Means and methods for diagnosing pancreatic cancer in a subject based on a metabolite panel

Abstract

The present invention relates to the field of diagnostic methods. Specifically, the present invention contemplates a method for diagnosing pancreas cancer in a subject and, preferably for differentiating between pancreatic cancer and pancreatitis and a method for identifying whether a subject is in need for a therapy of pancreatic cancer. The invention also relates to tools for carrying out the aforementioned methods, such as diagnostic devices.

Claims

1. A method for treating a subject in need of a pancreas cancer therapy, comprising: (a) obtaining a sample of a subject suspected to suffer from pancreas cancer, wherein said sample is a blood, plasma, or serum sample, and then subjecting the sample to total lipids extraction; (b) determining, using mass spectrometry, from the sample of step (a) the amounts of a group of biomarkers said group comprising (i) at least the biomarkers of one of the combinations shown in Table 1a, wherein the sphingomyelin(s), ceramide(s), and/or phosphorylated sphingobase(s) of Table 1a is/are selected from Table 1b or comprising (ii) at least the biomarkers shown in any one of Tables 17 to 26; (c) comparing the said amounts of the biomarkers with references, whereby pancreas cancer is diagnosed; (d) identifying a subject in need of a pancreas cancer therapy if said subject is diagnosed to suffer from pancreas cancer; and (e) treating the subject with a cancer therapy selected from the group consisting of surgery, radiotherapy, or drug treatment.

2. The method of claim 1, wherein said references are derived from a sample of a subject or a group of subjects known to suffer from pancreatic cancer.

3. The method of claim 1, wherein said references are derived from a sample of a subject or a group of subjects known not to suffer from pancreatic cancer.

4. The method of claim 1, wherein said group of biomarkers comprises at least one further biomarker selected from Table 16.

5. The method of claim 1, wherein said group of biomarkers is the group shown in any one of Tables 2 to 15.

6. The method of claim 1, wherein the said group of biomarkers further comprises CA19-9.

7. The method of claim 1, wherein said diagnosing comprises differentiating between pancreatic cancer and pancreatitis.

8. The method of claim 1, wherein said pancreas cancer is pancreas adenocarcinoma.

9. The method of claim 1, wherein steps (b) and (c) are performed using a device comprising: (a) an analyzing unit for the said sample of the subject comprising a detector for the amounts of a group of biomarkers said group comprising (i) at least the biomarkers of one of the combinations shown in Table 1a, wherein the sphingomyelin(s), ceramide(s), and/or phosphorylated sphingobase(s) of Table 1a is/are selected from Table 1b or comprising (ii) at least the biomarkers shown in any one of Tables 17 to 26, said detector comprising a mass spectrometry device and allowing for the determination of the amounts of the biomarkers of the said group of biomarkers in the sample; and operatively linked thereto, (b) an evaluation unit comprising a data processing unit and a data base, said data base comprising a stored reference and said data processing unit having tangibly embedded an algorithm for carrying out a comparison of the amounts of the biomarkers of the group of biomarkers determined by the analyzing unit and the stored reference and for generating an output information based on which the diagnosis can be established.

Description

EXAMPLES

(1) The invention will now be illustrated by the following Examples which are not intended to restrict or limit the scope of this invention.

Example 1: Patients, Plasma and Serum Preparation

(2) A total of 474 patients with pancreatic cancer, chronic pancreatitis, liver cirrhosis and healthy blood donors were enrolled in the clinical study. In this retrospective case control study samples of: 158 patients suffering from pancreatic ductal adenocarcinoma (PDAC), 159 samples of chronic pancreatitis (CP) patients, samples of 80 patients suffering from liver cirrhosis (LC) and 77 samples of healthy volunteers (blood donors, BD) were included. All patients or their legal representatives gave their written informed consent and the local ethics review boards approved the protocol. Patients were consecutively recruited from two centers. After blood drawing and centrifugation, plasma or serum samples were collected in Eppendorf tubes and stored at 80 C. for further analysis. Sample processing was performed according to the institutional standard operating procedure. Exclusion criteria were a concomitant malignant disease, curative treatment of malignant disease less than 2 years of recruitment to the trial, concomitant cystic diseases of the pancreas, pregnancy or patients unable to give informed consent

Example 2: Metabolite Profiling

(3) MxP Broad and MxP Steroids

(4) Three types of mass spectrometry analyses were applied to all samples. GC-MS (gas chromatography-mass spectrometry; Agilent 6890 GC coupled to an Agilent 5973 MSSystem, Agilent, Waldbronn, Germany) and LC-MS/MS [liquid chromatography-MS/MS; Agilent 1100 HPLCSystem (Agilent, Waldbronn, Germany) coupled to an Applied Biosystems API4000 MS/MSSystem (Applied Biosystems, Darmstadt, Germany)] were used for broad profiling [van Ravenzwaay, B. et al. The use of metabolomics for the discovery of new biomarkers of effect. Toxicol Lett 172, 21-8 (2007]. Solid phase extraction-LC-MS/MS [SPE-LC-MS/MS; Symbiosis Pharma (Spark, Emmen, Netherlands) coupled to an Applied Biosystems API4000 MS/MSSystem (Applied Biosystems, Darmstadt, Germany)] was used for the determination steroid levels. Fractionation and derivatisation of samples and detection technologies have been previously described [van Ravenzwaay, B. et al. The use of metabolomics for the discovery of new biomarkers of effect. Toxicol Lett 172, 21-8 (2007, Roessner, U., Wagner, C., Kopka, J., Trethewey, R. N. & Willmitzer, L. Technical advance: simultaneous analysis of metabolites in potato tuber by gas chromatography-mass spectrometry. Plant J23, 131-42 (2000), Mutch, D. M. et al. Metabolite profiling identifies candidate markers reflecting the clinical adaptations associated with Roux-en-Y gastric bypass surgery. PLoS One 4, e7905 (2009)]. Proteins were removed from plasma samples by precipitation. Subsequently polar and non-polar fractions were separated for both GC-MS and LC-MS/MS analyses by adding water and a mixture of ethanol and dichloromethane. For GC-MS analyses, the non-polar fraction was treated with methanol under acidic conditions to yield the fatty acid methyl esters derived from both free fatty acids and hydrolyzed complex lipids. The polar and non-polar fractions were further derivatized with O-methyl-hydroxyamine hydrochloride (20 mg/ml in pyridine, 50 II) to convert oxo-groups to O-methyloximes and subsequently with a silylating agent (MSTFA, 50 II) before GC-MS analysis. For LC-MS/MS analyses, both fractions were reconstituted in appropriate solvent mixtures. High performance LC (HPLC) was performed by gradient elution using methanol/water/formic acid on reversed phase separation columns. Mass spectrometric detection technology was applied as described in the U.S. Pat. No. 7,196,323, which allows targeted and high sensitivity Multiple Reaction Monitoring profiling in parallel to a full screen analysis. Steroids and their related metabolites were measured by online SPE-LC-MS/MS.

