Supplementary Information

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1 Supplementary Information Detection and differential diagnosis of colon cancer by a cumulative analysis of promoter methylation Qiong Yang 1,3*, Ying Dong 2,3, Wei Wu 1, Chunlei Zhu 1, Hui Chong 1, Jiangyang Lu 2*, Dehai Yu 1 Libing Liu 1, Fengting Lv 1, Shu Wang 1* 1 Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing , P. R. China. yangqiong@iccas.ac.cn; wangshu@iccas.ac.cn 2 The First Hospital Affiliated to General Hospital of the Chinese People s Liberation Army, Beijing , P. R. China. lujy@263.com 3 Equal contributions to this work 1

2 Supplementary Figure S1. Standard curves for quantitative determination of the degree of DNA methylation Supplementary Figure S1. The degree of methylation indicates the percentage of methylation of the CDKN2A/P16 gene promoter. The samples showing different degrees of methylation are prepared by mixing known fully methylated and unmethylated DNA samples. 2

3 Supplementary Table S1. Comparision of CCP-based FRET method and bisulfite sequencing for detecting the methylation degree of CDKN2A/p16 gene promoter Sample CCP-based FRET method Bisulfite sequencing Caco2 cell line 100% 100% Colo320 cell line 97% 100% HT29 cell line 96% 100% HEK293cell line 0% 0% 1# Normal colon tissue Can not be detected 11 % 2# Normal colon tissue 0% 2.7 % 4 # Adenoma 63% 52% 5# Adenoma 9% 0 % 8# Adenoma 6% 0 % 11# Adenoma 33% 20% 15# Adenoma 14% 7.5% 17# Adenoma 10% 0 % 26# Adenoma 3% 0 % 31# Carcinoma 68% 66 % 32# Carcinoma 68% 53 % 33# Carcinoma 12% 0 % 34# Carcinoma 10% 0 % 35# Carcinoma 31% 10 % 3

4 Supplementary Table S2. Statistical significance of each of the colon cancer-related gene promoter methylation in 80 cases. Colon-related gene P value a Carcinoma+Adenoma VS. Normal Carcinoma VS. Adenoma VIM < MGMT CDKN2A/p16 <0.001 <0.001 MLH ESR1 < APC < TMEFF2/HPP <0.001 a Statistical significance was analyzed by the Mann-Whitney U test. 4

5 Supplementary Table S3. Validation of the CCP-FRET system and discrimination equations in 30 blind cases using three combined methylation alterations (VIM, APC and CDKN2A/P16). Samples Detection results from CCP-FRET and discrimination equation Pathologic diagnosis P81 Positive Carcinoma P82 Positive Carcinoma P83 Positive Carcinoma P84 Positive Carcinoma P85 Positive Carcinoma P86 Positive Carcinoma P87 Positive Carcinoma P88 Positive Carcinoma P89 Positive Carcinoma P90 Positive Carcinoma P91 Positive Adenoma P92 Positive Adenoma P93 Negative (False) Adenoma P94 Positive Adenoma P95 Positive Adenoma P96 Negative (False) Adenoma P97 Positive Adenoma P98 Positive Adenoma P99 Negative (False) Adenoma P100 Positive Adenoma P101 Negative Normal P102 Negative Normal P103 Negative Normal P104 Negative Normal P105 Negative Normal P106 Negative Normal P107 Positive (False) Normal P108 Negative Normal P109 Positive (False) Normal P110 Positive (False) Normal Correct rate 80% 5

6 Supplementary Table S4. Validation of the differential diagnosis in 20 blind patient cases using two combined methylation alterations (TMEFF2/HPP1 and CDKN2A/P16). Samples Detection results from CCP-based FRET and Pathologic diagnosis discrimination equation P81 Carcinoma Carcinoma P82 Carcinoma Carcinoma P83 Carcinoma Carcinoma P84 Carcinoma Carcinoma P85 Carcinoma Carcinoma P86 Carcinoma Carcinoma P87 Carcinoma Carcinoma P88 Carcinoma Carcinoma P89 Carcinoma Carcinoma P90 Carcinoma Carcinoma P91 Adenoma Adenoma P92 Adenoma Adenoma P93 Adenoma Adenoma P94 Carcinoma (False) Adenoma P95 Adenoma Adenoma P96 Adenoma Adenoma P97 Adenoma Adenoma P98 Adenoma Adenoma P99 Adenoma Adenoma P100 Adenoma Adenoma Correct rate 97.6% 6

7 Supplementary Table S5. Comparing the sensitivity of combination and single biomarker in differential diagnosis Detection mode Sensitivity (P value) Specificity Single TMEFF2/HPP1 80% (=0.0373) 92% Signal CDKN2A/P16 22% (<0.001) 92% Combination of two biomarkers 94% 92% P, Chi-square test is used to compare the different significance of detection sensitivity of combined methylation alterations to that of single one. n carcinoma = 50; n adenoma = 50. 7

