CLC and IFNAR1 are differentially expressed and a global immunity score is distinct between early- and late-onset colorectal cancer.

Colorectal cancer (CRC) incidence increases with age, and early onset of the disease is an indication of genetic predisposition, estimated to cause up to 30% of all cases. To identify genes associated with early-onset CRC, we investigated gene expression levels within a series of young patients with CRCs who are not known to carry any hereditary syndromes (n=24; mean 43 years at diagnosis), and compared this with a series of CRCs from patients diagnosed at an older age (n=17; mean 79 years). Two individual genes were found to be differentially expressed between the two groups, with statistical significance; CLC was higher and IFNAR1 was less expressed in early-onset CRCs. Furthermore, genes located at chromosome band 19q13 were found to be enriched significantly among the genes with higher expression in the early-onset samples, including CLC. An elevated immune content within the early-onset group was observed from the differentially expressed genes. By application of outlier statistics, H3F3A was identified as a top candidate gene for a subset of the early-onset CRCs. In conclusion, CLC and IFNAR1 were identified to be overall differentially expressed between early- and late-onset CRC, and are important in the development of early-onset CRC.

The testicular germ cell tumour transcriptome.

Testicular germ cell tumours (TGCTs) are characterized by young age of onset and a complex pattern of histological subtypes. Transcriptomic studies have tried to uncover the gene expression patterns underlying this. Here, we present a systematic review of transcriptome studies of TGCTs of adolescents and young adults and identify genes common across the various studies, both for TGCTs in general as well as the histological subtypes, hence elucidating both transcriptional changes associated with malignant transformation and differentiation patterns. A meta-analysis of this type adds power and significance to the genes thus found, where most studies have included only a limited number of samples. Both known (KRAS, MYCN and TPD52) and novel (CCT6A, IGFBP3 and SALL2) cancer genes are implicated in TGC tumorigenesis. Gene expression patterns characteristic to embryonic stem cells are also found deregulated in TGC tumorigenesis. This is reflected in how pluripotent embryonal carcinoma cells commonly differentiate into a variety of embryonic and extra-embryonic histological types, each with unique transcriptomes. The embryonal carcinomas in particular are found to overexpress pluripotency genes, while gene signatures for seminomas, teratomas and yolk sac tumours were also identified. This underlines the distinctive transcriptomic programme across histological subtypes, especially striking given that the TGCT genome is largely similar across the same subtypes.

Frequent promoter hypermethylation of the O6-Methylguanine-DNA Methyltransferase (MGMT) gene in testicular cancer.

Testicular germ cell tumours are classified into two major histological subgroups, seminomas and nonseminomas. All tumours display several recurrent chromosomal aberrations, but few target genes have been identified. Previous studies have shown that genome-wide hypermethylation of CpG islands is significantly more prevalent in nonseminomas than in seminomas. We have studied two potential target genes in testicular cancer. A series of 70 tumours were analysed for methylation of CpG sites in the O(6)-methylguanine-DNA methyltransferase (MGMT) gene promoter, and in exon 1alpha of the cyclin-dependent kinase inhibitor 2A gene (CDKN2A). In addition, eight microsatellite markers within and flanking these genes at chromosome arms 10q and 9p, respectively, were analysed for allelic imbalances. Allele alterations were frequently seen at 9p loci (47 out of 70, 67%), but none of the tumours (none out of 55) showed methylation of CDKN2A. On the other hand, a high frequency of MGMT promoter methylation (32 out of 69, 46%) was found, as well as allelic imbalances at 10q markers (50 out of 70, 71%). A significantly higher methylation frequency was found in nonseminomas (24 out of 35, 69%) compared to seminomas (eight out of 33, 24%) (P=0.0003, Fisher's exact test). Immunohistochemical analysis of the MGMT protein in a subgroup (n=20) of the testicular tumours supported the hypothesis of gene silencing being the functional consequence of the promoter methylation. In summary, our data suggest that inactivation of MGMT contributes to development of nonseminomatous testicular cancer.

Epigenetic and genetic features of 24 colon cancer cell lines.

