MC-seq is a scalable, CLIA-approvable methylation assay that is currently not widely used in cancer research
EWAS studies in cancer patients have identified interindividual variability in the epigenome, and the recent availability of affordable EWAS technologies have led to a rapid increase in epigenetic biomarker studies aimed at identifying differential methylation features that could be predictive of clinical outcome. The most commonly used platforms are array-based, like the Illumina Human 450K and Infinium MethylationEPIC arrays, which provide limited coverage of CpG sites across the epigenome. Whole genome bisulfite sequencing (WGBS) is the most comprehensive method for epigenome profiling, capturing 28 million CpGs. However, the cost, intensive workflow, and need for high quality and quantity of DNA input significantly limit its clinical translatability, particularly in cancer treatment. MC-Seq has emerged as a promising intermediary between arrays and WGBS, using NGS to capture significantly more CpGs than array-based platforms, while having the advantage of being more high-throughput and affordable than WGBS. We and others have compared CpG coverage and efficiency of different methylation quantification platforms [17, 20, 21]. A recent publication from our group has demonstrated that MC-Seq is a more reliable and efficient platform for epigenome profiling than array-based platforms like the EPIC array. When the EPIC array and MC-Seq were compared in peripheral blood mononuclear cell samples, MC-Seq captured significantly more CpGs in coding regions and CpG islands than the EPIC array. The EPIC array captured 846,464 CpG sites per sample, whereas MC-Seq captured 3,708,550 CpG sites per sample. Of the 472,540 CpG sites captured by both platforms, there was high correlation (r = 0.98–0.99) in methylation status . Moreover, while the EPIC array is enriched for genes with known roles in carcinogenesis, MC-Seq quantifies methylation in a more agnostic manner and profiles 3–4 times more CpGs than the EPIC array, allowing for a higher chance of discovering novel epigenetic modifications in cancer. Furthermore, the coverage areas within each gene were more comprehensive than the EPIC array and other commonly used methylation analysis techniques, like PCR or pyrosequencing. Herein, we demonstrated that MC-Seq captured significantly more CpG sites within functional gene regions, owing to the higher overall profiling capability of this technique.
Oral SCC is an epigenetically-regulated cancer with promising methylation biomarker candidates
Methylation studies on OSCC patients [6–16] including our own studies [7, 8] have demonstrated that methylation is a common event and highlighted specific genes for mechanistic studies. For example, a EWAS using the Illumina Human 450K array on 108 head and neck SCC patients of multiple sub-sites including oral cavity identified hypermethylation and inactivation of key tumor suppressor genes . Clinical translation of these methylation biomarker studies has been limited due to: 1) combining OSCC with other head and neck cancer sub-sites (i.e., oropharynyx, hypopharynx, larynx), which creates a heterogeneous cohort that fails to recognize OSCC as a distinct clinical disease, and 2) relying solely on array-based platforms, which query a limited number of CpGs. As a result, none of these studies have produced a methylation biomarker with high prognostic performance.
In addition to being a distinct clinical subsite from other head and neck sites, the oral cavity is an easily accessible anatomic site for non-invasive biopsy techniques. Clinical translation of a biomarker requires that it can be measured during treatment. Waiting until after tumor removal for the formalin-fixed, paraffin-embedded (FFPE) tissues delays potentially necessary treatment. Researchers have used both saliva and brush swabs to noninvasively sample OSCC cells at the time of diagnosis. In our own studies, we have used saliva to identify methylation biomarkers of OSCC. We demonstrated that a multi-gene panel could be constructed using either a methylation array or Methylight, a polymerase chain reaction (PCR) technique [7, 8]. However, we and others have shown that concordance of methylation between saliva and cancer tissue is highly variable [22, 23].
Brush swabs and MC-Seq represent a noninvasive method to quantify methylation biomarkers
Our approach of using brush swabs and MC-Seq to determine the methylation signature at the time of diagnosis has a high potential for clinical translatability. We demonstrated in this study that brush swab and tissue biopsies from matched sites had highly correlated methylation signatures. Furthermore, the DNA quality and quantity from brush swab samples were adequate to perform MC-SEq. Mapping efficiency was equivalent between tissues and brush swabs. Given the high correlation between the paired tissues and brush swabs, and the satisfactory DNA yield, brush swabs could serve as a clinically robust surrogate to tissue biopsies. One previous study has assessed the reliability of brush swab DNA for MC-Seq compared to the Human 450K array , drawing similar conclusions to our study  that MC-Seq offered broader coverage of CpG sites and that sample-based correlation was high (r = 0.98) between the two platforms. However, they did not compare brush swab to underlying tissue collection. To our knowledge our study represents the first to directly compare the epigenome-wide signature of matched brush swabs and tissues, with the results having important implications in OSCC biomarker research.