Chromosomal Instability Phenotype Research Articles

The CpG island methylator phenotype (CIMP) in colorectal cancer.

It is clear that colorectal cancer (CRC) develops through multiple genetic and epigenetic pathways. These pathways may be determined on the basis of three molecular features: (i) mutations in DNA mismatch repair genes, leading to a DNA microsatellite instability (MSI) phenotype, (ii) mutations in APC and other genes that activate Wnt pathway, characterized by chromosomal instability (CIN) phenotype, and (iii) global genome hypermethylation, resulting in switch off of tumor suppressor genes, indicated as CpG island methylator phenotype (CIMP). Each of these pathways is characterized by specific pathological features, mechanisms of carcinogenesis and process of tumor development. The molecular aspects of these pathways have been used clinically in the diagnosis, screening and management of patients with colorectal cancer. In this review we especially describe various aspects of CIMP, one of the important and rather recently discovered pathways that lead to colorectal cancer.

Interactions of Apoptotic Cells with Macrophages in Radiation-Induced Bystander Signaling

Nontargeted effects that result in ongoing cellular and tissue damage show genotype-dependency in murine models with CBA/Ca, but not C57BL/6, exhibiting sensitivity to induced genomic instability. In vivo, radiation exposure is associated with genotype-dependent macrophage activation, and these cells are a source of bystander signaling involving cytokines and reactive oxygen and nitrogen species. The mechanisms responsible for macrophage activation and production of damaging bystander signals after irradiation are unclear. Macrophages from CBA/Ca exhibit an M1 (proinflammatory) phenotype compared to the M2 (anti-inflammatory) phenotype of C57BL/6 macrophages. Using the murine RAW264.7 macrophage-like cell line, we show that the ability of macrophages to interact with apoptotic cells and their responses to interaction varies significantly according to macrophage phenotype. Nonstimulated and M2 macrophages induce anti-inflammatory markers arginase and TGFβ after engulfment of apoptotic cells. In contrast, M1 macrophages do not induce anti-inflammatory responses, but express the proinflammatory markers NOS2, IL-6, TNFα, superoxide and NO, able to contribute to a damaging microenvironment. Macrophages stimulated with both inflammatory and anti-inflammatory agents prior to exposure to apoptotic cells induce a mixed response. The results indicate a complex cross-talk between macrophages and apoptotic cells and demonstrate that phagocytic clearance of apoptotic cells induced by genotoxic stress can produce microenvironmental responses consistent with the induction of a chromosomal instability phenotype in sensitive CBA/Ca mice with M1 macrophage activation, but not in resistant C57BL/6 mice with M2 macrophage activation. Modulation of macrophage phenotypes may represent a novel approach for reducing the nontargeted effects of radiation.

General aspects of colorectal cancer.

Colorectal cancer (CRC) is one of the main causes of death. Cancer is initiated by several DNA damages, affecting proto-oncogenes, tumour suppressor genes, and DNA repairing genes. The molecular origins of CRC are chromosome instability (CIN), microsatellite instability (MSI), and CpG island methylator phenotype (CIMP). A brief description of types of CRC cancer is presented, including sporadic CRC, hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndromes, familiar adenomatous polyposis (FAP), MYH-associated polyposis (MAP), Peutz-Jeghers syndrome (PJS), and juvenile polyposis syndrome (JPS). Some signalling systems for CRC are also described, including Wnt-β-catenin pathway, tyrosine kinase receptors pathway, TGF-β pathway, and Hedgehog pathway. Finally, this paper describes also some CRC treatments.

Single nucleotide polymorphism array analysis of microsatellite-stable, diploid/near-diploid colorectal carcinomas without the CpG island methylator phenotype

