Assessing DNA Damage Ex Vivo Induced by Chemotherapy Targeting Topoisomerase Inhibitors in Acute Leukemias.

Our previous experiments on leukemic cell lines demonstrated that the intensity of immunofluorescence (IF) that is proportional to the level of the phosphorylated histone H2AX (γH2AX) reveals the frequency of DNA double-strand breaks (DSBs) in chromatin induced by DNA topoisomerase I and II (topo I and II) inhibitors. The aim of the present study was to explore whether ex vivo assessment of the extent of DNA damage in leukemic blasts induced during chemotherapy is feasible. The long term goal is to develop a rapid assay to assess the effectiveness of chemotherapy early in the course of treatment of leukemia. Mononuclear cells were isolated from blood samples of 1 AML and 10 ALL patients with peripheral blasts, 4 of whom were, at diagnosis, high-risk ALL while 4 were in relapse. The median age of the patients was 17 yrs (2–25 yrs) and all were entered into an anthracycline-containing drug regimen. Samples were collected:

• prior to drug administration; and,

• one hour after completion of the drug infusion.

The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation occurring at sites of DSBs, which could be detected immunocytochemically and measured by multiparameter flow cytometry in conjunction with DNA content. In the only patient not responding to treatment, one of 4 relapsed ALL patients, there was no induction of DSBs and no activation of ATM. In all patients phosphorylation of ATM appeared to be strongly linked to induction of DSBs after treatment targeting topo inhibitors. In a pilot study of 3 adult patients with acute leukemia (two AML and one ALL in relapse) treated with the topo II inhibitor Mitoxantrone (MTX), ATM activation was strongly linked to induction of DSBs 1 h post drug infusion. ATM activation and induction of DSBs was several times higher in two adult responders in comparison to one non responder to therapy. A new approach to detect DNA damage in leukemic cell by flow cytometry using gating strategy which combines CD45 expression on the cell surface with side scatter (SS) is being developed and was applied in 5 cases. This approach will be useful when the proportion of blasts in peripheral blood is low. Although the number of patients is very small in this study, the results suggest that a cytometric assay that reveals the extent of DNA damage based on detection of H2AX phosphorylation during induction therapy targeting topo I and II inhibitors may provide an early prognostic marker.