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.

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.

Background: 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.The methylation status of eleven genes (ADAMTS1,CDKN2A,CRABP1,HOXA9,MAL,MGMT,MLH1,NR3C1,PTEN,RUNX3, andSCGB3A1) 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),BRAF-,KRAS-, andTP53mutation status.Results: 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 ofADAMTS1,MAL, andMGMTwere 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. HypermethylatedCRABP1, MLH1,NR3C1,RUNX3, andSCGB3A1were shown to be identifiers of carcinomas with microsatellite instability. In agreement with the CIMP concept, MSI and mutatedBRAFwere associated with samples harboring hypermethylation of several target genes.Conclusion: MethylatedADAMTS1,MGMT, andMALare suitable as markers for early tumor detection.

Background: The epigenetics of ovarian carcinogenesis remains poorly described. We have in the present study investigated the promoter methylation status of 13 genes in primary ovarian carcinomas (n = 52) and theirin vitromodels (n = 4; ES-2, OV-90, OVCAR-3, and SKOV-3) by methylation-specific polymerase chain reaction (MSP). Direct bisulphite sequencing analysis was used to confirm the methylation status of individual genes. The MSP results were compared with clinico- pathological features.Results: Eight out of the 13 genes were hypermethylated among the ovarian carcinomas, and altogether 40 of 52 tumours were methylated in one or more genes. Promoter hypermethylation ofHOXA9,RASSF1A,APC,CDH13,HOXB5,SCGB3A1 (HIN-1),CRABP1, andMLH1was found in 51% (26/51), 49% (23/47), 24% (12/51), 20% (10/51), 12% (6/52), 10% (5/52), 4% (2/48), and 2% (1/51) of the carcinomas, respectively, whereasADAMTS1,MGMT,NR3C1,p14^ARF, andp16^INK4awere unmethylated in all samples. The methylation frequencies ofHOXA9andSCGB3A1were higher among relatively early-stage carcinomas (FIGO I-II) than among carcinomas of later stages (FIGO III-IV;P= 0.002,P= 0.020, respectively). The majority of the early-stage carcinomas were of the endometrioid histotype. Additionally,HOXA9hypermethylation was more common in tumours from patients older than 60 years of age (15/21) than among those of younger age (11/30;P= 0.023). Finally, there was a significant difference inHOXA9methylation frequency among the histological types (P= 0.007).Conclusion: DNA hypermethylation of tumour suppressor genes seems to play an important role in ovarian carcinogenesis andHOXA9,HOXB5,SCGB3A1, andCRABP1are identified as novel hypermethylated target genes in this tumour type.

Early detection of the highly aggressive malignancy cholangiocarcinoma (CCA) remains a challenge but has the potential to render the tumor curable by surgical removal. This study evaluates a biomarker panel for the diagnosis of CCA by DNA methylation analyses of biliary brush samples. The methylation status of 13 candidate genes (CDO1, CNRIP1, DCLK1, FBN1, INA, MAL, SEPT9, SFRP1, SNCA, SPG20, TMEFF2, VIM, and ZSCAN18) was investigated in 93 tissue samples (39 CCAs and 54 nonmalignant controls) using quantitative methylation‐specific polymerase chain reaction. The 13 genes were further analyzed in a test series of biliary brush samples (15 CCAs and 20 nonmalignant primary sclerosing cholangitis controls), and the methylation status of the four best performing markers was validated (34 CCAs and 34 primary sclerosing cholangitis controls). Receiver operating characteristic curve analyses were used to evaluate the performance of individual biomarkers and the combination of biomarkers. The 13 candidate genes displayed a methylation frequency of 26%‐82% in tissue samples. The four best‐performing genes (CDO1, CNRIP1, SEPT9, and VIM) displayed individual methylation frequencies of 45%‐77% in biliary brushes from CCA patients. Across the test and validation biliary brush series, this four‐gene biomarker panel achieved a sensitivity of 85% and a specificity of 98%, with an area under the receiver operating characteristic curve of 0.944. Conclusion: We report a straightforward biomarker assay with high sensitivity and specificity for CCA, outperforming standard brush cytology, and suggest that the biomarker panel, potentially in combination with cytological evaluation, may improve CCA detection, particularly among primary sclerosing cholangitis patients. (Hepatology 2015;61:1651–1659)

Background: Methylation of the O⁶-methylguanine-DNA methyltransferase (MGMT) gene promoter is a favorable prognostic factor in glioblastoma patients. However, reported methylation frequencies vary significantly partly due to lack of consensus in the choice of analytical method. Method: We examined 35 low- and 99 high-grade gliomas using quantitative methylation specific PCR (qMSP) and pyrosequencing. Gene expression level of MGMT was analyzed by RT-PCR. Results: When examined by qMSP, 26% of low-grade and 37% of high-grade gliomas were found to be methylated, whereas 97% of low-grade and 55% of high-grade gliomas were found methylated by pyrosequencing. The average MGMT gene expression level was significantly lower in the group of patients with a methylated promoter independent of method used for methylation detection. Primary glioblastoma patients with a methylated MGMT promoter (as evaluated by both methylation detection methods) had approximately 5 months longer median survival compared to patients with an unmethylated promoter (log-rank test; pyrosequencing P = .02, qMSP P = .06). One third of the analyzed samples had conflicting methylation results when comparing the data from the qMSP and pyrosequencing. The overall survival analysis shows that these patients have an intermediate prognosis between the groups with concordant MGMT promoter methylation results when comparing the two methods. Conclusion: In our opinion, MGMT promoter methylation analysis gives sufficient prognostic information to merit its inclusion in the standard management of patients with high-grade gliomas, and in this study pyrosequencing came across as the better analytical method.