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Acute lymphoblastic leukaemia (ALL) is the most common cancer of childhood. Here, we map emerging evidence suggesting that children with ALL at the time of diagnosis may have a delayed maturation of the gut microbiome compared with healthy children. This finding may be associated with early-life epidemiological factors previously identified as risk indicators for childhood ALL, including caesarean section birth, diminished breast feeding and paucity of social contacts. The consistently observed deficiency in short-chain fatty-acid-producing bacterial taxa in children with ALL has the potential to promote dysregulated immune responses and to, ultimately, increase the risk of transformation of preleukaemic clones in response to common infectious triggers. These data endorse the concept that a microbiome deficit in early life may contribute to the development of the major subtypes of childhood ALL and encourage the notion of risk-reducing microbiome-targeted intervention in the future..

INTRODUCTION: Translation of genome-wide association study (GWAS) findings into preventive approaches is challenged by the identification of the causal risk variants and the understanding of the biological mechanisms by which they act. We present using allelic expression (AE) ratios to perform quantitative case-control analysis as a novel approach to identify risk associations, causal regulatory variants, and target genes. METHODS: Using the breast cancer (BC) risk locus 17q22 to validate this approach, we measured AE ratios in normal breast tissue samples from controls and cases, as well as from unmatched blood samples. Then we used in-silico and in-vitro analysis to map and functionally characterised candidate causal variants. RESULTS: We found a significant shift in the AE patterns of STXBP4 (rs2628315) and COX11 (rs17817901) in the normal breast tissue of cases and healthy controls. Preferential expression of the G-rs2628315 and A-rs17817901 alleles, more often observed in cases, was associated with an increased risk for BC. Analysis of blood samples from cases and controls found a similar association. Furthermore, we identified two putative cis-regulatory variants - rs17817901 and rs8066588 - that affect a miRNA and a transcription factor binding site, respectively. CONCLUSION: We propose causal variants and target genes for the 17q22 BC risk locus and show that using AE ratios in case-control association studies is helpful in identifying risk and mapping causal variants..

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BACKGROUND: Treatment on risk adapted intensive pediatric protocols has improved outcome for teenagers and young adults (TYA) with T-cell acute lymphoblastic leukemia (T-ALL). Understanding the biology of disease in this age group and the genetic basis of relapse is a key goal as patients with relapsed/refractory disease have poor outcomes with conventional chemotherapy and novel molecular targets are required. This study examines the question of whether TYA T-ALL has a specific biological-molecular profile distinct from pediatric or adult T-ALL. METHODS: Genomic characterization was undertaken of a retrospective discovery cohort of 80 patients aged 15-26 years with primary or relapsed T-ALL, using a combination of Genome-Wide Human SNP Array 6.0, targeted gene mutation and promoter methylation analyses. Findings were confirmed by MLPA, real-time quantitative PCR, and FISH. Whole Exome Sequencing was performed in 4 patients with matched presentation and relapse to model clonal evolution. A prevalence analysis was performed on a final data set of 1,792 individual cases to identify genetic lesions with age specific frequency patterns, including 972 pediatric (1-14 years), 439 TYA (15-24 years) and 381 adult (≥25 years) cases. These cases were extracted from 19 publications with comparable genomic data identified through a PubMed search. RESULTS: Genomic characterization of this large cohort of TYA T-ALL patients identified recurrent isochromosome 7q i(7q) in our discovery cohort (n = 3). Prevalence analysis did not identify any age specific genetic abnormalities. Genomic analysis of 6 pairs of matched presentation - relapsed T-ALL established that all relapses were clonally related to the initial leukemia. Whole exome sequencing analysis revealed recurrent, targetable, mutations disrupting NOTCH, PI3K/AKT/mTOR, FLT3, NRAS as well as drug metabolism pathways. CONCLUSIONS: All genetic aberrations in TYA T-ALL occurred with an incidence similar or intermediate to that reported in the pediatric and adult literature, demonstrating that overall TYA T-ALL exhibits a transitional genomic profile. Analysis of matched presentation - relapse supported the hypothesis that relapse is driven by the Darwinian evolution of sub-clones associated with drug resistance (NT5C2 and TP53 mutations) and re-iterative mutation of known key T-ALL drivers, including NOTCH1..

The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription. MYCN loss led to growth arrest, sensitizing cells for apoptosis following CDK2 inhibition. In MYCN-amplified neuroblastoma, MYCN invaded active enhancers, driving a transcriptionally encoded adrenergic gene expression program that was selectively reversed by CYC065. MYCN overexpression in mesenchymal neuroblastoma was sufficient to induce adrenergic identity and sensitize cells to CYC065. CYC065, used together with temozolomide, a reference therapy for relapsed neuroblastoma, caused long-term suppression of neuroblastoma growth in vivo, highlighting the clinical potential of CDK9/2 inhibition in the treatment of MYCN-amplified neuroblastoma..

ETV6-RUNX1 (E/R) fusion gene, arising in utero from translocation t(12;21)(p13:q22), is the most frequent alteration in childhood acute lymphoblastic leukemia (ALL). However, E/R is insufficient to cause overt leukemia since it generates a clinically silent pre-leukemic clone which persists in the bone marrow but fails to out-compete normal progenitors. Conversely, pre-leukemic cells show increased susceptibility to transformation following additional genetic insults. Infections/inflammation are the most accredited triggers for mutations accumulation and leukemic transformation in E/R+ pre-leukemic cells. However, precisely how E/R and inflammation interact in promoting leukemia is still poorly understood. Here we demonstrate that IL6/TNFα/ILβ pro-inflammatory cytokines cooperate with BM-MSC in promoting the emergence of E/R+ Ba/F3 over their normal counterparts by differentially affecting their proliferation and survival. Moreover, IL6/TNFα/ILβ-stimulated BM-MSC strongly attract E/R+ Ba/F3 in a CXCR2-dependent manner. Interestingly, E/R-expressing human CD34+ IL7R+ progenitors, a putative population for leukemia initiation during development, were preserved in the presence of BM-MSC and IL6/TNFα/ILβ compared to their normal counterparts. Finally, the extent of DNA damage increases within the inflamed niche in both control and E/R-expressing Ba/F3, potentially leading to transformation in the apoptosis-resistant pre-leukemic clone. Overall, our data provide new mechanistic insights into childhood ALL pathogenesis..

V(D)J recombination is essential to generate antigen receptor diversity but is also a potent cause of genome instability. Many chromosome alterations that result from aberrant V(D)J recombination involve breaks at single recombination signal sequences (RSSs). A long-standing question, however, is how such breaks occur. Here, we show that the genomic DNA that is excised during recombination, the excised signal circle (ESC), forms a complex with the recombinase proteins to efficiently catalyze breaks at single RSSs both in vitro and in vivo. Following cutting, the RSS is released while the ESC-recombinase complex remains intact to potentially trigger breaks at further RSSs. Consistent with this, chromosome breaks at RSSs increase markedly in the presence of the ESC. Notably, these breaks co-localize with those found in acute lymphoblastic leukemia patients and occur at key cancer driver genes. We have named this reaction "cut-and-run" and suggest that it could be a significant cause of lymphocyte genome instability..

Acute lymphoblastic leukemia (ALL) develops in the bone marrow in the vicinity of stromal cells known to promote tumor development and treatment resistance. We previously showed that the cyclooxygenase (COX) inhibitor indomethacin prevents the ability of stromal cells to diminish p53-mediated killing of cocultured ALL cells in vitro, possibly by blocking the production of prostaglandin E2 (PGE2). Here, we propose that PGE2 released by bone marrow stromal cells might be a target for improved treatment of pediatric ALL. We used a xenograft model of human primary ALL cells in nonobese diabetic-scid IL2rγnull mice to show that indomethacin delivered in the drinking water delayed the progression of ALL in vivo. The progression was monitored by noninvasive in vivo imaging of the engrafted leukemic cells, as well as by analyses of CD19+CD10+ leukemic blasts present in spleen or bone marrow at the termination of the experiments. The indomethacin treatment increased the level of p53 in the leukemic cells, implying that COX inhibition might reduce progression of ALL by attenuating protective paracrine PGE2 signaling from bone marrow stroma to leukemic cells..