Cell lines are invaluable biomedical research tools, and recent literature has emphasized the importance of genotype authentication and characterization. In the present study, 24 out of 27 cell line identities were confirmed by short tandem repeat profiling. The molecular phenotypes of the 24 colon cancer cell lines were examined, and microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) were determined, using the Bethesda panel mononucleotide repeat loci and two epimarker panels, respectively. Furthermore, the BRAF, KRAS and PIK3CA oncogenes were analyzed for mutations in known hotspots, while the entire coding sequences of the PTEN and TP53 tumor suppressors were investigated. Nine cell lines showed MSI. Thirteen and nine cell lines were found to be CIMP positive, using the Issa panel and the Weisenberger et al. panel, respectively. The latter was found to be superior for CIMP classification of colon cancer cell lines. Seventeen cell lines harbored disrupting TP53 mutations. Altogether, 20/24 cell lines had the mitogen-activated protein kinase pathway activating mutually exclusive KRAS or BRAF mutations. PIK3CA and PTEN mutations leading to hyperactivation of the phosphoinositide 3-kinase/AKT pathway were observed in 13/24 cell lines. Interestingly, in four cell lines there were no mutations in neither BRAF, KRAS, PIK3CA nor in PTEN. In conclusion, this study presents molecular features of a large number of colon cancer cell lines to aid the selection of suitable in vitro models for descriptive and functional research.

SPG20, a novel biomarker for early detection of colorectal cancer, encodes a regulator of cytokinesis.

Colorectal cancer is a common disease with high mortality. Suitable biomarkers for detection of tumors at an early curable stage would significantly improve patient survival. Here, we show that the SPG20 (spastic paraplegia-20) promoter, encoding the multifunctional Spartin protein, is hypermethylated in 89% of colorectal carcinomas, 78% of adenomas and only 1% of normal mucosa samples. SPG20 methylation was also present in a pilot series of stool samples and corresponding tumors from colorectal cancer patients. SPG20 promoter hypermethylation resulted in loss of mRNA expression in various cancer types and subsequent depletion of Spartin. We further showed that Spartin downregulation in cancer cells resulted in cytokinesis arrest, which was reversed when SPG20 methylation was inhibited. The present study identifies SPG20 promoter hypermethylation as a biomarker suitable for non-invasive detection of colorectal cancer, and a possible mechanism for cytokinesis arrest in colorectal tumorigenesis.

Novel epigenetically deregulated genes in testicular cancer include homeobox genes and SCGB3A1 (HIN-1).

Testicular germ cell tumours (TGCTs) are classified into two main histological subgroups: seminomas and non-seminomas. The latter comprise several subtypes: embryonal carcinomas, yolk sac tumours, choriocarcinomas, and teratomas. These embryonal and extra-embryonal-like differentiation lineages represent a caricature of early normal development, and inactivation of gene expression through promoter hypermethylation may therefore be of particular importance in germ cell tumourigenesis. The promoter methylation status of ten candidate genes-CDH13, DLX6, EMX2, HOXA9, HOXB5, MSX1, MSX2, RASSF1A, RUNX3, and SCGB3A1 (alias HIN-1)-was assessed by methylation-specific PCR in seven intratubular germ cell neoplasias and 55 primary TGCTs. Furthermore, by a discovery-based global approach, comparing cDNA microarray expression profiles of two germ cell tumour cell lines before and after treatment with the demethylating agent 5-aza-2'-deoxycytidine, a gene list of potentially epigenetic targets was identified, from which CGGBP1, CGRRF1, SMARCC2, SORBS1, and XPA were analysed further. Overall, the non-seminomas were significantly more often methylated than were seminomas (p < 0.001). The three most frequently methylated genes among this subtype were SCGB3A1 (54%), RASSF1A (29%), and HOXA9 (26%). CDH13 and HOXB5 were methylated at low frequencies (10-15%), and EMX2, MSX1, RUNX3, SORBS1, and XPA only rarely (<10%). In conclusion, this study has identified several novel epigenetically deregulated target genes in TGCT development, including homeobox genes and SCGB3A1, suggesting that epigenetic inactivation of key genes in normal development also has an important role in TGCTs.