Leukemic blasts with the paroxysmal nocturnal hemoglobinuria phenotype in children with acute lymphoblastic leukemia

Abstract

It has been proposed that genomic instability is essential to account for the multiplicity of mutations often seen in malignancies. Using the X-linked PIG-A gene as a sentinel gene for spontaneous inactivating somatic mutations, we previously showed that healthy individuals harbor granulocytes with the PIG-A mutant (paroxysmal nocturnal hemoglobinuria) phenotype at a median frequency (f) of ∼12 × 10(-6). Herein, we used a similar approach to determine f in blast cells derived from 19 individuals with acute lymphoblastic leukemia (ALL) and in immortalized Epstein-Barr virus-transformed B-cell cultures (human B-lymphoblastoid cell lines) from 19 healthy donors. The B-lymphoblastoid cell lines exhibited a unimodal distribution, with a median f value of 11 × 10(-6). In contrast, analysis of the f values for the ALL samples revealed at least two distinct populations: one population, representing approximately half of the samples (n = 10), had a median f value of 13 × 10(-6), and the remaining samples (n = 9) had a median f value of 566 × 10(-6). We conclude that in ALL, there are two distinct phenotypes with respect to hypermutability, which we hypothesize will correlate with the number of pathogenic mutations required to produce the leukemia.

Figure 1

Flow cytometry pseudocolor dot plot analyses of controls. Fluorescein isothiocyanate (FITC) and Alexa 488 register on FL1 (horizontal axis) and reflect the density of the GPI-linked proteins (CD55 and CD59) and the GPI anchor itself, respectively, on the surface of the cell. Phycoerythrin (PE) registers on FL2 (vertical axis), reflecting the density of CD45, a non–GPI-linked membrane protein. GPI⁻ cells register in the top left quadrant, and GPI⁺ cells register in the top right quadrant. A: Peripheral blood lymphocytes (PBLs) isolated from a patient with PNH. There are two distinct populations representing GPI⁺ and GPI⁻ cells. B: A spontaneously arising GPI⁻ clone of the Jurkat cell line registering in the top left quadrant. C: A representative BLCL derived from a healthy donor (BLCL 12): most of the cells are GPI⁺, with a small but distinct subpopulation of GPI⁻ cells registering in the top left quadrant. The frequency of these spontaneously arising phenotypic variants is 24 × 10⁻⁶ in this example. D: Unstained thawed blasts from a patient with ALL.

Figure 2

Flow cytometry pseudocolor dot plot analyses of samples derived from ALL blast populations. A and B: Representative examples of samples with a low frequency of spontaneously arising GPI⁻ phenotypic variants (patients 7 and 17, respectively). C: An example of a sample with an intermediate-sized population of GPI⁻ phenotypic variants (patient 11). D–F: Representative examples of samples exhibiting a very high frequency of GPI⁻ phenotypic variants (patients 5, 14, and 19, respectively). FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Figure 3

Histogram of f values for BLCL and ALL samples. A: Using a λ value of 0.25 in a Box-Cox transformation, f values for the BLCLs are unimodal and nearly symmetrical, and they fall on a nearly straight line in a q-q plot, suggesting that these values are near normally distributed. B: There is no transformation that could produce a unimodal symmetrical distribution for the f values measured in ALL samples. Using a log transformation of the f values, it is seen that the distribution is bimodal or trimodal.