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Ovarian minimal residual disease in chronic myeloid leukaemia
Reproductive BioMedicine Online (2014) 28, 255– 260
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Ovarian minimal residual disease in chronic myeloid leukaemia Ronit Abir a,b,*, Adina Aviram c,d, Meora Feinmesser e,b, Jerry Stein f,b, Isaac Yaniv f,b, Doris Parnes c,d, Avi Ben-Haroush a,b, Dror Meirow g,b, Esther Rabizadeh c,d, Benjamin Fisch a,b a Infertility and IVF Unit, Beilinson Women Hospital, Rabin Medical Center, Petach Tikva, Israel; b Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; c Hematology Laboratory, Rabin Medical Center, Petah Tikva, Israel; d Hemato-Oncology Laboratory, Felsenstein Medical Research Center, Tel Aviv University, Petah Tikva, Israel; e Department of Pathology, Rabin Medical Center, Beilinson Hospital, Petah Tikva, Israel; f Department of Pediatric Hematology Oncology, Schneider Children’s Medical Center of Israel, Petah Tikva, Israel; g IVF Unit, Division of Obstetrics and Gynecology, Sheba Medical Center, Tel Hashomer, Israel
* Corresponding author. E-mail address: [email protected] (R Abir). Ronit Abir, PhD graduated from the Hebrew University of Jerusalem, Israel and then worked as a research fellow at Hammersmith Hospital, London on aspects related to fertility preservation. Dr Abir is the director of the Fertility Preservation Program and Research Laboratory at the Infertility and IVF Unit, Beilinson Women Hospital, Rabin Medical Center. Her current research interests focus on in-vitro culture of human primordial follicles, involvement of growth factors and other genes in early folliculogenesis, development of human fetal follicles, methods to improve outcomes of human ovarian tissue grafting and cryopreservation of ovarian tissue from young cancer patients. She is also a senior lecturer on reproduction and embryology and histology of the female reproductive system at Sackler Faculty of Medicine, Tel Aviv University.
Abstract The options for fertility preservation include cryopreservation of ovarian tissue. Although transplantation of cryopre-
served–thawed ovarian tissue in cancer survivors has resulted in live births, there is evidence of malignancy involvement in ovarian tissue, especially in leukaemia. The objectives of this study were to investigate the involvement of chronic myeloid leukaemia (CML) in ovaries by both pathological/immunohistochemical methods and PCR for the identification of the Philadelphia chromosome (BCR-ABL transcripts). The patient was a survivor of paediatric CML whose ovaries were cryopreserved. The patient became infertile and requested ovarian reimplantation in adulthood. Pathological examinations of ovarian tissue with immunohistochemical stainings, quantitative PCR and two-step nested PCR were applied to identify BCR-ABL transcripts. Despite the lack of positive pathological/immunohistochemical evidence, PCR and two-step nested PCR revealed that the ovary was contaminated by malignant minimal residual CML. Survivors of childhood CML may harbour minimal residual disease in the ovaries. This finding stresses the danger of reseeding cancer by ovarian grafting, especially in patients with leukaemia. If ovarian grafting is considered, reimplantation should be preceded by examination of ovarian samples both pathologically and by molecular techniques. On the basis of molecular findings, ovarian autografting was not recommended in this case report. RBMOnline ª 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: cancer seeding by ovarian grafting, chronic myeloid leukaemia, ovarian transplantation, pathological/immunohistochemical examination, PCR, Philadelphia chromosome
1472-6483/$ - see front matter ª 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2013.10.011
256
Introduction Chronic myeloid leukaemia (CML) is the most common myeloproliferative disease, although it is relatively rare in paediatric patients (Cotta and Bueso-Ramos, 2007). Originating in abnormal pluripotent bone marrow stem cells, CML usually manifests as a hyperproliferative blood profile and splenomegaly. This includes elevated white blood cells specifically immature cells (blasts) as well as abnormal cells of the myeloid megakaryocyte cell lineages. Molecular studies have reported a reciprocal chromosomal translocation between chromosomes 9q34 and 22p11 in haematopoietic cells in at least 95% of the patients with CML yielding the Philadelphia chromomsome (Ph; Cotta and Bueso-Ramos, 2007; Ernst and Hochhaus, 2012; Ernst et al., 2011; Kishore and Marin, 2011; Sudoyo and Hardi, 2011). Ph harbours the Breakpoint Cluster Region (BCR)–ABL1 (c-ABL oncogene) fusion gene which encodes a tyrosine kinase (TK) oncogenic protein. Juxtaposition of the BCR domain and the ABL’s tyrosine kinase results in deregulated proliferation and apoptosis of haematopoietic progenitors and the clinical and morphologic manifestation of CML (Ernst and Hochhaus, 2012; Kishore and Marin, 2011). BCR-ABL transcripts can be rapidly identified by very sensitive techniques such as fluorescence in-situ hybridization or PCR (Ernst et al., 2011; Kishore and Marin, 2011; Saijo, 2012; Sudoyo and Hardi, 2011; Vardiman, 2009), by which the presence of single malignant cells can be detected. Improvements in anticancer treatment have led to increased survival of patients (Feigin et al., 2008). However, therapy can cause premature ovarian failure in girls and women. The options for fertility preservation are limited, and often the only possibility is cryopreservation of ovarian tissue containing small immature ovarian follicles. So far, autologous transplantation of cryopreserved–thawed ovarian tissue in cancer survivors has resulted in approximately 25 live births (Donnez et al., 2013; Silber, 2012). As far as is known, there is only one report of a leukaemia survivor who underwent ovarian reimplantation (Chung et al., 2013). Indeed, there is increasing although as-yet limited evidence of ovarian involvement in various leukaemic diseases (Chung et al., 2013; Dolmans et al., 2010, 2013; Kyono et al., 2009; Meirow et al., 2008; Rosendahl et al., 2010, 2013). The aim of this report was to describe a survivor of paediatric CML who requested ovarian grafting for fertility restoration from her frozen–thawed tissue. However, although pathological or immunohistochemical findings were negative, PCR showed evidence of BCR-ABL1 fusion transcripts in the ovarian tissue. On the basis of these molecular findings, ovarian autografting was not recommended. However, this is a single case, and information from more cases is needed.
