JNCI Monographs - current issue

Overview of Mechanisms and Consequences of Chromosomal Translocation

Rabkin, C. S., Janz, S. — 2008-07-22

Brief Historical Sketch of Chromosomal Translocations and Tumors

Potter, M. — 2008-07-22

The discovery of chromosomes emerged from the cytological analysis of mitosis in the 1870s. At the turn of the 20th century, cytologists and geneticists established that chromosomes carried the hereditary material. In the early 20th century, Theodore Boveri, recognizing the nonequivalence of individual chromosomes, began thinking about the biological consequences of imbalances of chromosomal compositions in somatic cells and how these might explain the origin of cancer. Many of his predictions would have to wait for confirmation until the 1950–1960s, when mammalian cytogenetics became feasible with the use of ascites tumors as sources of metaphases. This advance coupled with the discovery of G banding by Caspersson and his associates led to finding characteristic recurring chromosomal abnormalities in certain kinds of tumors. Chromosomal translocations that were associated with promoter deregulations or the formation of novel fusion genes were the prime models. This continuing progress combined with dramatic advances in DNA structure, transcription, and repair have provided new insights into the role of this class of mutations in neoplastic development.

Mechanistic Aspects of Lymphoid Chromosomal Translocations

Lieber, M. R., Raghavan, S. C., Yu, K. — 2008-07-22

Chromosomal translocations require double-strand breakage at two sites, followed by joining of the ends. The joining is usually done by nonhomologous DNA end-joining, though homologous recombination and single-strand annealing play roles in cases where there is homology. The mechanism of breakage can be more difficult to understand at sites other than the antigen receptor loci. Some breakage events in pre–B or pre–T cells are due to the RAG proteins recognizing heptamer/nonamer-like sequences, but most breaks are not. Rather, some of these breaks are due to RAG nicking at non–B DNA conformations. Translocation events in mature B cells, when RAGs are not expressed, may be due to the activation-induced deaminase (AID). But AID only acts on single-stranded DNA, and it is not yet clear how this single-stranded DNA arises at some transcribed sites and not others. During the physiologic process of class switch recombination, R-loops form at transcribed class switch regions, thereby accounting for how single strandedness is facilitated at these sites of AID action.

The RAD5-dependent Postreplication Repair Pathway is Important to Suppress Gross Chromosomal Rearrangements

Myung, K., Smith, S. — 2008-07-22

Genome instability is characteristic of cancer cells. Although it frequently occurs during carcinogenesis, the mechanism underlying genome instability is not clearly understood. Recent extensive genetic analyses from different organisms have begun to reveal mechanisms for the suppression of genome instability in general DNA metabolisms including DNA replication, recombination, DNA repair, and signal transduction. One DNA repair pathway called postreplication repair (also known as DNA damage bypass) has been highlighted for its role in genome stability. Central to DNA damage bypass, proliferating cell nuclear antigen (PCNA) directs different pathways through its mono- or polyubiquitination and sumoylation. In this review, we will discuss template switching dictated by the PCNA polyubiquitination and its roles in the suppression of genome instabilities.

On the Contribution of Spatial Genome Organization to Cancerous Chromosome Translocations

Soutoglou, E., Misteli, T. — 2008-07-22

The formation of cancer translocations requires the physical interaction of the translocating chromosomes. It has been postulated that the nonrandom spatial organization of the genome within the cell nucleus contributes to determining the outcome of chromosomal translocation. Comparative analysis of the spatial arrangement of translocations partners and their frequency of translocation suggests that translocations occur preferentially among proximally positioned genome regions. This model makes predictions about mechanisms of translocations and the dynamic properties of genome regions in vivo. Elucidating the contribution of spatial genome organization to the formation of chromosome translocations is an integral part of understanding how translocations form in vivo and has provoked the interrogation of several fundamental aspects of genome cell biology, including tissue-specific differences in genome organization, dynamics of genomes in vivo, and the mechanisms that are determining genome organization in vivo.