(5) MxP Lipids

(6) Total lipids were extracted from plasma or serum by liquid/liquid extraction using chloroform/methanol. The lipid extracts were subsequently fractionated by normal phase liquid chromatography (NPLC) into eleven different lipid groups according to [Christie, W. W. Rapid separation and quantification of lipid classes by high performance liquid chromatography and mass (light-scattering) detection. J Lipid Res 26, 507-12 (1985)]. The fractions were analyzed by LCMS/MS using electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) with detection of specific multiple reaction monitoring (MRM) transitions for cholesterol esters (CE), free sterols (FS), sphingoymelins (SM), and ceramides (CER) respectively. Sphingosines and sphingosine-1-phosphates (SP) were analyzed by LC-MS/MS using electrospray ionization (ESI) with detection of specific multiple reaction monitoring (MRM) transitions as described by [Schmidt, H., Schmidt, R. & Geisslinger, G. LC-MS/MS-analysis of sphingosine-1-phosphate and related compounds in plasma samples. Prostaglandins Other Lipid Mediat 81, 162-70 (2006)]. The lipid classes Monoacylglycerides (MAG), Triacylglycerides (TAG), Phosphatidylcholines (PC), Phosphatidylserines (PS), Phosphatidylinositoles (PI), Lysophosphatidylcholines (LPC), Diacylglycerols (DAG), Free fatty acids (FFA) were measured by GC-MS. The fractions are analyzed by GC-MS after derivatization with TMSH (Trimethyl sulfonium hydroxide), yielding the fatty acid methyl esters (FAME) corresponding to the acyl moieties of the class-separated lipids. The concentrations of FAME from C14 to C24 are determined in each fraction. In the tables below, any of the abbreviations above is used as prefix for a metabolite to indicate that the respective metabolite has been derived from the respective lipid or lipid fraction.

Example 3: Data Set Analysis and Normalization

(7) Prior to statistical analysis, log 10 transformation of ratios was conducted to assure normaldistribution of data. The software R 2.8.1 (package nlme) was used for data analyses and visualizations. Statistical analysis was done by a simple linear model (ANOVA) with disease, age, gender, BMI, and sample storage time, and sample type (plasma or serum), if appropriate, as fixed effects. Classification analysis with Random Forest (Liaw and Wiener (2002). Classification and Regression by random Forest. R News 2(3), 18-22.) and Elastic Net (Zou and Hastie (2005) Regularization and variable selection via the elastic net, Journal of the Royal Statistical Society, Series B) was done on log 10 transformed data including CA 19-9 or excluding CA19-9, as indicated in the panels. Feature selection was done by a forward search approach where the metabolite best correlating with the residuals of the last model is added to the next model. The inbuilt feature selection of Elastic Net was not applied. We used 10-fold crossvalidation with the feature selection embedded to repeatedly build a model on nine out of ten training folds to then estimate the AUC in an unbiased way on the test fold.

(8) Afterwards the final set of metabolites was determined by retraining the classifier on the entire data. We analyzed three different data sets: (1) data of plasma samples, (2) data of serum samples, and (3) data of plasma and serum samples together. In the case (3), the sample type was used as fixed effect in the ANOVA analysis as described.

(9) To analyse the performance of our selected panels, a classifier was built with a random forest or elastic net analysis with these sets of metabolites and the cross validated classification performance was estimated with the area under the curve (AUC) of a receiver operating characteristic (ROC) analysis. Performance calculations were carried out with or without prior ANOVA correction of metabolite data for confounding factors (age, gender, BMI, and sample type (if appropriate)).

(10) All 95% confidence intervals for the AUC were derived upon the basis of the binormal model as described in Zhou, Obuchowski and McClish [Statistical Methods in Diagnostic Medicine (2011), 2nd Edition, by Zhou, Obuchowski and McClish.] The assumption of binormality was visually checked with a QQ-Plot.

(11) The core panel was selected from results of multivariate analyses (Random Forest=RF)) and univariate analyses (ANOVA, ROC) for differential diagnosis between pancreatic cancer and pancreatitis as follows:

(12) TABLE-US-00001 TABLE 1a Combinations of biomarkers forming biomarker groups (core panels) Lysophosphatidyleth- Com- anolamine Sphingo- Phosphorylated bination (C18:2) myelin Ceramide sphingobase 1 + + + + 2 + + + 3 + + + 4 + + 5 + + + 6 + + 7 + + + = biomarker present in the combination; the sphingolipid shall have two hydroxyl groups, therefore denoted with the d nomenclature, and a sphingobase chain length of 7 C-atoms,;

(13) In cases the biomarker candidates include CA19-9, this data may be applied as numerical or categorical data. Numerical CA19-9 data refer to the measured CA19-9 concentrations (U/ml) per subject. For the categorical data, a defined cut-off of 37 U/ml was used for dichotomization of the data into a low CA19-9 group 37 U/ml) and a high CA19-9 group (>37 U/ml); The analyses were done on the data of plasma samples, the data of serum samples and on a combined analysis of plasma and serum.

