Cytogenetically balanced translocations are associated with focal copy number alterations

Current cytogenetic methods (e.g., G-banding and multicolor chromosomal painting) allow detection of translocation events but lack the resolution to (a) locate the breakpoints precisely at the chromosome band level or (b) discriminate balanced translocations from translocations with copy number alterations not previously reported, or imperfectly balanced translocations. In this study, we demonstrate that cytogenetically balanced translocations are in fact frequently associated with segmental gain or loss of DNA. The recent development of a whole genome tiling path BAC array has enabled tiling resolution analysis of genomic segmental copy number status. Combining tiling resolution BAC array comparative genomic hybridization (array CGH) with G-Banding analysis and multicolor chromosomal painting approaches such as spectral karyotyping (SKY) facilitates high-resolution mapping of genomic alterations associated with imperfectly balanced translocations. Using a refined version of our CGH array we have deduced the copy number status throughout the genomes of three cytogenetically well-characterized prostate cancer cell lines (PC3, DU145, LNCaP) to determine whether translocations are associated with focal gains and losses of DNA. At 78 kb tiling resolution we identified the boundaries of 170, 80, and 34 known and novel copy number alterations (CNA) in these cell line genomes, respectively. Thirty-three of the 36 known translocations (92%, P < 0.001) in DU145 were associated with segmental CNA. Likewise, 80% (P < 0.001) of the known translocations showed association in LNCaP. Although many translocation breakpoints exhibit segmental alteration in PC3, the pattern of chromosomal rearrangements is too complex for use in comprehensive association with CNA boundaries. Our results reveal that imperfectly balanced translocations in tumor genomes are a phenomenon that occurs at frequencies much higher than previously demonstrated. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

In this issue of Epigenetics

The pathogenesis of acute myeloid leukemias involves complex molecular events triggered by diverse alterations of genomic DNA. A limited number of initiating lesions, such as chromosomal translocations generating fusion genes, are constantly identified in specific forms of leukemia and are critical to leukemogenesis. Leukemia fusion proteins derived from chromosomal translocations can mediate epigenetic silencing of gene expression. Epigenetic deregulation of the DNA methylation status and of the chromatin “histone code” at specific gene sites cooperate in the pathogenesis of leukemias. The neutralization of these crucial oncogenic events can revert the leukemia phenotype. Thus, their identification and the study of their molecular and biological consequences is essential for the development of novel and specific therapeutic strategies. In this context, we recently reported a link between the differentiation block of leukemia and the epigenetic silencing of the microRNA-223 gene by the AML1/ETO oncoprotein, the product of the t(8;21) the commonest AML-associated chromosomal translocation. This finding indicates microRNAs as additional epigenetic targets for leukemogenesis and for therapeutic intervention in leukemias.     

Epigenetic therapy of leukemia: An update

Carcinogenesis is classically thought to result from genetic alterations in DNA sequence such as deletions, mutations, or chromosomal translocations. These in turn may lead to the activation of oncogenes, inactivation of tumor suppressor genes or formation of chimeric oncoproteins. Epigenetics, in contrast, refers to a number of biochemical modifications of chromatin, either to DNA directly or to its associated protein complexes that affect gene expression without altering the primary sequence of DNA [Robertson KD, Wolffe AP. DNA methylation in health and disease. Nat Rev Genet 2000;1:11-9; Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128:683-92]. A fundamental difference between genetic and epigenetic alterations is the irreversible nature of genetic lesions whereas epigenetic ones are potentially reversible, allowing for therapeutic intervention. In the last decade, it has become apparent that epigenetic changes play an important role in cancer, particularly in leukemia. Significant advances have been made in the elucidation of these processes as well as in translating this knowledge to the clinic, as in the development of new prognostic biomarkers or targeted therapies. In this review, we will focus on recent advances in epigenetic therapy in leukemia.     

Potential G-quadruplex formation at breakpoint regions of chromosomal translocations in cancer may explain their fragility

Genetic alterations like point mutations, insertions, deletions, inversions and translocations are frequently found in cancers. Chromosomal translocations are one of the most common genomic aberrations associated with nearly all types of cancers especially leukemia and lymphoma. Recent studies have shown the role of non-B DNA structures in generation of translocations. In the present study, using various bioinformatic tools, we show the propensity of formation of different types of altered DNA structures near translocation breakpoint regions. In particular, we find close association between occurrence of G-quadruplex forming motifs and fragile regions in almost 70% of genes involved in rearrangements in lymphoid cancers. However, such an analysis did not provide any evidence for the occurrence of G-quadruplexes at the close vicinity of translocation breakpoint regions in nonlymphoid cancers. Overall, this study will help in the identification of novel non-B DNA targets that may be responsible for generation of chromosomal translocations in cancer.     