Materials and methods Case description The study was conducted at a tertiary medical centre. The protocol was approved by the local Human Investigations Committee (reference number 5875, approved 6 July 2010).
R Abir et al. The patient was diagnosed with CML by bone marrow biopsy at the age of 12 years (December 1999). Laboratory results included an elevated leukocyte count (486,000/ll) and peripheral blood positive for Ph. She had experienced one menstrual cycle with a single menstrual bleeding 2 months before CML diagnosis. She underwent immediate ovarian cryopreservation for fertility preservation before any form of anticancer therapy was initiated. At surgery, the only finding was an enlarged spleen, but the uterus and ovaries were of normal size and with a few follicles larger than 10 mm. Histological examination of her ovaries did not reveal infiltrations of CML (see Pathological and immunohistochemical evaluation). She was treated with hydoroxyurea for 2 months with a decrease of her leukocyte counts (to 5000/ll). In February 2000, she underwent conditioning for bone marrow transplantation with cyclophosphamide (Cytoxan, 120 mg/kg; Bristol-Myers Squibb Co–Mead Johnson and Co, Evansville, IN, USA) and total body irradiation of 1200 centiGrey in six doses with partial lung sparing. She then received bone marrow transplantation from her human leukocyte antigen-identical healthy brother. Post-transplant complications included acute graft versus host (GVH) disease grade II, treated with prednisone at 2 mg/kg/d for six weeks, which eliminated chronic GVH. Her blood count stabilized 14 d after transplantation. Since resolution of acute post-transplantation GVH, she has been well and she continues to have no evidence of BCR-ABL transcripts in her peripheral blood. The percentage of Ph-positive cells from 1 year after the transplantation until the time of writing was 3 (false-positive), while PCR in blood remained negative. She continues to be healthy and is checked routinely. Seven months after the transplantation, she was treated for 7 months with conjugated oestrogens (Premaril 0.625/d; Dexxon, Or Akiva, Israel). Since then she took various forms of oral contraceptives until her wedding at the age of 24. Around that time she stopped the oral contraceptives, became amenorrhoeic, with hot flushes and a single episode of blood spotting. Her hormonal profile included: anti-Mu ¨llerian hormone <0.4 ng/ml; FSH 103 IU/l and thyroid-stimulating hormone 6.2 lIU/ml. Endometrial thickness measured 2 mm. The dimensions of the right and left ovaries were 27 mm · 19 mm and 14 mm · 9 mm, respectively. Upon 17b-oestrogen induction (8 mg/d for nine d), the endometriium thickened to 7 mm.
Histological preparation for light microscopy and immunohistochemistry The histological preparation method for ovarian tissue has been described in detail previously (Abir et al., 2010). These specimens were examined in the Department of Pathology for leukaemic infiltrates. Tissue samples were immunoassayed using as primary antibodies rabbit polyclonal against human myeloperoxidase (1:3000, A0398; DakoCytomation, Glostrup, Denmark), mouse monoclonal antibodies against glycophorin A (1:400, M0819; DakoCytomation), mouse monoclonal antibodies against CD34 (1:100, Biogenex, Fremont, CA, USA), mouse monoclonal antibodies against CD68 (1: 500, M0876, DakoCytomation), mouse monoclonal antibodies against LCA/CD45 (1:250, M0876; DakoCytomation) and rabbit polyclonal antibodies against human Factor VIII
Ovarian minimal residual disease in CML (1:300, A0082; DakoCytomation). The samples were incubated with the primary antibodies for 45 min. The negative controls were incubated with phosphate buffered saline at pH 7 (Biological Industries, Beit Ha’emek, Israel). Thereafter, the samples were incubated with horseradish peroxide polymer conjugate against mouse, rabbit and guinea pig antibodies (SuperPicture HRP, 878963; Zymed Laboratories). Finally, the sections were incubated with a diaminobenzidine urea H2O2 solution (Sigma Fast tablets; Sigma, St Louis, MO, USA) and counterstained with Mayer’s haematoxylin (Pioneer Research Chemicals, Colchester, Essex, UK). All immuonostains were conducted in a Ventana Benchmark XT machine (Hoffmann-La Roche Ltd, Basel, Switzerland).
