Early and accurate detection of cholangiocarcinoma in patients with primary sclerosing cholangitis by methylation markers in bile

Background and Aims Primary sclerosing cholangitis (PSC) is associated with increased risk of cholangiocarcinoma (CCA). Early and accurate CCA detection represents an unmet clinical need as the majority of patients with PSC are diagnosed at an advanced stage of malignancy. In the present study, we aimed at establishing robust DNA methylation biomarkers in bile for early and accurate diagnosis of CCA in PSC. Approach and Results Droplet digital PCR (ddPCR) was used to analyze 344 bile samples from 273 patients with sporadic and PSC-associated CCA, PSC, and other nonmalignant liver diseases for promoter methylation of cysteine dioxygenase type 1, cannabinoid receptor interacting protein 1, septin 9, and vimentin. Receiver operating characteristic (ROC) curve analyses revealed high AUCs for all four markers (0.77-0.87) for CCA detection among patients with PSC. Including only samples from patients with PSC diagnosed with CCA 36 months) as controls, and remained high (83%) when only including patients with PSC and dysplasia as controls (n = 23). Importantly, the bile samples from the CCA-PSCPeer reviewe

DNA methylation biomarkers for colorectal cancer detection: CDO1, DCLK1, ZNF331 and ZSCAN18

Colorectal cancer is one of the most prevalent causes of cancer deaths worldwide, with an estimated 1500 deaths each year in Norway alone. Early detection of colorectal cancer may significantly reduce this number, and it is therefore of great interest to identify biomarkers that can be used in a reliable non-invasive test for early detection. Aberrant promoter DNA methylation has great potential as diagnostic biomarkers. They are both prevalent in cancer and have been shown to be an early event in tumor development. These changes can further be detected in feces and blood, materials suitable for non-invasive testing. The present thesis is part of an ongoing project where we have set up a step-wise experimental protocol to identify DNA methylation biomarkers for cholangiocarcinomas – a malignancy arising in the bile ducts. We and others have previously shown that gastrointestinal cancers frequently display similar epigenetic aberrations, and the main focus of this thesis was to evaluate whether the identified candidates could be used as biomarkers for early detection of colorectal cancer. From the cholangiocarcinoma approach, 43 genes were identified as potential epigenetically deregulated genes. These genes were investigated by methylation specific PCR (MSP) in cancer cell lines (n=24). Twelve- and thirteen genes were frequently hypermethylated in the cholangiocarcinoma- and colon cancer cell lines, respectively, and were selected for further analysis in a pilot of primary tumors and normal samples from the respective malignancies. Four genes CDO1, DCLK1, ZNF331, and ZSCAN18 were found to be methylated in ≥75% of colorectal cancer samples and simultaneously weakly methylated/ unmethylated in ≥80% of the normal samples. These genes were subjected to quantitative real-time MSP (qMSP). Methylation of at least one of the four genes was observed in 62 of the 65 colorectal cancers analyzed (95% sensitivity), and in two out of the 50 normal mucosa samples (96% specificity), with a combined area under the Receiver Operating Characteristics (ROC) curve (AUC) of 0.976. The only significant association when comparing methylation status with clinicopathological features and tumor phenotype, was observed between ZSCAN18 and microsatellite instability (MSI), indicating that aberrant methylation of the four genes is present in all tumor subtypes independent of age, gender and stage. A patent application covering these markers has been filed, and we are currently collaborating with an industrial partner to develop a non- invasive test for early detection of colorectal cancer based on these and previously published results. To conclude: CDO1, DCLK1, ZNF331, and ZSCAN18 have been identified as novel promising DNA methylation biomarkers for early detection of colorectal cancer

Evaluation of commercial kits for isolation and bisulfite conversion of circulating cell-free tumor DNA from blood

Abstract Background DNA methylation biomarkers in circulating cell-free DNA (cfDNA) have great clinical potential for cancer management. Most methods for DNA methylation analysis require bisulfite conversion, causing DNA degradation and loss. This is particularly challenging for cfDNA, which is naturally fragmented and normally present in low amounts. The aim of the present study was to identify an optimal combination of cfDNA isolation and bisulfite conversion kits for downstream analysis of DNA methylation biomarkers in plasma. Results Of the five tested bisulfite conversion kits (EpiJET Bisulfite Conversion Kit, EpiTect Plus DNA Bisulfite Kit (EpiTect), EZ DNA Methylation-Direct Kit, Imprint DNA Modification Kit (Imprint) and Premium Bisulfite Kit), the highest and lowest DNA yield and recovery were achieved using the EpiTect kit and the Imprint kit, respectively, with more than double the amount of DNA for the EpiTect kit. Of the three tested cfDNA isolation kits (Maxwell RSC ccfDNA Plasma Kit, QIAamp Circulating Nucleic Acid Kit (CNA) and QIAamp MinElute ccfDNA Mini Kit), the CNA kit yielded around twice as much cfDNA compared to the two others kits, although with more high molecular weight DNA present. When comparing various combinations of cfDNA isolation kits and bisulfite conversion kits, the CNA kit and the EpiTect kit were identified as the best-performing combination, resulting in the highest yield of bisulfite converted cfDNA from normal plasma, as measured by droplet digital PCR (ddPCR). As a proof of principle, this kit combination was used to process plasma samples from 13 colorectal cancer patients for subsequent ddPCR methylation analysis of BCAT1 and IKZF1. Methylation of BCAT1 and/or IKZF1 was identified in 6/10 (60%) stage IV patients and 1/3 (33%) stage III patients. Conclusions Based on a thorough evaluation of five bisulfite conversion kits and three cfDNA isolation kits, both individually and in combination, the CNA kit and the EpiTect kit were identified as the best-performing kit combination, with highest DNA yield and recovery across a range of DNA input amounts. The combination was successfully used for detection of clinically relevant DNA methylation biomarkers in plasma from cancer patients

Multiregional assessment of CIMP in primary colorectal cancers: Phenotype concordance but marker variability

Intratumor heterogeneity of colorectal cancers (CRCs) is manifested both at the genomic and epigenomic levels. Early genetic aberrations in carcinogenesis are clonal and present throughout the tumors, but less is known about the heterogeneity of the epigenetic CpG island methylator phenotype (CIMP). CIMP characterizes a subgroup of CRCs thought to originate from specific precursor lesions, and it is defined by widespread DNA methylation within promoter regions. In this work, we investigated CIMP in two to four multiregional samples from 30 primary tumors (n = 86 samples) using the consensus Weisenberger gene panel (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1). Twenty‐nine of 30 tumors (97%) showed concordant CIMP status in all samples, and percent methylated reference (PMR) values of all five markers had higher intertumor than intratumor variation (P value = 1.5e−09). However, a third of the CIMP+ tumors exhibited discrepancies in methylation status in at least one of the five gene markers. To conclude, CIMP status was consistent within primary CRCs, and it is likely a clonal phenotype. However, spatial discordances of the individual genes suggest that large‐scale analysis of multiregional samples could be of interest for identifying CIMP markers that are robust to intratumor heterogeneity

Additional file 1 of BladMetrix: a novel urine DNA methylation test with high accuracy for detection of bladder cancer in hematuria patients

Additional file 1. Supplementary data