(14) TABLE-US-00002 TABLE 1b Directions of biomarker regulation, preferred ceramides, sphingomyelins, and phosphorylated sphingobases Direction in pancreatic Metabolite cancer relative to pancreatitis Lysophosphatidylethanolamine (C18:2) down Ceramide (d17:1,C16:0) up Ceramide (d18:1,C18:0) up Ceramide (d18:1,C24:0) down Sphinganine-1-phosphate (d18:0) down Sphingomyelin (d17:1,C16:0) up Sphingomyelin (d17:1,C16:0) up Sphingomyelin (d17:1,C16:0) up Sphingomyelin (d17:1,C18:0) up Sphingomyelin (d17:1,C18:0) up Sphingomyelin (d17:1,C20:0) up Sphingomyelin (d17:1,C24:1) up Sphingomyelin (d18:1,C18:0) up Sphingomyelin (d18:1,C18:1) up Sphingomyelin (d18:1,C19:0) up Sphingomyelin (d18:1,C20:1) up Sphingomyelin (d18:1,C22:1) up Sphingomyelin (d18:1,C23:1) up Sphingomyelin (d18:1,C24:1) up Sphingomyelin (d18:1,C24:2) up Sphingomyelin (d18:2,C17:0) up Sphingomyelin (d18:2,C19:0) up Sphingomyelin (d18:2,C20:1) up Sphingomyelin (d18:2,C21:0) up Sphingomyelin (d18:2,C22:1) up Sphingomyelin (d18:2,C24:2) up Sphingosine-1-phosphate (d18:1) down

(15) TABLE-US-00003 TABLE 2 Panel Number 3, Algorithm ROC, Matrix Serum and plasma, CA19-9 excluded, no ANOVA Correction of data direction from Metabolite AUC estimate ANOVA Sphingomyelin (d18:1,C19:0) 0.7851 up Sphingomyelin (d18:2,C19:0) 0.7719 up Sphingomyelin (d17:1,C18:0) 0.7743 up Sphingomyelin (d17:1,C20:0) 0.7511 up Sphingomyelin (d17:1,C24:1) 0.7323 up Ceramide (d17:1,C16:0) 0.7278 up 1-Hydroxy-2-amino-(cis,trans)-3, 0.7259 up 5-octadecadiene (from sphingolipids) Lysophosphatidylethanolamine (C18:2) 0.7362 down Sphingomyelin (d17:1,C16:0) 0.735 up Sphingomyelin (d17:1,C18:0) 0.7286 up

(16) TABLE-US-00004 TABLE 3 Panel Number 6, Algorithm RF, Matrix Serum and plasma, CA19-9 excluded, with ANOVA correction of data direction from Metabolite AUC estimate ANOVA Sphingomyelin (d18:1,C19:0) 0.8 up Sphingomyelin (d18:2,C22:1) 0.8 up Citrulline 0.8 down Glycocholic acid 0.8 up O-Acetylcarnitine 0.8 down Eicosapentaenoic acid 0.8 down (C20:cis[5,8,11,14,17]5) Sphinganine-1-phosphate (d18:0) 0.8 down Serine 0.8 down Sphingomyelin (d17:1,C16:0) 0.8 up Lysophosphatidylethanolamine (C18:2) 0.8 down

(17) TABLE-US-00005 TABLE 4 Panel Number 7, Algorithm RF, Matrix Serum and plasma, CA19-9 numerical included, with ANOVA correction of data direction from Metabolite AUC estimate ANOVA Sphingomyelin (d18:2,C22:1) 0.9 up Aspartate 0.9 down O-Acetylcarnitine 0.9 down Hexanoylcarnitine 0.9 up Eicosapentaenoic acid 0.9 down (C20:cis[5,8,11,14,17]5) Sphinganine-1-phosphate (d18:0) 0.9 down Proline 0.9 down Sphingomyelin (d17:1,C16:0) 0.9 up Lysophosphatidylethanolamine (C18:2) 0.9 down CA19-9 0.9 up

(18) TABLE-US-00006 TABLE 5 Panel Number 8, Algorithm RF, Matrix Plasma, CA19-9 excluded, with ANOVA correction of data direction from Metabolite AUC estimate ANOVA Sphingomyelin (d18:2,C21:0) 0.87 up gamma-Tocopherol 0.87 down Sorbitol 0.87 down Citrulline 0.87 down Pregnenolone sulfate 0.87 down Pipecolic acid 0.87 down Propionylcarnitine 0.87 down alpha-Ketoglutarate 0.87 up Sphingomyelin (d17:1,C16:0) 0.87 up Lysophosphatidylethanolamine (C18:2) 0.87 down

(19) TABLE-US-00007 TABLE 6 Panel Number 9, Algorithm RF, Matrix Plasma, CA19-9 numerical included, with ANOVA correction of data direction from Metabolite AUC estimate ANOVA Propionylcarnitine 0.95 down Eicosapentaenoic acid 0.95 down (C20:cis[5,8,11,14,17]5) alpha-Ketoglutarate 0.95 up Proline 0.95 down Sphingomyelin (d18:1,C24:1) 0.95 up Phosphatidylcholine (C18:0,C22:6) 0.95 up Sphingomyelin (d17:1,C16:0) 0.95 up Lysophosphatidylethanolamine (C18:2) 0.95 down Sphingomyelin (d18:2,C17:0) 0.95 up CA19-9 0.95 up