Quantitative reverse-transcription PCR Total RNA was extracted from the cryopreserved ovary with TRI-reagent (Molecular Research Centre, Cincinnati, OH, USA) and transcribed into cDNA using the Maxima First Strand cDNA Synthesis Kit for quantitative reversetranscription PCR (qPCR; Fermentas Life Sciences, www. fermentas.com). Assessment of the amount of BCR-ABL M-bcr transcripts was performed by qPCR (Rotor Gene 3000) using TaqMan probe-based technology, primers and probes as described (Gabert et al., 2003). Absolute quantification was achieved using the Ipsogen plasmid standard curves. Quantification analysis was performed in accordance with the published recommendations of European LeukaemiaNet (Baccarani et al., 2009). Analysis was conducted twice in quadruplicates each time, with negative cDNA controls (from Ph-negative samples).
Results Pathological evaluation and follicular counts The ovarian specimens were negative for malignancy on pathological evaluation. Moreover, all immunohistochemical stainings did not yield evidence of malignancy. There were up to 20 primordial and primary follicles per ovarian section.
Quantitative reverse-transcription PCR The amplification plots of ABL and BCR-ABL are shown in Figure 1. The results are expressed as the ratio of BCR-ABL/ABL transcripts. The calculated ratio for the cryopreserved ovarian tissue sample was 0.05%. Qualitative two-step nested PCR was conducted twice in quadruplicates each time, and performed as described by the Report of BIOMED-1 concerted Action (Gabert et al., 2003). PCR products were separated on agarose gel (Figure 2). Of note, analysis of concurrent peripheral blood samples by the same technique showed no evidence of BCR-ABL transcripts.
Discussion This case report poses questions of acute clinical relevance: Does ovarian tissue harbour occult leukaemia? If it does,
257 does autotransplant of this tissue place the recipient at risk of disease relapse? This case report shows ovarian contamination by very low levels of minimal residual disease in CML demonstrated by qPCR, despite lack of pathological/immunohistochemical evidence. Molecular biology techniques of PCR and qPCR are extremely sensitive and may indicate the presence of single malignant cells. This notwithstanding, given that ovarian harvesting from this patient was performed before the administration of systematic therapy, the very low concentration of BCR-ABL transcripts that was detected may represent the presence of small amounts of peripheral blood in the harvested tissue. There are three methods for evaluation of malignancy involvement of ovaries. The first is conventional pathological examinations (Kyono et al., 2009), the second is sensitive molecular methods to detect the specific chromosomal abnormality of the minimal residual disease (both on RNA and DNA levels; Abir et al., 2010; Chung et al., 2013; Dolmans et al., 2010; Meirow et al., 2008; Rosendahl et al., 2010, 2013) and the third involves xenografting human ovarian tissue from cancer patients to immunodeficient mice and examining the malignant potential of the human ovary (Chung et al., 2013; Dolmans et al., 2010; Kim et al., 2001; Lotz et al., 2011; Rosendahl et al., 2010, 2011, 2013). The clinical relevance of contaminated minimal residual disease cells in the ovary is not completely clear (Passeque et al., 2003; Saijo, 2012; Sampieri and Fodde, 2012). Already in 1976, an autopsy study reported a relatively high incidence of leukaemic cell infiltration in the testis (48.5%) and in the ovary (58.1%; Kamiyama and Funata, 1976), suggesting the gonads are a sanctuary site for extramedullary leukaemic infiltration, with the ovary being a possible protection niche for leukaemic progenitors. Thus, both hormonal stimulation for IVF as well as ovarian tissue grafting in leukaemia survivors may potentially pose a high risk for disease relapse. However, once transplanted the capability of residual leukaemic cells to repopulate the bone marrow and initiate disease relapse is still unknown. At the same time, there are indications that haematopoiesis may also occur in the ovary and testis (Kamiyama and Funata, 1976; Passeque et al., 2003; Sampieri and Fodde, 2012). The existence of quiescent-dormant CML progenitor cells in ovarian tissue that are either therapy resistant or in tissue from patients after successful therapy should not be excluded, as the cell origin responsible for the disease is still unclear (Kamiyama and Funata, 1976; Kishore and Marin, 2011; Zon, 1995). Although it has not been proven, it is theoretically possible that the dormant state may be a protective mechanism that enhances cell survival under adverse conditions. If this is indeed the case, the ovarian tissue should not have been harvested in the first place with the intention of transplantation but for other putative fertility restoration methods such as in-vitro maturation of primordial follicles (Abir et al., 2006) or implantation of isolated follicles (Vanacker et al., 2012). Moreover, in leukaemia survivors after complete remission, ovarian tissue was positive for molecular markers but did not reseed malignancy in murine hosts or contain any viable malignant cells (Greve et al., 2012). It is, however, theoretically possible that cancer would have been eventually initiated after a longer transplantation period than 20 weeks.
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R Abir et al.
Figure 1 Quantitative reverse-transcription PCR for ABL (A) and BCR-ABL (B) detection. Typical fluorescence curves obtained for standards (dotted lines) and sample (unbroken lines).
