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Supplement materials

Metabolite identification

Tandem mass spectrometry (MS/MS) spectra data for metabolites were acquired using

AB Sciex TripleTOF 5600+. The acquired MS/MS spectra were matched against our in- house standard MS/MS spectral library and metabolite standards. The MS/MS spectra match score was calculated using dot-product algorithm ranging from 0-1. The cutoff for match score was set as 0.8. The MS/MS spectra match results were further manually checked to confirm the identification. All the m/z errors are less than 25 ppm and all the RT errors are less than 18 seconds. Figure S3 show that the MS/MS spectra of eight biomarkers and metabolite standards matched well.

There are two lipids that were identified through manual analysis of accurate mass and

MS/MS spectra. In brief, characteristic ion of phosphatidylcholine (184.0739, positive mode) was found in both MS/MS spectra, therefore, the two metabolites were identified as phosphatidylcholine (PC). The search of accurate mass against all PC lipids identifies the two lipids as PC(14:0/0:0) and PC(16:0/18:2). Then the chemical structures of other fragments were also inferred to further confirm the structure of PC(14:0/0:0) and

PC(16:0/18:2). The MS/MS spectra and the fragment interpretations are provided in the

Figures S4.

Secondly, the biomarkers whose MS/MS spectra were not matched in our in-house databases or cannot be interpreted by MS/MS spectra, were searched against online databases of HMDB (www.hmdb.ca), METLIN (http://metlin.scripps.edu/) and KEGG

(http://www.genome.jp/kegg/). The mass tolerance between the measured m/z values and the exact masses of the components of interest was set to within 30 ppm. The potential biomarkers were identified according to their molecular weights. The list of metabolites from in-house and online database match is shown in Table S2.

S1 According to Metabolomics Standards Initiative (Lloyd et al. 2007), the eight potential biomarkers which are identified using reference standards are the level one metabolite identification (the highest level of metabolite identification), and the two PCs which are interpreted according to their MS/MS spectra are level two metabolite identification. The six metabolites which are identified by searching in HMDB, METLIN and KEGG are level four metabolite identification.

S2 Table S1. Baseline and histopathologic characteristics of participant subjects in training and validation groups.

Training group Validation group

ESCC Controls ESCC Controls

Sample size 77 84 20 21

Age, mean(sd) 62.0 (7.0) 58.2 (6.8) 63.5 (6.0) 59.0 (5.4)

Gender Males, n (%) 49 (63.6) 39 (46.4) 13 (65.0) 10 (47.6)

TNM Stages*

Stage 0/Tis* 31 -- 8 --

StageⅠ 14 -- 3 --

StageⅡ 8 -- 3 --

StageⅢ 24 -- 6 -- *American joint Committee on Cancer (AJCC) TNM Classfication of Carcinoma of the Esophagus and Esophagogastric Junction (7th ed, 2010). Tis, Squamous carcinoma in situ.

S3 Table S2. The detailed information about the 16 serum metabolites.

No Polarity RT HMDB Identity Name Pathway 1 POS 250.857 HMDB00638 Dodecanoic acid Fatty acid 2 NEG 502.7525 HMDB60082 cis-9-Palmitoleic acid 3 NEG 645.362 HMDB00220 Palmitic acid biosynthesis 4 NEG 645.331 HMDB00207 Oleic acid 5 POS 281.238 HMDB00063 Cortisol --- 6 POS 39.087 HMDB00158 L-Tyrosine --- 7 POS 182.416 HMDB00929 L-Tryptophan 8 NEG 415.034 HMDB07855 LPA(18:1/0:0) Glycerophosp 9 POS 374.5935 HMDB10379 PC(14:0/0:0) 10 POS 209.0565 HMDB10386 LysoPC(18:2) holipid 11 POS 548.814 HMDB10405 LysoPC(24:0) metabolism

and choline 12 POS 203.407 HMDB10389 LysoPC(18:4) metabolism in

cancer 13 POS 476.0315 HMDB07930 PC(14:1/P-18:1) Glycerophosp 14 POS 244.205 HMDB07973 PC(16:0/18:2) 15 POS 541.8255 HMDB08814 PC(24:1/22:6) holipid

metabolism;

16 NEG 512.406 HMDB00673 Linoleic acid Linoleic acid

metabolism Abbreviations: Retention time (s, RT); Measured mass to charge ratio (m/z);

Table S3. The confirmation of the chemical structures of 8 serum metabolites using standard references.