(20) TABLE-US-00008 TABLE 7 Panel Number 12, Algorithm RF, Matrix Plasma, CA19-9 categorical included, with ANOVA correction of data direction from Metabolite AUC estimate ANOVA Sphingomyelin (d18:2,C21:0) 0.94 up gamma-Tocopherol 0.94 down Citrulline 0.94 down Creatinine 0.94 up Propionylcarnitine 0.94 down alpha-Ketoglutarate 0.94 up Proline 0.94 down Sphingomyelin (d17:1,C16:0) 0.94 up Lysophosphatidylethanolamine (C18:2) 0.94 down CA19-9 0.94 up

(21) TABLE-US-00009 TABLE 8 Panel Number 14, Algorithm RF, Matrix Plasma, CA19-9 categorical included, no ANOVA correction of data direction from Metabolite AUC estimate ANOVA gamma-Tocopherol 0.93 down Palmitoleic acid (C16:cis[9]1) 0.93 down Proline 0.93 down Ketoleucine 0.93 down Isocitrate 0.93 up Phosphatidylcholine (C18:0,C22:6) 0.93 up Sphingomyelin (d17:1,C16:0) 0.93 up Lysophosphatidylethanolamine (C18:2) 0.93 down Sphingomyelin (d18:2,C17:0) 0.93 up CA19-9 0.93 up

(22) TABLE-US-00010 TABLE 9 Panel Number 15, Algorithm RF, Matrix Serum and plasma, CA19-9 excluded, with ANOVA correction of data direction from Metabolite AUC estimate ANOVA Sphingomyelin (d18:1,C19:0) 0.8 up Sphingomyelin (d18:2,C22:1) 0.8 up Citrulline 0.8 down Glycocholic acid 0.8 up O-Acetylcarnitine 0.8 down Eicosapentaenoic acid 0.8 down (C20:cis[5,8,11,14,17]5) Sphinganine-1-phosphate (d18:0) 0.8 down Serine 0.8 down Sphingomyelin (d17:1,C16:0) 0.8 up Lysophosphatidylethanolamine (C18:2) 0.8 down

(23) TABLE-US-00011 TABLE 10 Panel Number 16, Algorithm RF, Matrix Serum and plasma, CA19-9 excluded, no ANOVA correction of data direction from Metabolite AUC estimate ANOVA Sphingomyelin (d18:1,C19:0) 0.85 up Coenzyme Q10 0.85 down Glycocholic acid 0.85 up Docosahexaenoic acid 0.85 up (C22:cis[4,7,10,13,16,19]6) Palmitoleic acid (C16:cis[9]1) 0.85 down Lignoceric acid (C24:0) 0.85 down 1,5-Anhydrosorbitol 0.85 down Dehydroepiandrosterone sulfate 0.85 down Lysophosphatidylethanolamine (C18:2) 0.85 down

(24) TABLE-US-00012 TABLE 11 Panel Number 17, Algorithm RF, Matrix Serum and plasma, CA19-9 categorical included, with ANOVA correction of data direction from Metabolite AUC estimate ANOVA Sphingomyelin (d18:2,C22:1) 0.88 up Cryptoxanthin 0.88 up Aspartate 0.88 down O-Acetylcarnitine 0.88 down Eicosapentaenoic acid 0.88 down (C20:cis[5,8,11,14,17]5) Sphinganine-1-phosphate (d18:0) 0.88 down Histidine 0.88 down Sphingomyelin (d17:1,C16:0) 0.88 up Lysophosphatidylethanolamine (C18:2) 0.88 down CA19-9 0.88 up

(25) TABLE-US-00013 TABLE 12 Panel Number 18, Algorithm RF, Matrix Serum and plasma, CA19-9 categorical included, no ANOVA correction of data direction from Metabolite AUC estimate ANOVA Sphingomyelin (d18:2,C19:0) 0.91 up Ceramide (d18:1,C24:0) 0.91 down Coenzyme Q10 0.91 down Sphinganine-1-phosphate (d18:0) 0.91 down Histidine 0.91 down Dehydroepiandrosterone sulfate 0.91 down TAG (C18:2,C18:2) 0.91 up Lysophosphatidylethanolamine (C18:2) 0.91 down Sphingomyelin (d17:1,C16:0) 0.91 up CA19-9 0.91 up

(26) TABLE-US-00014 TABLE 13 Panel Number 19, Algorithm RF, Matrix Serum and plasma, CA19-9 excluded, with ANOVA correction of data AUC direction from Metabolite estimate ANOVA Sphingomyelin (d18:1, C19:0) 0.81 up Sphingomyelin (d18:2, C22:1) 0.81 up Coenzyme Q9 0.81 down O-Acetylcarnitine 0.81 down Hexanoylcarnitine 0.81 up Palmitoleic acid (C16:cis[9]1) 0.81 down Histidine 0.81 down Uric acid 0.81 down TAG (C16:0, C18:1, C18:3) 0.81 up Lysophosphatidylethanolamine (C18:2) 0.81 down

(27) TABLE-US-00015 TABLE 14 Panel Number 20, Algorithm RF, Matrix Serum and plasma, CA19-9 excluded, no ANOVA correction of data AUC direction from Metabolite estimate ANOVA Sphingomyelin (d18:1, C19:0) 0.83 up Coenzyme Q10 0.83 down Uridine 0.83 down Trimethylamine-N-oxide (TMAO) 0.83 down Docosahexaenoic acid 0.83 up (C22:cis[4,7,10,13,16,19]6) Palmitoleic acid (C16:cis[9]1) 0.83 down Threonine 0.83 down Uric acid 0.83 down Lysophosphatidylethanolamine (C18:2) 0.83 down

(28) TABLE-US-00016 TABLE 15 Panel Number 22, Algorithm RF, Matrix Serum and plasma, CA19-9 categorical included, no ANOVA correction of data AUC direction from Metabolite estimate ANOVA Sphingomyelin (d18:2, C19:0) 0.91 up Sorbitol 0.91 down Coenzyme Q9 0.91 down Uridine 0.91 down Proline 0.91 down Uric acid 0.91 down Phosphatidylcholine (C18:0, C22:6) 0.91 up Lysophosphatidylethanolamine (C18:2) 0.91 down CA19-9 0.91 up