It should also be borne in mind that some cells might exhibit flexibility in responding to different stochastic influences in their developmental milieus (Affer et al., 2011). By contrast, the frequency of leukaemic stem cells in the blood or marrow of patients afflicted with this disease is between 0.2% and 1% of the leukaemic burden (Passeque et al., 2003). As such, the frequency of leukaemia-initiating cells in the ovarian tissue of the current patient was extremely low. Despite this, even after taking into account the results of Greve et al. (2012), certainty the occasional presence of clonogenic leukaemic progenitors cannot be excluded. It will, therefore, be safer to avoid as much as possible this danger by testing the ovaries by all means available for possible malignancy markers. It is noteworthy that, in a study conducted by our group (Abir et al., 2010), a molecular marker of Ewing sarcoma was found in the ovaries of one
girl, while others did not find any traces of malignancy in similar patients (Greve et al., 2013). Ovarian transplant followed by a brief period of treatment with a tyrosine kinase inhibitor such as imatinib may be considered. These drugs, however, do not always eradicate CML at the stem cell level, therefore theoretically leaving a reservoir of cancer stem cells in the transplanted tissue (Kamiyama and Funata, 1976; Kishore and Marin, 2011; Passeque et al., 2003; Sampieri and Fodde, 2012; Zon, 1995). It is also noteworthy that drugs such as hydroxyurea reduce the number of white blood cells (as reported in the present case), but do not damage the ovarian follicles. Therefore, it might be worthwhile to treat patients with hydroxyurea prior to ovarian collection and to reduce CML cell infiltration into the ovary (Chung et al., 2013).
Ovarian minimal residual disease in CML
Figure 2 Two-step nested quantitative reverse-transcription PCR. PCR products were analysed on a 2% agarose gel. Sample lanes 1–4 = cryopreserved ovarian tissue sample with a PCR product of 458 bp, typical for b3a2 transcripts (the breakpoint of BCR gene is downstream of exon 14); NEG = Philadelphia-negative sample derived from healthy control; NTC = no-template control; POS = Philadelphia-positive sample derived from a CML patient with a PCR product of 383 bp, typical for b2a2 transcripts (the breakpoint of BCR gene is downstream of exon 13); ABL = positive control for the gene ABL, demonstrating RNA integrity of the sample); Marker = 100-bp DNA size ladder.
The present report adds to the accumulating evidence of this danger regarding leukaemia. Ovarian leukaemia involvement was documented in a large pathological study (Kyono et al., 2009). The current study, however, is the fourth to identify leukaemia minimal residual disease in the ovary of a patient with CML in the presence of negative pathological findings (Dolmans et al., 2010; Meirow et al., 2008; Rosendahl et al., 2010). Ovarian leukaemia CML minimal residual disease has been reported in 13 patients (Dolmans et al., 2010; Meirow et al., 2008; Rosendahl et al., 2010) including the present case report. Minimal residual disease was identified not only in CML patients, but also in patients with acute lymphoblastic leukaemia (ALL; Dolmans et al., 2010; Rosendahl et al., 2010) and acute myeloblastic leukaemia (Rosendahl et al., 2010). Moreover, when tissue from all patients was transplanted into immunodeficient mice, a portion of the mice developed intraperitoneal leukaemic masses 6 months after grafting (Dolmans et al., 2010). There are reports of ovarian involvement also in nonleukaemia malignancies including Hodgkin’s lymphoma (Bittinger et al., 2011; Kyono et al., 2009), non-Hodgkin’s lymphoma (Kyono et al., 2009), breast cancer, pulmonary carcinoma, gastric carcinoma, colon carcinoma and Ewing sarcoma (Abir et al., 2010). In conclusion, the present report, together with previous studies (Dolmans et al., 2010; Kyono et al., 2009; Meirow et al., 2008; Rosendahl et al., 2010), suggests that CML may leave malignant traces in the ovary. Therefore, clinicians considering ovarian grafting in cancer survivors, especially in leukaemia survivors, should be cautious. So far,
259 ovarian tissue collected before bone marrow transplantation has been reimplanted into one CML survivor, after highly sensitive qPCR did not show evidence of leukaemic cells in the ovarian samples (Chung et al., 2013). The same group did not transplant ovarian tissue to another Phovarian positive patient. Ovarian samples from the patients should be examined both pathologically and by molecular techniques and possibly also by transplantation into immunodeficient mice. However, it is doubtful if the use of a murine host will be accepted for clinical purposes nor is the exact period of malignancy introduction clear. The bias of sampling error, however, can never be completely eliminated. It is clear that new methods will be needed to insure safer fertility restoration for patients with acute or chronic leukaemia. The methods include the development of an in-vitro maturation system for frozen–thawed primordial follicles (Abir et al., 2006) or transplantation of isolated ovarian follicles rather than of ovarian tissue (Vanacker et al., 2012). In summary, this study demonstrates the presence of occult leukaemia in a histologically normal ovary harvested from a patient with CML. The theoretical risk of cancer recurrence due to the presence of clonogenic leukaemia cells in the ovary has caused ovarian autotransplantation to be deferred in this patient.
Acknowledgements The authors are greatly indebted to Ms Gloria Ganzach from the Editorial Board of Rabin Medical Center, Beilinson Hospital and to Dr Yehezkel Lande from our department for the English editing. We are also indebted to our laboratory technician Ms Carmela Felz for assistance with the histological sectioning.
