Anthony M. Ford

Chromosome translocations and covert leukemic clones are generated during normal fetal development

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 (≈5%), protracted latency, and transgenic modeling all suggest that additional postnatal exposure and/or 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 immunophenotype/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.

Specific JAK2 mutation (JAK2R683) and multiple gene deletions in Down syndrome acute lymphoblastic leukemia

Children with Down syndrome (DS) have a greatly increased risk of acute megakaryoblastic leukemia (AMKL) and acute lymphoblastic leukemia (ALL). Both DS-AMKL and the related transient myeloproliferative disorder (TMD) have GATA1 mutations as obligatory, early events. To identify mutations contributing to leukemogenesis in DS-ALL, we undertook sequencing of candidate genes, including FLT3, RAS, PTPN11, BRAF, and JAK2. Sequencing of the JAK2 pseudokinase domain identified a specific, acquired mutation, JAK2R683, in 12 (28%) of 42 DS-ALL cases. Functional studies of the common JAK2R683G mutation in murine Ba/F3 cells showed growth factor independence and constitutive activation of the JAK/STAT signaling pathway. High-resolution SNP array analysis of 9 DS-ALL cases identified additional submicroscopic deletions in key genes, including ETV6, CDKN2A, and PAX5. These results infer a complex molecular pathogenesis for DS-ALL leukemogenesis, with trisomy 21 as an initiating or first hit and with chromosome aneuploidy, gene deletions, and activating JAK2 mutations as complementary genetic events.

RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia

The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL) cases, is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near breakpoints, incorporation of non-templated sequence at junctions, ∼30-fold enrichment at promoters and enhancers of genes actively transcribed in B cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single-cell tracking shows that this mechanism is active throughout leukemic evolution, with evidence of localized clustering and reiterated deletions. Integration of data on point mutations and rearrangements identifies ATF7IP and MGA as two new tumor-suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1–positive lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B cell differentiation.

Developmentally regulated rearrangement and expression of genes encoding the T cell receptor-T3 complex

Human leukemic cells corresponding to the earliest identifiable stages of intrathymic T cell differentiation lack cell surface expression of the T cell receptor(TCR alpha/beta)-T3 complex but transcribe TCR beta mRNA from either germ-line configuration (1/13) or partially (DJ) or fully (VDJ) rearranged (12/13) genes. These cells do not produce TCR alpha mRNA, but do contain T3 delta and T3 epsilon mRNA and accumulate T3 polypeptides, primarily in the perinuclear envelope. Equivalent normal T cells isolated from thymus have a predominantly germ-line configuration of TCR beta but contain intracellular T3 proteins. T3 gene expression is therefore a very early event in T cell differentiation. TCR alpha chain production appears to be the limiting maturation-linked event in the transport, assembly, and cell surface membrane insertion of the TCR alpha/beta-T3 complex.

Single cell mutational profiling and clonal phylogeny in cancer

The development of cancer is a dynamic evolutionary process in which intraclonal, genetic diversity provides a substrate for clonal selection and a source of therapeutic escape. The complexity and topography of intraclonal genetic architectures have major implications for biopsy-based prognosis and for targeted therapy. High-depth, next-generation sequencing (NGS) efficiently captures the mutational load of individual tumors or biopsies. But, being a snapshot portrait of total DNA, it disguises the fundamental features of subclonal variegation of genetic lesions and of clonal phylogeny. Single-cell genetic profiling provides a potential resolution to this problem, but methods developed to date all have limitations. We present a novel solution to this challenge using leukemic cells with known mutational spectra as a tractable model. DNA from flow-sorted single cells is screened using multiplex targeted Q-PCR within a microfluidic platform allowing unbiased single-cell selection, high-throughput, and comprehensive analysis for all main varieties of genetic abnormalities: chimeric gene fusions, copy number alterations, and single-nucleotide variants. We show, in this proof-of-principle study, that the method has a low error rate and can provide detailed subclonal genetic architectures and phylogenies.

The TEL-AML1 leukemia fusion gene dysregulates the TGF-β pathway in early B lineage progenitor cells

Chromosome translocation to generate the TEL-AML1 (also known as ETV6-RUNX1) chimeric fusion gene is a frequent and early or initiating event in childhood acute lymphoblastic leukemia (ALL). Our starting hypothesis was that the TEL-AML1 protein generates and maintains preleukemic clones and that conversion to overt disease requires secondary genetic changes, possibly in the context of abnormal immune responses. Here, we show that a murine B cell progenitor cell line expressing inducible TEL-AML1 proliferates at a slower rate than parent cells but is more resistant to further inhibition of proliferation by TGF-beta. This facilitates the competitive expansion of TEL-AML1-expressing cells in the presence of TGF-beta. Further analysis indicated that TEL-AML1 binds to a principal TGF-beta signaling target, Smad3, and compromises its ability to activate target promoters. In mice expressing a TEL-AML1 transgene, early, pre-pro-B cells were increased in number and also showed reduced sensitivity to TGF-beta-mediated inhibition of proliferation. Moreover, expression of TEL-AML1 in human cord blood progenitor cells led to the expansion of a candidate preleukemic stem cell population that had an early B lineage phenotype (CD34+CD38-CD19+) and a marked growth advantage in the presence of TGF-beta. Collectively, these data suggest a plausible mechanism by which dysregulated immune responses to infection might promote the malignant evolution of TEL-AML1-expressing preleukemic clones.