(29) TABLE-US-00017 TABLE 16 Additional Biomarkers for Panel Composition gamma-Tocopherol Coenzyme Q10 Sorbitol Aspartate Citrulline Coenzyme Q9 Pregnenolone sulfate Creatinine Creatine Glycocholic acid Trimethylamine-N-oxide (TMAO) Pipecolic acid Proline betaine O-Acetylcarnitine Carnosine Propionylcarnitine Hexanoylcarnitine Eicosapentaenoic acid (C20:cis[5,8,11,14,17]5) Docosahexaenoic acid (C22:cis[4,7,10,13,16,19]6) Palmitoleic acid (C16:cis[9]1) Lignoceric acid (C24:0) 1-Hydroxy-2-amino-(cis,trans)-3,5-octadecadiene (from sphingolipids) Pyruvate Threonine Mannose alpha-Ketoglutarate Serine Proline 3-Hydroxybutyrate 1,5-Anhydrosorbitol Histidine Ketoleucine 2-Hydroxybutyrate Isocitrate Dehydroepiandrosterone sulfate TAG (C18:2, C18:2) TAG (C16:0, C18:1, C18:3) Sphingosine-1-phosphate (d18:1) Kynurenine Tryptophan

(30) TABLE-US-00018 TABLE 17 Panel Number 1, Algorithm ANOVA, Matrix Serum and plasma, CA19-9 excluded, with ANOVA correction of data AUC direction from Metabolite estimate ANOVA Sphingomyelin (d18:1, C20:1) 0.68 up Sphingomyelin (d18:1, C22:1) 0.67 up Sphingomyelin (d18:1, C23:1) 0.71 up Sphingomyelin (d18:1, C24:2) 0.66 up Citrulline 0.63 down Coenzyme Q9 0.62 down Mannose 0.63 up Proline 0.66 down Histidine 0.66 down Uric acid 0.67 down

(31) TABLE-US-00019 TABLE 18 Panel Number 2, Algorithm ANOVA, Matrix Serum and plasma, CA19-9 excluded, with ANOVA correction of data AUC direction from Metabolite estimate ANOVA Sphingomyelin (d18:1, C18:0) 0.80 (Plasma, excl. up CA19-9) Sphingomyelin (d18:1, C18:1) 0.80 (Plasma, excl. up CA19-9) Sphingomyelin (d18:1, C20:1) 0.80 (Plasma, excl. up CA19-9) Sphingomyelin (d18:1, C22:1) 0.80 (Plasma, excl. up CA19-9) Sphingomyelin (d18:1, C23:1) 0.80 (Plasma, excl. up CA19-9) Sphingomyelin (d18:1, C24:2) 0.80 (Plasma, excl. up CA19-9) Ceramide (d18:1, C18:0) 0.80 (Plasma, excl. up CA19-9) Ceramide (d17:1, 016:0) 0.80 (Plasma, excl. up CA19-9) Coenzyme Q9 0.80 (Plasma, excl. down CA19-9) Proline 0.80 (Plasma, excl. down CA19-9)

(32) TABLE-US-00020 TABLE 19 Panel Number 4, Algorithm ROC, Matrix Plasma, CA19-9 excluded, no ANOVA correction of data AUC direction from Metabolite estimate ANOVA Sphingomyelin (d18:1, C19:0) 0.7819 up Sphingomyelin (d18:2, C19:0) 0.7862 up Sphingomyelin (d18:2, C20:1) 0.7537 up Sphingomyelin (d17:1, C16:0) 0.7325 up Sphingomyelin (d17:1, C18:0) 0.8133 up Sphingomyelin (d17:1, C20:0) 0.7545 up 2-Hydroxybutyrate 0.7065 up Phosphatidylcholine (C18:0, C22:6) 0.7769 up Sphingomyelin (d17:1, C16:0) 0.7652 up Sphingomyelin (d18:2, C17:0) 0.7657 up

(33) TABLE-US-00021 TABLE 20 Panel Number 5, Algorithm ROC, Matrix Serum, CA19-9 excluded, no ANOVA correction of data AUC direction from Metabolite estimate ANOVA Sphingomyelin (d18:1, C19:0) 0.781 up Sphingomyelin (d18:2, C19:0) 0.7577 up Sphingomyelin (d18:2, C21:0) 0.7217 up Sphingomyelin (d17:1, C18:0) 0.7406 up Sphingomyelin (d17:1, C20:0) 0.7478 up Sphingomyelin (d17:1, C24:1) 0.7427 up Ceramide (d17:1, C16:0) 0.7469 up Coenzyme Q10 0.7397 down Uric acid 0.7133 down Sphingomyelin (d17:1, C18:0) 0.7023 up

(34) TABLE-US-00022 TABLE 21 Panel Number 10, Algorithm RF, Matrix Serum, CA19-9 excluded, with ANOVA correction of data AUC direction from Metabolite estimate ANOVA Sphingomyelin (d18:1, C19:0) 0.75 up Sphingomyelin (d18:2, C24:2) 0.75 up Coenzyme Q10 0.75 down Coenzyme Q9 0.75 down Uridine 0.75 down Carnosine 0.75 down Sphinganine-1-phosphate (d18:0) 0.75 down 3-Hydroxybutyrate 0.75 down Histidine 0.75 down

(35) TABLE-US-00023 TABLE 22 Panel Number 11, Algorithm RF, Matrix Serum, CA19-9 numerical included, with ANOVA correction of data AUC direction from Metabolite estimate ANOVA Sphingomyelin (d18:2, C22:1) 0.91 up Proline betaine 0.91 up O-Acetylcarnitine 0.91 down Sphinganine-1-phosphate (d18:0) 0.91 down Uric acid 0.91 down TAG (C18:2, C18:2) 0.91 up Phosphatidylcholine (C16:0, C20:5) 0.91 down CA19-9 0.91 up