Induction of Chromosomal Translocations in Mouse and Human Cells Using Site-Specific Endonucleases

Weinstock, D. M., Brunet, E., Jasin, M. — 2008-07-22

Reciprocal chromosomal translocations are early and essential events in the malignant transformation of several tumor types, yet the precise mechanisms that mediate translocation formation are poorly understood. We review here the development of approaches to induce and recover translocations between two targeted DNA double-strand breaks (DSBs) in mammalian chromosomes. Using mouse cells, we find that nonhomologous end-joining readily mediates translocation formation between two DSBs generated by site-specific endonucleases. Translocations occur much less frequently, however, than intrachromosomal repair of a single DSB. Translocation junctions obtained with this approach have similar end modifications to translocation junctions found in human tumors, including deletions, insertions, and repair at short stretches of homology. These modifications are more extensive than repair junctions at a single DSB, suggesting that different factors may be involved in translocation formation and repair of a single DSB. Finally, we describe a novel approach to induce translocations in human cells. Translocation model systems provide an opportunity to study the involvement of mammalian DNA repair and signaling factors in the etiology of chromosomal rearrangements.

Characterization of MYC Translocations in Multiple Myeloma Cell Lines

Dib, A., Gabrea, A., Glebov, O. K., Bergsagel, P. L., Kuehl, W. M. — 2008-07-22

Translocations involving an MYC gene (c >> N >>L) are very late tumor progression events and provide a paradigm for secondary translocations in multiple myeloma. Using a combination of fluorescent in situ hybridization and comparative genomic hybridization arrays (aCGH), we have identified rearrangements of an MYC gene in 40 of 43 independent myeloma cell lines. A majority of MYC translocations involve an Ig locus (IgH > Ig >> Ig), but the breakpoints only infrequently occur near or within switch regions or V(D)J sequences. Surprisingly, about 40% of MYC translocations do not involve an Ig locus. The MYC translocations mostly are nonreciprocal translocations or insertions, often with the involvement of three chromosomes and sometimes with associated duplication, amplification, inversion, and other associated chromosomal abnormalities. High-density aCGH analyses should facilitate the cloning of MYC breakpoints, enabling the determination of their structures and perhaps elucidating how rearrangements not involving an Ig gene cause dysregulation of an MYC gene.

Structure and Consequences of IGH Switch Breakpoints in Burkitt Lymphoma

Guikema, J. E. J., Schuuring, E., Kluin, P. M. — 2008-07-22

The t(8;14) MYC/IGH breakpoint is the hallmark translocation of human Burkitt lymphoma (BL). The translocation breakpoint most often involves the immunoglobulin heavy-chain switch regions and is thought to be brought about by an aberrant class switch recombination (CSR) event. During CSR in normal germinal center B cells, DNA double-stranded breaks are introduced in Sµ and one of the downstream switch regions (S, S, or S) that are juxtaposed and ligated to form the switch junction, with deletion of the intervening DNA. In contrast, aberrant switch recombination in BL exclusively involves only one switch region, resulting in a perfect reciprocal translocation. A functional consequence of this type of translocation is that IgM expression from the chromosome affected by the translocation is not necessarily disrupted.

Genetic and Environmental Cofactors of Myc Translocations in Plasma Cell Tumor Development in Mice

Janz, S. — 2008-07-22

Peritoneal plasmacytomagenesis in inbred BALB/c mice affords an experimental model system for the study of the mechanism by which naturally occurring Myc (c-myc) translocations collaborate with host susceptibility factors and environmental influences in tumor development. Mouse plasmacytoma is initiated in ~80% of cases by a balanced chromosomal T(12;15)(Igh-Myc) translocation that results in a mode of Myc deregulation that renders the survival and outgrowth of the translocation-bearing tumor precursor exquisitely dependent upon factors provided by sustained inflammation (IL-6) and gut flora microbes. Tumor susceptibility genes of BALB/c, such as weak efficiency alleles of genes encoding p16^Ink4a and Frap (mTOR), are also required for plasmacytoma, although the pathways linking these genes with deregulated Myc and the environment have not yet been elucidated. The findings in mouse plasmacytoma may be relevant for hematopoietic neoplasms in human beings, in which leukemia- and lymphoma-associated chromosomal translocation (LLA-CT) is much more frequent than subsequent neoplasia. Just like T(12;15)-carrying B-lymphocytes and plasma cells in mice, the malignant transformation of LLA-CT–bearing blood cells in humans may be a rare occurrence that requires several genetic and environmental cofactors to take place.

