Little is known of the genetic architecture of cancer at the subclonal and single-cell level or in the cells responsible for cancer clone maintenance and propagation. Here we have examined this issue in childhood acute lymphoblastic leukaemia in which the ETV6-RUNX1 gene fusion is an early or initiating genetic lesion followed by a modest number of recurrent or 'driver' copy number alterations. By multiplexing fluorescence in situ hybridization probes for these mutations, up to eight genetic abnormalities can be detected in single cells, a genetic signature of subclones identified and a composite picture of subclonal architecture and putative ancestral trees assembled. Subclones in acute lymphoblastic leukaemia have variegated genetics and complex, nonlinear or branching evolutionary histories. Copy number alterations are independently and reiteratively acquired in subclones of individual patients, and in no preferential order. Clonal architecture is dynamic and is subject to change in the lead-up to a diagnosis and in relapse. Leukaemia propagating cells, assayed by serial transplantation in NOD/SCID IL2Rγ(null) mice, are also genetically variegated, mirroring subclonal patterns, and vary in competitive regenerative capacity in vivo. These data have implications for cancer genomics and for the targeted therapy of cancer.

Studies on monozygotic twins with concordant leukemia and retrospective scrutiny of neonatal blood spots of patients with leukemia indicate that chromosomal translocations characteristic of pediatric leukemia often arise prenatally, probably as initiating events. The modest concordance rate for leukemia in identical twins (Ϸ5%), protracted latency, and transgenic modeling all suggest that additional postnatal exposure and͞or genetic events are required for clinically overt leukemia development. This notion leads to the prediction that chromosome translocations, functional fusion genes, and preleukemic clones should be present in the blood of healthy newborns at a rate that is significantly greater than the cumulative risk of the corresponding leukemia. Using parallel reverse transcriptase-PCR and real-time PCR (Taqman) screening, we find that the common leukemia fusion genes, TEL-AML1 or AML1-ETO, are present in cord bloods at a frequency that is 100-fold greater than the risk of the corresponding leukemia. Single-cell analysis by cell enrichment and immunophenotype͞ fluorescence in situ hybridization multicolor staining confirmed the presence of translocations in restricted cell types corresponding to the B lymphoid or myeloid lineage of the leukemias that normally harbor these fusion genes. The frequency of positive cells (10 ؊4 to 10 ؊3 ) indicates substantial clonal expansion of a progenitor population. These data have significant implications for the pathogenesis, natural history, and etiology of childhood leukemia.C himeric fusion genes generated by chromosomal translocations are consistent genetic abnormalities in pediatric acute leukemia (1-3). DNA breaks and fusions occur in introns, and each patient's leukemic cells have a unique or clonotypic breakpoint, providing a specific, sensitive, and stable marker for tracking leukemic clones (4). Using this approach in identical twins with concordant leukemia (5-7) and, retrospectively, with neonatal blood spots (8-10), it was demonstrated that common fusion genes-MLL fusions in infants with acute lymphoblastic leukemia (ALL) and TEL-AML1 and AML1-ETO in children with ALL or acute myeloblastic leukaemia (AML), respectively-arise predominantly in utero, probably as initiating events, and are present before and at birth in circulating blood.The evolutionary, clonal development of pediatric cancers involves a sequence of two or more independent genetic events (11), and it therefore is unlikely that fusion genes, initiating the disease, would be sufficient. This supposition is supported by the modest concordance rate for ALL in monozygotic twin children, 5-10% (12), by protracted postnatal latency (up to 14 years) (7) and by the absence of overt signs of leukemia in mice transgenic for AML1-ETO (13) or TEL-AML1 (14). This finding leads to the prediction that chromosomal translocations, functional fusion genes, and preleukemic clones should be generated in stem cells during fetal hemopoiesis, and present in blood at birth, at a rate that substantially exceeds the k...

Understanding cancer pathogenesis requires knowledge of not only the specific contributory genetic mutations but also the cellular framework in which they arise and function. Here we explore the clonal evolution of a form of childhood precursor-B cell acute lymphoblastic leukemia that is characterized by a chromosomal translocation generating a TEL-AML1 fusion gene. We identify a cell compartment in leukemic children that can propagate leukemia when transplanted in mice. By studying a monochorionic twin pair, one preleukemic and one with frank leukemia, we establish the lineal relationship between these "cancer-propagating" cells and the preleukemic cell in which the TEL-AML1 fusion first arises or has functional impact. Analysis of TEL-AML1-transduced cord blood cells suggests that TEL-AML1 functions as a first-hit mutation by endowing this preleukemic cell with altered self-renewal and survival properties.