Backtracked analysis of preleukemic fusion genes and DNA repair foci in umbilical cord blood of children with acute leukemia
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Milan Škorvaga1, Matúš Durdík1, Pavol Košík1, Eva Marková1, Marek Holop2, Miroslav Kubeš2, Judita Puškáčová3, Alexandra Kolenová3 and Igor Belyaev1
1Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovak Republic
2Eurocord-Slovakia, Bratislava, Slovak Republic
3Children’s Hematology and Oncology Clinic, Faculty of Medicine, Comenius University, Bratislava, Slovak Republic
Igor Belyaev, email: Igor.Beliaev@savba.sk
Keywords: leukemia; umbilical cord blood; preleukemic fusion genes; imaging flow cytometry; translocations
Received: November 09, 2017 Accepted: March 13, 2018 Published: April 10, 2018
The first event in origination of many childhood leukemias is a specific preleukemic fusion gene (PFG) that arises, often in utero, in hematopoietic stem/progenitor cells (HSPC) from misrepaired DNA double strand break (DSB). An immanently elevated level of DSB and impaired apoptosis may contribute to origination and persistence of PFG and donor cell-derived leukemia in recipients of allogeneic transplantation of umbilical cord blood (UCB). We investigated DSB, apoptosis and PFG in the backtracked UCB cells of leukemic patients. RNA from UCB of three patients with acute lymphoblastic leukemia, patient with acute megakaryoblastic leukemia and Down syndrome, and four healthy children was screened for common PFG by RT-qPCR. Presence of PFG was validated by sequencing. Endogenous γH2AX and 53BP1 DNA repair foci, cell populations, and apoptosis were analyzed in UCB CD34+/- cells with imaging and standard flow cytometry. We found MLL2-AF4 and BCR-ABL (p190) fusion genes in UCB of two out from four pediatric patients, apparently not detected at diagnosis, while UCB cells of TEL-AML1+ ALL patient were tested negative for this PFG and no PFG were detected in UCB cells of healthy children. No significant difference in DNA damage and apoptosis between UCB CD34+/- cells from healthy children and leukemic patients was observed, while Down syndrome trisomy increased DNA damage and resulted in distribution of cell populations resembling transient abnormal myelopoiesis. Our findings indicate increased genetic instability in UCB HSPC of leukemic patients and may be potentially used for diagnostics and exclusion of possibly affected UCB from transplantation.
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