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Abstract

The CDKN2A tumor-suppressor locus on chromosome band 9p21, which encodes p16(INK4A), a negative regulator of cyclin-dependent kinases, and p14(ARF1), an activator of TP53, is inactivated in many human cancers by point mutation, promoter hypermethylation, and, often, deletion. Homozygous deletions are unusually prevalent at this locus in very different human cancers. In the present study, we compared deletions in squamous cell carcinoma of the head and neck (SCCHN) cell lines to those in T-cell acute lymphatic leukemia (T-ALL), glioma, and bladder carcinoma (TCC) cell lines. Of 14 SCCHN lines, 10 showed homozygous deletions of CDKN2A, one displayed promoter hypermethylation with gene silencing, and one had a frameshift deletion in exon 2. Many deletion ends were in or proximal to the repetitive sequence clusters flanking the locus. Breakpoint junctions displayed variable microhomologies or insertions characteristic of DNA repair by nonhomologous end-joining. In general, deletions were much smaller in SCCHN than in TCC and glioma. In T-ALL, breakpoints were near consensus sites for recombination mediated by RAG (recombination activating genes) enzymes, and the structure of the junctions was consistent with this mechanism. We suggest that different mechanisms of CDKN2A deletion prevail in different human cancers. Aberrant RAG-mediated recombination may be responsible in T-ALL, and exuberant DNA repair by nonhomologous end-joining is the likely prevailing mechanism in SCCHN, but a distinct mechanism in TCC and glioma remains to be elucidated.

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... Array-based genome-wide profiling studies have shown copy number aberrations (CNAs) to be common, mostly comprising deletions and affecting genes involved in B-lymphocyte development and differentiation 5 , such as CDKN2A, PAX5, BTG1, TBL1XR1, RAG1, RAG2 and the wild-type copy of ETV6. The presence of V(D)J recombination sequence motifs close to these CNAs has suggested a role for aberrant RAG endonuclease targeting at these loci [6][7][8][9][10] , but these studies have been limited to the analysis of a small number of annotated breakpoints at specific genes. ...
... RAG endonucleases bind DNA at RSS motifs and cleave DNA at the boundary between the RSS and the flanking coding sequence, thereby generating two blunt and two hairpin ends that are held in close proximity to each other by the RAG complex 13 . Processing of these ends often involves the addition of non-templated sequence (NTS) at the breakpoint by terminal deoxynucleotidyl transferase (TdT) in a process that results in further diversification of the V(D)J locus 14 Clustering of deletion breakpoints adjacent to RSS sites or motifs approximating the conserved RSS DNA sequence 17 in lymphoid genes has provided some evidence of off-target RAG activity in leukemias [6][7][8][9][10]18 . ...
... Within each locus, deletions that did not satisfy our criteria for annotation as having RSS-like sequences were observed to cluster with structural variations that did have a nearby RSS motif ( Fig. 3d and Supplementary Table 12). Not unexpectedly, the genes disrupted in these clustered and reiterated deletions are among the most frequently targeted in ALL, including CDKN2A, BTG1, TBL1XR1, RAG1, RAG2 and BTLA [8][9][10]19,29 . ...
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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.
... Not only in this particular situation DNA replication patterns could be disturbed, with normally late-replicating DNA shifting towards earlier periods within S-phase. Barrier functions of repeat DNA in the genome could be alleviated, allowing DNA processing during repair and Holiday junctions formed during recombination to pass through stretches of DNA that are less accessible in normal cells [36]. ...
... In a similar fashion, hypomethylation of retroelement sequences dispersed in the genome could facilitate illegitimate recombination. In favor of this idea, L1 sequences are enriched at the ends of 3p14.1 and 9p21 deletions in carcino- mas [36, 98, 99] and homozygous deletions arise preferentially in chromosomal regions with high LINE content [100]. It has also been suggested that L1 and HERV sequences are involved in the formation of double-minute circular chromosomes in cancer cells [101, 102]. ...
... While deletion ends are indeed often located in or near L1 sequences and particularly L1 clusters, the breakpoints invariably show hallmarks of DNA double-strand break repair by nonhomologous end-joining (NHEJ). Typically, one end of the deletion is located in or close to an L1 sequence, while the other end is provided by an unrelated single copy or repeat sequence [36, 99, 103]. Such structures also appeared as occasional end products of repair of DNA double-strand breaks induced by a restriction endonuclease at a specific chromosomal site [104] . ...
Article
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Retrotransposons like L1 are silenced in somatic cells by a variety of mechanisms acting at different levels. Protective mechanisms include DNA methylation and packaging into inactive chromatin to suppress transcription and prevent recombination, potentially supported by cytidine deaminase editing of RNA. Furthermore, DNA strand breaks arising during attempted retrotranspositions ought to activate cellular checkpoints, and L1 activation outside immunoprivileged sites may elicit immune responses. A number of observations indicate that L1 sequences nevertheless become reactivated in human cancer. Prominently, methylation of L1 sequences is diminished in many cancer types and full-length L1 RNAs become detectable, although strong expression is restricted to germ cell cancers. L1 elements have been found to be enriched at sites of illegitimate recombination in many cancers. In theory, lack of L1 repression in cancer might cause transcriptional deregulation, insertional mutations, DNA breaks, and an increased frequency of recombinations, contributing to genome disorganization, expression changes, and chromosomal instability. There is however little evidence that such effects occur at a gross scale in human cancers. Rather, as a rule, L1 repression is only partly alleviated. Unfortunately, many techniques commonly used to investigate genetic and epigenetic alterations in cancer cells are not well suited to detect subtle effects elicited by partial reactivation of retroelements like L1 which are present as abundant, but heterogeneous copies. Therefore, effects of L1 sequences exerted on the local chromatin structure, on the transcriptional regulation of individual genes, and on chromosome fragility need to be more closely investigated in normal and cancer cells.
... In previous studies, breakpoints for interstitial 9p21 deletions in lymphoid leukemia were found clustered at a few defined sites (i.e., BCSs) that have nucleotide sequences similar to recombination signal sequences (RSSs) for V(D)J recombination (Cayuela et al., 1997;Kitagawa et al., 2002;Raschke et al., 2005). The results indicated that DSBs were illegitimately produced by the RAG complex. ...
... Therefore, the chromatin for the BCSs was thought to take a configuration(s) making them susceptible to digestion by DNA endonucleases such as restriction enzymes and RAG proteins. Breakpoints for 9p21 deletions in nonhematological malignancies were not mapped at specific DNA sequences (Florl and Schulz, 2003;Sasaki et al., 2003;Raschke et al., 2005). Therefore, genomic features enhancing DSBs were unclear. ...
Article
A restriction enzyme, MspI, was introduced into cultured human cells as a probe to detect genomic regions susceptible to DNA double-strand breaks (DSBs). A 2 h exposure to MspI at a concentration of 8 U/mul produced DSBs at MspI sites in more than 80% of HeLa cells. The sensitivity to digestion was examined on chromosomal DNAs for the region containing the p16 tumor suppressor gene and two other related genes, p14ARF and p15, by Southern blot hybridization analysis and linker-mediated capture of DNA fragments digested in vivo. DNAs for the promoter regions of the three genes, respectively, were sensitive to MspI digestion in HeLa cells, while DNA for the p16 promoter region was less sensitive in lung cancer cells with hypermethylation of the region. Breakpoints for interstitial 9p21 deletions removing the p16/p14ARF/p15 locus in a variety of human cancers were significantly over-represented in the three sensitive regions. The results suggest that the MspI sensitivity in vivo of each genomic region reflects its susceptibility to DSBs that trigger chromosome aberrations in human cells. This method could help us understand the pathogenic significance of differential susceptibility to DSBs among genomic regions in human carcinogenesis.
... Furthermore, the single major product of the PCR reactions was resolved by agarose gel electrophoresis, excised, extracted and sequenced ( Figure 5B). The breakpoint cloned is in agreement with two other reports ( Figure 5B) [37,38]. ...
... Our approach can also ease traditional labor-intensive experiments that aim to understand how genomic breakpoints are generated during cancer development, particularly in primary tumors. Although illegitimate V(D)J recombination may be responsible for creating CDKN2A deletions in acute lymphoblastic leukemia, more breakpoint sequence data will be needed for other types of cancers to delineate the molecular mechanisms [37,38,47,48]. Furthermore, the technique described in this paper can be used not only for deletion mapping, but it can also be applied to map other types of genomic rearrangement, such as translocations and inversions. ...
Article
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CDKN2A (encodes p16(INK4A) and p14(ARF)) deletion, which results in both Rb and p53 inactivation, is the most common chromosomal anomaly in human cancers. To precisely map the deletion breakpoints is important to understanding the molecular mechanism of genomic rearrangement and may also be useful for clinical applications. However, current methods for determining the breakpoint are either of low resolution or require the isolation of relatively pure cancer cells, which can be difficult for clinical samples that are typically contaminated with various amounts of normal host cells. To overcome this hurdle, we have developed a novel approach, designated Primer Approximation Multiplex PCR (PAMP), for enriching breakpoint sequences followed by genomic tiling array hybridization to locate the breakpoints. In a series of proof-of-concept experiments, we were able to identify cancer-derived CDKN2A genomic breakpoints when more than 99.9% of wild type genome was present in a model system. This design can be scaled up with bioinformatics support and can be applied to validate other candidate cancer-associated loci that are revealed by other more systemic but lower throughput assays.
... Taken together, these alterations result not only in proliferative advantages but also in increased susceptibility to the accumulation of additional genetic alterations that contribute to tumor progression and acquisition of more aggressive phenotypes. In almost all tumors (1)(2)(3)(4)(5), these cell-cycle defects are mediated by the inactivation of a region located in humans at chromosome arm 9p21. ...
Article
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The 9p21 locus, encoding three important tumor suppressors (p16/CDKN2A, p14/ARF, and p15/CDKN2B), is a major target of inactivation in the pathogenesis of many human tumors. To explore, at high resolution, the frequency and size of alterations affecting this locus in adult BCR-ABL1-positive acute lymphoblastic leukemia (ALL) and to investigate their prognostic value, 112 patients (101 de novo and 11 relapsed cases) were analyzed by genome-wide single-nucleotide polymorphism arrays and gene candidate deep exon sequencing. Paired diagnosis-relapse samples were further available and analyzed for 19 (19%) cases. CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 25% of newly diagnosed patients, respectively. Deletions were monoallelic in 72% of cases, and in 43% of them, the minimal overlapping region of the lost area spanned only the CDKN2A/B gene locus. An analysis conducted at relapse showed an increase in the detection rate of CDKN2A/ARF loss (47%) compared with the time of diagnosis (P = 0.06). Point mutations within the 9p21 locus were found at very low levels, with only a nonsynonymous substitution in the exon 2 of CDKN2A. Of note, deletions of CDKN2A/B were significantly associated with poor outcomes in terms of overall survival (P = 0.0206), disease free-survival (P = 0.0010), and cumulative incidence of relapse (P = 0.0014). Inactivation of the 9p21 locus by genomic deletion is a frequent event in BCR-ABL1-positive ALL. Deletions are frequently acquired during leukemia progression and are a poor prognostic marker of long-term outcomes.
