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

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

Phospholipase C Isozymes Are Deregulated in Colorectal Cancer – Insights Gained from Gene Set Enrichment Analysis of the Transcriptome

Colorectal cancer (CRC) is one of the most common cancer types in developed countries. To identify molecular networks and biological processes that are deregulated in CRC compared to normal colonic mucosa, we applied Gene Set Enrichment Analysis to two independent transcriptome datasets, including a total of 137 CRC and ten normal colonic mucosa samples. Eighty-two gene sets as described by the Kyoto Encyclopedia of Genes and Genomes database had significantly altered gene expression in both datasets. These included networks associated with cell division, DNA maintenance, and metabolism. Among signaling pathways with known changes in key genes, the “Phosphatidylinositol signaling network”, comprising part of the PI3K pathway, was found deregulated. The downregulated genes in this pathway included several members of the Phospholipase C protein family, and the reduced expression of two of these, PLCD1 and PLCE1, were successfully validated in CRC biopsies (n = 70) and cell lines (n = 19) by quantitative analyses. The repression of both genes was found associated with KRAS mutations (P = 0.005 and 0.006, respectively), and we observed that microsatellite stable carcinomas with reduced PLCD1 expression more frequently had TP53 mutations (P = 0.002). Promoter methylation analyses of PLCD1 and PLCE1 performed in cell lines and tumor biopsies revealed that methylation of PLCD1 can contribute to reduced expression in 40% of the microsatellite instable carcinomas. In conclusion, we have identified significantly deregulated pathways in CRC, and validated repression of PLCD1 and PLCE1 expression. This illustrates that the GSEA approach may guide discovery of novel biomarkers in cancer

DNA Sequence Profiles of the Colorectal Cancer Critical Gene Set KRAS-BRAF-PIK3CA-PTEN-TP53 Related to Age at Disease Onset

The incidence of colorectal cancer (CRC) increases with age and early onset indicates an increased likelihood for genetic predisposition for this disease. The somatic genetics of tumor development in relation to patient age remains mostly unknown. We have examined the mutation status of five known cancer critical genes in relation to age at diagnosis, and compared the genomic complexity of tumors from young patients without known CRC syndromes with those from elderly patients. Among 181 CRC patients, stratified by microsatellite instability status, DNA sequence changes were identified in KRAS (32%), BRAF (16%), PIK3CA (4%), PTEN (14%) and TP53 (51%). In patients younger than 50 years (n = 45), PIK3CA mutations were not observed and TP53 mutations were more frequent than in the older age groups. The total gene mutation index was lowest in tumors from the youngest patients. In contrast, the genome complexity, assessed as copy number aberrations, was highest in tumors from the youngest patients. A comparable number of tumors from young (<50 years) and old patients (>70 years) was quadruple negative for the four predictive gene markers (KRAS-BRAF-PIK3CA-PTEN); however, 16% of young versus only 1% of the old patients had tumor mutations in PTEN/PIK3CA exclusively. This implies that mutation testing for prediction of EGFR treatment response may be restricted to KRAS and BRAF in elderly (>70 years) patients. Distinct genetic differences found in tumors from young and elderly patients, whom are comparable for known clinical and pathological variables, indicate that young patients have a different genetic risk profile for CRC development than older patients

Improved prognostication of glioblastoma beyond molecular subtyping by transcriptional profiling of the tumor microenvironment

Glioblastoma (GBM), the most aggressive form of brain cancer, is characterized by a high level of molecular heterogeneity, and infiltration by various immune and stromal cell populations. Important advances have been made in deciphering the microenvironment of GBMs, but its association with existing molecular subtypes and its potential prognostic role remain elusive. We have investigated the abundance of infiltrating immune and stromal cells in silico, from gene expression profiles. Two cohorts, including in-house normal brain and glioma samples (n = 70) and a large sample set from TCGA (n = 393), were combined into a single exploratory dataset. A third independent cohort (n = 124) was used for validation. Tumors were clustered based on their microenvironment infiltration profiles, and associations with known GBM molecular subtypes and patient outcome were tested a posteriori in a multivariable setting. We identified a subset of GBM samples with significantly higher abundances of most immune and stromal cell populations. This subset showed increased expression of both immune suppressor and immune effector genes compared to other GBMs and was enriched for the mesenchymal molecular subtype. Survival analyses suggested that tumor microenvironment infiltration pattern was an independent prognostic factor for GBM patients. Among all, patients with the mesenchymal subtype with low immune and stromal infiltration had the poorest survival. By combining molecular subtyping with gene expression measures of tumor infiltration, the present work contributes with improving prognostic models in GBM

Protein expression of BIRC5, TK1, and TOP2A in malignant peripheral nerve sheath tumours - A prognostic test after surgical resection.

No consensus treatment regime exists beyond surgery for malignant peripheral nerve sheath tumours (MPNST), and the purpose of the present study was to find new approaches to stratify patients with good and poor prognosis and to better guide therapeutic intervention for this aggressive soft tissue cancer. From a total of 67 MPNSTs from Scandinavian patients with and without neurofibromatosis type 1, 30 MPNSTs were investigated by genome-wide RNA expression profiling and 63 MPNSTs by immunohistochemical (IHC) analysis, and selected genes were submitted to analyses of disease-specific survival. The potential drug target genes survivin (BIRC5), thymidine kinase 1 (TK1), and topoisomerase 2-alpha (TOP2A), all encoded on chromosome arm 17q, were up-regulated in MPNST as compared to benign neurofibromas. Each of them was found to be independent prognostic markers on the gene expression level, as well as on the protein level. A prognostic profile was identified by combining the nuclear expression scores of the three proteins. For patients with completely resected tumours only 15% in the high risk group were alive after two years, as compared to 78% in the low risk group. In conclusion, we found a novel protein expression profile which identifies MPNST patients with inferior prognosis even after assumed curative surgery. The tested proteins are drug targets; therefore the expression profile may provide predictive information guiding the design of future clinical trials. Importantly, as the effect is seen on the protein level using IHC, the biomarker panel can be readily implemented in routine clinical testing