarian specimens collected at diagnosis of leukemia and in 10 collected at morphological remission induced by chemotherapy. Morphological bone marrow remission was defined as the restoration of normal hematopoiesis with a blast cell fraction of <5% by light microscopic examination. Bone marrow MRD status was analyzed by flow cytometry or qPCR and correlated with the simultaneously measured quantitative MRD in the ovarian tissue.
The study material consisted of nine consecutive ovarian samples from deceased childhood leukemia patients who had participated in a research project for fertility preservation at the Helsinki University Central Hospital during 1999–2012 and had a leukemia-specific marker that enabled qPCR analysis of MRD in the cryopreserved ovarian tissue. Ovarian samples from surviving girls (n=11) and those from the deceased girls with no leukemia-specific marker (n=2) were excluded form the study. The patients underwent ovarian cryopreservation before hematopoietic stem cell transplantation at morphological bone marrow remission. Informed consent was obtained from the guardians and age-appropriate patients. The Research Ethics Committee of Helsinki University Hospital approved the study.
The adult study material consisted of seven ovarian samples from young women with leukemia who had their ovarian tissue cryopreserved as part of the clinical fertility preservation programs in Tampere and Oslo University Central Hospitals. Seven women with a leukemia-specific PCR marker were identified and all gave their informed consent for leukemia contamination analysis. Ovarian biopsy for the adult patients was preferably collected at diagnosis. Clinical patient and treatment characteristics, time in remission and time without bone marrow MRD before ovarian biopsy are shown in Table 1.
Ovarian tissue cryopreservation was performed by using the slow freezing method and propanediol or ethylene glycol as cryoprotectant.9, 10 Entire cryopreserved cryovials containing 3–6 ovarian fragments of 2 × 2 mm or 1–2 fragments of 5 × 5 mm in size were processed for qPCR. Small, thawed tissue pieces (1 × 1 mm and 5 × 5 mm) were analyzed for patients 6 and 14. Detailed information on the molecular genetic analysis is accessible in the Supplementary information.
All the six ovarian samples collected at the diagnosis showed evidence of ovarian MRD varying between 0.2% and 57% (Table 1). The degree of leukemic contamination showed no association with the type of leukemia or whether the MRD was measured by RNA- or DNA-based qPCR methods. Two out of the 10 patients, whose specimens were collected at remission, showed positive ovarian MRD (Table 1). Patient No. 16 had ovarian MRD at the level of 0.2% with no simultaneous bone marrow MRD detectable by flow cytometry (<0.1%) or qPCR (<0.001%) and repeated bone marrow samples negative for MRD for 17 weeks before ovarian biopsy (Table 1). The other patient (No 15) who had ovarian MRD below the quantitative range (<0.01%, see Table 1) showed simultaneous bone marrow MRD at the level of 0.2% analyzed by flow cytometry. Of the eight patients with negative ovarian MRD, one patient simultaneously had positive bone marrow MRD at the level of 1% by flow cytometry (patient No. 12).
All the samples yielded sufficient amounts of nucleic acids to be assayed according to the EAC and EuroMRD recommendations (Supplementary information) and sensitivities of the assays ranged from 0.0003% to 0.06%. No significant differences in the qPCR analytical sensitivities of either DNA- or RNA-based approaches were observed among the four patients (patient Nos. 7, 11, 13 and 15) who had both DNA- and RNA-based MRD markers available (Table 1). For those patients (Nos. 5 and 6) who had two DNA-based leukemia-specific markers, very similar levels of ovarian MRD were measured by both markers (Table 1).
The present results provide novel MRD data for the discussion on the risk of ovarian leukemia contamination during morphological bone marrow remission. These results suggest that postponing the biopsy until bone marrow has cleared from MRD may result in less leukemic contamination in the cryopreserved ovarian tissue. The ovarian leukemia contamination has previously been evaluated by Dolmans et al.6 and Greve et al.11 The number of patients is low in both studies, but results suggest that about half of the evaluable patients had positive ovarian MRD as measured by PCR after initiation of major leukemia therapy. In the study of Dolmans et al.6, 2 patients out of 12 with acute leukemia had received a full cycle of intravenous chemotherapy before excision of the ovarian cortex. One patient had positive and one negative PCR result for residual leukemia cells. In the study of Greve et al.,11 17 out of 21 patients with acute leukemia were in morphological bone marrow remission. The ovarian cortex of four of these patients could be analyzed by PCR for residual leukemia cells. For two patients, the result was positive.
In the present study, ovarian MRD measured by qPCR was correlated with the bone marrow MRD status. Six of the seven patients had no ovarian MRD when in remission with no signs of MRD in repeated bone marrow samples 10–30 weeks before ovarian biopsy, whereas one out of the two who had a positive bone marrow MRD at the time of ovarian biopsy also had a positive ovarian MRD (Table 1). This suggests that antileukemic therapy able to induce MRD negativity in the marrow can also eliminate leukemic contamination from the ovarian specimens. Thus, the risk of leukemic contamination in the ovarian cortex can be reduced, but not completely abolished, if the fertility preservation is postponed until the MRD has disappeared from bone marrow.