... Interestingly, deletions ends were indeed preferentially located in or close to LINE-1 sequences, in support of the above hypotheses (Florl and Schulz 2003). Subsequent studies on the same type of deletions in squamous cell carcinomas of the head and neck yielded a different result (Raschke et al. 2005), i.e., deletion ends were preferentially found in proximate clusters of repetitive sequences (LTR and LINEs) flanking the CDKN2A locus. This finding suggests an unexpected mechanism by which hypomethylation of repeat sequences could affect genomic stability. ...
Article
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While specific genes are hypermethylated in the genome of cancer cells, overall methylcytosine content is often decreased as a consequence of hypomethylation affecting many repetitive sequences. Hypomethylation is also observed at a number of single-copy genes. While global hypomethylation is highly prevalent across all cancer types, it often displays considerable specificity with regard to tumor type, tumor stage, and sequences affected. Following an overview of hypomethylation alterations in various cancers, this review focuses on 3 hypotheses. First, hypomethylation at a single-copy gene may occur as a 2-step process, in which selection for gene function follows upon random hypo methylation. In this fashion, hypomethylation facilitates the adaptation of cancer cells to the ever-changing tumor tissue microenvironment, particularly during metastasis. Second, the development of global hypomethylation is intimately linked to chromatin restructuring and nuclear disorganization in cancer cells, reflected in a large number of changes in histone-modifying enzymes and other chromatin regulators. Third, DNA hypomethylation may occur at least partly as a consequence of cell cycle deregulation disturbing the coordination between DNA replication and activity of DNA methyltransferases. Finally, because of their relation to tumor progression and metastasis, DNA hypomethylation markers may be particularly useful to classify cancer and predict their clinical course.
... A similar deletion was found in some human squamous cell carcinomas of the head and neck. 33 It seems likely that a certain site where the genome is liable to be lost in the p16ink4a gene, and this may be associated with malignant transformation of some kinds of tumors. Generally, inactivation of p16ink4a is caused by homozygous deletions, methylations of the promoter or point mutations, and the frequencies and patterns of p16ink4a inactivation are different depending on the origins of tumors. ...
Article
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Transgenic rats expressing the pX gene of human T lymphocyte virus type-I (HTLV-I) under control of the rat lymphocyte-specific protein tyrosine kinase type-I promoter (lck-pX rats) developed benign epithelial thymomas. When the thymuses of newborn lck-pX rats were transplanted into the subcapsular space of the kidney in other thymectomized lck-pX rats, similar tumors developed in the transplanted thymuses. Following the tumor growth, dissemination in the abdominal cavity and distant metastasis occurred. The tumors were histopathologically similar to the original thymomas, but prominent nuclear atypia and high mitotic activity were present. The Ki-67 index was twice as high as that in the originals. The tumors were transplantable into the subcutis of lck-pX rats, although transplantation of the originals never succeeded. All evidence indicated that malignant transformation of thymoma was induced by the heterotopic transplantation. Expression of the pX transgene in the transformed tumors were significantly reduced. Among host genes, the expression of p16ink4a/ARF, which was significantly upregulated in the originals, was never detected in the transformed tumors. Genomic Southern blots and PCR suggest that homozygous deletion of the p16ink4a/ARF gene may play important roles in malignant transformation in this model. Our model described here is a useful unique model for in vivo malignant transformation.
... Our findings of the p16 INK4a /p14 ARF deletion in MPM seem consistent with other reports that the sizes of homozygous deletions vary individually in any given tissue type of malignancy. (51)(52)(53) Although it is very clear that p16 INK4a /p14 ARF is the most important target TSG at the 9p21.3 region, other genes in this homozygous deletion region should also be studied to determine whether any of them play a role in the development of MPM. ...
Article
Genome-wide array-based comparative genomic hybridization analysis of malignant pleural mesotheliomas (MPM) was carried out to identify regions that display DNA copy number alterations. Seventeen primary tumors and nine cell lines derived from 22 individuals were studied, some of them originating from the same patients. Regions of genomic aberrations observed in >20% of individuals were 1q, 5p, 7p, 8q24 and 20p with gains, and 1p36.33, 1p36.1, 1p21.3, 3p21.3, 4q22, 4q34-qter, 6q25, 9p21.3, 10p, 13q33.2, 14q32.13, 18q and 22q with losses. Two regions at 1p32.1 and 11q22 showed a high copy gain. The 1p32.1 region contained a protooncogene, JUN, and we further demonstrated overexpression of JUN with real-time polymerase chain reaction analysis. As MPM cell lines did not overexpress JUN, our findings suggested that induction of JUN expression was involved in the development of MPM cells in vivo, which also might result in gene amplification in a subset of MPM. Meanwhile, the most frequent alteration was the 9p21.3 deletion, which includes the p16(INK4a)/p14(ARF) locus. With polymerase chain reaction analysis, we determined the extent of the homozygous deletion regions of the p16(INK4a)/p14(ARF) locus in MPM cell lines, which indicated that the deletion regions varied among cell lines. Our results with array comparative genomic hybridization analysis provide new insights into the genetic background of MPM, and also give some clues to develop a new molecular target therapy for MPM.
... Furthermore, several epithelial cancer cell lines exhibited RAG and immunoglobulin transcription [203]. RAG mediated recombination may further be responsible for deletions at the CDKN2A (p16 INK4a ) tumor suppressor locus in T-cell acute lymphatic leukemia (T-ALL) cases [204]. The expression of AID, RAG and immunoglobulin transcripts and immunoglobulin proteins was recently reported in a set of diverse tumor cell lines and 66 different epithelial tissues of increasing malignancy [205]. ...
Article
It is well documented that viral genomes either inserted into the cellular DNA or co-replicating with it in episomal form can be lost from neoplastic cells. Therefore, "hit and run"-mechanisms have been a topic of longstanding interest in tumor virology. The basic idea is that the transient acquisition of a complete or incomplete viral genome may be sufficient to induce malignant conversion of host cells in vivo, resulting in neoplastic development. After eliciting a heritable change in the gene expression pattern of the host cell (initiation), the genomes of tumor viruses may be completely lost, i.e. in a hit and run-scenario they are not necessary for the maintenance of the malignant state. The expression of viral oncoproteins and RNAs may interfere not only with regulators of cell proliferation, but also with DNA repair mechanisms. DNA recombinogenic activities induced by tumor viruses or activated by other mechanisms may contribute to the secondary loss of viral genomes from neoplastic cells. Viral oncoproteins can also cause epigenetic dysregulation, thereby reprogramming cellular gene expression in a heritable manner. Thus, we expect that epigenetic scenarios of viral hit and run-tumorigenesis may facilitate new, innovative experiments and clinical studies in spite of the fact that the regular presence of a suspected human tumor virus in an early phase of neoplastic development and its subsequent regular loss have not been demonstrated yet. We propose that virus-specific "epigenetic signatures", i.e. alterations of the host cell epigenome, especially altered DNA methylation patterns, may help to identify viral hit and run-oncogenic events, even after the complete loss of tumor viruses from neoplastic cells.
... Although sequencing of these breakpoints was not performed, the results would be consistent with a common mechanism for the formation of deletions in this disorder, as has been previously proposed. 6 Three of the smaller deletions involved CDKN2A, but were distal to and did not include CDKN2B. This result is consistent with previous reports that found CDKN2A deleted in 65% of T-ALL patients, whereas CDKN2B was deleted in only 23% of patients. ...
Article
Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.
... 41 In addition, it has been proposed that certain structural features of the relevant chromosomal region contribute to its fragility (for example, proximity to LINE repeats) in human cells 39 and, interestingly, that different mechanisms of homozygous deletion (including non-homologous end-joining and less well-characterized variant mechanisms) predominate in different types of cancer. 42,43 If confirmed in human cell transformation models (for example, in 'stasis' bypass in normal early-passage human mammary epithelial cells), 44 our results using the SHD system may well have important implications for understanding the mechanisms of human radiation carcinogenesis, and risks inherent in IR exposure. ...
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Immortalization (senescence bypass) is a critical rate-limiting step in the malignant transformation of mammalian somatic cells. Human cells must breach at least two distinct senescence barriers to permit unfettered clonal evolution during cancer development: (1) stress- or oncogene-induced premature senescence (SIPS/OIS), mediated via the p16-Rb and/or ARF-p53-p21 tumour-suppressive pathways, and (2) replicative senescence triggered by telomere shortening. In contrast, because their telomerase is constitutively active, cells from small rodents possess only the SIPS/OIS barrier, and are therefore useful for studying SIPS/OIS bypass in isolation. Dermal fibroblasts from the Syrian hamster (SHD cells) are exceptionally resistant to spontaneous SIPS bypass, but it can be readily induced following exposure to a wide range of chemical and physical carcinogens. Here we show that a spectrum of carcinogen-specific mutational and epigenetic alterations involving the INK4A (p16), p53 and INK4B (p15) genes are associated with induced SIPS bypass. With ionizing radiation, immortalization is invariably accompanied by efficient biallelic deletion of the complete INK4/CDKN2 locus. In comparison, SHD cells immortalized by the powerful polycyclic hydrocarbon carcinogen benzo(a)pyrene display transversion point mutations in the DNA-binding domain of p53 coupled with INK4 alterations such as loss of expression of p15. Epimutational silencing of p16 is the primary event associated with immortalization by nickel, a human non-genotoxic carcinogen. As SIPS/OIS bypass is a prerequisite for the immortalization of normal diploid human epithelial cells, our results with the SHD model will provide a basis for delineating combinations of key molecular changes underpinning this important event in human carcinogenesis.Oncogene advance online publication, 12 March 2012; doi:10.1038/onc.2012.45.
... We found, for example, that deletions in ETV6, EBF1 and RB1 cooccur with BTG1 deletions, indicating that loss of normal BTG1 function may add to defects in pRb/E2F-mediated cell cycle and EBF1-mediated B-cell differentiation pathways. Illegitimate RAGmediated recombination has been suggested as the responsible mechanism for IKZF1 [19,20], CDKN2A/B [33,34] and LMO2 [35] deletions, as well as ETV6-RUNX1 translocations [30]. It remains to be established whether RAG-mediated recombination, or other mechanisms like CSR, are implicated in the occurrence of RB1 and EBF1 deletions. ...
