Transcriptome instability in colorectal cancer identified by exon microarray analyses: Associations with splicing factor expression levels and patient survival

Background Colorectal cancer (CRC) is a heterogeneous disease that, on the molecular level, can be characterized by inherent genomic instabilities; chromosome instability and microsatellite instability. In the present study we analyze genome-wide disruption of pre-mRNA splicing, and propose transcriptome instability as a characteristic that is analogous to genomic instability on the transcriptome level. Methods Exon microarray profiles from two independent series including a total of 160 CRCs were investigated for their relative amounts of exon usage differences. Each exon in each sample was assigned an alternative splicing score calculated by the FIRMA algorithm. Amounts of deviating exon usage per sample were derived from exons with extreme splicing scores. Results There was great heterogeneity within both series in terms of sample-wise amounts of deviating exon usage. This was strongly associated with the expression levels of approximately half of 280 splicing factors (54% and 48% of splicing factors were significantly correlated to deviating exon usage amounts in the two series). Samples with high or low amounts of deviating exon usage, associated with overall transcriptome instability, were almost completely separated into their respective groups by hierarchical clustering analysis of splicing factor expression levels in both sample series. Samples showing a preferential tendency towards deviating exon skipping or inclusion were associated with skewed transcriptome instability. There were significant associations between transcriptome instability and reduced patient survival in both sample series. In the test series, patients with skewed transcriptome instability showed the strongest prognostic association (P = 0.001), while a combination of the two characteristics showed the strongest association with poor survival in the validation series (P = 0.03). Conclusions We have described transcriptome instability as a characteristic of CRC. This transcriptome instability has associations with splicing factor expression levels and poor patient survival

Optimality of broken extremals

In this paper we analyse the optimality of broken Pontryagin extremal for an n-dimensional affine control system with a control parameter, taking values in a k- dimensional closed ball. We prove the optimality of broken normal extremals when n = 3 and the controllable vector fields form a contact distribution, and when the Lie algebra of the controllable fields is locally orthogonal to the singular locus and the drift does not belong to it. Moreover, if k = 2, we show the optimality of any broken extremal even abnormal when the controllable fields do not form a contact distribution in the point of singularity.Comment: arXiv admin note: text overlap with arXiv:1610.0675

Gene methylation profiles of normal mucosa, and benign and malignant colorectal tumors identify early onset markers

<p>Abstract</p> <p>Background</p> <p>Multiple epigenetic and genetic changes have been reported in colorectal tumors, but few of these have clinical impact. This study aims to pinpoint epigenetic markers that can discriminate between non-malignant and malignant tissue from the large bowel, i.e. markers with diagnostic potential.</p> <p>The methylation status of eleven genes (<it>ADAMTS1</it>, <it>CDKN2A</it>, <it>CRABP1</it>, <it>HOXA9</it>, <it>MAL</it>, <it>MGMT</it>, <it>MLH1</it>, <it>NR3C1</it>, <it>PTEN</it>, <it>RUNX3</it>, and <it>SCGB3A1</it>) was determined in 154 tissue samples including normal mucosa, adenomas, and carcinomas of the colorectum. The gene-specific and widespread methylation status among the carcinomas was related to patient gender and age, and microsatellite instability status. Possible CIMP tumors were identified by comparing the methylation profile with microsatellite instability (MSI), <it>BRAF</it>-, <it>KRAS</it>-, and <it>TP53 </it>mutation status.</p> <p>Results</p> <p>The mean number of methylated genes per sample was 0.4 in normal colon mucosa from tumor-free individuals, 1.2 in mucosa from cancerous bowels, 2.2 in adenomas, and 3.9 in carcinomas. Widespread methylation was found in both adenomas and carcinomas. The promoters of <it>ADAMTS1</it>, <it>MAL</it>, and <it>MGMT </it>were frequently methylated in benign samples as well as in malignant tumors, independent of microsatellite instability. In contrast, normal mucosa samples taken from bowels without tumor were rarely methylated for the same genes. Hypermethylated <it>CRABP1, MLH1</it>, <it>NR3C1</it>, <it>RUNX3</it>, and <it>SCGB3A1 </it>were shown to be identifiers of carcinomas with microsatellite instability. In agreement with the CIMP concept, MSI and mutated <it>BRAF </it>were associated with samples harboring hypermethylation of several target genes.</p> <p>Conclusion</p> <p>Methylated <it>ADAMTS1</it>, <it>MGMT</it>, and <it>MAL </it>are suitable as markers for early tumor detection.</p

The loss of NKX3.1 expression in testicular – and prostate – cancers is not caused by promoter hypermethylation