Colorectal carcinomas are considered to progress by chromosomal instability (CIN), or microsatellite instability (MSI) and/or epigenetic gene silencing; however, in previous studies we observed a small fraction of tumours without this molecular phenotype. To further investigate these 'X-type' tumours, neoplastic glands from five tumours were isolated by laser-capture microdissection and used for single nucleotide polymorphism (SNP) array analyses. DNA from our own low-passage primary colorectal carcinoma cell lines (n=9) was used for comparison. Two of these 'X-type' tumours had very low numbers of aberrations (totals of four and five, respectively), consisting of trisomies and arm amplifications. Conversely, aberrations were markedly more frequent in the control cases and three of the 'X-type' tumours (range, 11-40). These aberrations included deletions of chromosomes and chromosome arms, uniparental disomies (UPD), trisomies and arm amplifications. Recurrent microdeletions (<1 MB) were observed at 3p14.2 (FHIT), 16p13.2 (A2BP1) and 20p12.1 (MACROD2). Microsatellite analyses with polymorphic markers at five 'canonical' colorectal carcinoma loci demonstrated a complete loss of one allele in all but one case. When compared to the SNP arrays, concordant results were observed in 93% of tests; however, this was only if DNA from cell lines or laser-capture microdissections was used. In conclusion, colorectal carcinomas may develop without the classic molecular features of CIN, MSI and/or CpG island methylator phenotype (CIMP), but this is a rare event. UPD is frequent but does not define a separate molecular phenotype. Furthermore, our study supports the notion that SNP arrays are reliable for genome-wide detection of deletions and UPD, but discourages the use of microsatellite analyses to detect loss of heterozygosity with DNA from whole tissues.

A Screen for Selective Killing of Cells with Chromosomal Instability Induced by a Spindle Checkpoint Defect

BackgroundThe spindle assembly checkpoint is crucial for the maintenance of a stable chromosome number. Defects in the checkpoint lead to Chromosomal INstability (CIN), which is linked to the progression of tumors with poor clinical outcomes such as drug resistance and metastasis. As CIN is not found in normal cells, it offers a cancer-specific target for therapy, which may be particularly valuable because CIN is common in advanced tumours that are resistant to conventional therapy.Principal FindingsHere we identify genes that are required for the viability of cells with a CIN phenotype. We have used RNAi knockdown of the spindle assembly checkpoint to induce CIN in Drosophila and then screened the set of kinase and phosphatase genes by RNAi knockdown to identify those that induce apoptosis only in the CIN cells. Genes identified include those involved in JNK signaling pathways and mitotic cytoskeletal regulation.Conclusions/SignificanceThe screen demonstrates that it is feasible to selectively kill cells with CIN induced by spindle checkpoint defects. It has identified candidates that are currently being pursued as cancer therapy targets (e.g. Nek2: NIMA related kinase 2), confirming that the screen is able to identify promising drug targets of clinical significance. In addition, several other candidates were identified that have no previous connection with mitosis or apoptosis. Further screening and detailed characterization of the candidates could potentially lead to the therapies that specifically target advanced cancers that exhibit CIN.

Application of molecular techniques in the diagnosis, prognosis and management of patients with colorectal cancer: a practical approach

There has been an increasing role for molecular diagnostics in the diagnosis and management of cancer, and colorectal carcinoma is no exception. Recent molecular advances have elucidated 3 broad molecular subtypes of colorectal cancer, including chromosomal instability, microsatellite instability, and cytosine-phosphoguanine island methylator phenotype, which will be discussed. Also, the common syndromes associated with colorectal carcinoma will be reviewed with a focus on the differentiation between Lynch syndrome and microsatellite unstable tumors. Molecular biomarkers for predictive and prognostic markers are also becoming widely used, and due to the clinical use of monoclonal antibodies to the epidermal growth factor receptor, an emphasis is placed on that pathway.

Molecular events in primary and metastatic colorectal carcinoma: a review.

Colorectal cancer (CRC) is a heterogeneous disease, developing through a multipathway sequence of events guided by clonal selections. Pathways included in the development of CRC may be broadly categorized into (a) genomic instability, including chromosomal instability (CIN), microsatellite instability (MSI), and CpG island methylator phenotype (CIMP), (b) genomic mutations including suppression of tumour suppressor genes and activation of tumour oncogenes, (c) microRNA, and (d) epigenetic changes. As cancer becomes more advanced, invasion and metastases are facilitated through the epithelial-mesenchymal transition (EMT), with additional genetic alterations. Despite ongoing identification of genetic and epigenetic markers and the understanding of alternative pathways involved in the development and progression of this disease, CRC remains the second highest cause of malignancy-related mortality in Canada. The molecular events that underlie the tumorigenesis of primary and metastatic colorectal carcinoma are detailed in this manuscript.