Article
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Recurrent submicroscopic deletions in genes affecting key cellular pathways are a hallmark of pediatric acute lymphoblastic leukemia (ALL). To gain more insight into the mechanism underlying these deletions, we have studied the occurrence and nature of abnormalities in one of these genes, the B-cell translocation gene 1 (BTG1), in a large cohort of pediatric ALL cases. BTG1 was found to be exclusively affected by genomic deletions, which were detected in 65 out of 722 B-cell precursor ALL (BCP-ALL) patient samples (9%), but not in 109 T-ALL cases. Eight different deletion sizes were identified, which all clustered at the telomeric site in a hotspot region within the second (and last) exon of the BTG1 gene, resulting in the expression of truncated BTG1 read-through transcripts. The presence of V(D)J recombination signal sequences at both sites of virtually all deletions strongly suggests illegitimate RAG1/RAG2-mediated recombination as the responsible mechanism. Moreover, high levels of histone H3 lysine 4 trimethylation (H3K4me3), which is known to tether the RAG enzyme complex to DNA, were found within the BTG1 gene body in BCP-ALL cells, but not T-ALL cells. BTG1 deletions were rarely found in hyperdiploid BCP-ALLs, but were predominant in other cytogenetic subgroups, including the ETV6-RUNX1 and BCR-ABL1 positive BCP-ALL subgroups. Through sensitive PCR-based screening, we identified multiple additional BTG1 deletions at the subclonal level in BCP-ALL, with equal cytogenetic distribution which, in some cases, grew out into the major clone at relapse. Taken together, our results indicate that BTG1 deletions may act as "drivers" of leukemogenesis in specific BCP-ALL subgroups, in which they can arise independently in multiple subclones at sites that are prone to aberrant RAG1/RAG2-mediated recombination events. These findings provide further evidence for a complex and multiclonal evolution of ALL.
... This latter result suggests that deletions affecting these two groups arise by different mechanisms. Deletions in common fragile site genes can be induced by replicative stress and subsequent DNA damage and repair [22], deletions affecting CDKN2A/B have been suggested to arise from aberrant recombination or DNA repair by nonhomologous end-joining [23]. In support of the idea that these two types of deletions arise by separate mechanisms, we found that the frequency of co-occurrence of deletions in different common fragile site genes and co-occurrence of deletions in different tumor suppressor genes was greater than that of co-deletion of CFS and tumor suppressor genes (Figure 4C). ...
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One of the key questions about genomic alterations in cancer is whether they are functional in the sense of contributing to the selective advantage of tumor cells. The frequency with which an alteration occurs might reflect its ability to increase cancer cell growth, or alternatively, enhanced instability of a locus may increase the frequency with which it is found to be aberrant in tumors, regardless of oncogenic impact. Here we've addressed this on a genome-wide scale for cancer-associated focal deletions, which are known to pinpoint both tumor suppressor genes (tumor suppressors) and unstable loci. Based on DNA copy number analysis of over one-thousand human cancers representing ten different tumor types, we observed five loci with focal deletion frequencies above 5%, including the A2BP1 gene at 16p13.3 and the MACROD2 gene at 20p12.1. However, neither RNA expression nor functional studies support a tumor suppressor role for either gene. Further analyses suggest instead that these are sites of increased genomic instability and that they resemble common fragile sites (CFS). Genome-wide analysis revealed properties of CFS-like recurrent deletions that distinguish them from deletions affecting tumor suppressor genes, including their isolation at specific loci away from other genomic deletion sites, a considerably smaller deletion size, and dispersal throughout the affected locus rather than assembly at a common site of overlap. Additionally, CFS-like deletions have less impact on gene expression and are enriched in cell lines compared to primary tumors. We show that loci affected by CFS-like deletions are often distinct from known common fragile sites. Indeed, we find that each tumor tissue type has its own spectrum of CFS-like deletions, and that colon cancers have many more CFS-like deletions than other tumor types. We present simple rules that can pinpoint focal deletions that are not CFS-like and more likely to affect functional tumor suppressors.
... However, in many real tumors, further complications exist as these structural variants often do not have exact boundaries. In the CDKN2A region, deletion boundaries often vary over several hundred kilobases and even megabases (Raschke et al., 2005). This type of variation is even observed in deletions/translocations which result in fusion proteins; the TMPRSS2 and ETS family fusion in prostate cancer not only lacks specificity in the genes it hits (ERG and ETV1/4), but also as to which exons are joined together (Tomlins et al., 2005). ...
Article
Primer approximation multiplex PCR (PAMP) is a new experimental protocol for efficiently assaying structural variation in genomes. PAMP is particularly suited to cancer genomes where the precise breakpoints of alterations such as deletions or translocations vary between patients. The design of PCR primer sets for PAMP is challenging because a large number of primer pairs are required to detect alterations in the hundreds of kilobases range that can occur in cancer. These sets of primers must achieve high coverage of the region of interest, while avoiding primer dimers and satisfying the physico-chemical constraints of good PCR primers. We describe a natural formulation of these constraints as a combinatorial optimization problem. We show that the PAMP primer design problem is NP-hard, and design algorithms based on simulated annealing and integer programming, that provide good solutions to this problem in practice. The algorithms are applied to a test region around the known CDKN2A deletion, which show excellent results even in a 1:49 mixture of mutated:wild-type cells. We use these test results to help set design parameters for larger problems. We can achieve near-optimal designs for regions close to 1 Mb. Contact:abashir@ucsd.edu Supplementary information: Supplementary data are available at Bioinformatics online.
... Deletions at this chromosomal region occur frequently in ovarian cancers 29 and in a number of other tumour types 17 likely reflecting the involvement of additional genes or the presence of alternative mutation mechanisms. 30 Indeed, while a role for p16 inactivation has been suggested in the pathogenesis of ovarian cancers and a correlation as also been established as a prognostic parameter, 31 whether CDKN2A/B loci are the only target of the chromosomal deletions remains to be determined. Using MLPA, chromosome 9p specific probes to further investigate the deleted alleles we demonstrated the homozygous loss of a several megabase-region containing a number of additional genes. ...
Article
The BRCA1 gene is responsible for a high number of hereditary breast and ovarian cancers that cluster in families with a strong genetic predisposition. Despite intense investigation, the accumulating findings on BRCA1 biological functions have not yet been translated into specific therapeutic approaches, also due to the lack of suitable experimental models. The purpose of this study was to establish and characterize cell cultures and xenografts from patients with BRCA1 -/- ovarian cancers. We derived two ovarian cancer cell lines, termed PD-OVCA1 and PD-OVCA2, both from patients previously treated with chemotherapy, that propagate in SCID mice as well as in vitro for a limited number of passages. Both cell lines expressed cytokeratins and the CA125 tumour marker. A detailed molecular characterization highlighted both constitutive and somatic genetic events that abrogate BRCA1 gene function. Both cell lines were shown to lose the wild type BRCA1 allele; intriguingly, these deletions were apparently accompanied by gain of one or more copies of the mutant alleles. Finally, a genomic profile of major chromosomal aberrations was obtained by the Multiplex Ligation-dependent Probe Amplification (MLPA) technique, which disclosed chromosomal imbalances targeting specific genes in each cell line. The PD-OVCA1 and PD-OVCA2 ovarian cancer cell lines will provide a valuable tool for new experimental models for the study of BRCA1-associated tumour biology.
... Previous analyses of CDKN2A deletion breakpoints found that the likely mechanism of repair was tissue-specific. Illegitimate V(D)J recombination was implicated in the majority of CDKN2A deletions in lymphoid leukemia, while evidence of NHEJ and MMEJ were seen in lung and other non-lymphoid cancers (Cayuela et al., 1997;Sasaki et al., 2003;Raschke et al., 2005). While microhomology was present at most breakpoints of both TransFlip mutations and interstitial deletions, the significance of the few bases involved seems insufficient to explain the mechanism, given the expected microhomology length of 1.35 bases genome-wide by chance alone. ...
Article
Pancreatic ductal adenocarcinoma (PDAC) is driven by the inactivation of the tumor suppressor genes (TSGs), CDKN2A (P16) and SMAD4 (DPC4), commonly by homozygous deletions (HDs). Using a combination of high density single-nucleotide polymorphism (SNP) microarray and whole genome sequencing (WGS), we fine-mapped novel breakpoints surrounding deletions of CDKN2A and SMAD4 and characterized them by their underlying structural variants (SVs). Only one third of CDKN2A and SMAD4 deletions (6 of 18) were simple interstitial deletions, rather, the majority of deletions were caused by complex rearrangements, specifically, a translocation on one side of the TSG in combination with an inversion on the other side. We designate these as "TransFlip" mutations. Characteristics of TransFlip mutations are: (1) a propensity to target the TSGs CDKN2A and SMAD4 (P < 0.005), (2) not present in the germline of the examined samples, (3) non-recurrent breakpoints, (4) relatively small (47 bp to 3.4 kb) inversions, (5) inversions can be either telomeric or centromeric to the TSG, and (6) non-reciprocal, and non-recurrent translocations. TransFlip mutations are novel complex genomic rearrangements with unique breakpoint signatures in pancreatic cancer. We hypothesize that they are a common but poorly understood mechanism of TSG inactivation in human cancer. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.
... We found, for example, that deletions in ETV6, EBF1 and RB1 cooccur with BTG1 deletions, indicating that loss of normal BTG1 function may add to defects in pRb/E2F-mediated cell cycle and EBF1-mediated B-cell differentiation pathways. Illegitimate RAGmediated recombination has been suggested as the responsible mechanism for IKZF1 [19,20], CDKN2A/B [33,34] and LMO2 [35] deletions, as well as ETV6-RUNX1 translocations [30]. It remains to be established whether RAG-mediated recombination, or other mechanisms like CSR, are implicated in the occurrence of RB1 and EBF1 deletions. ...
... In some cases of NTRK1 and NTRK3 fused tumors, an additional secondary cyclindependent kinase inhibitor 2A (CDKN2A) deletion has been described [16,17,28,31,43]. The CDKN2A tumor-suppressor locus on chromosome band 9p21 can be inactivated, and this locus is known to be prone to homozygous deletions in a wide range of human cancers [44]. The significance of this finding is unclear. ...
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Tropomyosin receptor kinase (TK) is encoded by the neurotrophic tyrosine receptor kinase genes (NTRK) 1, 2, and 3, whose activation plays an important role in cell cycle proliferation and survival. Fusions of one of these genes can lead to constitutive activation of TRK, which can potentially be oncogenic. NTRK fusions are commonly present in rare histologic tumor types. Among sarcomas, infantile fibrosarcoma shows NTRK fusion in more than 90% of the cases. Many other sarcoma types are also investigated for NTRK fusions. These fusions are druggable alteration of the agnostic type, meaning that all NTRK fused tumors can be treated with NTRK-inhibitors regardless of tumor type or tissue of origin. TRK-inhibitors have shown good response rates, with durable effects and limited side effects. Resistance to therapy will eventually occur in some cases, wherefore the next-generation TRK-inhibitors are introduced. The diagnosis of NTRK fused tumors, among them sarcomas, remains an issue, as many algorithms but no guidelines exist to date. Given the importance of this diagnosis, in this paper we aim to (1) analyze the histopathological features of sarcomas that correlate more often with NTRK fusions, (2) give an overview of the TRK-inhibitors and the problems that arise from resistance to the therapy, and (3) discuss the diagnostic algorithms of NTRK fused tumors with emphasis on sarcomas.