Acute lymphoblastic leukaemia (ALL) is the most common cancer of childhood. Although the overall survival of children with ALL is now more than 90%, leukaemia remains one of the leading causes of death from disease. In developed countries, the overall survival of patients with ALL has increased to more than 80%; however, those children cured from ALL still show a significant risk of short- and long-term complications as a consequence of their treatment. Accordingly, there is a need not only to develop new methods of diagnosis and prognosis but also to provide patients with less toxic therapies. MicroRNAs (miRNAs) are small ribonucleic acids (RNA), usually without coding potential, that regulate gene expression by directing their target messenger RNAs (mRNAs) for degradation or translational suppression. In paediatric ALL, several miRNAs have been observed to be overexpressed or underexpressed in patient cohorts compared to healthy individuals, while numerous studies have identified specific miRNAs that can be used as biomarkers to diagnose ALL, classify it into subgroups, and predict prognosis. Likewise, a variety of miRNAs identify as candidate targets for treatment, although there are numerous obstacles to overcome before their clinical use in patients. Here, we summarise the roles played by different miRNAs in childhood leukaemia, focussing primarily on their use as diagnostic tools and potential therapeutic targets, as well as a role in predicting treatment outcome. Finally, we discuss the potential roles of miRNA in immunotherapy and the novel contributions made by gut miRNAs to regulation of the host microbiome..

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Single-cell genetics were used to interrogate clonal complexity and the sequence of mutational events in STIL-TAL1+ T-ALL. Single-cell multicolour FISH was used to demonstrate that the earliest detectable leukaemia subclone contained the STIL-TAL1 fusion and copy number loss of 9p21.3 (CDKN2A/CDKN2B locus), with other copy number alterations including loss of PTEN occurring as secondary subclonal events. In three cases, multiplex qPCR and phylogenetic analysis were used to produce branching evolutionary trees recapitulating the snapshot history of T-ALL evolution in this leukaemia subtype, which confirmed that mutations in key T-ALL drivers, including NOTCH1 and PTEN, were subclonal and reiterative in distinct subclones. Xenografting confirmed that self-renewing or propagating cells were genetically diverse. These data suggest that the STIL-TAL1 fusion is a likely founder or truncal event. Therapies targeting the TAL1 auto-regulatory complex are worthy of further investigation in T-ALL..

SCL/TAL1 interrupting locus (STIL)-T-cell acute leukaemia (TAL1) fusion genes are present in approximately 11-27% of children with paediatric T-cell acute lymphoblastic leukaemia (T-ALL), but the developmental timing of the rearrangement is still unknown. To investigate whether the fusion gene can be detected in neonatal blood spots (NBSs) from paediatric patients diagnosed with T-cell ALL, we analysed DNA from 38 paediatric patients with T-ALL by nested polymerase chain reaction and electrophoresis. The STIL-TAL1 fusion gene was not detected in NBSs from any of the 38 patients with T-ALL, suggesting that STIL-TAL1 fusion genes are most probably postnatal events in paediatric T-ALL..

The origin of cancers is associated with etiology as well as therapeutics. Several studies reveal that malignancies in children can originate in utero. However, a diagnostic approach to distinguish between cancers initiated pre- or postnatally is absent. Here we identified a transcriptional factor FEV (fifth Ewing variant) that was expressed in fetal hematopoietic cells and became silent after birth. We characterized that FEV was essential for the self-renewal of hematopoietic stem cells (HSCs). We next found that FEV was expressed in most infant leukemia samples, but seldom in adult samples, in accord with the known prenatal origins of the former. We further determined the majority of pediatric acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) were FEV positive. Moreover, FEV knockdown markedly impaired the leukemia-propagating ability of leukemic stem cells. We therefore identified FEV is unique to fetal HSCs and stably expressed in leukemic cells of prenatal origin. It may also provide a tractable therapeutic target..

Childhood acute lymphoblastic leukaemia (ALL) with MLL rearrangement (MLL-r) is an aggressive disease still associated with a high mortality rate. Recent investigations have identified co-operating mutations in the RAS pathway and although the functional consequences of these mutations are not yet fully understood, aberrant regulation of RAS pathway signalling at both transcriptional and protein levels is observed. Studies investigating the efficacy of specific inhibitors of this pathway, e.g. MEK-inhibitors, have also achieved encouraging results. In this context, this mini-review summarizes the available data surrounding MLL-r infant ALL with RAS mutation in relation to other well-known features of this intriguing disease..

Induced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming "boosters" also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency..

Identical twins have provided unique insights on timing or sequence of genetic events in acute lymphoblastic leukaemia (ALL). To date, this has mainly focused on ALL with MLL or ETV6-RUNX1 fusions, with hyperdiploid ALL remaining less well characterised. We examined three pairs of monozygotic twins, two concordant and one discordant for hyperdiploid ALL, for single-nucleotide polymorphism (SNP)-defined copy number alterations (CNAs), IGH/L plus TCR gene rearrangements and mutations in NRAS, KRAS, FLT3 and PTPN11 genes. We performed whole exome sequencing in one concordant twin pair. Potential 'driver' CNAs were low, 0-3 per case, and all were different within a pair. One patient had an NRAS mutation that was lacking from leukaemic cells of the twin sibling. By exome sequencing, there were 12 nonsynonymous mutations found in one twin and 5 in the other, one of which in SCL44A2 was shared or identical. Concordant pairs had some identical IGH/L and TCR rearrangements. In the twin pair with discordant hyperdiploid ALL, the healthy co-twin had persistent low level hyperdiploid CD19+ cells that lacked a CNA present in the ALL cells of her sibling. From these data, we propose that hyperdiploid ALL arises in a pre-B cell in utero and mutational changes necessary for clinical ALL accumulate subclonally and postnatally..

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Studies on twins with concordant acute lymphoblastic leukemia (ALL) have revealed that ETV6-RUNX1 gene fusion is a common, prenatal genetic event with other driver aberrations occurring subclonally and probably postnatally. The fetal cell type that is transformed by ETV6-RUNX1 is not identified by such studies or by the analysis of early B-cell lineage phenotype of derived progeny. Ongoing, clonal immunoglobulin (IG) and cross-lineage T-cell receptor (TCR) gene rearrangements are features of B-cell precursor leukemia and commence at the pro-B-cell stage of normal B-cell lineage development. We reasoned that shared clonal rearrangements of IG or TCR genes by concordant ALL in twins would be informative about the fetal cell type in which clonal advantage is elicited by ETV6-RUNX1. Five pairs of twins were analyzed for all varieties of IG and TCR gene rearrangements. All pairs showed identical incomplete or complete variable-diversity-joining junctions coupled with substantial, subclonal and divergent rearrangements. This pattern was endorsed by single-cell genetic scrutiny in one twin pair. Our data suggest that the pre-leukemic initiating function of ETV6-RUNX1 fusion is associated with clonal expansion early in the fetal B-cell lineage..

Childhood acute lymphoblastic leukemia (ALL) can often be traced to a pre-leukemic clone carrying a prenatal genetic lesion. Postnatally acquired mutations then drive clonal evolution toward overt leukemia. The enzymes RAG1-RAG2 and AID, which diversify immunoglobulin-encoding genes, are strictly segregated in developing cells during B lymphopoiesis and peripheral mature B cells, respectively. Here we identified small pre-BII cells as a natural subset with increased genetic vulnerability owing to concurrent activation of these enzymes. Consistent with epidemiological findings on childhood ALL etiology, susceptibility to genetic lesions during B lymphopoiesis at the transition from the large pre-BII cell stage to the small pre-BII cell stage was exacerbated by abnormal cytokine signaling and repetitive inflammatory stimuli. We demonstrated that AID and RAG1-RAG2 drove leukemic clonal evolution with repeated exposure to inflammatory stimuli, paralleling chronic infections in childhood..

