Methylated circulating tumor DNA as a biomarker in cutaneous melanoma.

The detection and monitoring of circulating tumor DNA (ctDNA) in melanoma using mutation-detection techniques such as droplet digital PCR and targeted next-generation sequencing (NGS) panels has been well documented (reviewed in [1]). In melanoma, baseline ctDNA assessment predicts overall survival of stage III patients and longitudinal assessment predicts response and survival of stage IV patients treated with immune checkpoint inhibitors and targeted therapies [2–4]. ctDNA can also be used to monitor the appearance of treatment-resistant melanoma subclones [2], tumor heterogeneity [5], metabolic tumor burden [6] and differentiates true progression from pseudoprogression in melanoma patients treated with immunotherapy [7]. ctDNA can be detected in approximately 34% of stage III melanoma and 73% of stage IV melanoma [2,4]. Several customized melanoma-associated NGS mutation panels for ctDNA have been described [8,9], and these panels are designed to detect greater than 80% of melanomas with mutant allele frequency detection limits of only 0.1%. These sequencing panels have several limitations, however. They often yield lower coverage of guaninecytosine (GC)-rich DNA regions, and these regions can be extremely informative cancer markers. For instance, the TERT gene has a GC-rich promoter that is mutated in approximately 70% of melanomas [8]. The complete sequencing of large genes (such as the NF1 gene at >8 kb) is also difficult as the increased gene coverage comes at the expense of overall mutation detection sensitivity. Moreover, the significance of many, low-frequency mutations can be unclear and alterations in some genes, including the TP53 tumor suppressor gene, may not reflect tumor biology, but rather clonal hematopoiesis, a common age-associated phenomenon involving the expansion of nucleated blood cells with somatic mutations [10]. An alternative liquid biopsy approach for monitoring cancer, without prior knowledge of somatic mutation profiles, involves the detection of epigenetic DNA changes, such as methylation. Methylation of cytosine residues within CpG islands is important for the regulation of gene expression and is altered during the development and progression of many cancers, including melanoma. Importantly, aberrant methylation of CpG-rich gene promoters can be a very consistent feature of cancer [11] and thus, the analysis of methylated DNA in liquid biopsies is a rapidly emerging area of interest [12]. Patterns of DNA methylation can change during melanoma progression and the analysis of DNA methylation can provide valuable information related to the phenotypic behavior and stage of melanoma [13]. The analysis of ctDNA methylation is challenging due to the low amounts and highly fragmented nature of ctDNA. Typically, most methylation analysis workflows incorporate an initial bisulfite conversion step which preserves methylated cytosines prior to downstream sequencing. With limited amounts of ctDNA, it is imperative that the efficiency of bisulfite conversion nears 100% [14], and we have recently achieved bisulfite conversion efficiency of >99.5% when using 20 ng of ctDNA [15].