... The breakpoints of these deletions often localize in the recombination signal sequence (RSS) that is recognized by the RAG. The structure of the junctions is compatible with typical RAG double-strand DNA breaks [12][13][14][15][16]. However, the RSS sequences have not been found in all the breakpoints studied, raising the possibility that, in a small subset of lymphoid leukemias, the 9p21.3 ...
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Acute lymphoblastic leukemia (ALL) is a malignant clonal expansion of lymphoid hematopoietic precursors that exhibit developmental arrest at varying stages of differentiation. Similar to what occurs in solid cancers, transformation of normal hematopoietic precursors is governed by a multistep oncogenic process that drives initiation, clonal expansion and metastasis. In this process, alterations in genes encoding proteins that govern processes such as cell proliferation, differentiation, and growth provide us with some of the clearest mechanistic insights into how and why cancer arises. In such a scenario, deletions in the 9p21.3 cluster involving CDKN2A/ARF/CDKN2B genes arise as one of the oncogenic hallmarks of ALL. Deletions in this region are the most frequent structural alteration in T-cell acute lymphoblastic leukemia (T-ALL) and account for roughly 30% of copy number alterations found in B-cell-precursor acute lymphoblastic leukemia (BCP-ALL). Here, we review the literature concerning the involvement of the CDKN2A/B genes as a prognosis marker of good or bad response in the two ALL subtypes (BCP-ALL and T-ALL). We compare frequencies observed in studies performed on several ALL cohorts (adult and child), which mainly consider genetic data produced by genomic techniques. We also summarize what we have learned from mouse models designed to evaluate the functional involvement of the gene cluster in ALL development and in relapse/resistance to treatment. Finally, we examine the range of possibilities for targeting the abnormal function of the protein-coding genes of this cluster and their potential to act as anti-leukemic agents in patients.
... The breakpoints of these deletions often localize in the recombination signal sequence (RSS) that is recognized by the RAG. The structure of the junctions is compatible with typical RAG double-strand DNA breaks [12][13][14][15][16]. However, the RSS sequences have not been found in all the breakpoints studied, raising the possibility that, in a small subset of lymphoid leukemias, the 9p21.3 ...
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Acute lymphoblastic leukemia (ALL) is a malignant clonal expansion of lymphoid hema-topoietic precursors that exhibit developmental arrest at varying stages of differentiation. Similar to what occurs in solid cancers, transformation of normal hematopoietic precursors is governed by a multistep oncogenic process that drives initiation, clonal expansion and metastasis. In this process, alterations in genes encoding proteins that govern processes such as cell proliferation, differentiation , and growth provide us with some of the clearest mechanistic insights into how and why cancer arises. In such a scenario, deletions in the 9p21.3 cluster involving CDKN2A/ARF/CDKN2B genes arise as one of the oncogenic hallmarks of ALL. Deletions in this region are the most frequent structural alteration in T-cell acute lymphoblastic leukemia (T-ALL) and account for roughly 30% of copy number alterations found in B-cell-precursor acute lymphoblastic leukemia (BCP-ALL). Here, we review the literature concerning the involvement of the CDKN2A/B genes as a prognosis marker of good or bad response in the two ALL subtypes (BCP-ALL and TALL). We compare frequencies observed in studies performed on several ALL cohorts (adult and child), which mainly consider genetic data produced by genomic techniques. We also summarize what we have learned from mouse models designed to evaluate the functional involvement of the gene cluster in ALL development and in relapse/resistance to treatment. Finally, we examine the range of possibilities for targeting the abnormal function of the protein-coding genes of this cluster and their potential to act as anti-leukemic agents in patients.
... CDKN2A is a tumor suppressor gene located on chromosome 9p21 that encodes the cell cycle inhibitor protein p16 [1]. Genetic alterations of this gene are frequently observed in various types of human cancers [1][2][3]. With regard to brain tumors and prognosis, homozygous deletion of the CDKN2A gene has been reported to be associated with shortened survival in isocitrate dehydrogenase (IDH)-mutant glioma patients [4][5][6][7][8]. ...
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Background: CDKN2A is a tumor suppressor gene that encodes the cell cycle inhibitor protein p16. Homozygous deletion of the CDKN2A gene has been associated with shortened survival in isocitrate dehydrogenase (IDH)-mutant gliomas. This study aimed to analyze the prognostic value of p16 and to evaluate whether p16 immunohistochemical staining could be used as a prognostic marker to replace CDKN2A genotyping in diffuse gliomas. Materials and methods: p16 immunohistochemistry was performed on tissue microarrays of 326 diffuse gliomas with diagnoses that reflected IDH-mutations and 1p/19q codeletion status. The results were divided into three groups (negative, focal expression, overexpression) according to the presence and degree of p16 expression. Survival analysis was performed to assess the prognostic value of p16 expression. Results: A loss of p16 expression predicted a significantly worse outcome in all glioma patients (n=326, p<.001), in the IDH-mutant glioma patients (n=103, p=.010), and in the IDH-mutant astrocytoma patients (n=73, p=.032). However, loss of p16 expression did not predict the outcome in the IDH-wildtype glioma patients (n=223, p=.121) or in the oligodendroglial tumor patients with the IDH-mutation and 1p/19q codeletion (n=30, p=.457). Multivariate analysis showed the association was still significant in the IDH-mutant glioma patients (p=.008; hazard ratio [HR], 2.637; 95% confidence interval [CI], 1.295 to 5.372) and in the IDH-mutant astrocytoma patients (p=.001; HR, 3.586; 95% CI, 1.649 to 7.801). Interestingly, patients who presented with tumors with p16 overexpression also had shorter survival times than did patients with tumors with p16 focal expression in the whole glioma (p< .001) and in IDH-mutant glioma groups. (p=.046). Conclusion: This study suggests that detection of p16 expression by immunohistochemistry can be used as a useful surrogate test to predict prognosis, especially in IDH-mutant astrocytoma patients.
... Even so, off-target RAG-mediated DNA breaks are reported in lymphoid malignancies, mostly identified by the presence of cryptic RSS target sites. These include deletions of IKZF1 (IKAROS family zinc finger 1), BTG1 (B-cell translocation gene 1), and CDKN2A (cyclin dependent kinase inhibitor 2A) [20][21][22], and fusions such a P2RY8-CRLF2 in acute lymphoblastic leukemia (ALL). Fusions involving antigen receptors have been attributed to RAG and/or AID activity, such as a BCL2-IGH [23], IGH-MYC, and IGH-BCL6 [24] in lymphoma, and IGH-CRLF2 in pediatric ALL [25]. ...
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Blast crisis of chronic myeloid leukemia is associated with poor survival and the accumulation of genomic lesions. Using whole-exome and/or RNA sequencing of patients at chronic phase (CP, n = 49), myeloid blast crisis (MBC, n = 19), and lymphoid blast crisis (LBC, n = 20), we found 25 focal gene deletions and 14 fusions in 24 patients in BC. Deletions predominated in LBC (83% of structural variants). Transcriptional analysis identified the upregulation of genes involved in V(D)J recombination, including RAG1/2 and DNTT in LBC. RAG recombination is a reported mediator of IKZF1 deletion. We investigated the extent of RAG-mediated genomic lesions in BC. Molecular hallmarks of RAG activity; DNTT-mediated nucleotide insertions and a RAG-binding motif at structural variants were exclusively found in patients with high RAG expression. Structural variants in 65% of patients in LBC displayed these hallmarks compared with only 5% in MBC. RAG-mediated events included focal deletion and novel fusion of genes associated with hematologic cancer: IKZF1, RUNX1, CDKN2A/B, and RB1. Importantly, 8/8 patients with elevated DNTT at CP diagnosis progressed to LBC by 12 months, potentially enabling early prediction of LBC. This work confirms the central mutagenic role of RAG in LBC and describes potential clinical utility in CML management.
... P15 and p16 block the cell cycle at the G1/S phase by inhibition of cyclin CDK2/4/6 complexes, which help to maintain the inhibitory function of the Rb, whilst p14 influences cell division at the S/G2 phase by suppressing p53 [8]. The disturbance of the Rb and p53 signaling network through CDKN2A and CDKN2B deletions, mutations, aberrant repressions or epigenetic silencing results in the acquisition of abnormal self-renewing capabilities, proliferation advantage and genomic instability, leading to tumor progression [9]. Deletions followed by promoter methylation are the most common causes of CDKN2A and CDKN2B inactivation in ALL, but neither specific breakpoints for deletions nor any specific methylation profiles have been described [10][11][12][13]. ...
... Remarkably, in some cancers, over half of the chromosomal aberrations that span the CDKN2A locus are large homozygous deletions (HDs) [25][26][27][28]. CDKN2 A HDs have been reported in several cancers (we only name a few) [29][30][31][32][33]66] including a recent pan-cancer distribution of of CDKN2A homozygous deletions. Here, we built on these previous studies and asked whether the breakpoints of these deletions distrubute equally on either side of the CDKN2A locus and attempted to identify a potential reason for the braks. ...
Article
Here we present and describe data on homozygous deletions (HD) of human CDKN2 A and neighboring regions on the p arm of Chromosome 9 from cancer genome sequences deposited on the online Catalogue of Somatic Mutations in Cancer (COSMIC) database. Although CDKN2 A HDs have been previously described in many cancers, this is a pan-cancer report of these aberrations with the aim to map the distribution of the breakpoints. We find that HDs of this locus have a median range of 1,255,650bps. When the deletion breakpoints were mapped on both the telomere and centromere proximal sides of CDKN2A, most of the telomere proximal breakpoints concentrate to a narrow region of the chromosome which includes the gene MTAP. The centromere proximal breakpoints of the deletions are distributed over a wider chromosomal region. Furthermore, gene expression analysis shows that the deletions that include the CDKN2A region also include the MTAP region and this observation is tissue independent. We propose a model that may explain the origin of the telomere proximal CDKN2A breakpoints Finally, we find that HD distributions for at least three other loci, RB1, SMAD4 and PTEN are also not random.
... P15 and p16 block the cell cycle at the G1/S phase by inhibition of cyclin CDK2/4/6 complexes, which help to maintain the inhibitory function of the Rb, whilst p14 influences cell division at the S/G2 phase by suppressing p53 [8]. The disturbance of the Rb and p53 signaling network through CDKN2A and CDKN2B deletions, mutations, aberrant repressions or epigenetic silencing results in the acquisition of abnormal self-renewing capabilities, proliferation advantage and genomic instability, leading to tumor progression [9]. Deletions followed by promoter methylation are the most common causes of CDKN2A and CDKN2B inactivation in ALL, but neither specific breakpoints for deletions nor any specific methylation profiles have been described [10][11][12][13]. ...