Identify Name Polarit Measured m/z Measured RT (s) Accurate Standard RT m/z error (ppm) RT error (s)

y m/z Dodecanoic acid POS 218.2166 223.0 218.2115 247.1 23.4 -17.1 cis-9-Palmitoleic NEG 253.2173 466.0 253.2173 467.5 -0.1 -1.5

S4 acid Palmitic acid NEG 255.2331 635.9 255.2330 635.6 -0.39 0.3 Oleic acid NEG 281.2487 635.9 281.2486 635.8 -0.36 0.1 Cortisol POS 363.2165 268.2 363.2166 268.0 -0.26 0.2 L-Tyrosine POS 182.0814 102.0 182.0812 116.2 1.1 -14.2 L-Tryptophan POS 205.0975 168.4 205.0972 168.3 1.46 0.1 Linoleic acid NEG 279.2330 481.5 279.2330 484.1 0 -2.6

Abbreviations: Retention time (s, RT); Measured mass to charge ratio (m/z). m/z error was calculated using (Measured m/z – Standard m/z) *106/ Standard m/z, and RT error was calculated using (Measured RT – Standard RT).

S5 Table S4. ROC analysis using random forest model that combines 16 biomarkers to diagnose ESCC (stage 0, I-II and III) in the validation data.

Samples R Prevalence1 Prevalence2 ASE% (95%CI) SP% (95%CI) PPV% NPV% PPV% NPV% O Controls (N=21) vs 085.00 (70.00~100.00) 90.48 (76.19~100.00) 0.89 99.98 8.27 99.83 U ESCC (N=20) . Stage 0 (N=8) 0.881 100.00 (100.00~100.00) 71.43 (52.38~90.48) 0.35 100.0 3.41 100.00

(0.754~1.000) 0 0.881 83.33 (50.00~100.00) 90.48 (76.19~100.00) 0.87 99.98 8.12 99.81 Stage Ⅰ-Ⅱ (N=6) (0.743~1.000) 0.929 100.00 (100.00~100.00) 90.48 (76.19~100.00) 1.04 100.0 9.59 100.00 Stage Ⅲ (N=6) (0.824~1.000) 0 1 Assume ESCC prevalence as 1/1000 in city of Feicheng. 2 Assume ESCC prevalence as 1/100 in high-risk population or in clinical practice. AUC, area under ROC curve; Sensitivity, SE; Specificity, SP; Positive predictive value, PPV; Negative predictive value, NPV

S6 Supplement Figures

Figure S1. Typical UHPLC-QTOF/MS chromatograms of serum samples for ESCC and healthy controls subjects, in ESI +/- modes. Figure S2. The PCA performed on the whole samples including ESCC patients, healthy controls, and QC samples. Figure S3. The MS/MS spectra of 8 biomarkers matched well with corresponding metabolite standards. Black represent the MS/MS spectra of biomarkers from serum samples and red represent the MS/MS spectra of standards. Figure S4. The MS/MS spectra, the interpretation of each of fragments and the chemical structures of two lipids: (A) PC(14:0/0:0); (B) PC(16:0/18:2).The red ion is the characteristic ion of phosphatidylcholine (PC). Figure S5. The relative intensity of 16 differential metabolites in ESCC patients versus healthy controls. s

Figure S6. Overview of pathway analysis using MetaboAnalyst. A list of compound labels (HMDB IDs) are input into MetaboAnalyst and MetaScape. The parameters of pathway analysis are set as: pathway library=Homo sapiens (human); Over representation analysis=Hypergeometric Test; Pathway Topology Analysis=Relative-betweeness centrality. Figure S7. The comparison of statistical values of selected biomarkers between age and gender-adjusted and unadjusted data