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

Objective: 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. Methods: 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. Results: Genome-wide screening isolated SIM1, MIR129-2, NR1I2, and HOXB-AS4, as specifically methylated in pancreatic cancer cells. Bisulfite pyrosequencing validated that one or more of three genes (SIM1, MIR129-2, and NR1I2) 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 KRAS mutant allele frequency (R = 0.79). Conclusion: The DNA methylation marker is useful to estimate the pancreatic cancer cell fraction in DNA samples.研究目的：胰腺癌以致密间质为典型特征。为评估共存非癌细胞对分子分析的干扰程度，本研究旨在开发一种可评估DNA样本中癌细胞占比的DNA甲基化标记物。 方法：采用癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据库中的22份胰腺癌组织与9份非癌组织的微阵列数据开展全基因组筛选；验证阶段纳入31份手术切除肿瘤样本（含10份导管内乳头状黏液性肿瘤（Intraductal Papillary Mucinous Neoplasm, IPMN）与21份胰腺癌样本）、4份正常样本及26份非肿瘤样本；通过亚硫酸氢盐焦磷酸测序完成基因特异性甲基化分析。 结果：全基因组筛选获得在胰腺癌细胞中特异性甲基化的SIM1、MIR129-2、NR1I2及HOXB-AS4基因。经亚硫酸氢盐焦磷酸测序验证，22份（71.0%）肿瘤样本（含8份IPMN与14份胰腺癌样本）中存在至少1个上述3个基因（SIM1、MIR129-2及NR1I2）的甲基化修饰，而26份（86.7%）正常及非肿瘤样本中这三个基因均呈低甲基化水平。据此，上述三个基因共同构成了胰腺癌癌细胞比例的检测标记物。通过该标记物估算的癌细胞比例与基于KRAS突变等位基因频率得到的估算结果高度相关（R=0.79）。 结论：本研究开发的DNA甲基化标记物可有效用于评估DNA样本中的胰腺癌细胞占比。