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The inactivation of tumor suppressor genes located within 9p21 locus (CDKN2A, CDKN2B) occurs in up to 30% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), but its independent prognostic significance remains controversial. In order to investigate the prognostic impact of deletions and promoter methylation within 9p21, 641 children with newly diagnosed BCP-ALL using methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) were investigated. A total of 169 (26.4%) microdeletions in 9p21 were detected, of which 71 were homozygous. Patients with CDKN2A homozygous deletions were older at diagnosis (p < .001), more frequently steroid resistant (p = .049), had higher WBC count (p < .001), higher MRD at Day 15 (p = .013) and lower relapse-free survival [p = .028, hazard ratio: 2.28 (95% confidence interval: 1.09–4.76)] than patients without these alterations. CDKN2A homozygous deletions coexisted with IKZF1 and PAX5 deletions (p < .001). In conclusion, CDKN2A homozygous deletions, but not promoter methylation, are associated with poor response to treatment and increased relapse risk of pediatric BCP-ALL.
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Copy number variations (CNVs) which include deletions, duplications, inversions, translocations, and other forms of chromosomal re-arrangements are common to human cancers. In this report we investigated the pattern of these variations with the goal of understanding whether there exist specific cancer signatures. We used re-arrangement endpoint data deposited on the Catalogue of Somatic Mutations in Cancers (COSMIC) for our analysis. Indeed, we find that human cancers are characterized by specific patterns of chromosome rearrangements endpoints which in turn result in cancer specific CNVs. A review of the literature reveals tissue specific mutations which either drive these CNVs or appear as a consequence of CNVs because they confer an advantage to the cancer cell. We also identify several rearrangement endpoints hotspots that were not previously reported. Our analysis suggests that in addition to local chromosomal architecture, CNVs are driven by the internal cellular or nuclear physiology of each cancer tissue.
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A feasible method to detect somatic copy number deletion (SCND) of tumor suppressor genes is still absent up to date. To overcome the obstacle of SCND detection, we analyzed genomic coordinates of gene deletion fragments using the Catalogue Of Somatic Mutation In Cancer (COSMIC) datasets. Interstitial base-resolution deletion/fusion coordinates for CDKN2A were extracted from published articles and our whole genome sequencing (WGS) datasets. The results showed that estimated common deletion regions (CDRs) were observed in many tumor suppressor genes, such as ATM, CDKN2A, FAT1, miR31HG, PTEN, and RB1 in the SNP array-based COSMIC datasets. A 5.1-kb base-resolution CDR could be identified in >90% cancer samples by sequencing. Using the true CDKN2A CDR as a PCR target, a multiplex quantitative PCR assay P16-Light was programmed to detect CDKN2A gene copy number with a lower detection limit of 20%. P16-Light was further confirmed with WGS as the gold standard among cancer tissue samples from 139 patients. In conclusion, CDRs are common in many tumor suppressor genes. The 5.1-kb CDKN2A CDR was found in >90% cancers containing CDKN2A deletion. The CDKN2A CDR was used a potential target for developing the P16-Light assay to detect CDKN2A SCND and amplification for routine clinical practices.
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Phosphatase and tensin homolog (PTEN)-inactivating mutations and/or deletions are an independent risk factor for relapse of T-cell acute lymphoblastic leukemia (T-ALL) patients treated on Dutch Childhood Oncology Group or German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia protocols. Some monoallelic mutated or PTEN wild-type patients lack PTEN protein, implying that additional PTEN inactivation mechanisms exist. We show that PTEN is inactivated by small deletions affecting a few exons in 8% of pediatric T-ALL patients. These microdeletions were clonal in 3% and subclonal in 5% of patients. Conserved deletion breakpoints are flanked by cryptic recombination signal sequences (cRSSs) and frequently have non-template-derived nucleotides inserted in between breakpoints, pointing to an illegitimate RAG recombination-driven activity. Identified cRSSs drive RAG-dependent recombination in a reporter system as efficiently as bona fide RSSs that flank gene segments of the T-cell receptor locus. Remarkably, equivalent microdeletions were detected in thymocytes of healthy individuals. Microdeletions strongly associate with the TALLMO subtype characterized by TAL1 or LMO2 rearrangements. Primary and secondary xenotransplantation of TAL1-rearranged leukemia allowed development of leukemic subclones with newly acquired PTEN microdeletions. Ongoing RAG activity may therefore actively contribute to the acquisition of preleukemic hits, clonal diversification, and disease progression.
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The most common pediatric malignancy is acute lymphoblastic leukemia (ALL), of which T-cell ALL (T-ALL) comprises 10-15% of cases. T-ALL arises in the thymus from an immature thymocyte as a consequence of a stepwise accumulation of genetic and epigenetic aberrations. Crucial biological processes such as differentiation, self-renewal capacity, proliferation, and apoptosis are targeted and deranged by several types of neoplasia-associated genetic alteration, for example translocations, deletions, and mutations of genes that code for proteins involved in signaling transduction, epigenetic regulation, and transcription. Epigenetically, T-ALL is characterized by gene expression changes caused by hypermethylation of tumor suppressor genes, histone modifications, and miRNA and lncRNA abnormalities. Although some genetic and gene expression patterns have been associated with certain clinical features, such as immunophenotypic subtype and outcome, none has of yet generally been implemented in clinical routine for treatment decisions. The recent advent of massive parallel sequencing technologies has dramatically increased our knowledge of the genetic blueprint of T-ALL, revealing numerous fusion genes as well as novel gene mutations. The challenges now are to integrate all genetic and epigenetic data into a coherent understanding of the pathogenesis of T-ALL and to translate the wealth of information gained in the last few years into clinical use in the form of improved risk stratification and targeted therapies. Here, we provide an overview of pediatric T-ALL with an emphasis on the acquired genetic alterations that result in this disease. This article is protected by copyright. All rights reserved.
Chapter
The t(12;21)(p13;q22) translocation is the most frequently occurring single genetic abnormality in pediatric leukemia. This translocation results in the fusion of the ETV6 and RUNX1 genes. Since its discovery in the 1990s, the function of the ETV6-RUNX1 fusion gene has attracted intense interest. In this chapter, we will summarize current knowledge on the clinical significance of ETV6-RUNX1, the experimental models used to unravel its function in leukemogenesis, the identification of co-operating mutations and the mechanisms responsible for their acquisition, the function of the encoded transcription factor and finally, the future therapeutic approaches available to mitigate the associated disease.
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Background: Lung adenocarcinoma (LUAD) possesses a poor prognosis with a low 5-year survival rate even for stages I-III resected patients, it is thus critical to understand the determinants that affect the survival and discover new potentially prognostic biomarkers. Somatic copy number alterations (CNAs) are major source of genomic variations driving tumor evolution, CNAs screening may identify prognostic biomarkers. Methods: Oncoscan MIP array was used to analyze the patterns of CNAs on formalin fixed paraffin embedded(FFPE) tumor specimens from 163 consecutive stage I-III resected LUAD patients, 145 out of which received platinum-based adjuvant chemotherapy. Results: Of the 163 patients, 91(55.8%) were recurred within 3 years after surgery. The most common aberrations in our cohort were 1q, 5p, 5q, 7p, 8q, 14p, 16p, 17q, 20q for copy number gains and 8p, 9p, 13p, 16q, 18q for losses. The GISTIC2 analysis produced 45 amplification peaks and 40 deletion peaks, involving some reported genes TERT, EGFR, MYC, CCND1, CDK4, MDM2, ERBB2, NKX2-1, CCNE1, and CDKN2A, most of which were consistent with TCGA database. The amplifications of 12p12.1 (CMAS, GOLT1B, YS2, LDHB, RECQL, ETNK1, IAPP, PYROXD1, KRAS) and KDM5A were correlated with worse prognosis in our cohort, this result was further validated in 506 LUAD patients from TCGA. In addition, 163 patients could be well-classified into five groups, and the clinical outcomes were significantly different based on threshold copy number at reoccurring alteration peaks. Among the 145 patients who received adjuvant chemotherapy, focal amplification of ERBB2 and deletion of 4q34.3 were found to be specific in relapsed patients, this result was validated in an independent group of Imielinski et al., demonstrating these two CNAs may contribute to resected LUAD recurrence after adjuvant chemotherapy. Conclusion: This study suggests that CNAs profiling may be a potential prognostic classifier in resected LAUD patients. Amplifications of 12p12.1 and KDM5A might be prognostic biomarkers for LUAD, and amplification of ERBB2 and deletion of 4q34.3 predicted early relapse after adjuvant chemotherapy. These novel findings may provide implication for better implementation of precision therapy for lung cancer patients.
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Clinical management and risk stratification of B-lymphoblastic leukemia/ lymphoma (B-ALL/LBL) depend largely on identification of chromosomal abnormalities obtained using conventional cytogenetics and Fluorescence In Situ Hybridization (FISH) testing. In the last few decades, testing algorithms have been implemented to support an optimal risk-oriented therapy, leading to a large improvement in overall survival. In addition, large scale genomic studies have identified multiple aberrations of prognostic significance that are not routinely tested by existing modalities. However, as chromosomal microarray analysis (CMA) and next-generation sequencing (NGS) technologies are increasingly used in clinical management of hematologic malignancies, these abnormalities may be more readily detected. In this article, we have compiled a comprehensive, evidence-based review of the current B-ALL literature, focusing on known and published subtypes described to date. More specifically, we describe the role of various testing modalities in the diagnosis, prognosis, and therapeutic relevance. In addition, we propose a testing algorithm aimed at assisting laboratories in the most effective detection of the underlying genomic abnormalities.
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Alterations of the CDKN2A locus on chromosome 9p21 encoding the p16INK4A cell cycle regulator and the p14ARF1 p53 activator proteins are frequently found in bladder cancer. Here, we present an analysis of 86 transitional cell carcinomas (TCC) to elucidate the mechanisms responsible for inactivation of this locus. Multiplex quantitative PCR analysis for five microsatellites around the locus showed that 34 tumors (39%) had loss of heterozygosity (LOH) generally encompassing the entire region. Of these, 17 tumors (20%) carried homozygous deletions of at least one CDKN2A exon and of flanking microsatellites, as detected by quantitative PCR. Analysis by restriction enzyme PCR and methylation-specific PCR showed that only three specimens, each with LOH across 9p21, had bona fide hypermethylation of the CDKN2A exon 1 CpG-island in the remaining allele. Like most other specimens, these three specimens displayed substantial genome-wide hypomethylation of DNA as reflected in the methylation status of LINE L1 sequences. The extent of DNA hypomethylation was significantly more pronounced in TCC with LOH and/or homozygous deletions at 9p21 than in those without (26% and 28%, respectively, on average, versus 11%, p < 0.0015). No association of LOH or homozygous deletions at 9p21 with tumor stage or grade was found. The data indicate that DNA hypermethylation may be rare in TCC and that deletions are the most important mechanism for inactivation of the CDKN2A locus. The predominance of allelic loss may be explained by its correlation with genome-wide DNA hypomethylation, which is thought to favor chromosomal instability and illegitimate recombination.