BACKGROUND: Recent studies have demonstrated that the NKX3.1 protein is commonly down-regulated in testicular germ cell tumors (TGCTs) and prostate carcinomas. The homeobox gene NKX3.1 maps to chromosome band 8p21, which is a region frequently lost in prostate cancer, but not in TGCT. Mutations have not been reported in the NKX3.1 sequence, and the gene is hypothesized to be epigenetically inactivated. In the present study we examined the methylation status of the NKX3.1 promoter in relevant primary tumors and cell lines: primary TGCTs (n = 55), intratubular germ cell neoplasias (n = 7), germ cell tumor cell lines (n = 3), primary prostate adenocarcinomas (n = 20), and prostate cancer cell lines (n = 3) by methylation-specific PCR and bisulphite sequencing. RESULTS AND CONCLUSIONS: Down-regulation of NKX3.1 expression was generally not caused by promoter hypermethylation, which was only found in one TGCT. However, other epigenetic mechanisms, such as modulation of chromatin structure or modifications of histones, may explain the lack of NKX3.1 expression, which is seen in most TGCTs and prostate cancer specimens

Distinct high resolution genome profiles of early onset and late onset colorectal cancer integrated with gene expression data identify candidate susceptibility loci

<p>Abstract</p> <p>Background</p> <p>Estimates suggest that up to 30% of colorectal cancers (CRC) may develop due to an increased genetic risk. The mean age at diagnosis for CRC is about 70 years. Time of disease onset 20 years younger than the mean age is assumed to be indicative of genetic susceptibility. We have compared high resolution tumor genome copy number variation (CNV) (Roche NimbleGen, 385 000 oligo CGH array) in microsatellite stable (MSS) tumors from two age groups, including 23 young at onset patients without known hereditary syndromes and with a median age of 44 years (range: 28-53) and 17 elderly patients with median age 79 years (range: 69-87). Our aim was to identify differences in the tumor genomes between these groups and pinpoint potential susceptibility loci. Integration analysis of CNV and genome wide mRNA expression data, available for the same tumors, was performed to identify a restricted candidate gene list.</p> <p>Results</p> <p>The total fraction of the genome with aberrant copy number, the overall genomic profile and the <it>TP53 </it>mutation spectrum were similar between the two age groups. However, both the number of chromosomal aberrations and the number of breakpoints differed significantly between the groups. Gains of 2q35, 10q21.3-22.1, 10q22.3 and 19q13.2-13.31 and losses from 1p31.3, 1q21.1, 2q21.2, 4p16.1-q28.3, 10p11.1 and 19p12, positions that in total contain more than 500 genes, were found significantly more often in the early onset group as compared to the late onset group. Integration analysis revealed a covariation of DNA copy number at these sites and mRNA expression for 107 of the genes. Seven of these genes, <it>CLC, EIF4E</it>, <it>LTBP4, PLA2G12A, PPAT</it>, <it>RG9MTD2</it>, and <it>ZNF574</it>, had significantly different mRNA expression comparing median expression levels across the transcriptome between the two groups.</p> <p>Conclusions</p> <p>Ten genomic loci, containing more than 500 protein coding genes, are identified as more often altered in tumors from early onset versus late onset CRC. Integration of genome and transcriptome data identifies seven novel candidate genes with the potential to identify an increased risk for CRC.</p

Hypermethylated MAL gene – a silent marker of early colon tumorigenesis

Background Tumor-derived aberrantly methylated DNA might serve as diagnostic biomarkers for cancer, but so far, few such markers have been identified. The aim of the present study was to investigate the potential of the MAL (T-cell differentiation protein) gene as an early epigenetic diagnostic marker for colorectal tumors. Methods Using methylation-specific polymerase chain reaction (MSP) the promoter methylation status of MAL was analyzed in 218 samples, including normal mucosa (n = 44), colorectal adenomas (n = 63), carcinomas (n = 65), and various cancer cell lines (n = 46). Direct bisulphite sequencing was performed to confirm the MSP results. MAL gene expression was investigated with real time quantitative analyses before and after epigenetic drug treatment. Immunohistochemical analysis of MAL was done using normal colon mucosa samples (n = 5) and a tissue microarray with 292 colorectal tumors. Results Bisulphite sequencing revealed that the methylation was unequally distributed within the MAL promoter and by MSP analysis a region close to the transcription start point was shown to be hypermethylated in the majority of colorectal carcinomas (49/61, 80%) as well as in adenomas (45/63, 71%). In contrast, only a minority of the normal mucosa samples displayed hypermethylation (1/23, 4%). The hypermethylation of MAL was significantly associated with reduced or lost gene expression in in vitro models. Furthermore, removal of the methylation re-induced gene expression in colon cancer cell lines. Finally, MAL protein was expressed in epithelial cells of normal colon mucosa, but not in the malignant cells of the same type. Conclusion Promoter hypermethylation of MAL was present in the vast majority of benign and malignant colorectal tumors, and only rarely in normal mucosa, which makes it suitable as a diagnostic marker for early colorectal tumorigenesis

A universal assay for detection of oncogenic fusion transcripts by oligo microarray analysis