(36) TABLE-US-00024 TABLE 23 Panel Number 13, Algorithm RF, Matrix Plasma, CA19-9 categorical included, no ANOVA correction of data AUC direction from Metabolite estimate ANOVA Sphingomyelin (d17:1, C18:0) 0.96 up Ceramide (d18:1, C24:0) 0.96 down Sphinganine-1-phosphate (d18:0) 0.96 down Pyruvate 0.96 down Proline 0.96 down Histidine 0.96 down Isocitrate 0.96 up Phosphatidylcholine (C18:0, C22:6) 0.96 up Sphingomyelin (d18:2, C17:0) 0.96 up CA19-9 0.96 up

(37) TABLE-US-00025 TABLE 24 Panel Number 21, Algorithm RF, Matrix Serum and Plasma, CA19-9 categorical included, with ANOVA correction of data AUC direction from Metabolite estimate ANOVA Sphingomyelin (d18:1, C19:0) 0.88 up Sphingomyelin (d18:2, C22:1) 0.88 up Coenzyme Q9 0.88 down Creatine 0.88 down O-Acetylcarnitine 0.88 down Hexanoylcarnitine 0.88 up Proline 0.88 down Uric acid 0.88 down TAG (C18:2, C18:2) 0.88 up CA19-9 0.88 up

(38) TABLE-US-00026 TABLE 25 Panel Number 23, Algorithm ANOVA, Matrix Serum and Plasma, CA19-9 excluded, with ANOVA correction of data AUC direction from Metabolite estimate ANOVA Sphingomyelin (d18:1, C18:0) 0.76 up Sphingomyelin (d18:1, C18:1) 0.76 up Sphingomyelin (d18:1, C20:1) 0.76 up Sphingomyelin (d18:1, C22:1) 0.76 up Sphingomyelin (d18:1, C23:1) 0.76 up Lysophosphatidylethanolamine 0.76 down (C18:2) Coenzyme Q9 0.76 down Ceramide (d17:1, C16:0) 0.76 up Ceramide (d18:1, C18:0) 0.76 up Prolin 0.76 down

(39) TABLE-US-00027 TABLE 26 Panel Number 24, Algorithm elastic net, Matrix Plasma, CA19-9 included, no ANOVA Correction of data AUC direction from Metabolite estimate ANOVA CA19-9 0.93 up Sphingomyelin (d17:1, C18:0) 0.93 up Phosphatidylcholine (C18:0, C22:6) 0.93 up Proline 0.93 down Sphingosine-1-phosphate (d18:1) 0.93 down alpha-Ketoglutarate 0.93 up Palmitoleic acid (C16:cis[9]1) 0.93 down Kynurenine 0.93 up Tryptophan 0.93 down Serine 0.93 down