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We have recently shown that the multiple tumor suppressor gene 1 (MTS1 ) encoding the p16(INK4a) and p19(ARF) cell-cycle inhibitors is inactivated by deletion or disruption in most human T-cell acute lymphoblastic leukemias (T-ALLs), representing the most frequent genetic event thus far described in this disease. To analyze the mechanism of these chromosomal events, we used cloning, sequencing, and/or polymerase chain reaction mapping to study 15 rearrangements occurring in the MTS1 locus. We found that these breakpoints occur in two clusters (MTS1(bcralpha) and MTS1(bcrbeta) ). The three rearrangements occurring in MTS1(bcralpha) correspond to a recurrent recombination juxtaposing 5' MTS2 exon 1 and 5' MTS1 exon 1alpha sequences. Breakpoints for 10 of 12 rearrangements within MTS1(bcrbeta) are located at a polymorphic (CA) repeat, suggesting that this sequence might play a role in the clustering. For both MTS1(bcralpha) and MTS1(bcrbeta), sequence analyses and PCR mapping experiments show that the tightly clustered breakpoints are located in the vicinity of heptamers whose sequence is similar to those involved in the V(D)J recombination. Moreover, short deletions, GC-rich random nucleotide additions, and clone-specific junctional sequences are present in all cases, further suggesting that the rearrangements are due to illegitimate V(D)J recombinase activity. These data are the first demonstration that a tumor suppressor gene can be inactivated by the V(D)J recombinational mechanism. Moreover, they reinforce the view that this process, normally required for T-cell differentiation, plays a crucial role in the pathogenesis of T-ALL.
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We studied 11 head and neck squamous carcinoma (HNSC) cell lines and 46 primary tumors for p16 gene status by protein, mRNA, and DNA genetic/epigenetic analyses to determine the incidence, the mechanism(s), and the potential biological significance of its inactivation. Of the 11 cell lines, only 1 showed intact p16 and 10 lacked its protein and mRNA; DNA analysis of these 10 cell lines showed 2 homozygous deletions, 6 methylations at exon 1 and 2, and 2 with no detectable abnormalities. In primary tumors, 16 (34.7%) of the 46 showed detectable p16 protein and mRNA; of these, 12 had no DNA abnormalities and 4 had only exon 2 methylation. Loss of p16 expression was found in three tumors with concurrent mutation at exon 2 and methylation at exon 2 (two) and both 1 and 2 (one). Of the 30 tumors that lacked p16 protein, 27 also lacked mRNA, 1 had detectable p16 mRNA, and 2 failed RT-PCR amplification. Twenty-two of the thirty tumors showed DNA alterations and eight manifested no abnormalities; DNA alterations comprised 6 homozygous deletions, 2 concurrent mutations and methylation of exon 2, and 13 with methylation at exon 1 and exons 1 and 2 (12 with methylation only and 1 with mutation) at exon 1. Except for patients' gender (P = 0.02), no significant correlation between p16 and clinicopathological factors was observed. We conclude that in HNSC 1) intragenic p16 alterations are infrequent events, 2) methylation of exon 1 constitutes a common mechanism in silencing the p16 gene, 3) p16 inactivation may play an important role in the early development and progression of HNSC, and 4) no association between p16 alterations and conventional clinicopathological factors was noted in this cohort.
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Loss of WRN causes the genomic instability progeroid syndrome, Werner syndrome. WRN encodes a multifunctional nuclear protein with 3'-->5' exonuclease and 3'-->5' helicase activities. Linear plasmids with noncompatible ends introduced to Werner syndrome cells underwent extensive deletions at nonhomologous joining ends, particularly at the 3' protruding single-stranded end. This extensive deletion phenotype was complemented by wild-type WRN. These results suggest that WRN can out-compete other exonucleases that participate in double-strand break repair or stabilize the broken DNA end.
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To understand molecular pathways underlying 9p21 deletions, which lead to inactivation of the p16/CDKN2A, p14/ARF, and/or p15/CDKN2B genes, in lymphoid leukemia, 30 breakpoints were cloned from 15 lymphoid leukemia cell lines. Seventeen (57%) breakpoints were mapped at five breakpoint cluster sites, BCS-LL1 to LL5, each of <15 bp. Two breakpoint cluster sites were located within the ARF and CDKN2Bloci, respectively, whereas the remaining three were located >100 kb distal to the CDKN2A, ARF, andCDKN2B loci. The sequences of breakpoint junctions indicated that deletions in the 11 (73%) cell lines were mediated by illegitimate V(D)J recombination targeted at the five BCS-LL and six other sites, which contain sequences similar to recombination signal sequences for V(D)J recombination. An extrachromosomal V(D)J recombination assay indicated that BCS-LL3, at which the largest number of breakpoints (i.e. five breakpoints) was clustered, has a V(D)J recombination potential 150-fold less than the consensus recombination signal sequence. Three other BCS-LLs tested also showed V(D)J recombination potential, although it was lower than that of BCS-LL3. These results indicated that illegitimate V(D)J recombination, which was targeted at several ectopic recombination signal sequences widely distributed in 9p21, caused a large fraction of 9p21 deletions in lymphoid leukemia.
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Interstitial deletions of the chromosome 9p21 segment encoding the p16/CDKN2A tumor suppressor gene (i.e., 9p21 deletions) are frequently observed in a variety of human cancers. A majority of these deletions in lymphoid leukemia have been indicated to be mediated by illegitimate V(D)J recombination. In the present study, to elucidate the molecular processes of 9p21 deletions in nonlymphocytic malignancies, breakpoints for these deletions were analysed in 21 lung cancer cell lines and 32 nonlymphocytic cancer cell lines of nine other histological types. In all, 32 breakpoints in 21 lung cancer cell lines and 56 breakpoints in 32 nonlung cancer cell lines were mapped in a 450-kb segment encompassing the CDKN2A locus with a 10-kb resolution. The largest number of breakpoints (i.e., seven breakpoints in lung cancer and 12 breakpoints in nonlung cancers) was mapped in a 10-kb region containing the CDKN2A gene. More precise mapping of these seven and 12 breakpoints revealed that none of these breakpoints were located within 50-bp intervals to each other in this 10 kb region. Cloning and sequencing of breakpoints in 18 representative cell lines (six lung and 12 nonlung cancers) further revealed that there were no significant homologies among breakpoints in these 18 cell lines. In 11 (61%) cell lines, 1-5-bp nucleotides were overlapped at breakpoint junctions. These results indicate that DNA double-strand breaks triggering 9p21 deletions do not occur at specific DNA sequences, although they preferentially occur in or near the CDKN2A locus. It was also indicated that two broken DNA ends are rejoined by nonhomologous end-joining repair, preferentially utilizing microhomologies of DNA ends, in the occurrence of 9p21 deletions.
Article
Loss of CDKN2A expression was demonstrated by immunohistochemistry in 87% of oral and oropharyngeal squamous cell carcinoma (OSCC) primary tumor samples. By contrast, DNA studies showed a much lower frequency of loss of the CDKN2A gene. Point mutations and promoter methylation of CDKN2A were seen in 7% and 23%, respectively, of primary tumors. Loss of heterozygosity analysis using a dense set of 9p markers showed allelic imbalance that included CDKN2A in only 31% of samples, but a further 47% showed loss at loci near CDKN2A with apparent retention of CDKN2A. No tumor with any allelic imbalance expressed CDKN2A, whether or not the imbalance appeared to involve the CDKN2A locus. We interpret these data as showing partially overlapping deletions on the two 9p homologues, with homozygous deletion of CDKN2A masked by amplification of contaminating stromal material. Our data show that inactivation of the CDKN2A gene products is a near‐universal step in the development of oral and oropharyngeal squamous cell carcinomas, and we suggest that homozygous deletion is the most common mechanism of inactivation. The CDKN2A locus may be particularly prone to deletion because it encodes two unrelated tumor suppressor proteins, CDKN2A (p16INK4a) and p19ARF, and deletion, but not point mutation or methylation, would inactivate both gene products. However, our results also suggest that complex patterns of allelic imbalance in primary squamous carcinomas in general may not provide reliable evidence for the existence of multiple tumor suppressor genes within a single chromosomal region. Genes Chromosomes Cancer 25:16–25, 1999. © 1999 Wiley‐Liss, Inc.
Article
The tumor suppressor gene p16, when altered, has been shown to play a role in oncogenesis in many different tumor types including head and neck cancer. The goal of this study was to analyse alterations to p16 in squamous cell carcinoma (SCC) of the head and neck and to correlate these with clinical outcome. RNA was isolated from 26 SCC head and neck tumors and from 24 matched controls. A reverse transcription polymerase chain reaction was utilized to generate p16 cDNA, which was sequenced and analysed for alterations. In the 26 patient group 58% of the tumors had a p16 alteration, which were characterized by: 8 deletions, 1 insertion/deletion, 4 point mutations and 2 with no p16 expression. In 24 matched normal tissue samples there were no p16 alterations. Those patients with p16 alterations appear to have survival rates comparable to those without p16 alterations, although patients with p16 alterations appear to have more recurrences.
Article
Loss of CDKN2A expression was demonstrated by immunohistochemistry in 87% of oral and oropharyngeal squamous cell carcinoma (OSCC) primary tumor samples. By contrast, DNA studies showed a much lower frequency of loss of the CDKN2A gene. Point mutations and promoter methylation of CDKN2A were seen in 7% and 23%, respectively, of primary tumors. Loss of heterozygosity analysis using a dense set of 9p markers showed allelic imbalance that included CDKN2A in only 31% of samples, but a further 47% showed loss at loci near CDKN2A with apparent retention of CDKN2A. No tumor with any allelic imbalance expressed CDKN2A, whether or not the imbalance appeared to involve the CDKN2A locus. We interpret these data as showing partially overlapping deletions on the two 9p homologues, with homozygous deletion of CDKN2A masked by amplification of contaminating stromal material. Our data show that inactivation of the CDKN2A gene products is a near-universal step in the development of oral and oropharyngeal squamous cell carcinomas, and we suggest that homozygous deletion is the most common mechanism of inactivation. The CDKN2A locus may be particularly prone to deletion because it encodes two unrelated tumor suppressor proteins, CDKN2A (p16INK4a) and p19ARF, and deletion, but not point mutation or methylation, would inactivate both gene products. However, our results also suggest that complex patterns of allelic imbalance in primary squamous carcinomas in general may not provide reliable evidence for the existence of multiple tumor suppressor genes within a single chromosomal region. Genes Chromosomes Cancer 25:16–25, 1999. © 1999 Wiley-Liss, Inc.
