Backtracking leukemia to birth: Identification of clonotypic gene fusion sequences in neonatal blood spots

Abstract

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...

Backtracking to birth of the NUP98-HOXD13 gene fusion in an infant acute myeloid leukemia

“…This is in line with previous findings, where the number of pre-leukemic cells present on Guthrie cards has been estimated to be in the range of 1 in 200-4000 cells when the presence of the MLL-AF4 allele has been investigated. 8 However, there are no studies available that correlate the amount of pre-leukemic cells on Guthrie cards with the onset of disease in a given patient. This might be due to the fact that most pre-leukemic clones need secondary genetic hits to develop into a leukemic clone and enable to develop the disease.…”

Insights into the cellular origin and etiology of the infant pro-B acute lymphoblastic leukemia with MLL-AF4 rearrangement

“…MLL-AF4 arises prenatally during embryonic/fetal hematopoiesis as evidenced by the analysis of MLL clonotypic breakpoints in leukemic cells of monozygotic twins with ALL 10 and the detection of genomic MLL-AF4 fusion sequences in archived neonatal blood spots of infants who developed ALL. 11 Moreover, the exceptionally high concordance rate of leukemia in monozygotic twin infants, approaching 100%, 48,49 suggests that all necessary genetic events required for leukemogenesis, are accomplished prenatally. 49 The nature of the cells initially transformed by MLL-AF4 in utero is unknown.…”

“…Elegant studies on identical twins with concordant MLL-AF4 þ leukemia and retrospective analyses of the clonotypic MLLrearranged sequences of blast cells from young patients in their neonatal blood spots revealed a in utero origin of the MLL rearrangements. 10,11 Importantly, compelling information indicates that the MLL-AF4 does not suffice to promote leukemogenesis on its own and additional secondary genetic insults are required. 3,9 Epidemiological and genetic studies support the contention that the in utero origin of MLL rearrangements in infant leukemia may be the result of exposures, during pregnancy, to genotoxic compounds present in the maternal diet intake capable of inducing breaks in the MLL locus in the fetus but not in the mother who has functional DNA repair mechanisms in place.…”

Adenovirus DNA is detected at increased frequency in Guthrie cards from children who develop acute lymphoblastic leukaemia

“…Recent studies have shed light on the natural history of this disease. Identification of leukaemia-associated translocations in neonatal blood spots (Guthrie cards) of children who will develop leukaemia years later indicates that the initiating event(s) of ALL usually occur before birth (Gale et al, 1997;Wiemels et al, 1999). Epidemiologic studies (Kinlen, 1988;Kinlen and Doll, 2004) suggest that at least one step in the process of ALL development involves an infectious agent (reviewed in Greaves, 2006).…”