Characterisation of T-ALL Cells in Serial NOD/SCID Xenografts.

The treatment of paediatric T lineage ALL has improved considerably in recent years. However, a significant proportion of children continue to fail therapy and intensification of treatment regimens may only lead to an increase in the incidence of adverse risk effects. Therefore, it is necessary to increase our understanding of the early stages of development of this malignancy and its pathogenic mechanisms. In the current study we have established and characterised continuous xenografts from 6 patients with childhood T-ALL as a renewable source of leukaemia cells that retain the essential characteristics of the original disease. Unsorted cells from 6 children diagnosed with T-ALL were inoculated into the lateral tail vein of sublethally irradiated NOD/SCID mice. From two weeks post-inoculation onwards, engraftment levels in the peripheral blood (PB) were monitored by serial tail vein sampling and bone marrow (BM) aspirates were analysed from 4 weeks post inoculation. All animals were sacrificed no later than 10 weeks post inoculation. Human CD45⁺ cells were first detected 2 weeks after inoculation (1.6–5% CD45⁺) in animals injected with cells from two of the six patients. By week 4, CD45⁺ cells were detected in the PB from animals injected with cells from all 6 patients (2–22% CD45⁺) and higher levels were detected in the BM aspirates (5–52% CD45⁺). In each case, the levels of human cell engraftment increased with time and by week 10 significant levels of CD45⁺ cells were detected in NOD/SCID BM (30–99% CD45⁺). Flow cytometric and FISH analyses confirmed that the engrafted cells had a similar immunophenotype and karyotype as the diagnostic samples, confirming engraftment of leukaemia cells. BM cells harvested from the primary animals were subsequently used in serial transplantation studies to determine whether T-ALL could be transferred into serial recipients. To date, secondary and tertiary transplant experiments have resulted in similar levels of engraftment with no loss of NOD/SCID repopulating potential in all 6 cases. Cells from 2 patients have been used in quaternary, quinary and senary transplants with similar results (57–99% CD45⁺) and no changes observed in either the rate of engraftment nor the immunophenotype or karyotype of the passaged cells. Molecular analyses were performed on the diagnostic samples and cells from the established xenografts to screen for the presence of unique T cell receptor gamma (TCRG) gene rearrangements, present in approximately 95% of T-ALL patients. The PCR products were subjected to heteroduplex analysis for clonality assessment followed by sequencing of monoclonal products. The exact junctional rearrangement was established by comparison with all known human TCRG germline sequences. Results to date have indicated that the unique rearrangements detected at diagnosis were also found in cells harvested from primary, secondary and tertiary xenografts. Analyses of cells from successive xenografts are ongoing to determine whether the unique genotype remains stable. These findings demonstrate that the immunophenotype, karyotype and genotype of 6 paediatric T-ALL cases were preserved with multiple serial passages in NOD/SCID mice. These continuous xenografts retained the fundamental biological characteristics of the original disease and thus could be a valuable experimental model system to augment our knowledge of the biology and regulation of paediatric T-ALL and a credible tool for the preclinical evaluation of novel agents.