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SHORT COMMUNICATION
Ovarian minimal residual disease in chronic myeloid leukaemia Ronit Abir a,b,*, Adina Aviram c,d, Meora Feinmesser e,b, Jerry Stein f,b, Isaac Yaniv f,b, Doris Parnes c,d, Avi Ben-Haroush a,b, Dror Meirow g,b, Esther Rabizadeh c,d, Benjamin Fisch a,b a Infertility and IVF Unit, Beilinson Women Hospital, Rabin Medical Center, Petach Tikva, Israel; b Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; c Hematology Laboratory, Rabin Medical Center, Petah Tikva, Israel; d Hemato-Oncology Laboratory, Felsenstein Medical Research Center, Tel Aviv University, Petah Tikva, Israel; e Department of Pathology, Rabin Medical Center, Beilinson Hospital, Petah Tikva, Israel; f Department of Pediatric Hematology Oncology, Schneider Children’s Medical Center of Israel, Petah Tikva, Israel; g IVF Unit, Division of Obstetrics and Gynecology, Sheba Medical Center, Tel Hashomer, Israel
* Corresponding author. E-mail address: [email protected] (R Abir). Ronit Abir, PhD graduated from the Hebrew University of Jerusalem, Israel and then worked as a research fellow at Hammersmith Hospital, London on aspects related to fertility preservation. Dr Abir is the director of the Fertility Preservation Program and Research Laboratory at the Infertility and IVF Unit, Beilinson Women Hospital, Rabin Medical Center. Her current research interests focus on in-vitro culture of human primordial follicles, involvement of growth factors and other genes in early folliculogenesis, development of human fetal follicles, methods to improve outcomes of human ovarian tissue grafting and cryopreservation of ovarian tissue from young cancer patients. She is also a senior lecturer on reproduction and embryology and histology of the female reproductive system at Sackler Faculty of Medicine, Tel Aviv University.
Abstract The options for fertility preservation include cryopreservation of ovarian tissue. Although transplantation of cryopre-
served–thawed ovarian tissue in cancer survivors has resulted in live births, there is evidence of malignancy involvement in ovarian tissue, especially in leukaemia. The objectives of this study were to investigate the involvement of chronic myeloid leukaemia (CML) in ovaries by both pathological/immunohistochemical methods and PCR for the identification of the Philadelphia chromosome (BCR-ABL transcripts). The patient was a survivor of paediatric CML whose ovaries were cryopreserved. The patient became infertile and requested ovarian reimplantation in adulthood. Pathological examinations of ovarian tissue with immunohistochemical stainings, quantitative PCR and two-step nested PCR were applied to identify BCR-ABL transcripts. Despite the lack of positive pathological/immunohistochemical evidence, PCR and two-step nested PCR revealed that the ovary was contaminated by malignant minimal residual CML. Survivors of childhood CML may harbour minimal residual disease in the ovaries. This finding stresses the danger of reseeding cancer by ovarian grafting, especially in patients with leukaemia. If ovarian grafting is considered, reimplantation should be preceded by examination of ovarian samples both pathologically and by molecular techniques. On the basis of molecular findings, ovarian autografting was not recommended in this case report. RBMOnline ª 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: cancer seeding by ovarian grafting, chronic myeloid leukaemia, ovarian transplantation, pathological/immunohistochemical examination, PCR, Philadelphia chromosome
1472-6483/$ - see front matter ª 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2013.10.011
256
Introduction Chronic myeloid leukaemia (CML) is the most common myeloproliferative disease, although it is relatively rare in paediatric patients (Cotta and Bueso-Ramos, 2007). Originating in abnormal pluripotent bone marrow stem cells, CML usually manifests as a hyperproliferative blood profile and splenomegaly. This includes elevated white blood cells specifically immature cells (blasts) as well as abnormal cells of the myeloid megakaryocyte cell lineages. Molecular studies have reported a reciprocal chromosomal translocation between chromosomes 9q34 and 22p11 in haematopoietic cells in at least 95% of the patients with CML yielding the Philadelphia chromomsome (Ph; Cotta and Bueso-Ramos, 2007; Ernst and Hochhaus, 2012; Ernst et al., 2011; Kishore and Marin, 2011; Sudoyo and Hardi, 2011). Ph harbours the Breakpoint Cluster Region (BCR)–ABL1 (c-ABL oncogene) fusion gene which encodes a tyrosine kinase (TK) oncogenic protein. Juxtaposition of the BCR domain and the ABL’s tyrosine kinase results in deregulated proliferation and apoptosis of haematopoietic progenitors and the clinical and morphologic manifestation of CML (Ernst and Hochhaus, 2012; Kishore and Marin, 2011). BCR-ABL transcripts can be rapidly identified by very sensitive techniques such as fluorescence in-situ hybridization or PCR (Ernst et al., 2011; Kishore and Marin, 2011; Saijo, 2012; Sudoyo and Hardi, 2011; Vardiman, 2009), by which the presence of single malignant cells can be detected. Improvements in anticancer treatment have led to increased survival of patients (Feigin et al., 2008). However, therapy can cause premature ovarian failure in girls and women. The options for fertility preservation are limited, and often the only possibility is cryopreservation of ovarian tissue containing small immature ovarian follicles. So far, autologous transplantation of cryopreserved–thawed ovarian tissue in cancer survivors has resulted in approximately 25 live births (Donnez et al., 2013; Silber, 2012). As far as is known, there is only one report of a leukaemia survivor who underwent ovarian reimplantation (Chung et al., 2013). Indeed, there is increasing although as-yet limited evidence of ovarian involvement in various leukaemic diseases (Chung et al., 2013; Dolmans et al., 2010, 2013; Kyono et al., 2009; Meirow et al., 2008; Rosendahl et al., 2010, 2013). The aim of this report was to describe a survivor of paediatric CML who requested ovarian grafting for fertility restoration from her frozen–thawed tissue. However, although pathological or immunohistochemical findings were negative, PCR showed evidence of BCR-ABL1 fusion transcripts in the ovarian tissue. On the basis of these molecular findings, ovarian autografting was not recommended. However, this is a single case, and information from more cases is needed.
