Preservation of male fertility in childhood acute leukemia : An experimental study addressing novel strategies and putative risks

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Women's and Children's Health

Sammanfattning: Although treatment of cancer in young boys with cytotoxic agents has long been known to cause infertility in the survivors, the mechanisms underlying drug-induced damage to the immature testis remain unclear. A number of clinical issues associated with transplantation of testicular germ cells or tissues also require clarification. The present investigation was designed to address some of these questions. First, we examined how a cytotoxic drug causes damage to immature testes. Following a single intraperitoneal injection of doxorubicin and, in some cases, additional administration of amifostine to 6-, 16- and 24 day-old rats, the frequency of apoptosis in microsegments of seminiferous tubules was evaluated. Gonocytes and migrating spermatogonia were identified as the targets for doxorubicin, which caused an 8-fold increase in the apoptosis of these cell types in the youngest rats. Activation of p53 and caspase-8 were the key initial events involved in germ cell apoptosis. Amifostine was incapable of protecting germ cells from doxorubicin-induced apoptosis (Paper I). In order to explore transplantation of testicular germ cells as a strategy for preservation of fertility, acute T-lymphoblastic leukemia (ALL) induced experimentally in rats was employed as a model for children with ALL. When testicular cells isolated from the testes of leukemic rats were transplanted into the seminifereous tubules of syngenic healthy rats, all of the recipients died from donor-derived leukemia, indicating a high risk of reseeding tumor cells into patients. The development of novel strategies for elimination of tumor cells and/or of animal models for detection of contaminated leukemic cells is thus required (Paper II). For this purpose, two purification procedures based on surface markers, the flow cytometric sorting (FACS) and immunomagnetic cell sorting (MACS), were evaluated. First CD4/MHC-Cl I and Ep-CAM, were identified as appropriate markers for leukemic and germ cells, respectively Labeling these cells with fluorescent monoclonal antibodies against these markers allowed positive selection of germ cells and/or negative depletion of leukemic cells. A combination of these procedures yielded testicular cells free from leukemic cells, but the efficacy and safety of this approach are inadequate for clinical use. Variations in the immunophenotypes and other forms of heterogeneity among testicular leukemic cells, together with aggregation of leukemic and germ cells, were the critical factors that prevented efficient purification of germ cells by FACS (Paper III). The efficiency of MACS in removing leukemic testis cells labeled with microbeads conjugated with antibody against CD4 was then evaluated. With this procedure, contamination of the germ cell preparation obtained was only reduced 30% (as determined by FACS) and after injection of this preparation into healthy animals, all of the recipients died of leukemia. Clearly, MACS is incapable of eliminating leukemic cells from germ cell preparations (Paper IV). Finally, fresh or cryopreserved testicular tissues obtained from leukemic rats were grafted subcutaneously into syngenic PVG rats or nude mice to determine whether mature sperm can be produced by such grafts contaminated with leukemic cells as well as to evaluate the possibility of using nude mice for the detection of contaminated leukemic cells. All of the auto- and xenografted animals developed local tumors at the site of injection and/or generalized leukemia. The seminiferous tubules were destroyed by the leukemic cells and no gametes were retrieved. Thus, although testicular grafts contaminated with leukemic cells do not produce gametes, xenotransplantation of testicular tissue into nude mice may provide a valuable biological indictor of leukemic cell contamination in testicular biopsies (Paper V). Our data provide highly relevant preclinical information concerning cytotoxic damage to the immature testis and transplantation of testicular cells and testicular tissue following treatment for leukemia, and should help in the design of strategies for preservation of fertility in boys treated for cancer. The xenograft model offers a clinical tool for assessing tumor cell contamination in testicular biopsies from boys with cancer designated for use in restoring fertility.

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