The mystery of chromosomal translocations in cancer

Chromosomal translocations in human cancer may result in products that can be suppressed by targeting drugs. An example is bcr-abl tyrosine kinase in chronic myelogenous leukemia that can be treated with imatinib mesylate. However, the mechanisms of translocations or exchanges of chromosomal segments are virtually unknown. In this summary, chromosomal translocations in human cancer are compared with 'crossing over' of chromosomal segments occurring during the first meiotic division. Several proposed mechanisms of the exchange of DNA between and among chromosomes are discussed. The conditions that appear essential for these events to occur are listed. Among them are proximity of the involved DNA segments, mechanisms of excising the target DNA, its transport to the new location, and integration into the pre-existing chromosome. The conclusion based on extensive review of the literature is that practically nothing is known about the mechanism of 'crossing over' or translocation. Based on prior work on normal human cells, it is suggested that only one of the two autosomes participates in these events that may include loss of heterozygozity, another common abnormality in human cancer.     

Functional copy-number alterations in cancer

Understanding the molecular basis of cancer requires characterization of its genetic defects. DNA microarray technologies can provide detailed raw data about chromosomal aberrations in tumor samples. Computational analysis is needed (1) to deduce from raw array data actual amplification or deletion events for chromosomal fragments and (2) to distinguish causal chromosomal alterations from functionally neutral ones. We present a comprehensive computational approach, RAE, designed to robustly map chromosomal alterations in tumor samples and assess their functional importance in cancer. To demonstrate the methodology, we experimentally profile copy number changes in a clinically aggressive subtype of soft-tissue sarcoma, pleomorphic liposarcoma, and computationally derive a portrait of candidate oncogenic alterations and their target genes. Many affected genes are known to be involved in sarcomagenesis; others are novel, including mediators of adipocyte differentiation, and may include valuable therapeutic targets. Taken together, we present a statistically robust methodology applicable to high-resolution genomic data to assess the extent and function of copy-number alterations in cancer.     

Fragile and unstable chromosomes in cancer: causes and consequences

Cancer cells commonly exhibit various forms of genetic instability, such as changes in chromosome copy number, translocations and point mutations in particular genes. Although transmissible change seems to be an essential part of the neoplastic process, the extent to which DNA instability is a cause rather than a consequence of cancer is unclear. Chromosomal fragile sites have been proposed to be not only susceptible to DNA instability in cancer cells, but also associated with genes that contribute to the neoplastic process. Mutation at fragile site loci might therefore have a causative role in cancer. Recent studies on one class of human chromosomal fragile sites show that instability at fragile site loci can functionally contribute to tumor cell biology.     

S1 nuclease hypersensitive sites in an oligopurine/oligopyrimidine DNA from the t(10;14) breakpoint cluster region.

Recurring chromosomal translocations are frequently seen in cancers, especially in leukemias and lymphomas. The genes affected by these chromosomal translocations appear to play an important role in oncogenesis. The mechanism underlying the formation of chromosomal translocation is a subject under extensive study. In chromosomal translocations involving the Ig and TCR loci, complete heptamer-spacer-nonamer signal motifs are usually present at the break of the Ig and TCR genes, indicating the involvement of V-D-J recombinase(s). On the other hand, in only about 50% of the cases signal motif sequences have been found at the break in the other participating chromosome, suggesting that different mechanisms may be involved in the scission of the corresponding chromosome. Here we report the identification of an oligopurine/oligopyrimidine DNA in the t(10;14) breakpoint cluster region associated with T-cell acute lymphoblastic leukemia. S1 nuclease mapping revealed multiple S1 hypersensitive sites in the oligopurine/oligopyrimidine DNA. These data suggest a role for oligopurine/oligopyrimidine sequences (non-B DNA) in the formation of chromosomal translocation.     

Circulating nucleic acids (CNAs) and cancer—A survey

It has been known for decades that it is possible to detect small amounts of extracellular nucleic acids in plasma and serum of healthy and diseased human beings. The unequivocal proof that part of these circulating nucleic acids (CNAs) is of tumor origin, initiated a surge of studies which confirmed and extended the original observations. In the past few years many experiments showed that tumor-associated alterations can be detected at the DNA and RNA level. At the DNA level the detection of point mutations, microsatellite alterations, chromosomal alterations, i.e. inversion and deletion, and hypermethylation of promoter sequences were demonstrated. At the RNA level the overexpression of tumor-associated genes was shown. These observations laid the foundation for the development of assays for an early detection of cancer as well as for other clinical means.     