Strikingly, one of the ovarian samples, collected after 17 weeks of continuous molecular bone marrow remission, still contained a significant level of leukemic contamination (0.2%). Thus, even during long-term molecular remission some extramedullary infiltrations may exist in ovarian tissue, and leukemia-free ovarian specimens for fertility preservation cannot be guaranteed.
International guidelines have supported an early intervention with a biopsy before anticancer therapy.12 This is because several reports have shown that chemotherapy can negatively affect the oocyte quality,13 primordial follicle pool14 and vascularization.15 Thus, clinically, the ovarian tissue would need to be collected at the time of maximal disease burden when the ovarian tissue most probably is harboring malignant cells. In the present study, ovarian MRD was detected at the range of 0.2–57% in all samples collected at the diagnosis. This confirms the earlier reports, suggesting that ovarian material collected at the time of overt leukemia should be regarded as contaminated and not be used for re-implantation procedures.6, 7, 8, 11
In conclusion, our data indicate that postponing the fertility preservation measures to the time of leukemia remission with no bone marrow MRD results in less or no leukemic contamination in the ovarian material. However, the correlation between the bone marrow and ovarian MRD is not complete, and significant leukemic contamination may be present in ovarian tissue during marrow remission. Further studies will be needed to evaluate whether the ovarian samples with positive MRD contain viable malignant cells and whether the primordial follicles collected stay viable and will initiate maturation, when transplanted or cultured.
Barrett A, Nicholls J, Gibson B . Late effects of total body irradiation. Radiother Oncol 1987; 9: 131–135.
Wallace WH . Oncofertility and preservation of reproductive capacity in children and young adults. Cancer 2011; 117: 2301–2310.
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: 437–450.
Jadoul P, Dolmans MM, Donnez J . Fertility preservation in girls during childhood: is it feasible, efficient and safe and to whom should it be proposed? Hum Reprod Update 2010; 16: 617–630.
Sauvat F, Binart N, Poirot C, Sarnacki S . Preserving fertility in prepubertal children. Horm Res 2009; 71 (Suppl 1): 82–86.
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: 2908–2914.
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: 1007–1013.
Rosendahl M, Andersen MT, Ralfkiær E, Kjeldsen L, Andersen MK, Andersen CY . Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia. Fertil Steril 2010; 94: 2186–2190.
Hovatta O, Silye R, Krausz T, Abir R, Margara R, Trew G et al. Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants. Hum Reprod 1996; 11: 1268–1272.
Oskam IC, Babak A, Santos RR . Histologic and ultra-structural features of cryopreserved ovine ovarian tissue: deleterious effect of 1,2-propanediol applying different thawing protocols. Fertil Steril 2010; 93: 2764–2766.
Greve T, Clasen-Linde E, Andersen MT, Andersen MK, Sørensen SD, Rosendahl M et al. Cryopreserved ovarian cortex from patients with leukemia in complete remission contains no apparent viable malignant cells. Blood 2012; e-pub ahead of print 18 June 2012 doi:10.1182/blood-2012-01-403022.
Anderson RA . Policy and Practice sub-Group of the British Fertility Society. A strategy for future reproductive services for survivors of cancer. Hum Fertil 2003; 6: 113–115.
Raz A, Fisch B, Okon E, Feldberg D, Nitke S, Raanani H et al. Possible direct cytotoxicity effects of cyclophosphamide on cultured human follicles: an electron microscopy study. J Assist Reprod Genet 2002; 19: 500–506.
Morgan S, Anderson RA, Gourley C, Wallace WH, Spears N . How do chemotherapeutic agents damage the ovary? Hum Reprod Update 2012; 18: 525–535.
Meirow D, Dor J, Kaufman B, Shrim A, Rabinovici J, Schiff E et al. Cortical fibrosis and blood-vessels damage in human ovaries exposed to chemotherapy. Potential mechanisms of ovarian injury. Hum Reprod 2007; 22: 1626–1633.
We are grateful to Jan Delabie and Helen Vålerhaugen from Division of Pathology, Oslo University Hospital for acquisition of the data of the two Norwegian patients. This study was supported by grants from the Swedish Barncancerfonden, the Finnish Cancer Society, the Finnish Foundation for Pediatric Research, the Paulo Foundation, the Nona and Kullervo Väre Foundation and the Academy of Finland.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Leukemia website
About this article
Cite this article
Jahnukainen, K., Tinkanen, H., Wikström, A. et al. Bone marrow remission status predicts leukemia contamination in ovarian biopsies collected for fertility preservation. Leukemia 27, 1183–1185 (2013). https://doi.org/10.1038/leu.2012.279
Mechanisms of extramedullary relapse in acute lymphoblastic leukemia: Reconciling biological concepts and clinical issues
Blood Reviews (2019)
A protocol to isolate and qualify purified human preantral follicles in cases of acute leukemia, for future clinical applications
Journal of Ovarian Research (2018)
First delivery in a leukemia survivor after transplantation of cryopreserved ovarian tissue, evaluated for leukemia cells contamination
Fertility and Sterility (2018)
British Journal of Haematology (2017)
Leukemia & Lymphoma (2016)