Supplementary Figures from Predicting the Grade of Dysplasia of Pancreatic Cystic Neoplasms Using Cyst Fluid DNA Methylation Markers

Supplementary Figure S1. 1-D droplet digital PCR analysis plot showing detection of methylated target molecules in serially diluted DNA samples. Supplementary Figure S2. (A) Correlation between input amounts of bisulfite converted DNA and the number of methylated SOX17 molecules detected in four representative surgically aspirated cyst fluid samples. Supplementary Figure S3. (A) MSP analysis using pancreatic cancer cell lines, HPDE cell line, and normal pancreatic tissues. Supplementary Figure S4. (A) Schematic presentation of the 5' lesion of SOX17 gene with the primer location of MSP and bisulfite sequencing. Vertical lines represent each CpG dinucleotide. Supplementary Figure S5. Relative mRNA expression for seven marker genes in 11 pancreatic cancer cell lines with the level of HPDE cells set 1.0 as reference. Supplementary Figure S6. Relative mRNA expression for seven marker genes before and after the induction of 5-Aza-2′-deoxycytidine and/or trichostatin A using 3 pancreatic cancer cell lines of MIA PaCa-2 (A), Capan-1 (B), and AsPC-1 (C). Data are represented as the mean {plus minus} standard deviation. Supplementary Figure S7. AUC values for predicting the presence of malignancy within a cyst for each marker gene among discovery set (n = 29, white bar) and validation set (n = 154, gray bar) and merged cases (n = 183, black bar). Supplementary Figure S8. Representative 1-D plot results of ddQMSP assay for methylated SOX17 and ACTB reference using the pancreatic cyst fluid samples. Supplementary Figure S9. Quantification of methylated levels in IPMN with high-grade dysplasia and invasive cancer, stratified by the extensiveness of high-grade dysplasia lesion and depth of invasive lesion. Supplementary Figure S10. Extracted DNA quantity from the surgically aspirated cyst fluid samples in each diagnostic group. Supplementary Figure S11. AUC values for predicting the presence of high-grade dysplasia or invasive cancer within a cyst among merged 183 patients using the different measurement unit of quantification for methylation level assessment. Error bars indicate 95% confidence interval. Supplementary Figure S12. Correlation between patient age and the number of methylated target molecules per ng of cyst fluid DNA for six marker genes among IPMN with IGD cases (A) and SCN cases (B). R2 means coefficient of determination. Supplementary Figure S13. Comparison of methylated level between the samples collected in Johns Hopkins Hospital (JHH) and those in Asan Medical Center (AMC). Supplementary Figure S14. Pathological diagnosis of pancreatic cystic tumors in each risk group. Supplementary Figure S15. Comparison of methylated levels across the different cyst type of IPMN, MCN and SCN. Supplementary Figure S16. (A) Prevalence of KRAS colon 12 and GNAS codon 201 mutations in 103 surgically aspirated cyst fluid samples with IPMN diagnosis. Supplementary Figure S17. AUC values for predicting the presence of high-grade dysplasia or invasive cancer within a cyst among 103 IPMN cases stratified by KRAS or GNAS status.