Article
Growth of cancer cells is characterized by accelerated passage through the cell cycle, which is often caused by deregulation of the G1→S transition. In this study the expression of G1→S transition regulatory molecules was analyzed in 32 transitional cell carcinoma specimens and fifteen normal tissues obtained by cystectomy or nephroureterectomy of mainly locally advanced tumors, as well as six bladder cancer cell lines. Expression of mRNAs for cyclins D1 and D2 and cyclin-dependent kinases (CDK) 2 and 4 was investigated by quantitative reverse transcription-poly-merase chain reaction. Overexpression of cyclin D1 compared to normal mucosa was observed in 3 tumors (9.4%), but in neither of the cell lines. All tumors with overexpression were moderately differentiated (G2) pT1 or pT2 tumors, and thus among the less advanced specimens. Cyclin D2 was not expressed in normal bladder mucosa or in tumors. The expression of CDK4 mRNA varied within the same range in mucosa, tumors, and cell lines. CDK2 mRNA expression varied more strongly and was diminished in individual tumors and in four cell lines. It is concluded that cyclin D1 overexpression can play an important role in the early stage of urothelial tumorigenesis, driving cell proliferation. Ectopic expression of cyclin D2 or amplification of CDK4 does not occur at a significant frequency in urothelial carcinomas. Different expression patterns of cyclin D1 and CDK2 indicate heterogeneity in the mechanisms of G1→S transition deregulation in individual bladder tumors which may elicit differences in their biological and clinical behavior.
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The establishment of connections between biochemical defects and clinical disease is a major goal of modern molecular genetics. In this review, we examine the current literature that relates defects in the two major DNA double-strand-break repair pathways – homologous recombination and nonhomologous end-joining – with the development of human tumors. Although definitive proof has yet to be obtained, the current literature is highly suggestive of such a link.
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Interstitial deletions of the short arm of chromosome 9 are associated with glioma, acute lymphoblastic leukemia, melanoma, mesothelioma, lung cancer, and bladder cancer. The distal breakpoints of the deletions (in relation to the centromere) in 14 glioma and leukemia cell lines have been mapped within the 400 kb IFN gene cluster located at band 9p21. To obtain information about the mechanism of these deletions, we have isolated and analyzed the nucleotide sequences at the breakpoint junctions in two glioma-derived cell lines. The A1235 cell line has a complex rearrangement of chromosome 9, including a deletion and an inversion that results in two breakpoint junctions. Both breakpoints of the distal inversion junction occurred within AT-rich regions. In the A172 cell line, a tandem heptamer repeat was found on either side of the deletion breakpoint junction. The distal breakpoint occurred 5' of IFNA2; the 256 bp sequenced from the proximal side of the breakpoint revealed 95% homology to long interspersed nuclear elements. One- and two-base-pair overlaps were observed at these junctions. The possible role of sequence overlaps, and repetitive sequences, in the rearrangement is discussed.
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The product of the p16/CDKN2 locus, p16ink4, negatively regulates the cell cycle through binding and inactivation of cyclin-dependent kinases (CDKs) 4 and 6. This locus is frequently targeted for deletion in cell lines and primary tumor tissues. In gliomas, although up to 50% do not have detectable expression of p16/CDKN2 protein or mRNA, often the gene is wild type in sequence. Here, we tested the hypothesis that transcriptional repression of p16/CDKN2 in gliomas may be mediated by aberrant methylation of the CpG island, which is in the 5' region of the locus. Partial rather than complete p16/CDKN2 methylation was detected in 24% (10 of 42) of the gliomas, regardless of tumor grade, but was not observed in normal brain (0 of 10). We tested whether this partial methylation could inhibit expression in a human tumor cell line in which suppressed p16/CDKN2 expression was associated with both methylation and tightly compacted chromatin around the p16/CDKN2 promoter. Exposure of these cells to 5-aza-2-deoxycytidine resulted in a dramatic increase in promoter accessibility and induction of p16/CDKN2 expression, indicating that chromatin structure, CpG island methylation, and p16/CDKN2 expression are intimately associated. Taken together, these data suggest that methylation occurs in only a subset of cells within gliomas and that the methylation-associated inactivation of p16/CDKN2 expression observed in many common human cancers may mechanistically result from structural changes in the chromatin containing the p16/CDKN2 locus.
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Recent investigations have demonstrated p53 and Rb alterations in a subset of transitional cell carcinoma (TCC). Further genetic changes during tumor progression include overexpression of the c-myc gene in a significant number of mainly invasive bladder tumors. To study the possible interactions between these genes in TCC, urothelial cancer cell lines were chosen as an in vitro model. Expression and mutation of p53 was studied in 15 bladder cancer cell lines by immunocytochemistry, Western blot, polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing of double stranded PCR products of exons 4, 5, 7 and 8 of genomic DNA. C-myc expression and gene structure were studied using Northern and Southern blot techniques Rb protein expression was analyzed by Western blot. Twelve of 15 cell lines showed either p53 mutations or abnormal protein expression. Consistent with previous studies, five cell lines did not express Rb protein. None of the cell lines studied retained both tumor suppressor genes in a functional form. The c-myc gene appeared to be intact in all cell lines and copy numbers were close to normal. Northern analysis demonstrated that all cell lines expressed c-myc mRNA but evidence for altered regulation was found in at least two cell lines. Our data suggest that amplification or translocation are not the underlying mechanism for c-myc overexpression in urothelial tumors. No correlation between loss of Rb protein and c-myc expression was observed. The results presented here for the cell lines match well those obtained in vivo. Thus, these cell lines may provide a suitable model for further analysis of molecular alterations in urothelial cancer.
Article
Most of the 5-methylcytosine in mammalian DNA resides in transposons, which are specialized intragenomic parasites that represent at least 35% of the genome. Transposon promoters are inactive when methylated and, over time, C-->T transition mutations at methylated sites destroy many transposons. Apart from that subset of genes subject to X inactivation and genomic imprinting, no cellular gene in a non-expressing tissue has been proven to be methylated in a pattern that prevents transcription. It has become increasingly difficult to hold that reversible promoter methylation is commonly involved in developmental gene control; instead, suppression of parasitic sequence elements appears to be the primary function of cytosine methylation, with crucial secondary roles in allele-specific gene expression as seen in X inactivation and genomic imprinting.
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In mammalian cells, nonhomologous end-joining is the predominant mechanism to eliminate DNA double strand breaks. Such events are at the origin of deletion mutagenesis and chromosomal rearrangements. The hallmark of Fanconi anemia, an inherited cancer prone disorder, is increased chromosomal breakage associated to over-production of deletions. Knowing that double strand breaks are at the origin of deletion mutagenesis, the question arises whether their processing is affected in FA. We set up a "host cell end-joining assay" to analyze the fate of double strand breaks into extrachromosomal substrates transiently replicated in normal and FA-D lymphoblasts. Although no difference in plasmid survival was found, blunt-ended breaks were sealed with significantly lower fidelity in FA cells, resulting in a higher deletion frequency and a larger deletion size. The results suggest that FA-D and FA-B gene products are likely to play a role in end-joining fidelity of specific DNA double strand breaks.
Article
Growth of cancer cells is characterized by accelerated passage through the cell cycle, which is often caused by deregulation of the G1-->S transition. In this study the expression of G1-->S transition regulatory molecules was analyzed in 32 transitional cell carcinoma specimens and fifteen normal tissues obtained by cystectomy or nephroureterectomy of mainly locally advanced tumors, as well as six bladder cancer cell lines. Expression of mRNAs for cyclins D1 and D2 and cyclin-dependent kinases (CDK) 2 and 4 was investigated by quantitative reverse transcription-polymerase chain reaction. Overexpression of cyclin D1 compared to normal mucosa was observed in 3 tumors (9.4%), but in neither of the cell lines. All tumors with overexpression were moderately differentiated (G2) pT1 or pT2 tumors, and thus among the less advanced specimens. Cyclin D2 was not expressed in normal bladder mucosa or in tumors. The expression of CDK4 mRNA varied within the same range in mucosa, tumors, and cell lines. CDK2 mRNA expression varied more strongly and was diminished in individual tumors and in four cell lines. It is concluded that cyclin D1 overexpression can play an important role in the early stage of urothelial tumorigenesis, driving cell proliferation. Ectopic expression of cyclin D2 or amplification of CDK4 does not occur at a significant frequency in urothelial carcinomas. Different expression patterns of cyclin D1 and CDK2 indicate heterogeneity in the mechanisms of G1-->S transition deregulation in individual bladder tumors which may elicit differences in their biological and clinical behavior.
Article
Loss of CDKN2A expression was demonstrated by immunohistochemistry in 87% of oral and oropharyngeal squamous cell carcinoma (OSCC) primary tumor samples. By contrast, DNA studies showed a much lower frequency of loss of the CDKN2A gene. Point mutations and promoter methylation of CDKN2A were seen in 7% and 23%, respectively, of primary tumors. Loss of heterozygosity analysis using a dense set of 9p markers showed allelic imbalance that included CDKN2A in only 31% of samples, but a further 47% showed loss at loci near CDKN2A with apparent retention of CDKN2A. No tumor with any allelic imbalance expressed CDKN2A, whether or not the imbalance appeared to involve the CDKN2A locus. We interpret these data as showing partially overlapping deletions on the two 9p homologues, with homozygous deletion of CDKN2A masked by amplification of contaminating stromal material. Our data show that inactivation of the CDKN2A gene products is a near-universal step in the development of oral and oropharyngeal squamous cell carcinomas, and we suggest that homozygous deletion is the most common mechanism of inactivation. The CDKN2A locus may be particularly prone to deletion because it encodes two unrelated tumor suppressor proteins, CDKN2A (p16INK4a) and p19ARF, and deletion, but not point mutation or methylation, would inactivate both gene products. However, our results also suggest that complex patterns of allelic imbalance in primary squamous carcinomas in general may not provide reliable evidence for the existence of multiple tumor suppressor genes within a single chromosomal region.
Article
The tumor suppressor gene p16, when altered, has been shown to play a role in oncogenesis in many different tumor types including head and neck cancer. The goal of this study was to analyse alterations to p16 in squamous cell carcinoma (SCC) of the head and neck and to correlate these with clinical outcome. RNA was isolated from 26 SCC head and neck tumors and from 24 matched controls. A reverse transcription polymerase chain reaction was utilized to generate p16 cDNA, which was sequenced and analysed for alterations. In the 26 patient group 58% of the tumors had a p16 alteration, which were characterized by: 8 deletions, 1 insertion/deletion, 4 point mutations and 2 with no p16 expression. In 24 matched normal tissue samples there were no p16 alterations. Those patients with p16 alterations appear to have survival rates comparable to those without p16 alterations, although patients with p16 alterations appear to have more recurrences.