Absence of p53 permits propagation of mutant cells following genotoxic damage

Much evidence has been gathered in support of a critical role for p53 in the cellular response to DNA damage. p53 dysfunction is associated with progression and poor prognosis of many human cancers and with a high incidence of tumours in p53 knockout mice. The absence of a p53-dependent G1 arrest that facilitates DNA repair or apoptosis might impact critically on clinical cancer in two ways. First, by abrogating the impact on therapy that operates via genotoxic damage and apoptosis; and second, by encouraging progression either by inducing genomic instability and DNA mis-repair or by permitting survival of mutants. However, experiments examining the relationship between p53 deficiency and mutation frequency have so far failed to confirm these predictions. The precise role played by p53 is therefore unclear. We now report use of a short term in vitro approach to assess the influence of p53 on radiation-induced mutations at the hprt locus in murine B cell precursors that are normally radiation ultrasensitive. We find a high number of hprt mutants among X-irradiated p53 null cells, which results from preferential survival as clonogenic mutants rather than from a p53-dependent increase in mutation rate. This result has important implications for genotoxic cancer therapy.

Defining the oncogenic function of the TEL/AML1 (ETV6/RUNX1) fusion protein in a mouse model

The t(12;21) translocation, generating the TEL/AML1 fusion protein, is the most common genetic lesion in childhood cancer. Using a bone marrow transplantation model, we demonstrate that TEL/AML1 expression impinges on normal hematopoietic differentiation, leading to the in vivo accumulation and persistence of an early progenitor compartment with a Sca1+/Kithi/CD11b+ phenotype and an increased self-renewal capacity, as documented by replating assays in vitro. Differentiation of these cells is not blocked, but the frequency of mature blood cells arising from TEL/AML1-transduced progenitors is low. Impaired differentiation is prominently observed in the pro-B-cell compartment, resulting in an proportional increase in early progenitors in vivo, consistent with the t(12;21) ALL phenotype. Despite the accumulation of both multipotent and B-cell progenitors in vivo, no leukemia induction was observed during an observation period of over 1 year. These results are consistent with findings in twins with concordant ALL, showing that TEL/AML1 generates a preleukemic clone in utero that persists for several years in a clinically covert fashion. Furthermore, our studies showed that the pointed domain of TEL/AML1, which recruits transcriptional repressors and directs oligomerization with either TEL/AML1 or wild-type TEL, was essential for the observed differentiation impairment and could not be replaced with another oligomerization domain.

Immunoglobulin gene organisation and expression in haemopoietic stem cell leukaemia.

We have analysed the organisation and expression of mu genes in the granulocytic phase and in the lymphoid and myeloid blast crises of Philadelphia chromosome (Ph1) chronic granulocytic leukaemia (CGL), a leukaemia which is known to arise in multipotential stem cells. We find that mu chain gene rearrangement occurs exclusively in lymphoid blast crisis leading in some, but not all, cases to the synthesis of small amounts of cytoplasmic mu chains characteristic of early pre‐B lymphocytes. In Southern blots, only one or two rearranged mu chain genes are seen, suggesting that a clonal event leading to blast crisis can occur in a committed B cell precursor rather than in the multipotential stem cell precursor, in which the Ph1 chromosome originated. The pattern of mu gene rearrangement observed in Ph1 CGL blast crisis is compared with that in normal B cells, other B lineage malignancies, myeloid leukaemias and T cell leukaemias.

TEL Deletion Analysis Supports a Novel View of Relapse in Childhood Acute Lymphoblastic Leukemia

Purpose: TEL (ETV6)-AML1 (RUNX1) chimeric gene fusions are frequent genetic abnormalities in childhood acute lymphoblastic leukemia (ALL). They often arise prenatally as early events or initiating events and are complemented by secondary postnatal genetic events of which deletion of the non-rearranged, second TEL allele is the most common. This consistent sequence of molecular pathogenesis facilitates an analysis of the clonal origins of relapse in this leukemia, which has some unusual clinical features. Experimental Design: We compared the boundaries, by microsatellite mapping, of TEL deletions at relapse versus diagnosis in 15 informative patients. Moreover, we compared the relatedness of diagnostic and relapse clones using immunoglobulin and T-cell receptor genes rearrangements and clonotypic TEL-AML1 genomic fusion. Results: Five patients retained the apparent same size TEL deletion, seven had larger deletions, and three had smaller deletions at relapse. In all of the cases evaluated, the clonal relatedness of diagnostic and relapse cells was confirmed by the retention of clonotypic TEL-AML1 genomic sequence and/or at least one identical immunoreceptor gene rearrangement. Conclusions: These data provide further evidence that TEL deletions are secondary to TEL-AML1 fusions in ALL. They are compatible with the novel idea that in at least some cases of childhood ALL, remission occurs with persistence of a preleukemic “fetal” clone, and subsequent relapse reflects the emergence of a new subclone from this reservoir after an independent “second hit,” i.e., independent TEL deletion. To our knowledge, the study is the most extensive and comprehensive analysis of the relationship between diagnostic and relapse clones in childhood ALL presented thus far.