Background

In the context of precision medicine, the discovery of novel non-invasive biomarkers in clinical samples such as circulating cell-free DNA (ccfDNA) is crucial for improving the diagnosis, prognosis, predicting and monitoring the response to a patient-tailored treatment regimen against cancer and other diseases (Siravegna et al., 2017, Wan et al., 2017; Cabel et al., 2018; Barlebo Ahlborn and Østrup, 2019). The analysis of ccfDNA is challenging because the outcome depends on multiple pre-analytical steps and requires the development of methods for the detection of genetic and epigenetic variations that are present in limited quantity in highly fragmented samples (Lewis et al., 2015; Kumar et al., 2018).

Especially, methylated ccfDNA could be a promising non-invasive biomarker for cancer and potentially other diseases (Lehmann-Werman et al., 2016; Warton et al., 2016). In comparison to mutations and other genetic changes, DNA methylation changes occur early during carcinogenesis and are also present in a number of other complex non-neoplastic human diseases. Furthermore, DNA methylation changes are often restricted to a genomic region of limited size, for example the promoter associated CpG island or the transcription start site in contrast to genetic mutations that might be present all along the gene. The most commonly used methods for the analysis of DNA methylation patterns are based on a bisulfite reaction that converts cytosine bases into uracil bases while 5-methylcytosine bases are not converted (Frommer et al., 1992). Bisulfite conversion thus translates a DNA methylation difference into a sequence change. DNA methylation patterns are then read-out using next generation sequencing or microarrays for genome-wide analysis or Pyrosequencing, methylation-sensitive or methylation-specific PCR (MSP) methods for locus-specific analysis (Tost, 2016).

Different DNA methylation enrichment approaches for the analysis of low DNA methylation levels have been developed based on digestion or oligonucleotide probes (Liu et al., 2017; Campan et al., 2018; Distler, 2019). For example, the HeavyMethyl method which is based on enrichment of methylated DNA by blocker probes competing with the amplification primers on the target site, is used for a commercially available non-invasive blood based test (Molnár et al., 2015; Jung et al., 2018).

While MSP and MethyLight will specifically amplify methylated molecules in the presence of a 1,000 to 10,000 fold excess of unmethylated molecules, the efficient amplification relies on a pre-defined consistent methylation profile underlying the bindings sites for the amplification primers and the hydrolysis probe (for MethyLight), usually completely methylated molecules (Candiloro et al., 2011). However, in the presence of heterogeneous DNA methylation patterns, the assays fail to amplify partially methylated molecules and significantly underestimate the proportion of methylated samples (Alnaes et al., 2015).

Co-amplification at lower denaturation temperature PCR (COLD-PCR) approaches have been developed to enrich and analyze rare mutations including full, fast, ice (improved and complete enrichment), E-ice (Enhanced-ice) and temperature tolerant COLD-PCR and have been combined with several read-out technologies (Mauger et al., 2017). These protocols use a critical temperature (Tc) to selectively denature wild-type mutant heteroduplexes to allow the enrichment of rare mutations. More recently, fast-COLD-MS-PCR and E-ice-COLD-PCR, have been shown to allow the enrichment of unmethylated DNA and methylated DNA, respectively, from bisulfite-converted DNA (Castellanos-Rizaldos et al., 2014; Mauger et al., 2018).

E-ice-COLD-PCR uses a blocker probe that contains several LNA bases which selectively hybridizes to and thereby blocks the amplification of wild-type alleles and enriches mutant alleles followed by a Pyrosequencing assay for a sequence based read-out at single nucleotide resolution (How-Kit and Tost, 2015). For DNA methylation analysis, LNA blocker probes are designed to block unmethylated CpG sites on bisulfite-converted DNA enriching thus all other DNA methylation patterns except for completely unmethylated molecules (Mauger et al., 2018).

Therefore, the design of E-ice-COLD-PCR assays is fundamentally different from the more widely used MSP assays as it impedes the amplification of the normal, unmethylated state, but does not make any requirements on the degree or the patterns of DNA methylation in the amplified target region. All molecules different from the blocked pattern will be amplified and the subsequent sequencing-based analysis gives detailed information on the molecules that have been enriched. E-ice-COLD-PCR reaction can be easily multiplexed and the level of mutations or DNA methylation can be quantified using standard curves for the analysis of very low input material such as ccfDNA (Mauger et al., 2016 and 2018; Sefrioui et al., 2017).

The current protocol describes the development and implementation of an E-ice-COLD-PCR assay for the enrichment of low-abundant methylated DNA from a limited quantity of input sample and thus applicable to non-invasive blood-based tests. After bisulfite conversion, the E-ice-COLD-PCR reaction is performed followed by Pyrosequencing analysis. The optimization of E-ice-COLD-PCR includes assay design, optimization of the reaction and analysis of the amplification product.

While the presented protocol focusses on the technically more demanding detection of hypermethylation, blocker probes could also be used to block methylated DNA and enable the enrichment of unmethylated DNA in the presence of an excess of methylated DNA.