Supplementary Material for: Pancreatic Cancer Cell Fraction Estimation in a DNA Sample

<b><i>Objective:</i></b> Pancreatic cancers are characterized by dense stroma. To estimate the degree of interference by coexisting noncancer cells in molecular analyses, we aimed to develop a DNA methylation marker that assesses a cancer cell fraction in DNA samples. <b><i>Methods:</i></b> The microarray data of 22 pancreatic cancer tissues from the The Cancer Genome Atlas database and 9 noncancer tissues were used for genome-wide screening. Thirty-one surgical tumor samples (10 intraductal papillary mucinous neoplasms [IPMNs] and 21 pancreatic cancers), 4 normal, and 26 nontumor samples were used for validation. Gene-specific methylation analysis was conducted by bisulfite pyrosequencing. <b><i>Results:</i></b> Genome-wide screening isolated <i>SIM1</i>, <i>MIR129-2</i>, <i>NR1I2</i>, and <i>HOXB-AS4</i>, as specifically methylated in pancreatic cancer cells. Bisulfite pyrosequencing validated that one or more of three genes (<i>SIM1</i>, <i>MIR129-2</i>, and <i>NR1I2</i>) were methylated in 22 (71.0%) tumor samples (8 IPMNs and 14 cancers), and all showed low levels of methylation in 26 (86.7%) normal and nontumor samples. Therefore, the three genes collectively constituted one marker for a pancreatic cancer cell fraction. The cancer cell fraction estimated by the marker was highly correlated with that estimated using the <i>KRAS</i> mutant allele frequency (<i>R</i> = 0.79). <b><i>Conclusion:</i></b> The DNA methylation marker is useful to estimate the pancreatic cancer cell fraction in DNA samples.