Chromosomal Instability (CIN) Phenotype, CIN High or CIN Low, Predicts Survival for Colorectal Cancer

To examine whether chromosomal instability (CIN) phenotype, determined by the severity of CIN, can predict survival for stages II and III colorectal cancer (CRC). We determined microsatellite instability (MSI) and loss of heterozygosity (LOH) status in 1,103 patients (training [n = 845] and validation [n = 258] sets with stages II and III CRC). The LOH ratio was defined as the frequency of LOH in chromosomes 2p, 5q, 17p, and 18q. According to the LOH ratio, non-MSI high tumors were classified as CIN high (LOH ratio ≥ 33%) or CIN low (LOH ratio < 33%). CIN-high tumors were subclassified as CIN high (mild type; LOH ratio < 75%) or CIN high (severe type; LOH ratio ≥ 75%). We used microarrays to identify a gene signature that could classify the CIN phenotype and evaluated its ability to predict prognosis. CIN high showed the worst survival (P < .001), whereas there was no significant difference between CIN low and MSI high. CIN high (severe type) showed poorer survival than CIN high (mild type; P < .001). Multivariate analysis revealed that CIN phenotype was an independent risk factor for disease-free and overall survival, respectively, in both the training (P < .001 and P = .0155) and validation sets (P < .001 and P = .0076). Microarray analysis also revealed that survival was significantly poorer in those with the CIN-high than in the CIN-low gene signature (P = .0203). In a validation of 290 independent CRCs (GSE14333), the CIN-high gene signature showed significantly poorer survival than the CIN-low signature (P = .0047). The CIN phenotype is a predictive marker for survival and may be used to select high-risk patients with stages II and III CRC.

Abstract 1428: A CpG island methylator phenotype defines a clinically aggressive subgroup of posterior fossa ependymoma

Abstract Ependymoma is the third most common pediatric brain tumor and remains incurable in 45% of patients. It arises in the spinal cord, supratentorial brain, and most commonly in children, the posterior fossa (PF). We recently reported the identification of two molecularly and clinically distinct subgroups of PF ependymoma, which we named Group A and B. While patients with Group B tumors harbor a large number of gross chromosomal gains and losses (approx. 17 arm events per tumor) and have favorable prognoses (5 year PFS = 92%), patients with Group A tumors have balanced genomic profiles (approx. 1 arm event per tumor) with poor clinical outcomes (5 year PFS = 24%). We hypothesized that aberrant DNA methylation could be a mechanism driving the tumorigenesis of Group A PF ependymoma. To this end, we isolated methylated DNA in 92 ependymomas by Methyl Binding Domain 2 protein assisted recovery, and hybridized enriched DNA to promoter tiling arrays (Nimblegen). Using unsupervised hierarchical clustering we determined that the DNA methylation profiles of ependymoma were regionally specified, dividing tumors into subgroups according to their anatomical origin. Using both gene expression and DNA methylation platforms, we identified a subset of PF ependymoma, which clustered with spinal tumors, supporting the vast molecular differences between Group A and B PF ependymoma. We next compared the number of methylated genes identified in Group A versus B, and observed that Group A tumors exhibited a greater number of methylated genes at specific CpG islands, a feature described as a CpG island methylator phenotype (CIMP) in glioma, colon cancer, and breast cancer. We validated these findings in a non-overlapping cohort of 48 PF ependymomas, analyzed using a different array technology (Illumina Infinium 450K). Using various unsupervised clustering methods (HCL, K-MEANS, NMF, and SOM), we verified that Group A and B exhibited highly distinct DNA methylation profiles. Further, we confirmed that Group A tumours were defined by a greater overall number of methylated genes (A: 855, B: 233; Wilcoxon-Rank Sum Test), and a greater number of methylated genes per tumour (A: 511, B: 425; Wilcoxon-Rank Sum Test). We performed Gene Set Enrichment analysis and observed that many genes methylated in Group A exhibited a significant overlap with genes marked by the polycomb repressor (PRC2) complex in embryonic stem cells (p&amp;lt;0.0001, FDR&amp;lt;0.1%), a phenomenon seen in other cancer CIMPs. We propose two diverse mechanisms leading to tumourigenesis in Group A and B ependymoma. The greater number of chromosomal alterations in Group B suggests a Chromosomal Instability (CIN) phenotype, while the greater number of methylated CpG islands in Group A suggests a CpG island Methylator (CIMP) phenotype. Understanding these underlying mechanisms driving Group A and B pathogenesis may yield new leads for subgroup-specific treatments of PF ependymoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1428. doi:1538-7445.AM2012-1428