How the c-myc Promoter Works and Why It Sometimes Does Not

Levens, D. — 2008-07-22

The c-myc promoter is regulated by scores of signals, transcription factors, and chromatin components. The logic integrating these multiple signals remains unexplored. Recent evidence suggests that activated MYC expression is regulated in several phases: 1) conventional transcription factors trigger transcription by the RNA polymerase II (pol II) paused within the proximal promoter region. Concurrently (and probably consequently), newly arrived chromatin-remodeling complexes mobilize a nucleosome masking the far upstream element (FUSE), 1.7-kb upstream of the P2 start site; 2) binding by FUSE-binding proteins (first FBP3, then FBP); and 3) FBP-interacting repressor (FIR) binds FUSE and returns transcription to basal or steady-state levels. The recruitment and release of the FBPs and FIR is governed by FUSE-DNA conformation, itself controlled by dynamic supercoils propagated behind pol II. The organization and operation of the c-myc promoter make it difficult to inactivate, but sensitive to disturbances (translocations, viral insertions, amplification, and mutation) that disrupt the fine-tuning seen at its normal chromosomal context.

Age-Dependent Prevalence and Frequency of Circulating t(14;18)-Positive Cells in the Peripheral Blood of Healthy Individuals

Dolken, G., Dolken, L., Hirt, C., Fusch, C., Rabkin, C. S., Schuler, F. — 2008-07-22

Circulating t(14;18)-positive cells were detected by quantitative real-time polymerase chain reaction on DNA isolated from peripheral blood mononuclear cells (PBMNCs) from 644 healthy individuals between <1 and 91 years of age. In all, 45% of all samples (287/644) were positive, and 40% of the positive samples (114/287) contained more than one positive clone. The prevalence of t(14;18)-positive cells showed a strong correlation with age. A total of 36 cord blood samples and 48 PBMNCs from children <10 years were negative. The prevalence of circulating positive cells increased from the second to fifth decade of life from 20% to 66% and remained stable thereafter. Also the median frequency of circulating t(14;18)-positive cells as well as the prevalence of multiple clones showed an increase with age. In all, 4% (24/644) of all blood samples contained >1 positive cell in 25 000 cells, a finding restricted to healthy individuals >40 years. These results are discussed in relation to the low incidence of follicular lymphoma.

t(14;18) Translocations and Risk of Follicular Lymphoma

Rabkin, C. S., Hirt, C., Janz, S., Dolken, G. — 2008-07-22

The chromosomal translocation t(14;18)(q32;q21) is characteristic of follicular lymphoma and a frequent abnormality in other types of non-Hodgkin lymphoma (NHL). In healthy individuals, the same translocation may also be found in a small fraction of peripheral blood lymphocytes, the biological significance of which is beginning to be explored. Translocation prevalence and frequency are potential risk factors for developing NHL. Here, we review the published data and describe recent and ongoing work on this promising biomarker. We have a series of studies in four major areas: 1) t(14;18) prevalence and frequency in healthy individuals; 2) maturation of translocation-harboring cells; 3) effect of rituximab treatment on t(14;18) carriage; and 4) predictive and clonotypic relationship between t(14;18) and follicular lymphoma or other NHL. Further studies are warranted to increase understanding of this crucial molecular event in the development of hematopoietic malignancies. Potential applications include determination of elevated risk for lymphoma, early detection of disease, and identification of molecular targets for preventive interventions.

Clinical Significance of the Most Common Chromosome Translocations in Adult Acute Myeloid Leukemia

Mrozek, K., Bloomfield, C. D. — 2008-07-22

Acquired genetic alterations such as balanced and unbalanced chromosome aberrations and submicroscopic gene mutations and changes in gene expression strongly affect pretreatment features and prognosis of adults with acute myeloid leukemia (AML). The most frequent chromosome/molecular rearrangements, that is, t(8;21)(q22;q22)/RUNX1-RUNX1T1 and inv(16)(p13q22)/t(16;16)(p13;q22)/CBFB-MYH11 characteristic of core-binding factor (CBF) AML and t(15;17)(q22;q12~21)/PML-RARA characteristic of acute promyelocytic leukemia (APL), confer favorable clinical outcome when patients receive optimal treatment, that is, regimens that include high-dose cytarabine for CBF AML and all-trans-retinoic acid and/or arsenic trioxide for APL. Recently, mutations in such genes as KIT in CBF AML and FLT3 in APL have been correlated with clinical features and/or outcome of patients with these AML subtypes, and microarray gene expression profiling has been successfully used for diagnostic purposes and to provide biologic insights. These data underscore the value of genetic testing for common translocations for diagnosis, prognostication, and, increasingly, selecting therapy in acute leukemia.

