Epidemiological evidence has suggested that some pediatric leukemias may be initiated in utero and, for some pairs of identical twins with concordant leukemia, this possibility has been strongly endorsed by molecular studies of clonality. Direct evidence for a prenatal origin can only be derived by prospective or retrospective detection of leukemia-specific molecular abnormalities in fetal or newborn samples. We report a PCR-based method that has been developed to scrutinize neonatal blood spots (Guthrie cards) for the presence of numerically infrequent leukemic cells at birth in individuals who subsequently developed leukemia. We demonstrate that unique or clonotypic MLL-AF4 genomic fusion sequences are present and detectable in neonatal blood spots from individuals who were diagnosed with acute lymphoblastic leukemia at ages 5 months to 2 years and, therefore, have arisen during fetal hematopoiesis in utero. This result provides unequivocal evidence for a prenatal initiation of acute leukemia in young patients. The method should be applicable to other fusion genes in children with common subtypes of leukemia and will be of value in attempts to unravel the natural history and etiology of this major subtype of pediatric cancer.Epidemiological evidence suggests that exposures or events that occur prenatally or in infancy might play a role in the etiology of pediatric acute leukemia, the most common type of childhood cancer in developed countries (1-3). Being able to backtrack leukemic clones to the time of such events would have a considerable impact on our understanding of the natural history of the disease and on the design and interpretation of epidemiological studies. This requires access to both a leukemia-specific marker and, retrospectively, appropriate biological material. The only biological markers that can provide definitive identification of a leukemic clone are clonotypic alterations in DNA that are present in the leukemic cells at diagnosis, e.g., unique nonconstitutive mutations or rearrangements of genes (4-6). These provide specific and sensitive molecular markers for tracking the disease clone with the significant caveat that the mutant DNA sequence identified is not necessarily the initiating or first mutation in the leukemia and, therefore, might be absent at early stages of clonal evolution. The one readily available retrospective source of DNA from leukemic children is the Guthrie card or blood spot taken routinely by heel prick on most newborns (7,8). Normally used to detect evidence of inborn errors of metabolism, DNA from these spots has been used to detect constitutive mutations (9-11) and exogenous viral sequences (12, 13) using PCR but not, as far as we are aware, for acquired molecular abnormalities in leukemia or other cancers. We reasoned that blood spot DNA from individuals who developed leukemia at a young age would enable us to test the idea that the leukemic clone with its acquired molecular marker could have an in utero fetal origin and therefore be present, albeit at a...

Low folate intake as well as alterations in folate metabolism as a result of polymorphisms in the enzyme methylenetetrahydrofolate reductase (MTHFR) have been associated with an increased incidence of neural tube defects, vascular disease, and some cancers. Polymorphic variants of MTHFR lead to enhanced thymidine pools and better quality DNA synthesis that could afford some protection from the development of leukemias, particularly those with translocations. We now report associations of MTHFR polymorphisms in three subgroups of pediatric leukemias: infant lymphoblastic or myeloblastic leukemias with MLL rearrangements and childhood lymphoblastic leukemias with either TEL-AML1 fusions or hyperdiploid karyotypes. Pediatric leukemia patients (n ‫؍‬ 253 total) and healthy newborn controls (n ‫؍‬ 200) were genotyped for MTHFR polymorphisms at nucleotides 677 (C3 T) and 1,298 (A3 C). A significant association for carriers of C677T was demonstrated for leukemias with MLL translocations (MLL؉, n ‫؍‬ 37) when compared with controls [adjusted odd ratios (OR) ‫؍‬ 0.36 with a 95% confidence interval (CI) of 0.15-0.85; P ‫؍‬ 0.017]. This protective effect was not evident for A1298C alleles (OR ‫؍‬ 1.14). In contrast, associations for A1298C homozygotes (CC; OR ‫؍‬ 0.26 with a 95% CI of 0.07-0.81) and C677T homozygotes (TT; OR ‫؍‬ 0.49 with a 95% CI of 0.20 -1.17) were observed for hyperdiploid leukemias (n ‫؍‬ 138). No significant associations were evident for either polymorphism with TEL-AML1؉ leukemias (n ‫؍‬ 78). These differences in allelic associations may point to discrete attributes of the two alleles in their ability to alter folate and one-carbon metabolite pools and impact after DNA synthesis and methylation pathways, but should be viewed cautiously pending larger follow-up studies. The data provide evidence that molecularly defined subgroups of pediatric leukemias have different etiologies and also suggest a role of folate in the development of childhood leukemia.