Prenatal origin of acute lymphoblastic leukaemia in children

Summary

Background

There is little current insight into the natural history of childhood leukaemia or the timing of relevant mutational events. TEL-AML1 gene fusion due to chromosomal translocation is frequently seen in the common form of childhood acute lymphoblastic leukaemia. We investigated whether this abnormality arises prenatally.

Methods

We identified, by reverse-transcriptase PCR screening of blood or bone marrow, TEL-AML1 fusion in 12 children, plus a pair of identical twins, aged 2–5 years from Italy and the UK, who had newly diagnosed acute lymphoblastic leukaemia. We amplified and sequenced the genomic TEL-AML1 fusion gene with a long-distance inverse PCR method. Primers were designed that could be used in short-range PCR to screen for patient-specific, leukaemia clone-specific TEL-AML1 genomic fusion sequences in neonatal blood spots from each child.

Findings

We initially identified TEL-AML1 fusion sequences in blood spots from the identical twins, diagnosed with concordant acute lymphoblastic leukaemia at age 4 years, who shared a single or clonotypic TEL-AML1sequence that suggested prenatal origin in one twin. Three children were excluded because control genes could not be amplified. Of the other nine patients, six had positive blood spots. Blood spots that were classified as negative were uninformative.

Interpretation

Our findings showed that childhood acute lymphoblastic leukaemia is frequently initiated by a chromosome translocation event in utero. Studies in identical twins show however that such an event is insufficient for clinical leukaemia and that a postnatal promotional event is also required.

Introduction

Acute lymphoblastic leukaemia is the main type of leukaemia in children and the most common childhood cancer overall.¹ The disease is biologically and clinically heterogeneous, but one subtype, referred to as common acute lymphoblastic leukaemia, derived from B-cell progenitors, accounts for the peak incidence of acute lymphoblastic leukaemia between ages 2 years and 5 years2, 3 and for a large proportion of cases that are curable by combination chemotherapy.4, 5

There has been much speculation on the cause of childhood cancers, especially leukaemia, commonly in the context of apparent clusters of disease.6, 7 Ionising radiation is the only known cause of de-novo acute leukaemia in children, but is unlikely to represent a major causal pathway.⁸ Epidemiological studies have suggested a possible role of non-ionising radiation, some chemicals, and, especially, infection.8, 9, 10 What is generally missing from such studies is any insight into the natural history of the disease and the likely timing of key exposures and mutational events.

Clinically evident symptoms of childhood acute lymphoblastic leukaemia are generally present only for a few weeks before diagnosis,¹¹ and rarely for several months.¹² The development of acute lymphoblastic leukaemia probably therefore involves a covert or clinically silent phase between initiation and the onset of symptoms. Given the young age of most children who have acute lymphoblastic leukaemia and the latency expected for clonal evolution of cancer,³ it is possible that the disease originates before birth in utero. The only evidence to support this hypothesis has, however, been weak and indirect, since it was derived from epidemiological associations with radiation or viral exposures during pregnancy.13, 14, 15, 16

Molecular insights into childhood leukaemia have provided an opportunity to investigate the natural history of the disease more clearly. In particular, the consistent chromosomal and molecular genetic abnormalities provide stable and unique (or clonotypic) markers for tracking the leukaemic cells.³ Molecular studies on several pairs of identical twins, aged between 2 months and 14 years at diagnosis, have provided strong evidence that concordant acute lymphoblastic leukaemia arises in monozygotic twins after mutation and clonal expansion of one cell in one fetus in utero.17, 18, 19, 20 Since the disease in twins is not different, biologically or clinically, from that in singletons, at least some singletons are also likely to have prenatal initiation of leukaemia.

The concordance rate in monozygotic twins is not, however, known accurately, but is estimated to be around 5% (for children aged >1 year).21, 22 It follows that some additional event or exposure is required postnatally, for which discordance is the rule in twins, or that most childhood acute lymphoblastic leukaemia in twinned and non-twinned children is initiated postnatally.

Since this issue has a major bearing on the cause of childhood leukaemia, we aimed to find a direct and unambiguous test. We devised a way to identify the presence of clonotypic or patient-specific leukaemia fusion-gene sequences in neonatal blood spots or Guthrie cards. Neonatal blood samples are routinely used to screen for inherited metabolic disorders. The cards are commonly retained for many years, generally at room temperature, but have intact DNA that is amplifiable by PCR.²³ This source of DNA has been used to identify constitutive or inherited mutations23, 24 and exogenous viral sequences²⁵ in neonatal blood.

Lymphoblastic leukaemia in infants (aged < 1 year) is a highly malignant biological subtype that is frequently associated with fusion between the MLL and AF4 genes.²⁶ In three patients aged 5 months, 6 months, and 24 months at diagnosis, we showed, by retrospective assessment of neonatal blood spots, the presence of genomic MLL/AF4 sequences unique to the patients' leukaemic clone at birth. This result confirmed the prenatal origin of this type of leukaemia in the very young and established proof of principle for the method.²⁷

The most frequent chromosomal or molecular abnormalities in children who develop common acute lymphoblastic leukaemia at the age for peak incidence for the disease (2–5 years) are hyperdiploidy²⁸ and fusion of TEL-AML1 genes.²⁹ Only the latter is suitable as a PCR-amplifiable marker. To exploit TEL-AML1 fusion as a unique marker of the leukaemic clone, the genomic TEL-AML1 fusion sequence must be obtained. We were able to determine the sequence by molecularly cloning rearranged TEL fragments¹⁹ or by amplification of rearranged TEL-AML1 DNA by a long-distance inverse PCR method (LDI-PCR).³⁰

Based on the patient-specific clone-specific DNA fusion sequence, appropriate oligonucleotide primers can be designed for retrospective testing of Guthrie-card blood spots. This method is based on two premises: the chromosome translocation-generating TEL-AML1 fusion gene is an early or initiating event in common acute lymphoblastic leukaemia rather than a late or secondary event; and if TEL-AML1 fusion does occur in a fetal liver or bone-marrow cell in utero, the clonal progeny would spread into the blood by the time of birth and, moreover, would do so in sufficient concentrations for at least one cell to be present and detectable in a blood spot of about 30 μL. We believed that these premises were reasonable because we had previously shown the fetal origin and spread via blood of common acute lymphoblastic leukaemia TEL-AML1 in pairs of identical twins.19, 20 The degree of involvement of blood at birth in such cases remains unknown.

In this study we aimed to find out whether identification of the presence of the leukaemia-specific gene sequence in the blood at birth was possible in most cases.