Pilot Study of the Feasibility of Assessing DNA Damage and Apoptosis Induced by Chemotherapy in Children with Acute Leukemias.

The aim of the present study was to explore whether early assessment of the extent of DNA damage in leukemic blasts and incidence of apoptosis 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 7 ALL patients with peripheral blasts, 4 of whom were, at diagnosis, high-risk ALL while the remaining 3 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:

1. prior to drug administration; and,

2. one hour after completion of the drug infusion.

The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation (γH2AX) occurring at sites of DNA double-strand breaks (DSBs), which could be detected immunocytochemically and measured by multiparameter flow cytometry in conjunction with DNA content. The incidence of apoptosis was estimated 1 h after drug administration and also after 24 h ex vivo culturing, by identification of cells with DNA fragmentation detected by the TUNEL assay. In patients with complete clinical response (6 of 7) at the end of the induction phase, an increase of γH2AX (post treatment - pretreatment mean) was detected 1 h after the administration of drug treatment. The apoptotic index in this group after 24 h ex vivo culture was 1–16%. In the only patient not responding to treatment - one of 2 relapsed ALL patients -, there was no induction of DSBs or increase in apoptosis.

Although the number of patients is very small in this pilot study, the results suggest that a cytometric assay that reveals the extent of DNA damage based on detection of H2AX phosphorylation may provide an early prognostic marker.