Here, we report a high incidence of PAX5 abnormalities observed in 32/68 (47%) of patients with genetically unclassified childhood precursor B-cell acute lymphoblastic leukaemia (pre-B ALL). Various deletions, gains, mutations and rearrangements of PAX5 comprised 45%, 12%, 29% and 14%, respectively, of the abnormalities found. 28% of patients showed more than one abnormality of the gene, implying bi-allelic impairment of PAX5. Novel PAX5-RHOXF2, PAX5-ELK3 and PAX5-CBFA2T2 rearrangements, which lead to aberrant expression of PAX5, were also identified. PAX5 rearrangements demonstrated a complex mechanism of formation including concurrent duplications/deletions of PAX5 and its partner genes. Finally, the splice variant c.1013-2A>G, seen in two patients with loss of one PAX5 allele, was confirmed to be germ-line in one patient and somatic in the other. PAX5 alterations were also found to be clinically associated with a higher white blood cell count (P = 0·015). These findings contribute to the knowledge of PAX5 alterations and their role in the pathogenesis of pre-B ALL..

Cancer stem cells can escape therapeutic killing by adopting a quiescent or dormant state. The reversibility of this condition provides the potential for later recurrence or relapse, potentially many years later. We describe the genomics of a rare case of childhood BCR-ABL1-positive, B-cell precursor acute lymphoblastic leukemia that relapsed, with an acute myeloblastic leukemia immunophenotype, 22 years after the initial diagnosis, sustained remission and presumed cure. The primary and relapsed leukemias shared the identical BCR-ABL1 fusion genomic sequence and two identical immunoglobulin gene rearrangements, indicating that the relapse was a derivative of the founding clone. All other mutational changes (single-nucleotide variant and copy number alterations) were distinct in diagnostic or relapse samples. These data provide unambiguous evidence that leukemia-propagating cells, most probably pre-leukemic stem cells, can remain covert and silent but potentially reactivatable for more than two decades..

Infant T-cell acute lymphoblastic leukaemia (iT-ALL) is a very rare and poorly defined entity with a poor prognosis. We assembled a unique series of 13 infants with T-ALL, which allowed us to identify genotypic abnormalities and to investigate prenatal origins. Matched samples (diagnosis/remission) were analysed by single nucleotide polymorphism-array to identify genomic losses and gains. In three cases, we identified a recurrent somatic deletion on chromosome 3. These losses result in the complete deletion of MLF1 and have not previously been described in T-ALL. We observed two cases with an 11p13 deletion (LMO2-related), one of which also harboured a deletion of RB1. Another case presented a large 11q14·1-11q23·2 deletion that included ATM and only five patients (38%) showed deletions of CDKN2A/B. Four cases showed NOTCH1 mutations; in one case FBXW7 was the sole mutation and three cases showed alterations in PTEN. KMT2A rearrangements (KMT2A-r) were detected in three out of 13 cases. For three patients, mutations and copy number alterations (including deletion of PTEN) could be backtracked to birth using neonatal blood spot DNA, demonstrating an in utero origin. Overall, our data indicates that iT-ALL has a diverse but distinctive profile of genotypic abnormalities when compared to T-ALL in older children and adults..

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..

UNLABELLED: Although the ETV6-RUNX1 fusion is a frequent initiating event in childhood leukemia, its role in leukemogenesis is only partly understood. The main impact of the fusion itself is to generate and sustain a clone of clinically silent preleukemic B-cell progenitors (BCP). Additional oncogenic hits, occurring even several years later, are required for overt disease. The understanding of the features and interactions of ETV6-RUNX1-positive cells during this "latency" period may explain how these silent cells can persist and whether they could be prone to additional genetic changes. In this study, two in vitro murine models were used to investigate whether ETV6-RUNX1 alters the cellular adhesion and migration properties of BCP. ETV6-RUNX1-expressing cells showed a significant defect in the chemotactic response to CXCL12, caused by a block in CXCR4 signaling, as demonstrated by inhibition of CXCL12-associated calcium flux and lack of ERK phosphorylation. Moreover, the induction of ETV6-RUNX1 caused changes in the expression of cell-surface adhesion molecules. The expression of genes regulating the cytoskeleton was also affected, resulting in a block of CDC42 signaling. The abnormalities described here could alter the interaction of ETV6-RUNX1 preleukemic BCP with the microenvironment and contribute to the pathogenesis of the disease. IMPLICATIONS: Alterations in the expression of cytoskeletal regulatory genes and migration properties of BCP represent early events in the evolution of the disease, from the preleukemic phase to the clinical onset, and suggest new strategies for effective eradication of leukemia..

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..

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B-cell precursor childhood acute lymphoblastic leukemia with ETV6-RUNX1 (TELAML1) fusion has an overall good prognosis, but relapses occur, usually after cessation of treatment and occasionally many years later. We have investigated the clonal origins of relapse by comparing the profiles of genomewide copy number alterations at presentation in 21 patients with those in matched relapse (12-119 months). We identified, in total, 159 copy number alterations at presentation and 231 at relapse (excluding Ig/TCR). Deletions of CDKN2A/B or CCNC (6q16.2-3) or both increased from 38% at presentation to 76% in relapse, suggesting that cell-cycle deregulation contributed to emergence of relapse. A novel observation was recurrent gain of chromosome 16 (2 patients at presentation, 4 at relapse) and deletion of plasmocytoma variant translocation 1 in 3 patients. The data indicate that, irrespective of time to relapse, the relapse clone was derived from either a major or minor clone at presentation. Backtracking analysis by FISH identified a minor subclone at diagnosis whose genotype matched that observed in relapse similar to 10 years later. These data indicate subclonal diversity at diagnosis, providing a variable basis for intraclonal origins of relapse and extended periods (years) of dormancy, possibly by quiescence, for stem cells in ETV6-RUNX1(+) acute lymphoblastic leukemia. (Blood. 2011; 117(23): 6247-6254).

The timing and developmental sequence of events for BCR-ABL1(+) acute lymphoblastic leukemia (ALL), usually associated with IKAROS (IKZF1) deletions, are unknown. We assessed the status of BCR-ABL1 and IKZF1 genes in 2 pairs of monozygotic twins, one pair concordant, the other discordant for Philadelphia chromosome positive (Ph(+)) ALL. The twin pair concordant for ALL shared identical BCR-ABL1 genomic sequence indicative of monoclonal, in utero origin. One twin had IKZF1 deletion and died after transplantation. The other twin had hyperdiploidy, no IKZF1 deletion, and is still in remission 8 years after transplantation. In the twin pair discordant for ALL, neonatal blood spots from both twins harbored the same clonotypic BCR-ABL1 sequence. Low level BCR-ABL1(+) cells were present in the healthy co-twin but lacked the IKZF1 deletion present in the other twin's leukemic cells. The twin with ALL relapsed and died after transplantation. The co-twin remains healthy and leukemia free. These data show that in childhood Ph(+) ALL, BCR-ABL1 gene fusion can be a prenatal and possibly initiating genetic event. In the absence of additional, secondary changes, the leukemic clone remains clinically silent. IKZF1 is a secondary and probable postnatal mutation in these cases, and as a recurrent but alternative copy number change is associated with poor prognosis..