Modeling Chromosomal Translocations Using Conditional Alleles to Recapitulate Initiating Events in Human Leukemias

2008-07-22

Recurrent reciprocal chromosomal translocations are present in more than 50% of leukemias. A deeper understanding of how they affect cancer initiation is essential for evaluating the origins of cancer and the potential for therapy based on the translocation products. Mouse models of chromosomal translocations are required for this. Here we summarize three methodologies developed in our laboratory to model chromosomal translocations (knock-in, translocator, and invertor methods). We have used these models to study leukemias caused by fusions of the mixed lineage leukemia (MLL) gene and the Ews-ERG fusion gene to evaluate oncogenicity and elucidate some general principles about translocation products. We show that MLL fusions have the capacity to cause hematopoietic tumors only if expressed in permissive cells and that the Mll-Enl fusion can cause lineage reassignment if the chromosomal translocation occurs in lineage noncommitted progenitors. The leukemia-initiating cells generated by Mll fusions or by Ews-ERG fusion can be committed cells within the hematopoietic pathway. Our translocation mimic models are applicable to any human reciprocal chromosomal translocation.

NUP98-HOX Translocations Lead to Myelodysplastic Syndrome in Mice and Men

Slape, C., Lin, Y. W., Hartung, H., Zhang, Z., Wolff, L., Aplan, P. D. — 2008-07-22

The myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute myeloid leukemia (AML). A wide spectrum of genetic aberrations has been associated with MDS, including chromosomal translocations involving the NUP98 gene, most commonly leading to fusions of NUP98 with abd-b group HOX genes, including HOXD13. We used vav regulatory elements to direct expression of a NUP98-HOXD13 (NHD13) fusion gene in hematopoietic tissues. NHD13 transgenic mice faithfully recapitulate all the key features of MDS, including peripheral blood cytopenias, bone marrow dysplasia and apoptosis, and transformation to acute leukemia. The MDS that develops in NHD13 transgenic mice is highly lethal; within 14 months, 90% of the mice died of either leukemic transformation or severe anemia and leukopenia due to progressive MDS. These mice provide a preclinical model that can be used for the evaluation of MDS therapy and biology.

The Utility of t(14;18) in Understanding Risk Factors for Non-Hodgkin Lymphoma

2008-07-22

Characteristic chromosomal abnormalities are associated with specific histological subtypes of non-Hodgkin lymphoma (NHL). The chromosomal translocation t(14;18)(q32;q21) is one of the most common chromosomal abnormalities in NHL, occurring in 70%–90% of cases of follicular lymphoma, 20%–30% of diffuse large B-cell lymphoma, and 5%–10% of other less common subtypes. The t(14;18)-positive NHL may represent a homogenous group and, consequently, increase etiologic specificity in epidemiological studies. Although the t(14;18) has important clinical ramifications, its etiologic significance remains to be determined. Two population-based, case–control studies addressed this issue by evaluating potential risk factors for t(14;18)-positive and t(14;18)-negative subgroups of NHL. Both studies found that the association between pesticide exposures and risk of NHL was largely limited to t(14;18)-positive NHL cases. However, the findings regarding cigarette smoking, family history of hematopoietic cancer, and hair dye use were not entirely consistent. These results indicate that defining subgroups of NHL according to t(14;18) status may be useful for etiologic research, particularly for exposures that are genotoxic or may contribute to the development of NHL through pathways involving the t(14;18). Studies to further evaluate these associations and delineate the effects of various exposures in other genetically defined subgroups of NHL are warranted.

Chromosome Translocations in Workers Exposed to Benzene

2008-07-22

As benzene has been linked with elevated risk of both acute myeloid leukemia and lymphoma, we explored the effect of benzene exposure on levels of t(8;21), t(15;17), and t(14;18) translocations. Circulating lymphocytes of normal individuals also often contain t(14;18). Quantitative polymerase chain reaction analysis showed that 37 workers with benzene exposure had a decreased level of t(14;18) in their blood with only 16.2% having 10 or more copies of the t(14;18) BCL-2/IgH fusion gene/µg DNA, as opposed to 55% of 20 controls (P = .0063 by Fisher’s exact test). This decline may be related to the immunotoxicity to specific subtypes of circulating B-lymphocytes, but the data do not support the use of t(14;18) as a biomarker of increased lymphoma risk in benzene-exposed populations. None of 88 individuals (31 controls and 57 exposed) exhibited detectable t(8;21) transcripts, and while t(15;17) transcripts were detected in two individuals, the result is inconclusive as one was exposed and the other was unexposed.

