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

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.