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Chimeric fusion genes are highly prevalent in childhood acute lymphoblastic leukemia (ALL) and are mostly prenatal, early genetic events in the evolutionary trajectory of this cancer. ETV6-RUNX1-positive ALL also has multiple ( approximately 6 per case) copy number alterations (CNAs) as revealed by genome-wide single-nucleotide polymorphism arrays. Recurrent CNAs are probably "driver" events contributing critically to clonal diversification and selection, but at diagnosis, their developmental timing is "buried" in the leukemia's covert natural history. This conundrum can be resolved with twin pairs. We identified and compared CNAs in 5 pairs of monozygotic twins with concordant ETV6-RUNX1-positive ALL and 1 pair discordant for ETV6-RUNX1 positive ALL. We compared, within each pair, CNAs classified as potential "driver" or "passenger" mutations based upon recurrency and, where known, gene function. An average of 5.1 (range 3-11) CNAs (excluding immunoglobulin/T-cell receptor alterations) were identified per case. All "driver" CNAs (total of 32) were distinct within each of the 5 twin pairs with concordant ALL. "Driver" CNAs in another twin with ALL were all absent in the shared ETV6-RUNX1-positive preleukemic clone of her healthy co-twin. These data place all "driver" CNAs secondary to the prenatal gene fusion event and most probably postnatal in the sequential, molecular pathogenesis of ALL..

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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..

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. (Blood. 2009; 113: 646-648).

Rare cases of possible materno-fetal transmission of cancer have been recorded over the past 100 years but evidence for a shared cancer clone has been very limited. We provide genetic evidence for mother to offspring transmission, in utero, of a leukemic cell clone. Maternal and infant cancer clones shared the same unique BCR-ABL1 genomic fusion sequence, indicating a shared, single-cell origin. Microsatellite markers in the infant cancer were all of maternal origin. Additionally, the infant, maternally- derived cancer cells had a major deletion on one copy of chromosome 6p that included deletion of HLA alleles that were not inherited by the infant (i.e., foreign to the infant), suggesting a possible mechanism for immune evasion..

Purpose: Chimeric transcription factor ETV6/RUNX1 (TEL/AML1) is believed to cause pathologic block in lymphoid cell development via interaction with corepressor complex and histone deacetylase. We wanted to show the regulatory effect of ETV6/RUNX1 and its reversibility by histone deacetylase inhibitors (HDACi), as well as to identify potential ETV6/RUNX1-regulated genes.Experimental Design: We used luciferase assay to show the interaction of ETV6/RUNX1 protein, ETV6/RUNX1-regulated gene, and HDACi. To identify ETV6/RUNX1-regulated genes, we used expression profiling and HDACi in lymphoid cells. Next, using the flow cytometry and quantitative reverse transcription-PCR, we measured differentiation changes in gene and protein expression after HDACi treatment.Results: Luciferase assay showed repression of granzyme B expression by ETV6/RUNx1 protein and the reversibility of this effect by HDACi. Proving this regulatory role of ETV6/RUNX1, we identified, using complex statistical analysis, 25 genes that are potentially regulated by ETV6/RUNX1 protein. In four selected genes with known role in the cell cycle regulation (JunD, ACK1, PDGFRB, and TCF4), we confirmed expression changes after HDACi by quantitative analysis. After HDACi treatment, ETV6/RUNX1-positive cells showed immunophenotype changes resembling differentiation process compared with other leukemic cells (BCR/ABL, ETV6/PDGFRB positive). Moreover, ETV6/RUNX1-positive leukemic cells accumulated in G(1)-G(0) phase after HDACi whereas other B-lineage leukemic cell lines showed rather unspecific changes including induction of apoptosis and decreased proliferation.Conclusions: Presented data support the hypothesis that HDACi affect ETV6/RUNX1-positive cells via direct interaction with ETV6/RUNX1 protein and that treatment with HDACi may release aberrant transcription activity caused by ETV6/RUNX1 chimeric transcription factor..

Childhood leukemia is a common pediatric cancer in the developed world, the disease is biologically diverse and there is much discussion as to its causal mechanisms. Acute lymphoblastic leukemia (ALL) is the most common subtype and infants with ALL have a greatly increased risk of treatment failure. There are molecular and biological properties of leukemic cells that determine treatment outcome; these can usually be attributed to distinct genetic abnormalities that alter the normal proliferative and survival signals of hematopoietic cells. Experimental evidence for the existence of leukemic stem cells (LSC) has been obtained, and it is presumed that these cells arise from mutations in normal hematopoetic stem cells or progenitor cells, and they are difficult to eradicate. LSC seem to be surprisingly different from their normal counterparts and therefore are obvious new targets for drug therapy. Therapeutic concepts using monoclonal antibodies have substantially improved response rates in patients with malignant lymphomas and are currently being evaluated in other types of cancer..

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Stem and progenitor cells present attractive targets for transformation by leukemia-associated fusion genes generated by chromosomal translocation. The mechanism by which these fusion genes corrupt the transcriptional programs of these cellular compartments remains largely unknown. We have sought to gain insight into these issues through expressing TEL-AML1 and TEL-TRKC fusion genes in murine stem cells and recording effects on cell behavior in a transplant setting..

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(+)/Kit(hi)/CD11b(+) phenotype and an increased self- renewal capacity, as documented by replating assays in vitro. Differ entiation of these cells is not blocked, but the frequency of mature blood cells arising from TEL/AML1- transduced progenitors is low. Impa ired 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. Desp ite 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. ndings 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. Fur thermore, 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..

Rearrangements involving the MLL gene at 11q23 occur in a clinically relevant subgroup of patients with acute lymphoblastic leukemia (ALL) at all ages, and therefore their accurate identification at diagnosis is important. It has become commonplace to screen ALL patients for rearrangements of MLL using a dual-color fluorescence in situ hybridization (FISH) assay. We report on 12 ALL patients with an unusual FISH result consisting of the following signal pattern: one 5' green, no 3' red, and one/two fusion signals. This configuration is consistent with a MLL translocation and simultaneous deletion of 3' MLL-a well-established phenomenon-which has been interpreted as a positive result. G-banded and complementary metaphase FISH analyses confirmed an 11q23/MLL translocation in 8 of the 12 cases, whereas in one case, the identification of a del(11)(q23) was restricted to G-banded analysis only. In three cases, an MLL rearrangement was excluded by extensive FISH analysis and/or Southern blotting. In conclusion, the loss of the 3' MLL signal should not be assumed to be the result of a concurrent translocation and deletion event, and such aberrant FISH signal patterns should be investigated further by alternative methods for determining their MLL status..

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..

Identical infant twins with concordant leukemia were first described in 1882, and since that time many such pairs of infants and older children have been described. It has long been recognized that this situation offers a unique opportunity to identify aspects of the developmental timing, natural history, and molecular genetics of pediatric leukemia in general. We reviewed both the older literature and more recent molecular biologic studies that have uncovered the basis of concordance of leukemia. Molecular markers of clonality, including unique, genomic fusion gene sequences, have provided unequivocal evidence that twin pairs of leukemia have a common clonal origin. The only plausible basis for this, first suggested more than 40 years ago, is that following initiation of leukemia in one twin fetus, clonal progeny spread to the co-twin via vascular anastomoses within a single, monochorionic placenta. This explanation has been endorsed by the identification of clonotypic gene fusion sequences in archived neonatal blood spots of individuals who subsequently developed leukemia. These analyses of twin leukemias have thrown considerable light on the natural history of disease. They reveal a frequent prenatal origin and an early or initiating role for chromosome translocations. Further, they provide evidence for a variable and often protracted latency and the need, in childhood acute lymphoblastic leukemia (ALL)/acute myeloblastic leukemia (AML), for further postnatal exposures and/or genetic events to produce clinical disease. We argue that these insights provide a very useful framework for attempts to understand etiologic mechanisms..

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Comparative expression analysis of wild-typeETV6 in the disease state showed an absence of expression in ETV6-CBFA2 acute lymphoblastic leukaemia (ALL) when compared with non-ETV6-CBFA2 ALL and acute myeloid leukaemia. Fluorescent in-situ hybridization and loss of heterozygosity studies showed that 73% of the ETV6-CBFA2 samples had a fully or partially deleted second ETV6 allele, explaining the lack of wild-type expression in these patients. Although the second ETV6 allele was identified in the remaining patients, no ETV6 expression was detected. These observations support the hypothesis that loss of ETV6 expression is a critical secondary event for leukaemogenesis in ETV6-CBFA2 ALL..