<p>Abstract</p> <p>Background</p> <p>The ability to detect neoplasia-specific fusion genes is important not only in cancer research, but also increasingly in clinical settings to ensure that correct diagnosis is made and the optimal treatment is chosen. However, the available methodologies to detect such fusions all have their distinct short-comings.</p> <p>Results</p> <p>We describe a novel oligonucleotide microarray strategy whereby one can screen for all known oncogenic fusion transcripts in a single experiment. To accomplish this, we combine measurements of chimeric transcript junctions with exon-wise measurements of individual fusion partners. To demonstrate the usefulness of the approach, we designed a DNA microarray containing 68,861 oligonucleotide probes that includes oligos covering all combinations of chimeric exon-exon junctions from 275 pairs of fusion genes, as well as sets of oligos internal to all the exons of the fusion partners. Using this array, proof of principle was demonstrated by detection of known fusion genes (such as <it>TCF3:PBX1</it>, <it>ETV6:RUNX1</it>, and <it>TMPRSS2:ERG</it>) from all six positive controls consisting of leukemia cell lines and prostate cancer biopsies.</p> <p>Conclusion</p> <p>This new method bears promise of an important complement to currently used diagnostic and research tools for the detection of fusion genes in neoplastic diseases.</p

Cysteine-Rich Secretory Protein-3 (CRISP3) Is Strongly Up-Regulated in Prostate Carcinomas with the TMPRSS2-ERG Fusion Gene

A large percentage of prostate cancers harbor TMPRSS2-ERG gene fusions, leading to aberrant overexpression of the transcription factor ERG. The target genes deregulated by this rearrangement, however, remain mostly unknown. To address this subject we performed genome-wide mRNA expression analysis on 6 non-malignant prostate samples and 24 prostate carcinomas with (n = 16) and without (n = 8) TMPRSS2-ERG fusion as determined by FISH. The top-most differentially expressed genes and their associations with ERG over-expression were technically validated by quantitative real-time PCR and biologically validated in an independent series of 200 prostate carcinomas. Several genes encoding metabolic enzymes or extracellular/transmembrane proteins involved in cell adhesion, matrix remodeling and signal transduction pathways were found to be co-expressed with ERG. Within those significantly over-expressed in fusion-positive carcinomas, CRISP3 showed more than a 50-fold increase when compared to fusion-negative carcinomas, whose expression levels were in turn similar to that of non-malignant samples. In the independent validation series, ERG and CRISP3 mRNA levels were strongly correlated (rs = 0.65, p<0.001) and both were associated with pT3 disease staging. Furthermore, immunohistochemistry results showed CRISP3 protein overexpression in 63% of the carcinomas and chromatin immunoprecipitation with an anti-ERG antibody showed that CRISP3 is a direct target of the transcription factor ERG. We conclude that ERG rearrangement is associated with significant expression alterations in genes involved in critical cellular pathways that define a subset of locally advanced PCa. In particular, we show that CRISP3 is a direct target of ERG that is strongly overexpressed in PCa with the TMPRSS2-ERG fusion gene

Identification of an epigenetic biomarker panel with high sensitivity and specificity for colorectal cancer and adenomas

Background The presence of cancer-specific DNA methylation patterns in epithelial colorectal cells in human feces provides the prospect of a simple, non-invasive screening test for colorectal cancer and its precursor, the adenoma. This study investigates a panel of epigenetic markers for the detection of colorectal cancer and adenomas. Methods Candidate biomarkers were subjected to quantitative methylation analysis in test sets of tissue samples from colorectal cancers, adenomas, and normal colonic mucosa. All findings were verified in independent clinical validation series. A total of 523 human samples were included in the study. Receiver operating characteristic (ROC) curve analysis was used to evaluate the performance of the biomarker panel. Results Promoter hypermethylation of the genes CNRIP1, FBN1, INA, MAL, SNCA, and SPG20 was frequent in both colorectal cancers (65-94%) and adenomas (35-91%), whereas normal mucosa samples were rarely (0-5%) methylated. The combined sensitivity of at least two positives among the six markers was 94% for colorectal cancers and 93% for adenoma samples, with a specificity of 98%. The resulting areas under the ROC curve were 0.984 for cancers and 0.968 for adenomas versus normal mucosa. Conclusions The novel epigenetic marker panel shows very high sensitivity and specificity for both colorectal cancers and adenomas. Our findings suggest this biomarker panel to be highly suitable for early tumor detection

Systematic bioinformatic analysis of expression levels of 17,330 human genes across 9,783 samples from 175 types of healthy and pathological tissues

Our knowledge on tissue- and disease-specific functions of human genes is rather limited and highly context-specific. Here, we have developed a method for the comparison of mRNA expression levels of most human genes across 9,783 Affymetrix gene expression array experiments representing 43 normal human tissue types, 68 cancer types, and 64 other diseases. This database of gene expression patterns in normal human tissues and pathological conditions covers 113 million datapoints and is available from the GeneSapiens website