Abstract 4057: Quantitative assessment of 3D nuclear architecture in colon epithelial cells by micro-optical computed tomography

Abstract Colorectal cancer (CRC) is a commonly diagnosed cancer and the third leading cause of cancer-related deaths in the United States. About 50,000 people will die from this disease in 2011. CRCs develop through an accumulation of genetic and epigenetic aberrations and have been broadly classified into three molecular subtypes - chromosomal instability (CIN), microsatellite instability (MSI), and CpG Island Methylator Phenotype (CIMP). Research studies on CRC have predominantly focused on its molecular biological and genetic basis, but very little is known about the three-dimensional cell morphological variations between these subclasses. We investigated alterations in the three-dimensional (3D) nuclear architecture of the three molecular subtypes of CRC, comparing them to a benign adenoma, using representative epithelial cell line models: AAC1 (benign), AAC1-SB10 (CIN), HCT-116 (MSI) and RKO (CIMP). We applied micro-optical computed tomography (Cell-CT) to image one hundred individual hematoxylin-stained cells of each of the four types with submicron, isotropic spatial resolution. We performed automated 3D image analysis on the volumetric cell images, quantifying fifty descriptors (features) to characterize the nuclear morphology and coarse chromatin organization. We evaluated the statistical efficacy of our 3D features in distinguishing among the cell types. Our results demonstrated previously unreported morphological variations among the controls and three types of CRC. Relative to the benign adenoma cells, in contrast to CIN and MSI, the CIMP subtype exhibited markedly smaller cell and nuclear sizes, smaller nucleus-to-cytoplasm (NC) ratio and a predominantly smooth nuclear interior, lacking in clumpy chromatin. However, the CIMP cell nuclei exhibited the highest optical density among the four cell classes. MSI cells showed the clumpiest nuclear texture, followed by the benign adenoma cell nuclei. These results offer new perspectives on nuclear structure variations in benign colon adenoma and colorectal cancer cells, warranting further investigation. They suggest that the hypermethylation seen in the CIMP phenotype may result in demonstrable chromatin compaction with a concomitant reduction in nuclear size. The chromatin clumpiness observed in the MSI cells may reflect increased mutations at nuclear scaffold binding sites, which tend to be characterized by single and di-nucleotide repeats. We intend to pursue studies on human biopsy cells with ultimate goal of testing the efficacy of Cell-CT for CRC diagnosis, subtyping and staging. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4057. doi:1538-7445.AM2012-4057

Clinical aspect and molecular mechanism of DNA aneuploidy in gastric cancers

The biological characteristics of cancers depend mostly on genetic alterations in the cancer cells of individuals. Gastric cancers show a high frequency of DNA aneuploidy, a phenotype of chromosomal instability. Compared to diploid tumors, gastric carcinomas with aneuploidy have been shown to have high proliferative activity and high metastatic or invasive potential; these characteristics lead to a poor prognosis. It has been suggested that an abnormal spindle assembly checkpoint is involved in DNA aneuploidy, but the underlying mechanism is still unclear. This review, in order to determine whether gastric carcinomas that display aneuploidy are associated with a poorer prognosis than diploid tumors, and to discuss the biological mechanisms that induce aneuploidy, summarizes the results of studies on DNA ploidy in gastric cancer published in the English literature. Analysis of DNA ploidy in gastric cancer may provide clinically useful information from diagnostic, therapeutic, and prognostic standpoints. Further investigations may be needed to clarify the relationship between chromosome instability and DNA ploidy.