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A unique case of ALL in three monozygotic triplets diagnosed at the age of 24, 27 and 37 months is described. Archived bone marrow smears were available for molecular analysis of immunoglobulin heavy chain ( IGH ) and IG kappa genes and T-cell receptor ( TCR )- delta and gamma gene rearrangements. A shared IGH rearrangement was found in triplets "A" and "B", and an identical rearrangement of TCR-delta in triplets "B" and "C". These data suggest a common, monoclonal initiation of ALL in one of these three triplets, followed by dissemination of clonal progeny to the other twins via vascular anastomoses within the single, monochorionic placenta that they shared in utero . Differences in IGH rearrangements in diagnostic samples also indicates divergent subclonal evolution of the original "pre-leukaemic" clone..

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 ( approximately 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..

Approximately 20% of childhood B-precursor acute lymphoblastic leukemia (ALL) has a TEL-AML1 fusion gene, often in association with deletions of the nonrearranged TEL allele. TEL-AML1 gene fusion appears to be an initiating event and usually occurs before birth, in utero. This subgroup of ALL generally presents with low- or medium-risk features and overall has a very good prognosis. Some patients, however, do have relapses late or after the cessation of treatment, at least on some therapeutic protocols. They usually achieve sustained second remissions. Posttreatment relapses, or even very late relapses (5-20 years after diagnosis), in childhood ALL are clonally related to the leukemic cells at diagnosis (by IGH or T-cell receptor [TCR] gene sequencing) and are considered, therefore, to represent a slow re-emergence or escape of the initial clone seen at diagnosis. Microsatellite markers and fluorescence in situ hybridization identified deletions of the unrearranged TEL allele and IGH/TCR gene rearrangements were analyzed; the results show that posttreatment relapse cells in 2 patients with TEL-AML1-positive ALL were not derived from the dominant clone present at diagnosis but were from a sibling clone. In contrast, a patient who had a relapse while on treatment with TEL-AML1 fusion had essentially the same TEL deletion, though with evidence for microsatellite instability 5(') of TEL gene deletion at diagnosis, leading to extended 5(') deletion at relapse. It is speculated that, in some patients, combination chemotherapy for childhood ALL may fail to eliminate a fetal preleukemic clone with TEL-AML1 and that a second, independent transformation event within this clone after treatment gives rise to a new leukemia masquerading as relapse. (Blood. 2001;98:558-564).

We describe very uncommon phenotypic and cytogenetic findings in a 40-year-old female with blast phase of Philadelphia chromosome (Ph)-positive CML. In addition to the t(9:22)(q34:q11) that was detected in all metaphases, a t(11;17)(q23:q21) was identified in 15 of 20 metaphases. Reverse transcription-polymerase chain reaction showed the major and minor bcr/abl fusion transcripts in the cells from a bone marrow (BM) sample. Fluorescence in situ hybridization (FISH) analysis also showed that fusion signals of the bo and nhl probes were found in 95% of blastic cells and in 64% of neutrophils. MLL gene rearrangement was also detected in some blastic cells but not in neutrophils by FISH analysis. Phenotypically, blastic cells expressed mixed lineage antigens such as CD34, CD33, CD13. CD19, CD7, and CD41. Immunogenotypically. some population of BM cells showed monoclonal rearrangements of immunoglobulin heavy chain and T-cell receptor gamma chain genes by Southern blot analysis. Clinical course was aggressive, and therapy was poorly tolerated. Such findings seem to support an association between Ph and an abnormality of 11q23 with poor prognosis, and suggest that the expression of both abnormal genes may be related to this mixed lineage antigen-expressing leukemia. (C) 2001 Elsevier Science Inc. All rights reserved..

There is epidemiological evidence that infection may play a role in the etiology of childhood leukemia in particular common B cell precursor acute lymphoblastic leukemia. A panel of 20 leukemic samples (panel 1) was examined for the presence of four lymphotropic herpesviruses using conventional molecular techniques. A second independent panel of 27 leukemic samples (panel 2), along with 28 control peripheral blood samples from children with other forms of cancer, was tested for the presence of the same four viruses using sensitive realtime quantitative PCR, While herpesvirus genomes were detected, they were present at very low levels; detection rates and levels were similar in the leukemic and control panels. In addition we surveyed 18 leukemic samples (five from panel 1, six from panel 2 and a further seven samples not previously analyzed) using a degenerate PCR assay capable of detecting the genomes of known herpesviruses plus putative new members of the family. No novel herpesvirus genomes were detected suggesting that a herpesvirus is unlikely to be etiologically involved as a transforming agent in common acute lymphoblastic leukemia..

Acute leukemia (AL) in infants generally shows distinctive biologic features and has a poor prognosis..

The occurrence of childhood acute lymphoblastic leukemia (ALL) in 2 of 3 triplets provided a unique opportunity for the investigation of leukemogenesis and the natural history of ALL. The 2 leukemic triplets were monozygotic twins and shared an identical, acquired TEL-AML1 genomic fusion sequence indicative of a single-cell origin in utero in one fetus followed by dissemination of clonal progeny to the comonozygotic twin by intraplacental transfer. In accord with this interpretation, clonotypic TEL-AML1 fusion sequences could be amplified from the archived neonatal blood spots of the leukemic twins. The blood spot of the third, healthy, dizygotic triplet was also fusion gene positive in a single segment, though at age 3 years, his blood was found negative by sensitive polymerase chain reaction (PCR) screening for the genomic sequence and by reverse transcription-PCR, Leukemic cells in both twins had, in addition to TEL-AML1 fusion, a deletion of the normal, nonrearranged TEL allele, However, this genetic change was found by fluorescence in situ hybridization to be subclonal in both twins. Furthermore, mapping of the genomic boundaries of TEL deletions using microsatellite markers indicated that they were individually distinct in the twins and therefore must have arisen as independent and secondary events, probably after birth. These data support a multihit temporal model for the pathogenesis of the common form of childhood leukemia. (C) 2001 by The American Society of Hematology..

The t(12;21)(p13;q22) chromosomal translocation Is the most frequent illegitimate gene recombination in a pediatric cancer and occurs in approximately 25% of common acute lymphoblastic leukemia (cALL) cases. This rearrangement results in the in frame fusion of the 5'-region of the ETS-related gene, TEL (ETV6), to almost the entire acute myeloid leukemia 1 (AML1) (also called CBFA2 or PEBP2AB1) locus and expression of the TEL-AML1 chimeric protein. Although AML1 stimulates transcription, TEL-AML1 functions as a repressor of some AML1 target genes. In contrast to the wild type AML1 protein, both TEL and TEL-AML1 interact with N-coR, a component of the nuclear receptor corepressor complex with histone deacetylase activity. The interaction between TEL and N-coR requires the central region of TEL, which is retained In TEL-AML1, and TEL lacking this domain is impaired in transcriptional repression. Taken together, our results suggest that TEL-AML1 may contribute to leukemogenesis by recruiting N-CoR to AML1 target genes and thus imposing an altered pattern of their expression. (Blood, 2000;96:2557-2561) (C) 2000 by The American Society of Hematology..

The transcriptional activity of transcription factors is regulated by phosphorylation. The uncontrolled expression and constitutive activation of transcriptional regulators have been reported to cause malignant diseases. However, little is known about the phosphorylation status of tissue-specific transcription factors in human primary malignancies. Here we present the first insights into both protein expression and phosphorylation of transcription factors in a large-scale study of patients with acute myeloid leukemia (AML). We examined the expression and phosphorylation status of hematopoietic transcription factors PU.1 and C/EBP beta detected by the retarded mobility of the phosphorylated forms of the proteins. The rate of protein expression differed among French-American-British (FAB) subclasses. The expression of C/EBP beta and PU.1 were detectable in 77% and 61.%, respectively, of 90 AML samples examined. The expressed PU.1 and C/EBP beta was always accompanied with both phosphorylated and unphosphorylated forms of PU.1 and C/EBP beta, respectively. Statistical significance was observed between PU.1 expression (phosphorylation) and FAB classification (MO, M4, or M5 versus M2 or M3, P < .0001). PU.1 and C/EBP beta were simultaneously detected in all MO, M4, M5 and peripheral blood monocytes, whereas in M2 and M3, the expression of the 2 transcription factors varied among samples. Examination of protein expression and phosphorylation of these lineage-specific molecules may help us to understand the functional characteristics of AML. (C) 2000 The Japanese Society of Hematology..

