It has been proposed that hypermutability is necessary to account for the high frequency of mutations in cancer. However, historically, the mutation rate (μ) has been difficult to measure directly, and increased cell turnover or selection could provide an alternative explanation. We recently developed an assay for μ using PIG-A as a sentinel gene and estimated that its average value is 10.6 × 10 -7 mutations per cell division in B-lymphoblastoid cell lines (BLCLs) from normal donors. Here we have measured μ in human malignancies and found that it was elevated in cell lines derived from T cell acute lymphoblastic leukemia, mantle cell lymphoma, follicular lymphoma in transformed phase, and 2 plasma cell neoplasms. In contrast, μ was much lower in a marginal zone lymphoma cell line and 5 other plasma cell neoplasms. The highest μ value that we measured, 3286 × 10 -7 , is 2 orders of magnitude above the range we have observed in non-malignant human cells. We conclude that the type of genomic instability detected in this assay is a common but not universal feature of hematologic malignancies. IntroductionA model sentinel gene to measure spontaneous somatic mutations must be non-essential for growth or viability, and the mutant phenotype must be detectable among a vastly larger population of normal cells. Since a single mutation conferring loss of function could be complemented by the wild type allele on the homologous chromosome, most autosomal genes are not suitable for this purpose. However, due to hemizygosity in males and X-inactivation in females, a single mutation is sufficient to inactivate X-linked genes. For example, among lymphocytes in normal adults, resistance to 6-thioguanine occurs as a consequence of a spontaneous inactivating mutation in HPRT (Xq26-q27.2), at a frequency (f) of ∼ 2 -10 × 10 -6 [1].Corresponding author. David J. Araten, MD, NYU Cancer Center, 7 th Floor, 160 East 34 th Street, New York, NY 10016. 212-731-5186 (office); 212-731-5540 (fax). David.Araten@nyumc.org. There are no conflicts of interest to report.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. While f represents the proportion of mutants within a population, the mutation rate (μ) represents the probability of a new mutation per cell division. If mutations are growth-neutral, f will increase over time according to the formula Δf = μ × Δd, where d represents number of cell divisions. By analysis of f values for HPRT in human population groups of different ages and estimates for Δd in human lymphocytes in vivo, it has been proposed that the normal μ value for this gene is ∼8.4 ×...

The mutation rate (μ) is likely to be a key parameter in leukemogenesis, but historically, it has been difficult to measure in humans. The PIG-A gene has some advantages for the detection of spontaneous mutations because it is X-linked, and therefore only one mutation is required to disrupt its function. Furthermore, the PIG-A-null phenotype is readily detected by flow cytometry. Using PIG-A, we have now provided the first in vitro measurement of μ in myeloid cells, using cultures of CD34+ cells that are transduced with either the AML-ETO or the MLL-AF9 fusion genes and expanded with cytokines. For the AML-ETO cultures, the median μ value was ∼ 9.4 × 10-7 (range ∼ 3.6 to 23 × 10-7) per cell division. In contrast, few spontaneous mutations were observed in the MLL-AF9 cultures. Knockdown of p53 or introduction of mutant NRAS or FLT3 alleles did not have much of an effect on μ. Based on these data, we provide a model to predict whether hypermutability must occur in the process of leukemogenesis.

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.

The mutation rate (μ) is likely to be a critical parameter in leukemogenesis, but has not previously been measured in human myeloid cells. Using the PIG-A gene as a sentinel, we have recently measured the mutation rate in B-lymphoblastoid cell lines (BLCLs) and shown that it is elevated in cell lines derived from lymphoid malignancies. Here we have measured μ in normal human CD34+ cells derived from cord blood samples that have been transduced with an AML-ETO fusion gene, growing under the influence of cytokines. PIG-A is an X-linked gene essential for the synthesis of glycosylphosphatidylinositol (GPI). Due to X-inactivation in females and hemizygosity in males, a single mutation can produce the GPI (−) phenotype. From patients with PNH, it is known that a broad spectrum of mutations can inactivate PIG-A. Mutants do not express GPI-linked surface proteins such as CD55 or CD59 and do not bind to the FLAER reagent (which binds GPI directly), but do express transmembrane proteins (e.g. CD45). To measure the rate of new mutations in vitro, we first flow-sorted each culture to collect CD59(+) cells, in order to exclude pre-existing mutants. Then we expanded the collected cells over 3 weeks; using cell counts, we calculated population doublings in culture (d), which ranged from 5.1 to 7.4. Cells were then stained sequentially with biotinylated FLAER, murine anti-CD55 and anti-CD59 antibodies, rabbit anti-mouse PE and streptavidin-PE secondary conjugates, and CD45-FITC. Live cells were identified by forward and side scatter, propidium iodide exclusion, and expression of CD45. The mutant frequency (f) was calculated as the number of GPI (−) cells [which do not bind anti-CD55, CD59 or FLAER] divided by the total number of cells analyzed. An average of 1.2 million cells were analyzed to detect rare mutants. The mutation rate was calculated by the formula μ = f/d. Among 5 cultures derived from 2 different cord blood samples, the mean mutation rate was 12.4 × 10−7 (range 5.1 to 21.4 × 10−7) per cell division. This value is consistent with mathematical models of the mutation rates in human populations as well as our previous experimental determination of μ in BLCLs. We also analyzed 6 cell lines in which p53 had been disrupted by various methods, including shRNA, a dominant-negative p53 mutant, and introduction of HPV E6/E7. Overall, disruption of p53 resulted in a 30% increase in the mutation rate (p=.02). We conclude that spontaneous mutations in human myeloid cultures are rare but measurable and that disruption of p53 results in a modest increase in μ. We predict that the ability to measure this critical parameter in human hematopoietic cells will facilitate the investigation of the role of specific oncogene and tumor suppressor mutations in inducing hypermutability.