Mutability and mutational spectrum of chromosome transmission fidelity genes

It has been more than two decades since the original chromosome transmission fidelity (Ctf) screen of Saccharomyces cerevisiae was published. Since that time the spectrum of mutations known to cause Ctf and, more generally, chromosome instability (CIN) has expanded dramatically as a result of systematic screens across yeast mutant arrays. Here we describe a comprehensive summary of the original Ctf genetic screen and the cloning of the remaining complementation groups as efforts to expand our knowledge of the CIN gene repertoire and its mutability in a model eukaryote. At the time of the original screen, it was impossible to predict either the genes and processes that would be overrepresented in a pool of random mutants displaying a Ctf phenotype or what the entire set of genes potentially mutable to Ctf would be. We show that in a collection of 136 randomly selected Ctf mutants, >65% of mutants map to 13 genes, 12 of which are involved in sister chromatid cohesion and/or kinetochore function. Extensive screening of systematic mutant collections has shown that ~350 genes with functions as diverse as RNA processing and proteasomal activity mutate to cause a Ctf phenotype and at least 692 genes are required for faithful chromosome segregation. The enrichment of random Ctf alleles in only 13 of ~350 possible Ctf genes suggests that these genes are more easily mutable to cause genome instability than the others. These observations inform our understanding of recurring CIN mutations in human cancers where presumably random mutations are responsible for initiating the frequently observed CIN phenotype of tumors.

Cancer-associated alteration of pericentromeric heterochromatin may contribute to chromosome instability

Many tumors exhibit elevated chromosome mis-segregation termed chromosome instability (CIN), which is likely to be a potent driver of tumor progression and drug resistance. Causes of CIN are poorly understood but probably include prior genome tetraploidization, centrosome amplification and mitotic checkpoint defects. This study identifies epigenetic alteration of the centromere as a potential contributor to the CIN phenotype. The centromere controls chromosome segregation and consists of higher-order repeat (HOR) alpha-satellite DNA packaged into two chromatin domains: the kinetochore, harboring the centromere-specific H3 variant centromere protein A (CENP-A), and the pericentromeric heterochromatin, considered important for cohesion. Perturbation of centromeric chromatin in model systems causes CIN. As cancer cells exhibit widespread chromatin changes, we hypothesized that pericentromeric chromatin structure could also be affected, contributing to CIN. Cytological and chromatin immunoprecipitation and PCR (ChIP-PCR)-based analyses of HT1080 cancer cells showed that only one of the two HORs on chromosomes 5 and 7 incorporate CENP-A, an organization conserved in all normal and cancer-derived cells examined. Contrastingly, the heterochromatin marker H3K9me3 (trimethylation of H3 lysine 9) mapped to all four HORs and ChIP-PCR showed an altered pattern of H3K9me3 in cancer cell lines and breast tumors, consistent with a reduction on the kinetochore-forming HORs. The JMJD2B demethylase is overexpressed in breast tumors with a CIN phenotype, and overexpression of exogenous JMJD2B in cultured breast epithelial cells caused loss of centromere-associated H3K9me3 and increased CIN. These findings suggest that impaired maintenance of pericentromeric heterochromatin may contribute to CIN in cancer and be a novel therapeutic target.

A Novel Measure of Chromosome Instability Is An Independent Prognostic Factor in Multiple Myeloma