We analysed 67 samples from Brazilian children of diverse ethnic origins with acute lymphoblastic leukaemia (ALL) for the presence of the TEL-AML1 fusion gene transcripts using reverse transcription polymerase chain reaction (RT-PCR). All 12 positive cases (20% of the 60 B-cell precursor ALL) had common (CD10(+)) ALL with a mean age of 4 years (range 1-10 years). We conclude that the frequency, age, distribution and clinical features of the TEL-AML1 fusion gene-positive ALL is similar in the diverse ethnic backgrounds of the Brazilian children to that in other countries with predominantly white Caucasian or oriental ethnicity. Apparent exceptions to this generality are discussed..

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Background: Adult T cell leukemia lymphoma is a lymphoproliferative syndrome etiologically associated to human T cell lymphotropic virus type I. Aim: To describe the clinical and laboratory features of 26 Caucasian Chilean patients, with HTLV-I positive adult T-cell leukemia lymphoma (ATLL). Material and methods: Diagnostic criteria included clinical features, cell morphology, immunophenotype, HTLV-I serology and/or DNA analysis by Southern blot or PCR. Results: According to the clinical presentation 12 cases had the acute ATLL form, 6 had a lymphoma, 4 the chronic form and 4 bad Smoldering ATLL. The median presentation age was 50 years , younger than the Japanese patients, but significantly older than patients om other South American countries (eg Brasil, Jamaica, Colombia). The main clinical features: lymphadenopathy, skin lesions and hepatosplenomegaly, were similar in frequency to those of patients from other countries, except for the high incidence of associated neurological disease. Tropical Spastic Paraparests (TSP) in our series of ATLL, was seen in one third of the patients (8/26). A T-cell immunophenotype was shown in all 26 cases and HTLV-I serology was positive in 25/26 patients. Molecular analysis on the seronegative patient showed clonal integration of proviral HTLV-I DNA into the lymphocytes DNA, and thus he may have been a poor responder to the retroviral infection. Proviral DNA integration was also demonstrated in 15/16 patients being clonal in 10, polyclonal in 3 (all smoldering cases) and oligoclonal in one. Conclusions: ATLL in Chile has similar clinical and laboratory features es than the disease ill other parts of the world, except for a younger age than Japanese patients but older than those from other Latin American countries and a high incidence of patients with associated TSP. Detailed morphological and immunophenotypic analysis of the abnormal circulating lymphocytes, together with the documentation of HTLV-I by serology and/or DNA analysis are key tests for the identification of this disease..

Epidemiological evidence suggests that childhood leukaemia, and possibly common acute lymphoblastic leukaemia in particular, may have an infectious aetiology. Smith (1997 J Immunother 20: 89-100) recently suggested that the critical infectious event occurs during pregnancy, and identified the polyoma virus JC as a candidate agent. In the present study we investigated whether genomes from the JC virus, and closely related BK virus, could be detected in leukaemic cells. No positive results were obtained suggesting that JC virus is unlikely to play a direct role in leukaemogenesis. (C) 1999 Cancer Research Campaign..

We document an unusual case of HTLV-I positive adult T-cell leukaemia lymphoma (ATLL) in a 25 year old Chilean patient who presented with primary small intestinal involvement and during evolution developed a leukaemic phase. Duodenal biopsy showed infiltration by pleomorphic lymphoid cells with a CD45RO+ CD20- phenotype. Circulating lymphocytes had a convoluted nucleus and displayed a mature T-cell phenotype: CD2+, CD3+, CD4+, CD8-, CD25+, HLA-Dr+. HTLV-I serology was positive and HTLV-I retroviral sequences were demonstrated by PCR in the tissue. The patient was treated with chlorambucil and is well, disease free five years from diagnosis. Intestinal lymphoma as initial manifestation of ATLL is extremely uncommon, but when a T-cell lymphoma is detected in this localisation, in patients from a HTLV-I endemic area, retroviral studies are recommended in order to exclude an association with this retrovirus..

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The TEL (ETV6)-AML1 (CBFA2) gene fusion is the most common reciprocal chromosomal rearrangement in childhood cancer occurring in approximately 25% of the most predominant subtype of leukemia- common acute lymphoblastic leukemia. The TEL-AML1 genomic sequence has been characterized in a pair of monozygotic twins diagnosed at ages 3 years, 6 months and 4 years, 10 months with common acute lymphoblastic leukemia. The twin leukemic DNA shared the same unique (or clonotypic) but nonconstitutive TEL-AML1 fusion sequence. The most plausible explanation for this finding is a single cell origin of the TEL-AML fusion in one fetus in utero, probably as a leukemia-initiating mutation, followed by intraplacental metastasis of clonal progeny to the other twin. Clonal identity is further supported by the finding that the leukemic cells in the two twins shared an identical rearranged IGH allele. These data have implications for the etiology and natural history of childhood leukemia..

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The MLL gene is frequently rearranged in acute human leukemia of both the myeloid and lymphoid lineages. Using a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, we identified several abnormally spliced transcripts in which MLL exons were joined in an order different from the genomic orientation (scrambled exons). Mis-splicing of MILL was present in both normal and malignant tissues. Although the majority of these scrambled transcripts were joined accurately at consensus splice sites, there were several examples in which the junctions of exons spliced in aberrant order were at non-consensus sites. A number of features differentiate mis-splicing of MLL from the previously described cases of scrambled exons and circular RNAs. Some scrambled transcripts appear to be present in the polyadenylated fraction of RNA. No correlation of exon scrambling with exon skipping was found, and there was no particular tendency for the exons involved to be near large introns. Our data show that splicing of MLL is extremely complex. The presence of scrambled transcripts in both normal and leukemic cells, indistinguishable from transcripts resulting from genomic MLL rearrangements, precludes the use of nested RT-PCR as a screening method for detection of tandem duplication of MLL. (C) 1998 Elsevier Science B.V..

Acute leukemia has a high concordance rate in young identical twins and in infants this is known, from molecular analysis, to reflect an in utero origin in one twin followed by prenatal metastasis to the other twin via intraplacental anastomoses. The situation in older twins with leukemia has been less clear. We describe a pair of identical twins who were diagnosed with a T-cell malignancy at 9 and 11 years of age, one with T-cell non-Hodgkin's lymphoma and the other with T-cell acute lymphoblastic leukemia. Leukemic cells from the twins shared the same TCR beta gene rearrangement with an identical 11 bp N region. The most plausible interpretation of this result is that these malignancies were initiated in one twin fetus in utero, in a single T-lineage cell that had stable bi-allelic TCR beta rearrangements. Progeny of this cell then spread to the other twin before birth via shared placental vasculature. This was then followed by a 9- and 11-year preleukemic latent period before clinical disease manifestation as leukemia or lymphoma. This result has considerable implications for the etiology and natural history of pediatric leukemia. (C) 1997 by The American Society of Hematology..

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 G(1) 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..

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..