(40) TABLE-US-00028 TABLE 27 ANOVA Data for all Metabolites listed in any of Tables 1 to 26 for all matrices ANOVA result of pancreatic Direc- cancer relative to pancreatitis Matrix Metabolite tion Ratio p-value t-value Plasma 1,5-Anhydrosorbitol down 0.70 0.112184 1.60 Plasma 1-Hydroxy-2-amino- up 1.42 0.001078 3.33 (cis,trans)-3,5-octadecadiene (from sphingolipids) Plasma 2-Hydroxybutyrate up 1.37 0.02006 2.35 Plasma 3-Hydroxybutyrate up 1.56 0.162013 1.41 Plasma alpha-Ketoglutarate up 1.82 2.83E05 4.32 Plasma Aspartate down 0.98 0.803054 0.25 Plasma CA19-9 up 18.36 6.89E09 6.18 Plasma Carnosine down 0.61 0.140206 1.49 Plasma Ceramide (d17:1, C16:0) up 1.33 0.00966 2.62 Plasma Ceramide (d18:1, C18:0) up 1.44 0.010846 2.58 Plasma Ceramide (d18:1, C24:0) down 0.79 0.001509 3.23 Plasma Citrulline down 0.76 0.000856 3.40 Plasma Coenzyme Q10 down 0.85 0.165097 1.39 Plasma Coenzyme Q9 down 0.58 0.003463 2.97 Plasma Creatine down 0.75 0.004049 2.92 Plasma Creatinine up 1.03 0.63821 0.47 Plasma Cryptoxanthin down 0.87 0.553268 0.59 Plasma Dehydroepiandrosterone sulfate down 0.77 0.221202 1.23 Plasma Docosahexaenoic acid up 1.47 0.000276 3.72 (C22:cis[4,7,10,13,16,19]6) Plasma Eicosapentaenoic acid down 0.68 0.006485 2.76 (C20:cis[5,8,11,14,17]5) Plasma gamma-Tocopherol down 0.68 0.004717 2.87 Plasma Glycocholic acid up 6.15 0.000353 3.66 Plasma Hexanoylcarnitine up 1.14 0.295431 1.05 Plasma Histidine down 0.77 0.000324 3.68 Plasma Isocitrate up 1.26 0.008074 2.68 Plasma Ketoleucine down 0.83 0.146653 1.46 Plasma Lignoceric acid (C24:0) down 0.93 0.420195 0.81 Plasma Lysophosphatidylethanolamine (C18:2) down 0.61 1.44E06 5.02 Plasma Mannose up 1.33 0.023498 2.29 Plasma O-Acetylcarnitine up 1.07 0.547685 0.60 Plasma Palmitoleic acid (C16:cis[9]1) down 0.85 0.288324 1.07 Plasma Phosphatidylcholine down 0.90 0.032376 2.16 (C16:0, C20:5) Plasma Phosphatidylcholine up 1.26 8.59E05 4.04 (C18:0, C22:6) Plasma Pipecolic acid down 0.74 0.001085 3.33 Plasma Pregnenolone sulfate down 0.84 0.204908 1.27 Plasma Proline down 0.69 2.24E05 4.38 Plasma Proline betaine down 0.87 0.585038 0.55 Plasma Propionylcarnitine down 0.69 0.00053 3.54 Plasma Pyruvate down 0.93 0.367408 0.90 Plasma Serine down 0.96 0.4496 0.76 Plasma Sorbitol down 0.55 0.000401 3.63 Plasma Sphinganine-1-phosphate down 0.79 0.025867 2.25 (d18:0) Plasma Sphingomyelin (d17:1, C16:0) up 1.39 0.002201 3.11 Plasma Sphingomyelin (d17:1, C16:0) up 1.20 0.004553 2.88 Plasma Sphingomyelin (d17:1, C16:0) up 1.28 0.000338 3.67 Plasma Sphingomyelin (d17:1, C18:0) up 1.36 4.86E05 4.18 Plasma Sphingomyelin (d17:1, C18:0) up 1.21 0.001593 3.22 Plasma Sphingomyelin (d17:1, C20:0) up 1.34 0.001219 3.30 Plasma Sphingomyelin (d17:1, C24:1) up 1.59 4.28E05 4.21 Plasma Sphingomyelin (d18:1, C18:0) up 1.29 0.00202 3.14 Plasma Sphingomyelin (d18:1, C18:1) up 1.39 0.00092 3.38 Plasma Sphingomyelin (d18:1, C19:0) up 1.44 7.72E05 4.06 Plasma Sphingomyelin (d18:1, C20:1) up 1.62 0.000148 3.89 Plasma Sphingomyelin (d18:1, C22:1) up 1.77 5.47E05 4.15 Plasma Sphingomyelin (d18:1, C23:1) up 1.67 4.69E05 4.19 Plasma Sphingomyelin (d18:1, C24:1) up 1.19 0.017318 2.41 Plasma Sphingomyelin (d18:1, C24:2) up 1.58 0.000702 3.46 Plasma Sphingomyelin (d18:2, C17:0) up 1.15 0.005612 2.81 Plasma Sphingomyelin (d18:2, C19:0) up 1.37 0.000937 3.37 Plasma Sphingomyelin (d18:2, C20:1) up 1.39 0.000229 3.78 Plasma Sphingomyelin (d18:2, C21:0) up 1.29 0.007497 2.71 Plasma Sphingomyelin (d18:2, C22:1) up 1.62 3.12E06 4.84 Plasma Sphingomyelin (d18:2, C24:2) up 1.33 0.001829 3.17 Plasma TAG (C16:0, C18:1, C18:3) up 1.12 0.254012 1.14 Plasma TAG (C18:2, C18:2) up 1.03 0.787305 0.27 Plasma Threonine down 0.88 0.105825 1.63 Plasma Trimethylamine-N-oxide up 1.45 0.211712 1.25 (TMAO) Plasma Uric acid down 0.81 0.039553 2.08 Plasma Uridine down 0.93 0.36906 0.90 Serum 1,5-Anhydrosorbitol down 0.72 0.129242 1.53 Serum 1-Hydroxy-2-amino- up 1.24 0.086788 1.72 (cis,trans)-3,5-octadecadiene (from sphingolipids) Serum 2-Hydroxybutyrate down 0.97 0.849631 0.19 Serum 3-Hydroxybutyrate down 0.54 0.085235 1.73 Serum alpha-Ketoglutarate down 0.86 0.577279 0.56 Serum Aspartate down 0.78 0.081011 1.76 Serum CA19-9 up 8.33 9.03E05 4.02 Serum Carnosine down 0.67 0.054001 1.94 Serum Ceramide (d17:1, C16:0) up 1.39 0.00898 2.65 Serum Ceramide (d18:1, C18:0) up 1.42 0.018761 2.38 Serum Ceramide (d18:1, C24:0) down 0.93 0.306255 1.03 Serum Citrulline down 0.67 0.000306 3.73 Serum Coenzyme Q10 down 0.64 0.001002 3.35 Serum Coenzyme Q9 down 0.64 0.0233 2.29 Serum Creatine up 1.01 0.947844 0.07 Serum Creatinine down 0.85 0.118716 1.57 Serum Cryptoxanthin up 1.50 0.086784 1.72 Serum Dehydroepiandrosterone sulfate down 0.75 