8 Supplementary Table S6. Clinical characteristics of 50 carcinomas cases Patient Degree of tumor Serosa Lymph node CIMP Gender Age at surgery no. differentiation invasion metastasis 44 N Female 70 Well Negative Positive 35 N Male 57 Well Negative Negative 8 N Male 65 Well Negative Positive 1 N Male 66 Moderate Negative Positive 29 N Female 75 Moderate Positive Positive 39 N Male 84 Moderate Negative Negative 12 N Male 74 Moderate Negative Negative 13 N Male 67 Moderate Negative Negative 34 N Female 72 Moderate Negative Positive 37 N Female 63 Poor Negative Negative 43 N Female 70 Moderate Negative Negative 50 N Female 73 Poor Negative Negative 17 N Female 75 Moderate Positive Positive 20 L Female 77 Poor Positive Positive 25 L Female 55 Poor Negative Positive 45 L Male 65 Well Positive Negative 47 L Female 87 Moderate Negative Negative 2 L Male 52 Moderate Negative Negative 3 L Male 63 Moderate Positive Positive 6 L Male 74 Moderate Negative Negative 9 L Female 71 Moderate Negative Negative 14 L Female 66 Moderate Positive Positive 22 L Female 74 Moderate Negative Negative 23 L Male 73 Moderate Negative Negative 26 L Male 60 Poor Positive Positive 28 L Male 60 Moderate Negative Negative 46 L Female 66 Well Positive Positive 49 L Male 70 Moderate Negative Negative 7 H Male 75 Poor Positive Positive 15 H Male 57 Moderate Negative Positive 16 H Male 85 Moderate Positive Negative 19 H Male 42 Poor Positive Positive 24 H Female 50 Poor Positive Negative 27 H Female 78 Moderate Positive Negative 30 H Female 70 Moderate Positive Positive 32 H Male 74 Poor Positive Negative 33 H Female 75 Poor Positive Positive 36 H Female 35 Poor Negative Negative 38 H Female 87 Moderate Positive Negative 40 H Male 60 Moderate Positive Positive 41 H Female 85 Poor Negative Positive 8

9 42 H Female 59 Moderate Positive Positive 48 H Female 86 Moderate Positive Positive 4 H Female 68 Poor Negative Negative 5 H Female 60 Moderate Negative Positive 10 H Male 68 Poor Positive Positive 18 H Female 74 Well Positive Negative 21 H Female 79 Moderate Negative Negative 31 H Female 53 Well Negative Negative 11 H Male 56 Poor Positive Positive 9

10 Supplementary Table S7. Sequences of primers for amplifying the promoter regions of seven colon cancer-related genes Gene symbol Forward primer 5-3 Reverse primer (5-3 ) Product size (bp) Number of 5 CCGG3 site VIM GCCCTCGTTCGCCTCTTCT GGTGGACGTAGTCACGTAGC MGMT TGGCAAACTAAGGCACAGA GCATCCTCGCTGGACGC TMEFF2/ HPP1 GTTAGACCTTCTCTCGTCGC GAAAGGCTCCTCTGCATACG ESR1 GCATATGAGCTCGGGAGACC GCCGACACGCGAGCTCTGG CDKN2A/ P16 TCCTTCCTTGCCAACGCT GCCCCTCCTCTTTCTTCCTC APC GACAGAACAGCGAAGCAGTG AGGTGGGAAGACTACAATGC MLH1 TACGATGAGGCGGCGACAGA GCCAATAGGA GCAGAGATG

11 Supplementary Table S8. Sequences of primers for amplying the promoter regions in three CIMP-related genes Pimers Sequences (5-3 ) NEUROG1-F CAGCTTAGCCCGAGCCGA NEUROG1-R TTTATGCTCGCGGGAGGCC CACNA1G-F CAGGGGAAGCGGGACTCG CACNA1G-R ATCTGGTGGGCTCTAGGG CRABP1-F TGGAGGCTGAGGCACAAC CRABP1-R GCGGCGGCAGGTACGGACA 11

12 Supplementary Table S9. Sequences of primers for amplying the promoter regions in bisulfate-modified DNA Pimers Sequences (5-3 ) VIM-in-Forward TGGGATGGTAGTGGGAGG VIM-in-Reverse TAAACATAATCACATAACTC C VIM-out-Forward GGAGTAGGAAGGTTTGAGG VIM-out-Reverse AAATCCACCA AATCCTAC p16-in-forward GTA GGT GGG GAG GAG TTTAGT T p16-in-reverse CCCACCCTCTAA TAACCA ACCAA p16-out-forward GAGGGGGTAGGGGGATAT p16-out-reverse ACCAATCAACCAAAAACTCCATACTA 12

13 Supplementary Table S10. Correlation analysis of CIMP status and tumor location in 50 carcinomas cases Parameter CIMP N CIMP L CIMP H P Tumor location Proximal, n (%) 3 (23.08%) 2 (13.33%) 8 (36.36%) Distal, n (%) 10 (76.92%) 13 (86.67%) 14 (63.64%) CIMP, CpG island methylator phenotype; H, high; L, low; N, negative. Statistical significance is determined using Fisher's exact test. 13

14 Supplementary Methods To confirm the semi-quantitative results obtained from CCP-based FRET method, the methylation degrees of p16 CpG islands in 17 representative tissue samples and 4 cell lines were determined by using the bisulfite sequencing. 2 µg of the extracted DNA from cell lines, tissue samples were converted using an Epitect Bisulfite kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer s instructions. The nested -PCRs were then performed in a 25-µL reaction volume containing 1 µl of bisulfate modified DNA, 1 U of GoTaq Hot Start polymerase (Promega, Madison, USA ), 0.4 µm of each primer, 1.5 mm MgCl 2, 0.2 mm each dntp, and 1 GoTaq Flexi buffer. PCR for the sense strand of promoter areas in bisulfite-treated DNA was performed with the primers listed in the Supplementary Table 5. The reaction conditions were 95 C for 5 minutes, followed by 40 cycles at 95 C for 30 seconds and 58 C for 40 seconds, and final extension at 72 C for 10 minutes. The PCR fragments were inserted in to TA vector (Tiangen, Beijing, China) by a standard molecular experiment. About clones for each sample were picked up and cultured. Individual clones were sequenced by the deoxynucleotide chain-termination method using an ABI 310 Genetic analyzer (Applied Biosystems). 14