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We have compared the molecular architecture and function of the myeloperoxidase upstream enhancer in multipotential versus granulocyte-committed hematopoietic progenitor cells, We show that the enhancer is accessible in multipotential cell chromatin but functionally incompetent before granulocyte commitment. Multipotential cells contain both Pu1 and C-EBP alpha as enhancer-binding activities. Pu1 is unphosphorylated in both multipotential and granulocyte-committed cells but is phosphorylated in B lymphocytes, raising the possibility that differential phosphorylation may play a role in specifying its lymphoid versus myeloid functions. C-EBP alpha exists as multiple phosphorylated forms in the nucleus of both multipotential and granulocyte-committed cells. C-EBP beta is unphosphorylated and cytoplasmically localized in multipotential cells but exists as a phosphorylated nuclear enhancer-binding activity in granulocyte-committed cells. Granulocyte colony-stimulating factor-induced granulocytic differentiation of multipotential progenitor cells results in activation of C-EBP delta expression and functional recruitment of C-EBP delta and C-EBP beta to the nucleus, Our results implicate Pu1 and the C-EBP family as critical regulators of myeloperoxidase gene expression and are consistent with a model in which a temporal exchange of C-EBP isoforms at the myeloperoxidase enhancer mediates the transition from a primed state in multipotential cells to a transcriptionally active configuration in promyelocytes..

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We report detailed immunological, cytogenetic and molecular evidence for complete identity of the leukemic cell populations in monozygotic female twins with concordant leukemia diagnosed at two months of age. Both infants had early pre-B acute lymphoblastic leukemia with the (11;19)(q23;p13) chromosomal translocation. A common clonal origin of leukemia in these infants was suggested by the finding of identical oligoclonal heavy chain immunoglobulin gene rearrangements. Leukemic cell DNA was examined for 11q23 rearrangements by Southern blotting and restriction fragments of identical size were found in the two cases, in contrast to the diversity of rearrangements observed in other unrelated and nontwinned control infants with t(11;19)(q23;p13). Similar restriction fragments were absent in blood mononuclear DNA from both parents, liver tissue from one twin and remission bone marrow of the other, indicating that the 11q23 rearrangement was acquired and not inherited as a chromosomal abnormality or polymorphism. These findings provide a definitive evidence for intrauterine single cell origin, with twin to twin transmission, of concordant leukemia in this infant twin pair. (C) 1995 Wiley-Liss, Inc..

We describe the clinical and laboratory features in three Caucasian Chilean patients with tropical spastic paraparesis (TSP) associated with/or preceded by a lymphoproliferative disorder involving cutaneous lesions and localised lymphadenopathy. The neurological symptoms and signs were characteristic of TSP and CSF examination revealed the presence of oligoclonal bands. All three patients had a moderate leucocytosis (10-14 x 10(9)/l) with eosinophilia and a minority (2-4%) of circulating atypical polylobed or ATLL-like lymphocytes. Lymph node histology showed a diffuse pattern of infiltration (1 case) and marked expansion of the paracortical zone with convoluted lymphocytes and immunoblasts (2 cases). Skin biopsy demonstrated a dermal lymphoid infiltration with epidermotropism. Antibodies to HTLV-I were detected in the serum and CSF in the three patients and Southern blot analysis of peripheral blood mononuclear cells showed a monoclonal integration of HTLV-I proviral DNA in one case whereas in the two others the pattern was indicative of low level polyclonal integration. All three patients were treated with prednisolone and one with PUVA with transient partial response on the skin and neurological manifestations. Two patients died months to 5 years from presentation and the other is alive 12 years from diagnosis with active neurological and skin disease. The simultaneous occurrence of HTLV-I associated TSP with smouldering ATLL and a cutaneous ATLL or pre-leukaemic form is discussed..

We describe a case of neonatal mixed lineage leukaemia which presented with a dominant B progenitor lymphoblast population plus a minor monocytic component. Treatment of the patient with corticosteroid and Ara-C resulted in loss of lymphoblasts and a rapid (within 7 days) increase and dominance of the monocytic component. The common clonal origin of the two cell types was evident from the identical rearrangement in the MLL gene and a shared rearrangement of one IGH allele. In common with other neonatal or infant ALL with MLL gene rearrangements, this leukaemia may have originated in a common B-monocytic lineage stem cell during foetal haemopoiesis. The observations further suggest that the therapeutic impact of the MLL gene rearrangement is to some extent dependent on the cellular context in which it is expressed..

The myeloperoxidase (MPO) gene is selectively expressed during haemopoiesis in the granulocytic lineage. Compared with the erythroid (beta-globin) and B-cell (immunoglobulin) lineages, little is known of the regulatory sequences and transcription factors involved in the regulation of genes specific for granulopoiesis. We have approached this issue by identifying a strong enhancer for the murine MPO gene. A candidate enhancer region was mapped by the detection of a strong DNase I hypersensitive site, -3.4 to -3.2 kb upstream of the MPO gene. A 301 bp fragment encompassing the DNase I site was shown to have strong enhancer function in a transient assay following transfection of a reporter gene into a MPO-expressing cell (WEHI 3BD+), but was inactive in lymphoid cells. Analysis of sub-fragments revealed that the whole 301 bp fragment is required for maximal enhancer function..

We describe the clinical and laboratory features of nine patients born in Chile with HTLV-I-positive adult T-cell leukemia/lymphoma (ATLL). All were adults (median age 51 years) of Caucasian origin without evidence of Indian or foreign extraction and none had been out of the country. The main disease features were organomegaly, cutaneous lesions, hypercalcemia and leukemia with atypical polylobed lymphocytes displaying a CD2+/-, CD3+, CD4+, CD8-, CD7- T-cell phenotype. Eight patients presented with acute type ATLL and one had a chronic form lasting for 16 months prior to the development of the acute phase. Lymph node histology (three cases) was consistent with a T-cell non-Hodgkin's lymphoma (large and small cells). Antibodies to HTLV-I were detected by ELISA and particle agglutination in the serum from eight of nine patients. DNA analysis showed HTLV-I proviral DNA in all seven cases investigated, including the single serologically negative patient. In five cases, HTLV-I was monoclonally integrated and in one case oligoclonal. In the seventh case viral DNA clonal status was ambiguous. Response to therapy was poor and median survival was 3 months (range 2-20 months). This study provides further evidence that HTLV-I is endemic in Chile, a non-tropical country where the two main diseases associated with HTLV-I, ATLL and TSP, are found..

In addition to the growth hormone gene (hGH-N) itself, the human growth hormone (hGH) locus contains four related genes, namely hGH-V and hCS-L, -A and -B, which have appeared very recently in evolution and are specifically expressed in placenta. With the aim of identifying the regulatory elements responsible for this placental-specific expression, we have mapped the DNaseI hypersensitive sites present at the hGH gene cluster in a placental cell line (BeWo) that expresses the hGH-V and hCS genes. Our results reveal a complex pattern of hypersensitive sites distributed along the hGH locus, most of which appear to be cell type-specific. Thus, we have identified placental-specific hypersensitive sites within the first intron of the hGH-N and hGH-V genes, but not in the equivalent regions of the hCS genes. In addition, we have found several placental-specific hypersensitive sites downstream of the hCS-L and hCS-A genes, which might reflect the presence of enhancer elements similar to that located downstream of the hCS-B gene (Walker et al. (1990) J. Biol. Chem. 265, 12940). Comparison of BeWo cells with a placental cell line (JEG-3) which does not express the hGH-V and hCS genes revealed a very similar pattern of hypersensitive sites, suggesting that the sites detected are established before the onset of transcription. Our results indicate that the transition to an active hGH locus in placental cells requires multiple alterations in chromatin structure, and provide a framework for the molecular analysis of the regulatory elements and mechanisms mediating such processes..

THE majority (approximately 75%) of infant acute leukaemias have a reciprocal translocation between chromosome 11q23 and one of several partner chromosomes1. The gene at 11q23 (named MLL, ALL-1, HRX or HTRX-1; refs 2-6) has been cloned and shares homology with the Drosophila developmental gene trithorax3-5. Rearrangements of this gene (called HRX here) occur in introns and cluster in a region of approximately 10 kb; individual patients have different breakpoints3-10. Here we describe three pairs of infant twins with concordant leukaemia who each share unique (clonal) but non-constitutive HRX rearrangements in their leukaemic cells, providing evidence that the leukaemogenic event originates in utero and unequivocal support for the intra-placental 'metastasis' hypothesis for leukaemia concordance in twins11..

