Abstract 3849: Analysis of the mutation rate in T lymphoblastic leukemia cell lines

Abstract

Abstract To account for the high number of somatic mutations in cancer, it has been hypothesized that genomic instability is necessary for malignant transformation. We previously demonstrated that PIG-A is a useful reporter gene for quantitating somatic mutations. PIG-A encodes an enzyme that is essential for the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor, by which a set of membrane proteins are covalently linked to the cell surface. PIG-A is X-linked, and a broad spectrum of mutations occurring in only one allele can produce the GPI (-) phenotype. We have demonstrated occult populations of spontaneously arising blood cells with PIG-A mutations in normal individuals, and shown that among ex vivo blasts from patients with ALL, the frequency (f) of GPI(-) cells appeared trimodal, with some samples overlapping the normal range, whereas others were orders of magnitude higher. f values in T cell leukemias generally fell in the middle group. Here, we have studied a panel of cell lines derived from patients with T cell ALL and measured the mutation rate per cell division (μ) as an assessment of genomic instability. First, cells were stained with anti-CD59-FITC and sorted by collecting the upper 50th percentile, to eliminate pre-existing mutants. This purified GPI(+) population was counted and expanded in culture for 18 days, and then recounted to estimate cell divisions (d) in vitro. To estimate the frequency of new mutants arising in culture, the cells were first incubated with the FLAER reagent conjugated to Alexa 488, then a mixture of anti-CD55 and anti-CD59 antibodies, then a rabbit anti-mouse FITC secondary conjugate, followed by PE conjugated antibodies specific for transmembrane proteins(CD45 or HLA). Cells were gated based on light scatter, exclusion of propidium iodide, and expression of the transmembrane proteins; GPI(-) negative cells were defined as having <4% of the FITC/Alexa fluorescence as the population overall. Mutation rate was calculated by the formula μ = f divided by d. We found that one sample, derived from the cell line P12, demonstrated a mutation rate of 17.5 × 10−7 per cell division, whereas for the cell lines CEM, CUTTL, LOUCY, and HPBALL, μ ranged from 50 to 60 × 10−7. For comparison, we sorted, expanded, and analyzed cultures of T cells derived from 4 normal donors, stimulated with PHA, cytokines, and bead-immobilized antibodies, and here μ was generally lower, ranging from 1.5 × 10−7 to 18 × 10−7 (p <0.04, 2-sided non parametric test). The subtle elevation in μ in T cell ALL was in contrast to cell lines that we have studied from Burkitt's lymphomas, where the distribution was bimodal and where the μ values were all either below 25 × 10−7 (4 samples) or > 200 × 10−7(3 samples, p = 0.01, Fisher's exact test). We conclude that T cell ALL samples may demonstrate a more mild form of genomic instability than in Burkitt's lymphoma, and that genomic instability, as determined by this assay, is not absolutely required for leukemogenesis. Citation Format: David J. Araten, Erik Sherman, Aaron Etra. Analysis of the mutation rate in T lymphoblastic leukemia cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3849. doi:10.1158/1538-7445.AM2015-3849