Abstract 1824 Introduction.Chromosomal abnormalities are prevalent in multiple myeloma (MM) and have been useful in delineating disease subtypes and prognosis groups. Globally, a dichotomous developmental pathway based on ploidy status seems to exist. The accumulation of chromosomal abnormalities in MM often gives rise to complex copy number profiles which are difficult to be captured due to the limitations of karyotyping in a relatively non-proliferative tumor. It is therefore unclear if genomic complexity as a reflection of chromosomal instability (CIN) is of biological and clinical relevance in MM. In this study, we introduce a novel measure of CIN, chromosome instability genome event count (CINGEC), validated its prognostic relevance in myeloma, and subsequently derived a gene expression (GEP) signature, CINGECS, investigated the gene contents of it to elucidate biological mechanisms related to CIN, and assessed its association with survival in the context of other gene expression based prognostic factors. Method.The rationale for CINGEC is that the more unstable is a genome, the more genome events does it harbor irrespective of the size of altered segments. Hence, we first obtain a copy number level sequence from a segmentation result of copy number profile signals from aCGH or SNP chip. We developed an algorithm to estimate the minimum number of genome events to account for the observed complex genome profiles. CINGEC is the sum of genome event counts from all autosomal chromosomes. The GEP signature, CINGECS, is derived from a public dataset that has both aCGH and GEP (GSE26849 and GSE26760, n=246) by performing differential gene expression analysis (SAM, p<0.001, q<0.001, fold-change 2) using GEP data between samples grouped into the top and bottom quartiles based on their CINGEC obtained from the analysis of aCGH data. The biological mechanisms distinctive between high and low CINGEC samples are assessed with biological pathways such as Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). Results.We first show that CINGEC is associated with MM patient survival by analyzing a previously published Mayo Clinic aCGH dataset (n=64, Chng WJ et al. Leukemia 2010; 24: 833–842). Furthermore, CINGEC is a stronger prognostic factor than another aCGH-based measure of CIN derived from breast cancer, the Genome Instability Index (Chin et al. Genome Biol 2007;8; R215). The CINGECS comprise of 160 differentially expressed genes with 144 up-regulated and 16 down-regulated. KEGG pathway and GO analysis of this set of genes showed enrichment by those involved in (1) cell cycle checkpoints and progression such as cell cycle, cell division, spindle organization, mitosis, (2) DNA damage responses such as response to DNA damage stimulus, DNA repair, nucleotide-excision repair, DNA gap filling, and (3) generic cancer related processes such as DNA replication, cell proliferation. Finally, CINGECS is shown to be significantly associated with poorer overall as well as progression free survival in both newly diagnosed (UAMS TT2 dataset, n=351, GSE 2658) and relapsed patients (Millenium Apex Dataset, n=188, GSE9782). Furthermore, it is found to be a significant prognostic signature independent of diverse GEP signatures known to be associated with survival for MM patients, including the Proliferation Index, the Centrosome Index, the UAMS 70-gene High-Risk Signature, the IFM High-Risk Signature, the IL-6 signature derived from cell lines, as well as GEP-based signatures of genomic instability derived from other cancers such as the CIN70 signature (Carter et al. Nat Genet 2006; 38:1043–1048) and CINSARC67 (Chibon et al. Nat Med 2010; 16: 781–787). Conclusions.We conclude that CINGEC can account for CIN with high resolution copy number profile data. Also, its corresponding GEP signature, CINGECS, potentially encompasses changes reflective of both cause and consequences of the CIN phenotype and is an independent prognostic signature in MM. Disclosures:Fonseca:Consulting:Genzyme, Medtronic, BMS, Amgen, Otsuka, Celgene, Intellikine, Lilly Research Support: Cylene, Onyz, Celgene: Consultancy, Research Funding.

TheIn VivoExpression of Radiation-Induced Chromosomal Instability Has an Inflammatory Mechanism

Ionizing radiation is unequivocally leukemogenic and carcinogenic, and this is generally attributed to DNA damage arising as a consequence of deposition of energy in the cell nucleus at the time of exposure. However, nontargeted effects, in which DNA damage is produced in nonirradiated cells as a consequence of cell signaling processes, indicate additional mechanisms. Radiation-induced chromosomal instability, a nontargeted effect with the potential to produce pathological consequences, is characterized by an increased rate of chromosome aberrations many generations after the initial insult. In this study, using a mouse model that has been well characterized with respect to its susceptibility to both radiation-induced chromosomal instability and acute myeloid leukemia, we investigated whether the underlying signaling mechanism was an inflammatory process by studying the effects of a nonsteroidal anti-inflammatory drug. Treated mice showed significant reduction in expression of the chromosomal instability phenotype 100 days postirradiation associated with reduced expression of inflammatory markers. The data support the hypothesis that the radiation-induced chromosomal instability phenotype is not an intrinsic property of the cells but a consequence of inflammatory processes having the potential to contribute secondary damage expressed as nontargeted and delayed radiation effects.

The Complete Spectrum of Yeast Chromosome Instability Genes Identifies Candidate CIN Cancer Genes and Functional Roles for ASTRA Complex Components