The principal regulator of erythropoiesis is the glycoprotein erythropoietin, which interacts with a specific cell surface receptor (EpoR). A study aimed at analyzing EpoR gene regulation has shown that both pluripotent embryonal stem cells and early multipotent hematopoietic cells express EpoR transcripts. Commitment to nonerythroid lineages (e.g., macrophage or lymphocytic) results in the shutdown of EpoR gene expression, whereas commitment to the erythroid lineage is concurrent with or followed by dramatic increases in EpoR transcription. To determine whether gene activity could be correlated with chromatin alterations, DNase-hypersensitive sites (HSS) were mapped. Two major HSS located in the promoter region and within the first intron of the EpoR gene are present in all embryonal stem and hematopoietic cells tested, the intensities of which correlate well with EpoR expression levels. In addition, a third major HSS also located within the first intron of the EpoR gene is uniquely present in erythroid cells that express high levels of EpoR. Transfection assays show that sequences surrounding this major HSS impart erythroid cell-specific enhancer activity to a heterologous promoter and that this activity is at least in part mediated by GATA-1. These data, together with concordant expression levels of GATA-1 and EpoR in both early multipotent hematopoietic and committed erythroid cells, support a regulatory role of the erythroid cell-specific transcription factor GATA-1 in EpoR transcription in these cells. However, the lack of significant levels of GATA-1 expression in embryonal stem cells implies an alternative regulatory mechanism of EpoR transcription in cells not committed to the hematopoietic lineage..

The clonal and immunophenotypic characteristics of blood leukemic cells from BCR/ABL p190 transgenic mice were investigated. All cell populations evaluated in vivo and in vitro had B-lymphocyte progenitor immunophenotypes. Immunoglobulin (J(H)) rearrangement patterns provided evidence for clonal diversification at different sites in vivo. Multiple clones were established in vitro from two of these mice (nos. 730 and 753). These cells expressed BCR/ABL p190 protein tyrosine kinase (PTK) and were highly malignant on transfer to secondary recipients. Cells independently cloned in vitro shared identical immunophenotypes and clonal IgH rearrangements, but these were distinct from those of the dominant clones in the mouse from which they were derived. Nevertheless, in vitro clones from mouse no. 753 had an abnormal karyotype (chromosome 14 trisomy) in common with the dominant clone in blood, providing evidence for a hierarchy or clonal selection in vivo and in vitro. Two sets of in vitro clones proliferated independently of exogenous growth factors and stroma and released autocrine interleukin 7 growth factor activity. These data provide evidence for rapid divergent clonal evolution and selection of B-cell progenitors initiated by BCR/ABL p190, followed by other, secondary genetic events mirroring similar changes in the equivalent, highly malignant human leukemia Philadelphia (Ph)-positive/B-precursor acute lymphoblastic leukemia (ALL)..

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The beta-globin locus control region (LCR) is characterized by erythroid-specific DNase I hypersensitive sites and is involved in the chromatin organization, transcriptional potentiation, developmental regulation, and replication timing of the entire beta-globin gene cluster. When and how the LCR is first activated during erythropoiesis is not known. Here we analyze the chromatin structure of the LCR during early hematopoietic differentiation using nontransformed, multipotential, growth factor-dependent, murine hematopoietic progenitor cells. We show that LCR hypersensitive sites characteristic of erythroid cells are present in three independent multilineage progenitors [FDCP (factor-dependent cell, Paterson)-mix A4, B6SUtA, and LyD9] under conditions of self-renewal. Induction of differentiation down a nonerythroid pathway causes a progressive loss of hypersensitivity in the LCR. These results show that the beta-globin LCR is in an active chromatin configuration prior to erythroid commitment and indicate a significant role for selective gene repression in lineage specification..

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Multipotential interleukin 3-dependent non-immortalized murine hemopoietic progenitor cells have DNase 1-hypersensitive sites in the immunoglobulin heavy-chain and CD3-delta enhancers and transcribe germ-line T-cell antigen receptor gamma-chain (TCR-gamma), but not IgM or TCR-beta, genes. Induction of myeloid differentiation in these cells closes down expression and/or transcriptional accessibility of the immunoglobulin heavy-chain and TCR-gamma genes. The CD3-delta enhancer region remains DNase I-hypersensitive but closes down in B cells. In embryonic stem cells and pan-mesodermal cells, these genes or enhancer regions are neither expressed nor DNase I-hypersensitive. These data suggest that lineage potential may be programmed, at least in part, by alterations in the accessibility or conformation of regulatory regions of genes and that some promiscuity of gene expression and/or accessibility can precede lineage commitment and maturation in progenitor cells induced to self-renew by interleukin 3..

Regressing atypical histiocytosis is a rare multifocal cutaneous tumor characterized by large, spontaneously regressing, ulcerating skin nodules. Although initially self-remitting, the condition may progress to systemic lymphoma..

This paper reports a case of adult T-cell leukemia/lymphoma associated with human T-cell lymphotropic virus type I (HTLV-I) diagnosed in a Chilean patient who developed after 1 1/2 years a crisis with a progressive sensorimotor polyneuropathy. Serum and cerebrospinal fluid HTLV-I antibody tests were positive and HTLV-I DNA was clonally integrated in peripheral lymphocytes. This case is unusual in having simultaneous neurological disease. Along with other recent data from South America, this suggests that the endemic area of HTLV-I may spread far beyond the Caribbean area..

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In acute lymphoblastic leukemia (ALL) diagnostic samples and cell lines with unequivocal B cell precursor (common) or T cell precursor immunophenotypes, there is inappropriate or cross-lineage IgH or T cell receptor beta gene (TCR beta) rearrangement in approximately 25% of the cases. The frequency of such rearrangements is lower in mature lymphoid neoplasms and acute myeloblastic leukemia. The most immature B lineage ALL ('null' ALL) has a much lower frequency of TCR gene rearrangement than the common variant of B cell precursor ALL and also has a high frequency of oligoclonal rearrangements of IgH genes. Non-T leukemic cells with inappropriately rearranged TCR beta gene did not necessarily have a rearranged TCR gamma gene. Inappropriately rearranged IgH or TCR genes are usually not expressed at the mRNA level, and the gene for the TCR associated protein T3 delta is not detectably expressed at the mRNA or protein level in leukemias classified unambiguously as non-T. Five cases of acute leukemia with ambiguous or mixed lineage immunophenotypes (myeloid + T or myeloid + B) are described. These five had diverse patterns of IgH, TCR beta, and TCR gamma rearrangement, and all expressed terminal transferase concomitantly with MY9 (CD33). The T3 delta gene was expressed in two cases, which also expressed other T cell markers indicating that coordinated lymphoid lineage programs had been initiated. The implications of these observations for lineage-associated regulation of genes during normal differentiation and leukemogenesis are discussed..

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An immunoglobulin polypeptide chain is encoded by multiple gene segments that lie far apart in germ-line DNA and must be brought together to allow expression of an immunoglobulin gene active in B lymphocytes. For the immunoglobulin heavy chain genes, one of many variable (V) region genes becomes joined to one of several diversity (D) segments which are fused to one of several joining (J) segments lying 5' of the constant region (C) genes. Here we show that the rearranged mu genes of an IgM-producing human B-lymphocyte cell line exhibit pancreatic deoxyribonuclease (DNase I) hypersensitive sites in the JH-C mu intron that are absent in naked DNA or the chromatin of other differentiated cell types. DNA sequence analysis reveals that the major hypersensitive site maps to a conserved region of the JH-C mu intron recently shown to function as a tissue-specific enhancer of heavy-chain gene expression. A similar association of an enhancer-like element with a DNase I hypersensitive site has been reported for the mouse immunoglobulin light-chain J kappa-C kappa intron. These results implicate disruption of local chromatin structure in the mechanism of immunoglobulin enhancer function..

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