The pediatric TEL/AML1-positive B-ALL subgroup displays fairly uniform clinical features, making it appropriate for studying the development of this sub-type of ALL. Comparison of gene expression profiles in TEL/AML1-positive patients with those in TEL/AML1-negative patients, whose blast cells do not contain any recurrent chromosomal rearrangement, is potentially informative about the molecular processes and pathogenesis of TEL/AML1. We first obtained microarray data for 26 B-ALL patients included in our prospective study. These patients constituted a homogeneous group, receiving an identical treatment according to the FRALLE 2000 trial. Gene expression analysis followed by gene enrichment analysis allowed us to identify five discrete enriched GO categories – cell differentiation, cell proliferation, apoptosis, cell motility and response to wounding – that highlighted the TEL/AML1 biological processes. The GO categories identified were associated with 14 annotated genes (RUNX1, TCFL5, TNFRSF7, CBFA2T3, CD9, SCARB1, TP53INP1, ACVR1C, PIK3C3, EGFL7, SEMA6A, CTGF, LSP1, TFPI); the expression patterns of these selected genes allowed clustering of the TEL/AML1-positive Set-A patients into one branch. The expression patterns of these genes, as assessed either by microarray experiments or real-time RT-PCR, were also able to cluster the TEL/AML1-positive patients of two independent sets, Set-B and -C, into one branch. Thus, even though the size of the initial set was relatively small, the filters applied were stringent enough to limit the number of false positives, leading to accuracy and subsequent validation of the 14 annotated genes. Furthermore, six of the 14 TEL/AML1-selected genes (TNFRSF7, CD9, TCFL5, PIK3C3, CBFA2T3, SEMA6A) had previously been reported to be associated with TEL/AML1 signatures found in more heterogeneous groups of ALL patients (including those with T-ALL, Bcr-Abl, E2A-PBX, or MLL) [13, 15]. The identification of the same genes through different experimental approaches (Agilent, Affymetrix, NCBI) and in different patient sets is a strong argument for their importance in TEL/AML1-positive leukemia process , and for the relevance of the additional eight newly identified genes (RUNX1, SCARB1, TP53INP1, ACVR1, EGFL7, CTGF, LSP1, TFPI). Some genes previously described as relevant, including TERF2 and EPOR, did not appear among the genes we selected. This might be due to differences in patient sets (we did not include hyperdiploid patients, with > 50 chromosomes), or to differences in the affinities of the probe sets used [14, 18]. Our findings reveal new target genes characterizing limited and specific biological pathways associated with TEL/AML1 pathogenesis. Further in vivo and in vitro investigations to assess their biological effects should contribute to a better understanding of the disease.
Models of ALL pathogenesis have suggested that two classes of cooperating mutations are required for acute leukemia to develop : one involved in impairment of differentiation and the other in cell proliferation and/or survival. We found that differentiation was not inhibited in TEL/AML1-positive ALL patients but, rather, was enhanced and characterized by the over-expression of differentiation genes (TCFL5, TNFRSF7, ACVRIC). This is in agreement with the report by Pine et al  that TEL/AML1 fusion preceded differentiation to pre-B cells and suggests that TEL/AML1 fusion occurs in a totipotent hematopoietic progenitor cell and directs cell differentiation towards the B-lineage. We also highlighted the activation of proliferation/survival oncogenic processes with the up-regulation of the RUNX1, CBFA2T3, PIK3CT, SCARB1 and TP53INP1 genes. Our study also implicated cell motility and response to wounding processes in the TEL/AML1 cluster. Cell migration capacity may be a clue to explaining the very late relapse events, which affect some TEL/AML1-positive ALL patients. Indeed, the good outcome expected for TEL/AML1-positive ALL children is offset by the relatively high rate of very late relapse, especially in non-hematopoietic sites such as the ovary [20, 21].
Additional genetic changes are very common in TEL/AML1-positive ALL patients; about 70% also present with deletion of the second TEL gene (ETV6) on the non-rearranged chromosome 12 [21, 22]. About half TEL/AML1-positive patients (7/16) displayed an additional loss of the TEL gene, suggesting that there may be other genetic abnormalities acting as secondary events for TEL/AML1 leukemogenesis or contributing to the outcome. Unlike the TEL gene, the AML1 gene (also named RUNX1 according to the HUGO nomenclature) was significantly over-expressed in the TEL/AML1 cluster. RUNX1 is a member of the Runt transcription factor family and targets key regulators of the hematopoiesis process (M-CSF R, IL3, neutrophil elastase, MPO, granzyme B, TCRs, and B-Cell receptors) through its DNA-binding domain . The transcriptional activity of RUNX1 depends on its dimerization with the non-DNA binding factor CBFβ, and on the recruitment of co-factors. The RUNX1 transcription complex thus acts either as a transcriptional activator or as a repressor depending on the nature of the co-factors. The TEL/AML1 fusion protein (ETV6/RUNX1) associated with the t(12;21) translocation acts as a repressor. However, few of the genes selected in our analysis were down-regulated. This suggests that either gene up-regulation is an indirect process, dependent on the down-regulation of transcriptional repressors mediated by the TEL/AML1 fusion protein, or that the repressor function of the TEL/AML1 fusion protein is counterbalanced by the presence of a normal RUNX1 protein. This later possibility is supported by our observation of RUNX1 over-expression in the TEL/AML1-positive group by microarray experiments and q-PCR. Expression and cytogenetic data from patients 9 and 17 indicate that RUNX1 over-expression is not due to the expression of the TEL/AML1 (ETV6/RUNX1) fusion gene, driven by the TEL promoter, but to the native RUNX1 gene. An increased RUNX1 copy number was also found in one third of the TEL/AML1 patients, and this may explain, at least in part, RUNX1 over-expression. Gene amplification is a common mechanism of oncogene deregulation, which occurs with RUNX1 through chromosome 21 polysomy, by the presence of a RUNX1 tandem repeat on der(21) or with additional RUNX1 copies on extra-chromosomal elements . The over-expression of the RUNX1 gene with no apparent amplification of the RUNX1 locus also suggests that there may be cryptic amplification undetectable by FISH-analysis or deregulation of the RUNX1 promoter. Conversely, promoter silencing or gene deletion despite over-representation of chromosome-21 could explain low expression of the RUNX1 gene. RUNX1 over-expression appeared to be characteristic of the TEL/AML1-positive patient group. Indeed, all patients with RUNX1 over-expression clustered together, including the patient 9, who had four copies of RUNX1 but no TEL/AML1 fusion. By contrast, TEL/AML1-positive patient 17, who presents no RUNX1 over-expression, did not segregate with the TEL/AML1-positive group. It is possible that the expression levels of RUNX1 could explain the clinical heterogeneity of t(12;21) ALL cases. Indeed, it has been suspected that a RUNX1 gene copy number of four is associated with ALL with good prognosis. By contrast, whereas the amplification of RUNX1 to a copy number greater than four, which has been estimated to be the case in 2% of all pediatric ALL and particularly those with no TEL/AML1 chromosomal aberration, may be characteristic of a subtype of B-ALL associated with a poor prognosis [25, 26]. If these findings were confirmed, the TEL/AML1 fusion transcript and RUNX1 expression level data could be used as stratifying therapeutical markers, with possible prognostic value.