Chromosome instability (CIN) is observed in most solid tumors and is linked to somatic mutations in genome integrity maintenance genes. The spectrum of mutations that cause CIN is only partly known and it is not possible to predict a priori all pathways whose disruption might lead to CIN. To address this issue, we generated a catalogue of CIN genes and pathways by screening ∼2,000 reduction-of-function alleles for 90% of essential genes in Saccharomyces cerevisiae. Integrating this with published CIN phenotypes for other yeast genes generated a systematic CIN gene dataset comprised of 692 genes. Enriched gene ontology terms defined cellular CIN pathways that, together with sequence orthologs, created a list of human CIN candidate genes, which we cross-referenced to published somatic mutation databases revealing hundreds of mutated CIN candidate genes. Characterization of some poorly characterized CIN genes revealed short telomeres in mutants of the ASTRA/TTT components TTI1 and ASA1. High-throughput phenotypic profiling links ASA1 to TTT (Tel2-Tti1-Tti2) complex function and to TORC1 signaling via Tor1p stability, consistent with the role of TTT in PI3-kinase related kinase biogenesis. The comprehensive CIN gene list presented here in principle comprises all conserved eukaryotic genome integrity pathways. Deriving human CIN candidate genes from the list allows direct cross-referencing with tumor mutational data and thus candidate mutations potentially driving CIN in tumors. Overall, the CIN gene spectrum reveals new chromosome biology and will help us to understand CIN phenotypes in human disease.

Abstract 3884: DNA mismatch repair defects, MSI, does not play major role in colon carcinogenesis in young African Americans

Abstract Back ground: African Americans (AAs) are known to display a higher rate and even earlier age of colorectal cancer (CRC) than in the general population. Genetically, 10-12% microsatellite instability (MSI) cases involved in CRC development. However, if it occurs below the age of 50 it could be suspicious of Lynch syndrome. Aims and methods: We aimed to assess the role of MSI in pathogenesis of sporadic colorectal cancer in young African American. The CRC patients in this study were less than 50 years and referred to Howard University Hospital from 2005 to 2008. The medical records (n=50) as well as the pathology reports were reviewed. The MSI status was assessed in a multiplex PCR targeting 5 markers: BAT25, BAT26, NR21, NR27 and NR24. The PCR products were analysed in an Applied Biosystems Gene Scan 3130 machine. Samples with two or more instable markers were labeled MSI-H, with one instable marker: MSI-L and no stable marker: MSS. The presence or absence of expression of four DNA MMR proteins: MLH1, PMS2, MSH2 and MSH6 were evaluated using immunohistochemistry on a Tissue Microarray (TMA, n=26, duplicate) consisting (stage1: 3; stage 2: 6; stage3: 5; stage4: 4).TMA IHC staining were read by two different pathologists who were not aware of the patient clinical status and were scored thus in an objective manner. Two patients were MSI-L and one MSI-H. The response to 5-FU based therapy was reported. Results: Twenty six patients [age, median: 44.5; age range: 26-49; male, 17 (65.4%)] were enrolled in the study. Seven out of 13 available data had family history of CRC. Fourteen CRC cases were left sided. MSI analysis revealed that out of 24 patients: one was MSI-H and two were MIS-L and the rest were MSS. The MSI high patient and one of the MSI low had family history of CRC. MLH1, PMS2, MSH2 and MSH6 were not expressed in 7 (26.9%), 1 (3.8%), 0 (0%) and 5 (19.2%) of tumors, respectively. IHC confirmed the MSI-H tumor. There was a recurrence with lung and brain metastases at 72 months in one of the MSI-L patients following adjuvant chemotherapy with 5 Fluorouracil and Leucovorin. Discussion: Our results show that MSI is not a major pathway in tumorgenesis of sporadic CRCs in young AAs and we may need to study chromosomal instability or CpG Island methylator phenotype as alternative pathways which occur in African Americans and make them present the disease at younger age and advanced stage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3884. doi:10.1158/1538-7445.AM2011-3884

Rad52 function prevents chromosome loss and truncation in Candida albicans

RAD52 is required for almost all recombination events in Saccharomyces cerevisiae. We took advantage of the heterozygosity of HIS4 in the Candida albicans SC5314 lineage to study the role of Rad52 in the genomic stability of this important fungal pathogen. The rate of loss of heterozygosity (LOH) at HIS4 in rad52-ΔΔ strains was ∼10(-3) , at least 100-fold higher than in Rad52(+) strains. LOH of whole chromosome 4 or truncation of the homologue that carries the functional HIS4 allele was detected in all 80 rad52-ΔΔ His auxotrophs (GLH -GL lab His(-)) obtained from six independent experiments. Isolates that had undergone whole chromosome LOH, presumably due to loss of chromosome, carried two copies of the remaining homologue. Isolates with truncations carried centric fragments of broken chromosomes healed by de novo telomere addition. GLH strains exhibited variable degrees of LOH across the genome, including two strains that became homozygous for all the heterozygous markers tested. In addition, GLH strains exhibited increased chromosomal instability (CIN), which was abolished by reintroduction of RAD52. CIN of GLH isolates is reminiscent of genomic alterations leading to cancer in human cells, and support the mutator hypothesis in which a mutator mutation or CIN phenotype facilitate more mutations/aneuploidies.

