Backtracking RAS mutations in high hyperdiploid childhood acute lymphoblastic leukemia

Abstract

High hyperdiploidy is the single largest subtype of childhood acute lymphoblastic leukemia (ALL) and is defined by the presence of 51-68 chromosomes in a karyotype. The 5 or more extra chromosomes characterizing this subtype are known to occur in a single mitotic event, prenatally. We screened for RAS mutations among 517 acute childhood leukemias (including 437 lymphocytic, of which 393 were B-cell subtypes) and found mutations in 30% of high hyperdiploids compared to only 10% of leukemias of other subtypes (P<0.0001). We assessed whether KRAS mutations occurred before birth using a PCR-restriction enzyme-mediated Taqman quantitative PCR reaction, and found no evidence for prenatal KRAS mutations in 14 patients tested. While RAS mutations were previously associated with prior chemical exposures in childhood and adult leukemias, in this study RAS-mutated cases were not significantly associated with parental smoking when compared to study controls. IGH rearrangements were backtracked in three RAS-positive patients (which were negative for KRAS mutation at birth) and found to be evident before birth, confirming a prenatal origin for the leukemia clone. We posit a natural history for hyperdiploid leukemia in which prenatal mitotic catastrophe is followed by a postnatal RAS mutation to produce the leukemic cell phenotype.

Copyright © 2010 Elsevier Inc. All rights reserved.

Figures

Figure 1. TaqMan + REMS-PCR Backtracking

Electrophoresis analysis of REMS-PCR. SW837 DNA and various dilutions in REH DNA are shown, followed by two lanes with no template (blank) and two lanes of REH DNA (25 ng) run without BstN1 enzyme. All lanes with DNA include 25 ng; dilutions indicate the amount of SW837 within a background of REH DNA. Arrows indicate (i) – PCR control DNA, (ii) - BstN1 restriction enzyme control product, and (iii) – diagnostic mutant KRAS band. A, SW837 DNA, 4,166 copies (25 ng); B, 417 copies of SW837 DNA diluted in 25 ng WT DNA; C, 42 copies SW837 DNA diluted in 25 ng WT DNA; D, 4 copies of SW837 DNA in 25 ng of WT DNA; E, 25 ng REH cell line DNA; Blank, no DNA added to reaction; Guthrie shows an example of one Guthrie patient with positive control amplification band and negative bands for BstN1 control and diagnostic band. B. Amplification plot for testing Guthrie Card DNA. All reactions were performed in triplicate with 1 mM of each diagnostic primer, 40 nM of both the PCR control primer set and the RE control primer set. Each line represents the amplification of A to D as above; Guthrie, 25 ng of a test Guthrie DNA from a patient who had a KRAS mutation, which does not have detectable mutant KRAS. REH DNA (lane E) and “Blank” lanes also did not yield amplification, similar to Guthrie (data not shown).