Materials and methods Case description The study was conducted at a tertiary medical centre. The protocol was approved by the local Human Investigations Committee (reference number 5875, approved 6 July 2010).
R Abir et al. The patient was diagnosed with CML by bone marrow biopsy at the age of 12 years (December 1999). Laboratory results included an elevated leukocyte count (486,000/ll) and peripheral blood positive for Ph. She had experienced one menstrual cycle with a single menstrual bleeding 2 months before CML diagnosis. She underwent immediate ovarian cryopreservation for fertility preservation before any form of anticancer therapy was initiated. At surgery, the only finding was an enlarged spleen, but the uterus and ovaries were of normal size and with a few follicles larger than 10 mm. Histological examination of her ovaries did not reveal infiltrations of CML (see Pathological and immunohistochemical evaluation). She was treated with hydoroxyurea for 2 months with a decrease of her leukocyte counts (to 5000/ll). In February 2000, she underwent conditioning for bone marrow transplantation with cyclophosphamide (Cytoxan, 120 mg/kg; Bristol-Myers Squibb Co–Mead Johnson and Co, Evansville, IN, USA) and total body irradiation of 1200 centiGrey in six doses with partial lung sparing. She then received bone marrow transplantation from her human leukocyte antigen-identical healthy brother. Post-transplant complications included acute graft versus host (GVH) disease grade II, treated with prednisone at 2 mg/kg/d for six weeks, which eliminated chronic GVH. Her blood count stabilized 14 d after transplantation. Since resolution of acute post-transplantation GVH, she has been well and she continues to have no evidence of BCR-ABL transcripts in her peripheral blood. The percentage of Ph-positive cells from 1 year after the transplantation until the time of writing was 3 (false-positive), while PCR in blood remained negative. She continues to be healthy and is checked routinely. Seven months after the transplantation, she was treated for 7 months with conjugated oestrogens (Premaril 0.625/d; Dexxon, Or Akiva, Israel). Since then she took various forms of oral contraceptives until her wedding at the age of 24. Around that time she stopped the oral contraceptives, became amenorrhoeic, with hot flushes and a single episode of blood spotting. Her hormonal profile included: anti-Mu ¨llerian hormone <0.4 ng/ml; FSH 103 IU/l and thyroid-stimulating hormone 6.2 lIU/ml. Endometrial thickness measured 2 mm. The dimensions of the right and left ovaries were 27 mm · 19 mm and 14 mm · 9 mm, respectively. Upon 17b-oestrogen induction (8 mg/d for nine d), the endometriium thickened to 7 mm.
Histological preparation for light microscopy and immunohistochemistry The histological preparation method for ovarian tissue has been described in detail previously (Abir et al., 2010). These specimens were examined in the Department of Pathology for leukaemic infiltrates. Tissue samples were immunoassayed using as primary antibodies rabbit polyclonal against human myeloperoxidase (1:3000, A0398; DakoCytomation, Glostrup, Denmark), mouse monoclonal antibodies against glycophorin A (1:400, M0819; DakoCytomation), mouse monoclonal antibodies against CD34 (1:100, Biogenex, Fremont, CA, USA), mouse monoclonal antibodies against CD68 (1: 500, M0876, DakoCytomation), mouse monoclonal antibodies against LCA/CD45 (1:250, M0876; DakoCytomation) and rabbit polyclonal antibodies against human Factor VIII
Ovarian minimal residual disease in CML (1:300, A0082; DakoCytomation). The samples were incubated with the primary antibodies for 45 min. The negative controls were incubated with phosphate buffered saline at pH 7 (Biological Industries, Beit Ha’emek, Israel). Thereafter, the samples were incubated with horseradish peroxide polymer conjugate against mouse, rabbit and guinea pig antibodies (SuperPicture HRP, 878963; Zymed Laboratories). Finally, the sections were incubated with a diaminobenzidine urea H2O2 solution (Sigma Fast tablets; Sigma, St Louis, MO, USA) and counterstained with Mayer’s haematoxylin (Pioneer Research Chemicals, Colchester, Essex, UK). All immuonostains were conducted in a Ventana Benchmark XT machine (Hoffmann-La Roche Ltd, Basel, Switzerland).