0.173751 1.37 Serum Docosahexaenoic acid up 1.02 0.878814 0.15 (C22:cis[4,7,10,13,16,19]6) Serum Eicosapentaenoic acid down 0.73 0.048572 1.99 (C20:cis[5,8,11,14,17]5) Serum gamma-Tocopherol down 0.89 0.459956 0.74 Serum Glycocholic acid up 1.37 0.455023 0.75 Serum Histidine down 0.72 0.006032 2.78 Serum Isocitrate down 0.86 0.075543 1.79 Serum Ketoleucine down 0.93 0.558542 0.59 Serum Lignoceric acid (C24:0) down 0.90 0.286922 1.07 Serum Lysophosphatidylethanolamine (C18:2) down 0.83 0.097566 1.67 Serum Mannose up 1.75 0.028419 2.21 Serum O-Acetylcarnitine down 0.64 0.001349 3.27 Serum Palmitoleic acid (C16:cis[9]1) down 0.68 0.019995 2.35 Serum Phosphatidylcholine down 0.85 0.055291 1.93 (C16:0, C20:5) Serum Phosphatidylcholine up 1.05 0.513731 0.65 (C18:0, C22:6) Serum Pipecolic acid up 1.07 0.521609 0.64 Serum Pregnenolone sulfate down 0.69 0.031915 2.17 Serum Proline down 0.75 0.004589 2.88 Serum Proline betaine up 1.16 0.556731 0.59 Serum Pyruvate down 0.76 0.266689 1.11 Serum Serine down 0.86 0.081561 1.75 Serum Sorbitol up 1.16 0.519633 0.65 Serum Sphinganine-1-phosphate down 0.74 0.031159 2.18 (d18:0) Serum Sphingomyelin (d17:1, C16:0) up 1.25 0.053968 1.94 Serum Sphingomyelin (d17:1, C16:0) up 1.16 0.143493 1.48 Serum Sphingomyelin (d17:1, C16:0) up 1.14 0.301942 1.04 Serum Sphingomyelin (d17:1, C18:0) up 1.33 0.008979 2.65 Serum Sphingomyelin (d17:1, C18:0) up 1.79 0.126103 1.54 Serum Sphingomyelin (d17:1, C20:0) up 1.36 0.00399 2.92 Serum Sphingomyelin (d17:1, C24:1) up 1.18 0.043725 2.03 Serum Sphingomyelin (d18:1, C18:0) up 1.28 0.047624 2.00 Serum Sphingomyelin (d18:1, C18:1) up 1.33 0.012649 2.52 Serum Sphingomyelin (d18:1, C19:0) up 1.33 0.000409 3.61 Serum Sphingomyelin (d18:1, C20:1) up 1.25 0.021854 2.32 Serum Sphingomyelin (d18:1, C22:1) up 1.31 0.020283 2.35 Serum Sphingomyelin (d18:1, C23:1) up 1.18 0.048637 1.99 Serum Sphingomyelin (d18:1, C24:1) up 1.07 0.305724 1.03 Serum Sphingomyelin (d18:1, C24:2) up 1.27 0.036538 2.11 Serum Sphingomyelin (d18:2, C17:0) up 1.07 0.54147 0.61 Serum Sphingomyelin (d18:2, C19:0) up 1.45 0.000637 3.49 Serum Sphingomyelin (d18:2, C20:1) up 1.38 0.002494 3.08 Serum Sphingomyelin (d18:2, C21:0) up 1.34 0.00694 2.74 Serum Sphingomyelin (d18:2, C22:1) up 1.32 0.001103 3.33 Serum Sphingomyelin (d18:2, C24:2) up 1.22 0.004057 2.92 Serum TAG (C16:0, C18:1, C18:3) up 1.52 0.003144 3.00 Serum TAG (C18:2, C18:2) up 1.49 0.00169 3.20 Serum Threonine down 0.81 0.026945 2.23 Serum Trimethylamine-N-oxide down 0.68 0.255352 1.14 (TMAO) Serum Uric acid down 0.66 0.001044 3.34 Serum Uridine down 0.79 0.025021 2.26 Serum + Plasma 1,5-Anhydrosorbitol down 0.68 0.010301 2.58 Serum + Plasma 1-Hydroxy-2-amino- up 1.28 0.002321 3.07 (cis,trans)-3,5-octadecadiene (from sphingolipids) Serum + Plasma 2-Hydroxybutyrate up 1.10 0.372449 0.89 Serum + Plasma 3-Hydroxybutyrate down 0.78 0.289097 1.06 Serum + Plasma alpha-Ketoglutarate up 1.19 0.245593 1.16 Serum + Plasma Aspartate down 0.85 0.046851 2.00 Serum + Plasma CA19-9 up 11.88 3.93E12 7.24 Serum + Plasma Carnosine down 0.68 0.020215 2.34 Serum + Plasma Ceramide (d17:1, C16:0) up 1.34 0.000362 3.61 Serum + Plasma Ceramide (d18:1, C18:0) up 1.43 0.000256 3.70 Serum + Plasma Ceramide (d18:1, C24:0) down 0.89 0.020191 2.34 Serum + Plasma Citrulline down 0.71 1.55E07 5.39 Serum + Plasma Coenzyme Q10 down 0.75 0.000589 3.47 Serum + Plasma Coenzyme Q9 down 0.62 0.000287 3.67 Serum + Plasma Creatine down 0.88 0.107085 1.62 Serum + Plasma Creatinine down 0.93 0.240945 1.17 Serum + Plasma Cryptoxanthin up 1.18 0.309544 1.02 Serum + Plasma Dehydroepiandrosterone sulfate down 0.74 0.042037 2.04 Serum + Plasma Docosahexaenoic acid up 1.19 0.035833 2.11 (C22:cis[4,7,10,13,16,19]6) Serum + Plasma Eicosapentaenoic acid down 0.68 0.000267 3.69 (C20:cis[5,8,11,14,17]5) Serum + Plasma gamma-Tocopherol down 0.81 0.033443 2.14 Serum + Plasma Glycocholic acid up 2.46 0.004039 2.90 Serum + Plasma Histidine down 0.73 5.28E06 4.63 Serum + Plasma Isocitrate up 1.00 0.958958 0.05 Serum + Plasma Ketoleucine down 0.89 0.198701 1.29 Serum + Plasma Lignoceric acid (C24:0) down 0.91 0.151 1.44 Serum + Plasma Lysophosphatidylethanolamine (C18:2) down 0.71 2.15E06 4.85 Serum + Plasma Mannose up 1.51 0.003417 2.95 Serum + Plasma O-Acetylcarnitine down 0.78 0.005213 2.81 Serum + Plasma Palmitoleic acid (C16:cis[9]1) down 0.75 0.007319 2.70 Serum + Plasma Phosphatidylcholine down 0.86 0.001625 3.18 (C16:0, C20:5) Serum + Plasma Phosphatidylcholine up 1.14 0.004201 2.88 (C18:0, C22:6) Serum + Plasma Pipecolic acid down 0.90 0.113584 1.59 Serum + Plasma Pregnenolone sulfate down 0.78 0.01873 2.36 Serum + Plasma Proline down 0.73 1.61E06 4.89 Serum + