The PIG-A Gene Is Hypermutable in Lymphoid Neoplasms.

Abstract

In neoplasms, the presence of chromosomal abnormalities and point mutations is suggestive of genomic instability, but could also be a consequence of selection. While genomic instability may increase the chances that a malignant population acquires adaptive mutations, extremely high mutation rates may not be compatible with cell survival. To investigate the role of genomic instability in lymphoid neoplasms, we have applied a new method for the quantification of the human mutation rate, using the PIG-A gene as a sentinel. PIG-A is essential for the biosynthesis of glycosylphosphatidylinositol (GPI) and is mutated in blood cells of patients with Paroxysmal Nocturnal Hemoglobinuria. A broad range of mutations can produce the GPI (−) phenotype, and because PIG-A is on the X-chromosome, the effect of a single mutation is detectable. Since a host of proteins require GPI for attachment to the cell surface, rare mutants are readily detected by flow cytometry. We have previously shown that PIG-A mutations arise spontaneously in normal donors, and we determined that the mutation rate in normal B cell lines ranges from 2 to 29 per 107 cell divisions. Here we analyzed cell lines derived from: a transformed low grade lymphoma harboring a t(14;18) translocation; a mantle cell lymphoma harboring a t(11;14) translocation; a marginal zone lymphoma; and T cell ALL. Cells were first stained with an antibody specific for CD59 (a representative GPI-linked protein) and pre-existing GPI (−) cells were eliminated from the population by flow cytometric sorting, by gating on the upper 50th percentile of the distribution curve. The collected GPI (+) cells were then returned to culture and the number of cell divisions (d) determined by cell counts. After 3–4 weeks, the frequency (f) of new mutants arising in culture was determined by flow cytometric analysis of a large number of cells (median 2.3 x 106). Cells were stained simultaneously with antibodies specific for at least 3 GPI-linked proteins (e.g. CD48, CD52, CD55, and CD59) as well as a transmembrane protein (e.g. HLA-DR or CD45) to identify live cells. FLAER and proaerolysin-- which bind to GPI-- were used to confirm the phenotype. The frequency of mutants was determined by the number of GPI(−) cells divided by the number of GPI(+) cells analyzed, and the mutation rate (μ) was calculated with the formula μ = f ÷ d. We demonstrated a high mutation rate in 3 out of the 4 cells lines: 1750 x 10−7 (transformed lymphoma), 335 x 10−7 (mantle cell lymphoma), 112 x 10−7 (T cell ALL). Of note, the mutation rate was normal (4 x 10−7) in the marginal zone lymphoma—consistent with this being an indolent neoplasm. These data support the hypothesis that an elevated mutation rate is part and parcel of aggressive neoplasms and demonstrate that a 2-log elevation in this parameter is compatible with cell survival. With this model, it may be possible to predict the development of mutations that confer chemotherapy drug resistance.