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...

We report the cloning and characterization of the entire AFX gene which fuses to MLL in acute leukemias with a t(X;11)(q13;q23). AFX consists of two exons and encodes for a protein of 501 amino acids. We found that normal B-and T-cells contain similar levels of AFX mRNA and that both the MLL/AFX as well as the AFX/MLL fusion transcripts are present in the cell line and the ANLL sample with a t(X;11)(q13;q23). The single intron of the AFX gene consists of 3706 nucleotides. It contains ®ve simple sequence repeats with lengths of at least 12 bps, a chi-like octamer sequence (GCA/TGGA/TGG) and several immunoglobulin heptamer-like sequences (GATAGTG) that are distributed throughout the entire AFX intron sequence. In the KARPAS 45 cell line the breakpoints occur at nucleotides 2913/2914 of the AFX intron and at nucleotides 4900/4901 of the breakpoint cluster region of the MLL gene. The AFX protein belongs to the forkhead protein family. It is highly homologous to the human FKHR protein, the gene of which is disrupted by the t(2;13)(q35;q14), a chromosome rearrangement characteristic of alveolar rhabdomyosarcomas. It is noteworthy that the t(X;11)(q13;q23) in the KARPAS 45 cell line and in one acute nonlymphoblastic leukemia (ANLL) disrupts the forkhead domain of the AFX protein exactly at the same amino acids as does the t(2;13)(q35;q14) in case of the FKHR protein. In addition, the 5'-part of the AFX protein contains a conserved hexapeptide motif (QIYEWM) that is homologous to the functionally important conserved hexapeptide QIYPWM upstream of the homeobox domain in Hox proteins. This motif mediates the co-operative DNA binding of Pbx family members and Hox proteins and, therefore, plays an important role in physiologic and oncogenic processes. In acute leukemias with a t(X;11)(q13;q23), this hexapeptide motif is separated from the remaining forkhead domain within the AFX protein. The predicted amino acid sequence of AFX di ers signi®cantly from the partial AFX protein sequence published previously (Genes, Chromosomes and Cancer, 1994, 11, 79 ± 84). This discrepancy can be explained by the occurrence of two sequencing errors in the earlier work at nucleotide number 783 and 844 (loss of a cytosine residue or guanosine residue, respectively) that lead to two reading frame shifts.

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Some chromosomal translocations involved in the origin of leukemias and lymphomas are due to malfunctions of the recombinatorial machinery of immunoglobulin and Tcell receptor-genes. This mechanism has also been proposed for translocations t(4;11)(q21;q23), which are regularly associated with acute pro-B cell leukemias in early childhood. Here, reciprocal chromosomal breakpoints in primary biopsy material of fourteen t(4;11)-leukemia patients were analysed. In all cases, duplications, deletions and inversions of less than a few hundred nucleotides indicative of malfunctioning DNA repair mechanisms were observed. We concluded that these translocation events were initiated by several DNA strand breaks on both participating chromosomes and subsequent DNA repair by`error-prone-repair' mechanisms, but not by the action of recombinases of the immune system.