Quantitative reverse-transcription PCR Total RNA was extracted from the cryopreserved ovary with TRI-reagent (Molecular Research Centre, Cincinnati, OH, USA) and transcribed into cDNA using the Maxima First Strand cDNA Synthesis Kit for quantitative reversetranscription PCR (qPCR; Fermentas Life Sciences, www. fermentas.com). Assessment of the amount of BCR-ABL M-bcr transcripts was performed by qPCR (Rotor Gene 3000) using TaqMan probe-based technology, primers and probes as described (Gabert et al., 2003). Absolute quantification was achieved using the Ipsogen plasmid standard curves. Quantification analysis was performed in accordance with the published recommendations of European LeukaemiaNet (Baccarani et al., 2009). Analysis was conducted twice in quadruplicates each time, with negative cDNA controls (from Ph-negative samples).
Results Pathological evaluation and follicular counts The ovarian specimens were negative for malignancy on pathological evaluation. Moreover, all immunohistochemical stainings did not yield evidence of malignancy. There were up to 20 primordial and primary follicles per ovarian section.
Quantitative reverse-transcription PCR The amplification plots of ABL and BCR-ABL are shown in Figure 1. The results are expressed as the ratio of BCR-ABL/ABL transcripts. The calculated ratio for the cryopreserved ovarian tissue sample was 0.05%. Qualitative two-step nested PCR was conducted twice in quadruplicates each time, and performed as described by the Report of BIOMED-1 concerted Action (Gabert et al., 2003). PCR products were separated on agarose gel (Figure 2). Of note, analysis of concurrent peripheral blood samples by the same technique showed no evidence of BCR-ABL transcripts.
Discussion This case report poses questions of acute clinical relevance: Does ovarian tissue harbour occult leukaemia? If it does,
257 does autotransplant of this tissue place the recipient at risk of disease relapse? This case report shows ovarian contamination by very low levels of minimal residual disease in CML demonstrated by qPCR, despite lack of pathological/immunohistochemical evidence. Molecular biology techniques of PCR and qPCR are extremely sensitive and may indicate the presence of single malignant cells. This notwithstanding, given that ovarian harvesting from this patient was performed before the administration of systematic therapy, the very low concentration of BCR-ABL transcripts that was detected may represent the presence of small amounts of peripheral blood in the harvested tissue. There are three methods for evaluation of malignancy involvement of ovaries. The first is conventional pathological examinations (Kyono et al., 2009), the second is sensitive molecular methods to detect the specific chromosomal abnormality of the minimal residual disease (both on RNA and DNA levels; Abir et al., 2010; Chung et al., 2013; Dolmans et al., 2010; Meirow et al., 2008; Rosendahl et al., 2010, 2013) and the third involves xenografting human ovarian tissue from cancer patients to immunodeficient mice and examining the malignant potential of the human ovary (Chung et al., 2013; Dolmans et al., 2010; Kim et al., 2001; Lotz et al., 2011; Rosendahl et al., 2010, 2011, 2013). The clinical relevance of contaminated minimal residual disease cells in the ovary is not completely clear (Passeque et al., 2003; Saijo, 2012; Sampieri and Fodde, 2012). Already in 1976, an autopsy study reported a relatively high incidence of leukaemic cell infiltration in the testis (48.5%) and in the ovary (58.1%; Kamiyama and Funata, 1976), suggesting the gonads are a sanctuary site for extramedullary leukaemic infiltration, with the ovary being a possible protection niche for leukaemic progenitors. Thus, both hormonal stimulation for IVF as well as ovarian tissue grafting in leukaemia survivors may potentially pose a high risk for disease relapse. However, once transplanted the capability of residual leukaemic cells to repopulate the bone marrow and initiate disease relapse is still unknown. At the same time, there are indications that haematopoiesis may also occur in the ovary and testis (Kamiyama and Funata, 1976; Passeque et al., 2003; Sampieri and Fodde, 2012). The existence of quiescent-dormant CML progenitor cells in ovarian tissue that are either therapy resistant or in tissue from patients after successful therapy should not be excluded, as the cell origin responsible for the disease is still unclear (Kamiyama and Funata, 1976; Kishore and Marin, 2011; Zon, 1995). Although it has not been proven, it is theoretically possible that the dormant state may be a protective mechanism that enhances cell survival under adverse conditions. If this is indeed the case, the ovarian tissue should not have been harvested in the first place with the intention of transplantation but for other putative fertility restoration methods such as in-vitro maturation of primordial follicles (Abir et al., 2006) or implantation of isolated follicles (Vanacker et al., 2012). Moreover, in leukaemia survivors after complete remission, ovarian tissue was positive for molecular markers but did not reseed malignancy in murine hosts or contain any viable malignant cells (Greve et al., 2012). It is, however, theoretically possible that cancer would have been eventually initiated after a longer transplantation period than 20 weeks.
258
R Abir et al.
Figure 1 Quantitative reverse-transcription PCR for ABL (A) and BCR-ABL (B) detection. Typical fluorescence curves obtained for standards (dotted lines) and sample (unbroken lines).