Article
We have sequenced 870 kilobases of the FHIT/FRA3B locus, from FHIT intron 3 to intron 7. The locus is AT rich (61.5%) and Alu poor (6. 2%), and it apparently does not harbor other genes. In a detailed analysis of the 308-kilobase region between FHIT exon 5 and the telomeric end of intron 3, a region known to encompass a human papillomavirus-16 integration site and two clusters of aphidicolin-induced chromosome 3p14.2 breakpoints, we have precisely mapped 10 deletion and translocation endpoints in cancer-derived cell lines relative to positions of specific repetitive elements, regions of high genome flexibility and aphidicolin-induced breakpoints. Conclusions are (i) that aphidicolin-induced breakpoint clusters fall close to high-flexibility sequences, suggesting that these sequences contribute directly to aphidicolin-induced fragility; (ii) that 9 of the 10 FHIT allelic deletions in cancer cell lines resulted in loss of exons, with 7 deletion endpoints near long interspersed nuclear elements or long terminal repeat elements; and (iii) that cancer-specific deletions encompass multiple high-flexibility genomic regions, suggesting that fragile breaks may occur at these regions, whereas repair of the breaks involves homologous pairing of flanking sequences with concomitant deletion of the damaged fragile sequence.
Article
DNA double-strand breaks (DSB) are considered to be critical primary lesions in the formation of chromosomal aberrations. DSB may be induced by exogenous agents, such as ionizing radiation, but also occur spontaneously during cellular processes at quite significant frequencies. To repair this potentially lethal damage, eukaryotic cells have evolved a variety of repair pathways related to homologous and illegitimate recombination, also called non-homologous DNA end joining, which may induce small scale mutations and chromosomal aberrations. In this paper we review the major cellular sources of spontaneous DSB and the different homologous and illegitimate recombination repair pathways, with particular focus on their potential to induce chromosomal aberrations.
Article
The common fragile site, FRA3B, has been shown to be a site of frequent homozygous deletions in some cancers, resulting in loss of expression of the associated FHIT gene. It has been proposed that FHIT is a tumor suppressor gene that is inactivated as a result of the instability of FRA3B in tumorigenesis. More recently, deletions at other common fragile sites, FRA7G and FRA16D, have been identified in a small number of cancer cell lines. Here, we have mapped and molecularly characterized the frequently observed common fragile site FRAXB, located at Xp22.3. Like other common fragile sites, it spans a large genomic region of approximately 500 kb. Three known genes, including the microsomal steroid sulfatase locus (STS), map within the fragile site region. We examined FRAXB and four other fragile sites (FRA3B, FRA7G, FRA7H, FRA16D), and several associated genes, for deletions and aberrant transcripts in a panel of cancer cell lines and primary tumors. Deletions within FRAXB were seen in 4/27 (14.8%) of the primary tumors and cell lines examined. Three of the 21 (14.3%) cell lines examined were characterized by loss of expression of one or more FRAXB-associated genes. Moreover, all of the fragile sites examined were characterized by genomic deletions within the fragile site regions in one or more tumors or cell lines, including FRAXB, which is not associated with any known tumor suppressor genes or activity. Our results further support the hypothesis that common fragile sites and their associated genes are, in general, unstable in some cancer cells.
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Head and neck cancers include neoplasms of the oral cavity, pharynx, and larynx. The risk of these cancers is strongly associated with smoking and alcohol ingestion. There have been important advances in understanding of the molecular pathogenesis and progression of head and neck cancer and also in approaches to therapy, which include innovations in surgery, radiation therapy, and cytotoxic-drug therapy.
Article
Aberrations of the p53 tumor suppressor gene are common events in squamous cell carcinomas of the head and neck (SCCHN). However, reported frequencies range considerably, and the predictive value of aberrant p53 is continuing to be an issue of controversy. These inconsistencies are possibly caused by methodical limitations. In cell lines established from 23 SCCHN, we performed sequence analysis of p53 mRNA and genomic DNA, as well as protein detection using immunohistochemistry and Western blotting. Of the 23 SCCHN, 21 (91%) tumors have aberrant or no p53, including four aberrations (19%) located outside the usually examined exons 5 to 9. A second allele is present in 11, and the respective transcript in 4 tumors. Expression of protein could be detected in only 12 of the 21 p53 aberrant tumors (57%). Depending on the methodical approach applied, a considerable number of SCCHN may be misclassified regarding their p53 status.
Article
Genomic imbalances were investigated in 15 T-cell acute lymphoblastic leukemia cell lines using the comparative genomic hybridization (CGH) technique. In addition, in vitro response to the cytostatic drug doxorubicin was evaluated by means of a growth inhibition assay. The number of significant DNA copy number alterations (CNAs) varied from 0 to 16 per cell line and the number of additional alterations with borderline significance was in a range of 0-7. Three of the cell lines had a total number of genomic changes of >/=20, five had 11-19, and eight had </=10 CNAs. One cell line did not show any imbalance at all. Among the significant CNAs, losses of genomic material were slightly more frequent than gains (60:49). Gains dominated among the borderline alterations compared to losses (45:9). CNAs common to all cell lines examined were not found. The most frequent genomic imbalance (gain of 6q23) was shared by 9 of the 15 cell lines. A significant loss of 18q23 was observed in eight lines, however, often close to borderline significance. Seven of the cell lines were characterized by a loss of the entire short arm of chromosome 9 or parts of it with 9p21 as minimal band of overlap. Interestingly, cell lines with a 9p21 deletion exhibited twice the number of gains and 1.6 times the number of losses per line as compared with the cell lines without this deletion. A consistent pattern of the CNAs accompanying the 9p deletion could not be detected, although some associations were more obvious than others, e.g., gains of 6q22 in five cell lines with del(9p21), of 6p21 in four lines combined with a gain of 6q22, and of 20q in four cell lines, of 1p32 in three of these seven lines, a loss of 14q32 in three of them. Three of the lines with 9p deletion had gains of 6p21, 6q22 and 20q in common. Enh(6q22) or dim(14q32), respectively, were found in only one of the nine cell lines without the 9p deletion. Based on the dose response curves of the cell lines for doxorubicin, eight doxorubicin-sensitive cell lines had an inhibition concentration 50% (IC50) <10 nM (CCRF-CEM2, JURKAT, KE-37, MOLT-3, MOLT-4, P12-Ichikawa, PEER, and RPMI-8402) and seven doxorubicin-resistant cell lines had an IC50 >10 nM (BE-13, CCRF-CEM1, HUT-78, J-Jhan, Karpas-45, MOLT-17, and PF-382). The average number of CNAs per cell line was higher in the sensitive than in the resistant group (total 13.1:8.5; significant CNAs 9.1:5.8).
Article
The majority of malignant tumors exhibit complex genomic aberrations, and it has been suggested that an elevated rate of chromosome mutation is present in neoplastic cells. This chromosomal evolution is probably initiated at a preinvase stage and then proceeds continuously but at variable rates during tumor progression. Even though the initiating events may vary among neoplasms, the end point is often a similar set of cytogenetic and cytological events. These include an excessive shortening of telomeric repeats, disrupting the normal mitotic process and triggering chromosomal breakage-fusion-bridge events and gross genomic imbalances. Telomere dysfunction may also cause a failure of cytokinesis, resulting in tetraploidization and the accumulation of supernumerary centrosomes. In turn, this could cause multipolar cell division and gross aberrations in chromosome number. Several well-known cancer risk factors could contribute to these processes. For instance, breakage-fusion-bridge instability can be induced directly by clastogenic substances, whereas papilloma virus infection has been shown to dysregulate centrosome function. A prerequisite for the induction of chromosome instability through these mechanisms is partial inactivation of the DNA damage response and the mitotic checkpoint functions. Also, overexpression of telomerase appears to play a crucial role for moderating the rate of chromosomal evolution.
Article
The CDKN2A tumor-suppressor gene in chromosome band 9p21 encoding CDKN2A (also known as p16, INK4A), a negative regulator of cyclin-dependent kinases, and p14(ARF1), an activator of TP53, is inactivated in many human cancers by point mutations, promoter hypermethylation, or deletions. Homozygous deletions predominate in certain cancer types (e.g., bladder cancers). To understand why deletions are unusually prevalent at this locus, deletions in bladder and renal cancer cell lines were mapped in detail and several deletion breakpoints cloned. Deletions were interstitial and encompassed 0.1 to >30 Mb. Most deletion breakpoints were located in or close to LINE-1 retrotransposon clusters. Therefore, deletions of CDKN2A may be facilitated by the presence of LINE-1 clusters that flank the locus. All cloned junctions were products of non-homologous recombination and consistently contained exact 2-bp microhomologies. Microhomologies are otherwise hallmarks of DNA double-strand break repair by non-homologous end joining, but the consistent size found at the CDKN2A deletion junctions is difficult to reconcile with the known properties of this process. Therefore, an unknown mechanism appears to be involved in the generation of CDKN2A deletions during carcinogenesis.
Article
The carboxyl-terminal modulator protein (CTMP) has been identified as a negative regulator of protein kinase B/Akt. Aberrant Akt signaling is frequently observed in glioblastomas, the most common and most malignant glial brain tumors. Because loss of CTMP function and/or expression may remove the inhibitory effects on Akt and promote tumorigenesis, we studied 93 primary glioblastomas and nine glioblastoma cell lines for CTMP deletion, mutation, promoter hypermethylation, and mRNA expression. None of the tumors or cell lines had CTMP-homozygous deletions or coding sequence mutations. However, CTMP mRNA expression was lower by at least 50% relative to non-neoplastic brain tissue in 37 (40%) glioblastomas and six (67%) glioma cell lines. Reduced CTMP mRNA levels were closely associated with hypermethylation of the CTMP promoter. Furthermore, treatment of CTMP-hypermethylated A172 glioma cells with the demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A resulted in partial demethylation of the CTMP promoter and increased CTMP mRNA expression. Thus, epigenetic downregulation of CTMP transcription is a common aberration in glioblastomas.
Article
Epigenetic modifications of cytosine residues in DNA and the amino termini of histone proteins have emerged as key mechanisms in chromatin remodeling, impacting both the transcriptional regulation and the establishment of chromosomal domains. Using the chromatin immunoprecipitation (ChIP) assay, we demonstrate that aberrantly silenced genes in cancer cells exhibit a heterochromatic structure that is characterized by histone H3 lysine 9 (H3-K9) hypermethylation and histone H3 lysine 4 (H3-K4) hypomethylation. This aberrant heterochromatin is incompatible with transcriptional initiation but does not inhibit elongation by RNA polymerase II. H3-K9 methylation may, therefore, play a role in the silencing of tumor-suppressor genes in cancer. Treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR), previously known for its ability to inhibit cytosine methylation, induced a rapid and substantial remodeling of the heterochromatic domains of the p14ARF/p16INK4a locus in T24 bladder cancer cells, reducing levels of dimethylated H3-K9 and increasing levels of dimethylated H3-K4 at this locus. In addition, 5-Aza-CdR increased acetylation and H3-K4 methylation at the unmethylated p14 promoter, suggesting it can induce chromatin remodeling independently of its effects on cytosine methylation.
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