Testing in Vitro Sensitivity of Minimal Residual Disease in Pediatric Acute Lymphoblastic Leukemia.

Abstract 2543

Acute lymphoblastic leukemia (ALL) is the most frequent malignant disease in children and adolescents. Although improved risk grouping, anti-cancer treatment, and supportive care have resulted in survival rates above 80 %, 15–20 % of patients experience a relapse, which is associated with an inferior prognosis. Relapse is caused by persistence of minimal residual disease (MRD) primarily in the bone marrow during chemotherapy, and methods for identification of drugs that are capable of eliminating the MRD cell population is necessary, but not currently available. In vitro sensitivity testing has been carried out at the time of diagnosis and shows predictive value of treatment outcome and correlation to MRD levels. However, the in vitro sensitivity profiles have not been applicable for treatment stratification, since the vast majority of leukemic cells present at diagnosis are rather chemosensitive, as opposed to the MRD population. Thus, the more sensitive clones mask the more resistant ones. We hypothesized that in vitro sensitivity testing of the more resistant MRD cells remaining in bone marrow after the induction therapy could help to stratify patients to individualized chemotherapy.

The greatest challenge in carrying out in vitro sensitivity testing on MRD cell populations is the low number of leukemic cells available, i.e. 0.1–5%. Traditionally, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flourometric microculture cytotoxicity assays have been used for in vitro sensitivity testing. They, however, need several million cells for in vitro sensitivity testing of 3 drugs. We aimed at developing a cell death assay applicable on small ALL cell populations. We show that a flow cytometry based assay using annexin V (reflecting exposure of phophatidylserine on the outer cell membrane during early apoptosis) and 7-Aminoactinomycin D (reflecting cell membrane rupture during late apoptosis) staining is a reliable method for evaluating early and late stages of cell death in small ALL cell populations. Both in ALL cell lines REH (B-cell precursor (BCP) ALL, t(12;21), Jurkat (t-ALL), RS4;11 (BCP ALL, t(4;11)) and Nalm-6 (BCP ALL, t(5;12)) and in primary ALL cell samples the results are highly reproducible and show the same relative sensitivity profile as when a large number of cells are used. This cell death assay only demands a total of 20.000 cells to determine the in vitro sensitivity for 3 drugs run in triplicates at 4 concentrations. This assay is thus applicable for sorted MRD populations accounting for 0.1% of the mononuclear cells in the bone marrow.