Circulating nucleic acids (CNAs) and cancer--a survey

It has been known for decades that it is possible to detect small amounts of extracellular nucleic acids in plasma and serum of healthy and diseased human beings. The unequivocal proof that part of these circulating nucleic acids (CNAs) is of tumor origin, initiated a surge of studies which confirmed and extended the original observations. In the past few years many experiments showed that tumor-associated alterations can be detected at the DNA and RNA level. At the DNA level the detection of point mutations, microsatellite alterations, chromosomal alterations, i.e. inversion and deletion, and hypermethylation of promoter sequences were demonstrated. At the RNA level the overexpression of tumor-associated genes was shown. These observations laid the foundation for the development of assays for an early detection of cancer as well as for other clinical means.     

Dissecting DNA hypermethylation in cancer

There is compelling evidence to support the importance of DNA methylation alterations in cancer development. Both losses and gains of DNA methylation are observed, thought to contribute pathophysiologically by inactivating tumor suppressor genes, inducing chromosomal instability and ectopically activating gene expression. Lesser known are the causes of aberrant DNA methylation. Recent studies have pointed out that intrinsic gene susceptibility to DNA methylation, environmental factors and gene function all have an intertwined participation in this process. Overall, these data support a deterministic rather than a stochastic mechanism for de novo DNA methylation in cancer. In this review article, we discuss the technologies available to study DNA methylation and the endogenous and exogenous factors that influence the onset of de novo methylation in cancer.     

Maternal gametic transmission of translocations or inversions of human chromosome 11p15.5 results in regional DNA hypermethylation and downregulation of CDKN1C expression

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with genetic or epigenetic alterations in one of two imprinted domains on chromosome 11p15.5. Rarely, chromosomal translocations or inversions of chromosome 11p15.5 are associated with BWS but the molecular pathophysiology in such cases is not understood. In our series of 3 translocation and 2 inversion patients with BWS, the chromosome 11p15.5 breakpoints map within the centromeric imprinted domain, 2. We hypothesized that either microdeletions/microduplications adjacent to the breakpoints could disrupt genomic sequences important for imprinted gene regulation. An alternate hypothesis was that epigenetic alterations of as yet unknown regulatory DNA sequences, result in the BWS phenotype. A high resolution Nimblegen custom microarray was designed representing all non-repetitive sequences in the telomeric 33 Mb of the short arm of human chromosome 11. For the BWS-associated chromosome 11p15.5 translocations and inversions, we found no evidence of microdeletions/microduplications. DNA methylation was also tested on this microarray using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. This high-resolution DNA methylation microarray analysis revealed a gain of DNA methylation in the translocation/inversion patients affecting the p-ter segment of chromosome 11p15, including both imprinted domains. BWS patients that inherited a maternal translocation or inversion also demonstrated reduced expression of the growth suppressing imprinted gene, CDKN1C in Domain 2. In summary, our data demonstrate that translocations and inversions involving imprinted domain 2 on chromosome 11p15.5, alter regional DNA methylation patterns and imprinted gene expression in cis, suggesting that these epigenetic alterations are generated by an alteration in "chromatin context".     

Oncogenic genes and human malignancy.

All vertebrates possess a series of genes which are homologs of the oncogenic genes of acute transforming retroviruses. Two lines of evidence suggest that these genes may play a role in the development of human malignancy: (1) DNA from a variety of human tumors transforms NIH 3T3 mouse fibroblasts and the transforming genes from a number of carcinomas, sarcomas, and hematological malignancies have been identified as members of a family of genes, the ras family, closely related to the oncogenic genes of the Harvey and Kirsten murine sarcoma viruses; and (2) correlations exist between the chromosomal localizations of certain oncogenes and the chromosomal breakpoints in specific translocations and deletions in certain human malignancies. In three separate hematological malignancies, alterations in more than one oncogenic gene may be involved in the neoplastic process.     

Amplification of cellular oncogenes in cancer cells

Regulatory or structural alterations of cellular oncogenes have been implicated in the causation of various cancers. Oncogene alteration by point mutations can result in a protein product with strongly enhanced oncogenic potential. Aberrant expression of cellular oncogenes may be due to tumor-specific chromosomal translocations that dysregulate the normal functions of a proto-oncogene. Amplification of cellular oncogenes can also augment their expression by increasing the amount of DNA template available for the production of mRNA. It appears that amplification of certain oncogenes is a common correlate of the progression of some tumours and also occurs as a rare sporadic event affecting various oncogenes in different types of cancer. Amplified copies of oncogenes may or may not be associated with chromosomal abnormalities signifying DNA amplification: double minute chromosomes and homogeneously staining chromosomal regions. Amplified oncogenes, whether sporadic or tumour type-specific, are expressed at elevated levels, in some cases in cells where their diploid forms are normally silent. Increased dosage of an amplified oncogene may contribute to the multistep progression of at least some cancers.