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Supplementary Figure S1. 1-D droplet digital PCR analysis plot showing detection of
methylated target molecules in serially diluted DNA samples. Four replicates of each dilution
were performed. Horizontal pink bars indicate the threshold line and green (ACTB) or blue (7
marker genes) clusters represent “positive” droplets; gray clusters indicates “negative” droplets.
NTC, non-template control; UM, unmethylated.
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. R2 = coefficient of determination. Data are represented as the mean
± standard deviation. (B) The ratio of methylated SOX17 molecules to ACTB molecules at 1–
10 ng input DNA using the same samples. Data are represented as the mean ± standard deviation.
(C, D) Inter-day assay variation in methylated DNA levels measured on different days. The
number of methylated SOX17 molecules per L of cyst fluid (C) and the ratio of methylated
SOX17 molecules to ACTB molecules (D) measured on different days. % CV = coefficient of
variation (Standard deviation / Mean value × 100 (%).
CV, coefficient of variation
Supplementary Figure S3. (A) MSP analysis using pancreatic cancer cell lines, HPDE cell
line, and normal pancreatic tissues. Each column represents the methylation status of seven
marker genes. Black boxes denote methylated, white boxes indicate unmethylated, gray boxes
indicate both methylated and unmethylated. (B, C) Representative results of agaroge gel
electrophoresis after MSP analysis for seven marker genes in pancreatic cell lines and (B),
normal pancreatic tissue (C). “Methylated ” refers to a 100% methylated bisulfite conversion
DNA sample used as a positive control with and “Unmehtylated” refers to a 100% unmethylated
negative control DNA sample after bisulfite conversion.
M, methylated; U, unmethylated.
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. Rectangles indicate exon lesion with white (untranslated lesion) and black
(translated lesion) color. (B) Heat-map representation of the extent of SOX17 gene methylation
at individual 40 CpGs. Each rectangle represents a single CpG and each row represents a sample.
The extent of methylation is represented over the range of 0% (Green) to 100% (Red). The
percentage of the number of methylated CpGs to total analyzed CpGs from 12 clones in each
sample is shown in right side. The heat-map was constructed using R software. (C) Ratio of
hypermethylated SOX17 gene/-actin determined by ddQMSP assay. Error bar indicates
Poisson 95% confidence limits. (D) Correlation of methylation level by ddQMSP and bisulfite
sequencing in 11 pancreatic cancer cell lines and HPDE-immortalized pancreatic ductal
epithelial cell line. R2 means coefficient of determination.
BS, bisulfite sequencing; MSP, methylation specific PCR; F, forward primer; P, Probe; R,
reverse primer.
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. Black, white, and
gray bars means the samples with methylated, partially methylated, and unmethylated status
determined by MSP, respectively. Data are represented as the mean ± standard deviation.
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 ±
standard deviation.
5-Aza-dC, 5-Aza-2′-deoxycytidine; TSA, trichostatin A
*P < 0.05, **P < 0.01, ***P < 0.001.
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). Measurement unit of quantification is
methylated target molecules per L of cyst fluid. Error bars indicate 95% confidence interval.
Supplementary Figure S8. Representative 1-D plot results of ddQMSP assay for methylated
SOX17 and ACTB reference using the pancreatic cyst fluid samples. Horizontal pink bars
indicate the threshold lines as 6,000 fluorescence amplitudes for SOX17 signals (blue) and
3,500 for ACTB signals (green).
IPMN, intraductal papillary mucinous neoplasm; INV, invasive cancer; HGD, high-grade
dysplasia; IGD, intermediate-grade-dysplasia; SCN, serous cystic neoplasm.
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. The longer horizontal bar represents the median value and shorter ones
represents values of the 75th and 25th percentiles, respectively.
HGD, high-grade dysplasia; INV, invasive cancer.
Supplementary Figure S10. Extracted DNA quantity from the surgically aspirated cyst fluid
samples in each diagnostic group. The box represents the 25th to 75th percentiles, and the line
within the box is the median value.
IPMN; intraductal papillary mucinous neoplasm; INV, invasive cancer; HGD, high-grade
dysplasia; IGD, intermediate-grade dysplasia; LGD, low-grade dysplasia; MCN, mucinous
cystic neoplasm; SCN, serous cystic neoplasm.
**P < 0.01
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.
IPMN, intraductal papillary mucinous neoplasm; IGD, intermediate-grade dysplasia; SCN,
serous cystic neoplasm.
Supplementary Figure S13. Comparison of methylated level between the samples collected
in Johns Hopkins Hospital (JHH) and those in Asan Medical Center (AMC). The longer
horizontal bar represents the median value and shorter ones represents values of the 75th and
25th percentiles, respectively.
Supplementary Figure S14. Pathological diagnosis of pancreatic cystic tumors in each risk
group.
INV, invasive cancer; HGD, high-grade dysplasia.
Supplementary Figure S15. Comparison of methylated levels across the different cyst type of
IPMN, MCN and SCN. The longer horizontal bar represents the median value and shorter ones
represents values of the 75th and 25th percentiles, respectively.
***P < 0.001, N.S.; not significant.
Supplementary Figure S16. (A) Prevalence of KRAS colon 12 and GNAS codon 201 mutations
in 103 surgically aspirated cyst fluid samples with IPMN diagnosis. (B) Prevalence of highgrade dysplasia and invasive cancer and the mutant prevalence of KRAS and GNAS in 103
IPMN cyst fluid samples stratified by the number of methylated genes. (C, D) The methylation
score (average of the number of methylated genes) across IPMN cyst fluids stratified by
histologic grade (n =113, C) or KRAS and GNAS mutation status (n = 103, D). Each plot means
individual value and long horizontal bar indicates the methylation score.
*** P < 0.001
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.
Error bars indicate 95% confidence interval.