Oncogenèse colorectale

Recent progress in the field of molecular biology has allowed us to identify at least two different molecular mechanisms implicated in colorectal carcinogenesis (CRC): chromosomal instability (CIN) and genetic instability. Even though the two molecular mechanisms differ, their signalling pathways, implicated in malignant transformation of colonic epithelial cells, appear to be similar. The most frequent group of CRC, which represents 80% of sporadic CRC, is characterized by allelic losses on the short arm of chromosome 17 and 8 and on the long arm of chromosome 5, 18 and 22. These allelic losses are associated with mutations in TP53, APC, SMAD2 and SMAD4 genes. All of these alterations are grouped under the phenotype CIN. A genetic instability termed MSI (microsatellite instability), which results from a mismatch repair (MMR) deficiency, appears in 12-15% of CRC cases. The presence of MMR deficiency leads to the accumulation of mutations in genes controlling cell cycle and apoptosis (TGFBRII, BAX or CASPASE5). More recently, the existence of a third phenotype was suggested. The main alteration associated with this group of tumors is the hypermethylation of the promoter region of numerous genes, leading to their inactivation. An activating mutation of BRAF is frequently associated with this phenotype. As described above, CRC shows genetic heterogeneity, however the consequences in terms of signalling pathway alterations are similar. For example, the activation of Wnt signalling pathways can result from the inactivation of the APC gene in the CIN phenotype or from an activating mutation in the β-catenin gene in MSI tumors. The inactivation of TGFβ pathways is also present in both tumor types and is driven by SMAD4, and more rarely by a SMAD2 inactivating mutation in CIN tumors, or by the existence of a frame-shift mutation occurring in a polyG coding track of the TGFβ (transforming growth factor) receptor type II in MSI tumors. The RAS-MAP kinase pathway is activated by KRAS mutations in CIN tumors or by BRAF mutations in MSI tumors. The p53 pathway is inactivated by TP53 inactivation in CIN tumors or by BAX inactivating mutations in MSI tumors.

Abstract B52: Predictor of distinct genetic alterations in African Americans with colon cancer by genomic analysis

Abstract Purpose: Chromosomal instability (CIN), microsatellite instability (MIN), and CpG island methylation phenotype (CIMP) are known processes through which colorectal cancer (CRC) can occur. In a given tumor, one or more of these events might be responsible for tumor development. We previously reported the results of our MSI and CIMP analysis of colorectal tumors in African Americans (AA), a group at high risk. Here, we report our array comparative genomic hybridization (aCGH) population analysis of AA and Caucasian CRC and its implication for the understanding of CRC development in this population. Experimental design: We used 30 AA and 22 Caucasian CRC patients and applied aCGH (105k chip) to identify copy number aberrations in samples. In addition, we did a population comparative analysis with aCGH data in Caucasians as well as with a widely publicized list of colon cancer candidate genes (CAN genes). Bioinformatics analysis was done to calculate the chromosomal imbalance using Agilent Genomic Workbench 5.0. Results: There was an average of 30 aberrations per patient with all samples displaying some level of chromosomal instability. Analysis of DNA copy number of frequently altered chromosomes revealed that deletions occurred primarily in chromosomes 4, 8 and 18. Chromosomal duplications occurred in more than 50% of cases on chromosomes 7, 8, 13, 20 and X. The CIN profile showed some differences when compared to alterations in Caucasians. Conclusions: Prominent chromosome X amplification in male patients and chromosomes 4, 8 and 18 deletions were the primary targets in AAs. Most CAN genes were altered at high frequencies in AAs with EXOC4, EPHB6, GNAS, MLL3 and TBX22 as the most frequently deleted genes and HAPLN1, ADAM29, SMAD2 and SMAD4 as the most frequently amplified genes. CIN seems to be more prominent in AAs and may play a distinctive role in CRC development in this population. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B52.