Chromosomal Aberrations in Peripheral Blood Lymphocytes and Risk for Non-Hodgkin Lymphoma

2008-07-22

Chromosomal aberrations (CAs) are thought to be integrative biomarkers that reflect exposure to chromosome-damaging carcinogens and host factors. To investigate whether CAs indicate non-Hodgkin lymphoma (NHL) risk, we evaluated 200 metaphase spreads each for 67 incident low-grade, untreated NHL cases and 57 controls matched on age, sex, and storage time of cryopreserved lymphocytes. Hyperdiploidy of 47 chromosomes was statistically significantly associated with increased NHL risk with odds ratios of 1.4 (97% confidence interval [CI] = 0.6–3.5) and 3.5 (95% CI = 1.1–10.9) for medium and high levels of hyperdiploidy, respectively, compared to the lowest level (P-trend = .04). Hypodiploidy of 43 and 44 chromosomes increased NHL risk 3.3-fold (95% CI = 1.2–8.7) and 2.2 (95% CI = 1.0–5.2), respectively, compared to those without the event; total hypodiploidy was only moderately associated with risk. Chromosome and chromatid breaks were not associated with NHL risk. Our data suggest for the first time that aneuploidy identified in cultured, peripheral lymphocytes may be potential indicators of NHL risk.

Environmental and Genetic Susceptibility to MLL-Defined Infant Leukemia

Ross, J. A. — 2008-07-22

The study of rare cancers, including retinoblastoma, angiosarcoma, and vaginal clear cell carcinoma, has contributed greatly to our understanding of cancer mechanisms. Infants with leukemia may represent another important rare group. The majority of infants with leukemia have MLL gene rearrangements in their leukemia cells, and there is unequivocal laboratory evidence that these arise in utero. There is increasing evidence that environmental and genetic factors may contribute to the risk of MLL-defined infant leukemias. Because the infant exposure experience is only a small window in comparison to that of an individual who develops a malignancy in middle or late age, the pivotal factors responsible for this genetic anomaly may be easier to identify. With the largest case–control study of infant leukemia ever conducted underway in the Children's Oncology Group (COG AE24), there is a unique opportunity to integrate epidemiological data with laboratory data on MLL status and genotype.

Chromosomal Translocations in Childhood Leukemia: Natural History, Mechanisms, and Epidemiology

Wiemels, J. — 2008-07-22

The root causes of childhood leukemia will be discovered by understanding the mechanism of mutations in the context of the cell of origin and time in life of the child. Molecular studies using archival DNA samples and twins with concordant leukemia have demonstrated that most childhood leukemia translocation subtypes occur before to birth and occur in early progenitors. Translocation breakpoints typically harbor evidence of nonhomologous end-joining repair mechanisms, but in only a few examples are the causative mechanisms of breakage evident, such as V(D)J recombinase gene activation. Epidemiologic differences in the rates of translocations between populations may point to causal clues. Leukemia like all cancers is the product of two or more genetic and/or epigenetic events, and the natural history and mechanisms of these two events are likely independent, resulting in two or more "causes" of leukemia. Complementary mutations include point mutations, deletions, and epimutations, which have distinct associated causal mechanisms.

Germline Translocations in Mice: Unique Tools for Analyzing Gene Function and Long-Distance Regulatory Mechanisms

2008-07-22

Translocations have provided invaluable tools for identifying both cancer-linked genes and loci associated with heritable human diseases, but heritable human translocations are rare and few mouse models exist. Here we report progress on analysis of a collection of heritable translocations generated by treatment of mice with specific chemicals or radiation during late spermatogenic stages. The translocation mutants exhibit a range of visible phenotypes reflecting the disruption of coding sequences or the separation of genes from essential regulatory elements. The breakpoints of both radiation-induced and chemically induced mutations in these mice are remarkably clean, with very short deletions, duplications, or inversions in some cases, and ligation mediated by microhomology, suggesting nonhomologous end joining as the major path of repair. These mutations provide new tools for the discovery of novel genes and regulatory elements linked to human developmental disorders and new clues to the molecular basis of human genetic disease.