## 3 Results  
### Cohort and Gene Targets  
In this study, we focused on monitoring the methylation status of circulating free DNA in samples from 5 healthy individuals and 4 patients with advanced melanoma (Table 1). A panel of 7 genes was used in the methylation analyses: gap junction protein beta 2 (\(G\)\(B\)\(B\)), homeobox A9 (\(HOXA9\)), mesenchyme homeobox 2 (\(MEOX2\)), oligodendrocyte transcription factor 3 (\(OLIG3\)), paraoxonase 3 (\(PON3\)), Ras association domain family member 1 (\(RASSF1\)), and transcription factor AP2 beta (\(TFAP2B\)). These genes were chosen based on genome-wide methylation studies on melanoma and shown to be potential progression-related and/or prognostic biomarkers [24; 25; 36; 37; 38; 39; 40]. Furthermore, the hypermethylation of these genes (or closely related genes) in melanoma has been reported to be highly prevalent [39; 41; 42].  
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Subject**} & \multirow{2}{*}{**Age (years)**} & \multirow{2}{*}{**Current**} & \multirow{2}{*}{**Tumor Mutation**} & **ctDNA** & **wtDNA** & \multirow{2}{*}{**ctDNA Fraction**} & **Input Circulating** \\  & & & **Temperature** & & **(copies/mL)** & **(copies/mL** & **(MAF\%)** & **Free DNA for** \\  & & & **Sampling** & & **plasma)* & **plasma)* & **(MAF\%)** & **Bisulfite** \\ \hline Healthy 1 & 32 & No treatment & ND & ND & 3238 & ND & 23.56 \\ \hline Healthy 2 & 53 & No treatment & ND & ND & 2188 & ND & 18.22 \\ \hline Healthy 3 & 37 & No treatment & ND & ND & 2045 & ND & 17.33 \\ \hline Healthy 4 & 30 & No treatment & ND & ND & 1477 & ND & 14.67 \\ \hline Healthy 5 & 54 & No treatment & ND & ND & 1420 & ND & 15.11 \\ \hline Melanoma 1 & 76 & Pembro & BRAF V600E & 30,350 & 56,250 & 35 & 21.78 \\ \hline Melanoma 2a (PRE *) & 61 & Combi-DT & BRAF V600K & 4960 & 5800 & 46.1 & 15.00 \\ \hline Melanoma 2b (EDT **) & 61 & lpi + nivo & BRAF V600K & 1615 & 5800 & 21.8 & 20.80 \\ \hline Melanoma 3 & 75 & No treatment. & & & & & \\  & & Pre-ipi & & & & & \\ \hline Melanoma 4 & 38 & lpi + pembro & NRAS Q61K & 10,800 & 10,250 & 51.3 & 24.89 \\ \hline \hline \end{tabular}  
* Liquid biopsy obtained from patient 2 prior to immunotherapy treatment. *
* Liquid biopsy obtained from patient 2 while on immunotherapy treatment (nikovolumab + ipilimumab).
* Determined using ddPCR. ND, not determined; PRE, pre-treatment; EDT, early during treatment; MAF, mutant allele frequency; nivo, nivolumab (anti-PD1); pembro, pembrolizumab (anti-PD1); ppl, pinflammatory (anti-CTL4); comb-DT, combination ddafreshb (BRAF inhibited) and tranterinib (MEK inhibited); ct, circulating tumor; wt, wild-type.  
\end{table}
Table 1: Cohort used for methylation analysis.  
Initially, the ctDNA copy number/mL of plasma for the melanoma patients was determined using ddPCR analysis of previously established driver mutations in BRAF or NRAS (Table 1). ctDNA was detected in patient samples and ranged from 200-30,350 copies/mL plasma (Table 1). Longitudinal analysis of ctDNA from melanoma patient 2, revealed a 67% decrease in ctDNA after 3 weeks of immunotherapy (Table 1, compare melanoma patient 2 pre-treatment (PRE) and early during treatment (EDT) samples).