It should also be borne in mind that some cells might exhibit flexibility in responding to different stochastic influences in their developmental milieus (Affer et al., 2011). By contrast, the frequency of leukaemic stem cells in the blood or marrow of patients afflicted with this disease is between 0.2% and 1% of the leukaemic burden (Passeque et al., 2003). As such, the frequency of leukaemia-initiating cells in the ovarian tissue of the current patient was extremely low. Despite this, even after taking into account the results of Greve et al. (2012), certainty the occasional presence of clonogenic leukaemic progenitors cannot be excluded. It will, therefore, be safer to avoid as much as possible this danger by testing the ovaries by all means available for possible malignancy markers. It is noteworthy that, in a study conducted by our group (Abir et al., 2010), a molecular marker of Ewing sarcoma was found in the ovaries of one
girl, while others did not find any traces of malignancy in similar patients (Greve et al., 2013). Ovarian transplant followed by a brief period of treatment with a tyrosine kinase inhibitor such as imatinib may be considered. These drugs, however, do not always eradicate CML at the stem cell level, therefore theoretically leaving a reservoir of cancer stem cells in the transplanted tissue (Kamiyama and Funata, 1976; Kishore and Marin, 2011; Passeque et al., 2003; Sampieri and Fodde, 2012; Zon, 1995). It is also noteworthy that drugs such as hydroxyurea reduce the number of white blood cells (as reported in the present case), but do not damage the ovarian follicles. Therefore, it might be worthwhile to treat patients with hydroxyurea prior to ovarian collection and to reduce CML cell infiltration into the ovary (Chung et al., 2013).
Ovarian minimal residual disease in CML
Figure 2 Two-step nested quantitative reverse-transcription PCR. PCR products were analysed on a 2% agarose gel. Sample lanes 1–4 = cryopreserved ovarian tissue sample with a PCR product of 458 bp, typical for b3a2 transcripts (the breakpoint of BCR gene is downstream of exon 14); NEG = Philadelphia-negative sample derived from healthy control; NTC = no-template control; POS = Philadelphia-positive sample derived from a CML patient with a PCR product of 383 bp, typical for b2a2 transcripts (the breakpoint of BCR gene is downstream of exon 13); ABL = positive control for the gene ABL, demonstrating RNA integrity of the sample); Marker = 100-bp DNA size ladder.
The present report adds to the accumulating evidence of this danger regarding leukaemia. Ovarian leukaemia involvement was documented in a large pathological study (Kyono et al., 2009). The current study, however, is the fourth to identify leukaemia minimal residual disease in the ovary of a patient with CML in the presence of negative pathological findings (Dolmans et al., 2010; Meirow et al., 2008; Rosendahl et al., 2010). Ovarian leukaemia CML minimal residual disease has been reported in 13 patients (Dolmans et al., 2010; Meirow et al., 2008; Rosendahl et al., 2010) including the present case report. Minimal residual disease was identified not only in CML patients, but also in patients with acute lymphoblastic leukaemia (ALL; Dolmans et al., 2010; Rosendahl et al., 2010) and acute myeloblastic leukaemia (Rosendahl et al., 2010). Moreover, when tissue from all patients was transplanted into immunodeficient mice, a portion of the mice developed intraperitoneal leukaemic masses 6 months after grafting (Dolmans et al., 2010). There are reports of ovarian involvement also in nonleukaemia malignancies including Hodgkin’s lymphoma (Bittinger et al., 2011; Kyono et al., 2009), non-Hodgkin’s lymphoma (Kyono et al., 2009), breast cancer, pulmonary carcinoma, gastric carcinoma, colon carcinoma and Ewing sarcoma (Abir et al., 2010). In conclusion, the present report, together with previous studies (Dolmans et al., 2010; Kyono et al., 2009; Meirow et al., 2008; Rosendahl et al., 2010), suggests that CML may leave malignant traces in the ovary. Therefore, clinicians considering ovarian grafting in cancer survivors, especially in leukaemia survivors, should be cautious. So far,
259 ovarian tissue collected before bone marrow transplantation has been reimplanted into one CML survivor, after highly sensitive qPCR did not show evidence of leukaemic cells in the ovarian samples (Chung et al., 2013). The same group did not transplant ovarian tissue to another Phovarian positive patient. Ovarian samples from the patients should be examined both pathologically and by molecular techniques and possibly also by transplantation into immunodeficient mice. However, it is doubtful if the use of a murine host will be accepted for clinical purposes nor is the exact period of malignancy introduction clear. The bias of sampling error, however, can never be completely eliminated. It is clear that new methods will be needed to insure safer fertility restoration for patients with acute or chronic leukaemia. The methods include the development of an in-vitro maturation system for frozen–thawed primordial follicles (Abir et al., 2006) or transplantation of isolated ovarian follicles rather than of ovarian tissue (Vanacker et al., 2012). In summary, this study demonstrates the presence of occult leukaemia in a histologically normal ovary harvested from a patient with CML. The theoretical risk of cancer recurrence due to the presence of clonogenic leukaemia cells in the ovary has caused ovarian autotransplantation to be deferred in this patient.
Acknowledgements The authors are greatly indebted to Ms Gloria Ganzach from the Editorial Board of Rabin Medical Center, Beilinson Hospital and to Dr Yehezkel Lande from our department for the English editing. We are also indebted to our laboratory technician Ms Carmela Felz for assistance with the histological sectioning.
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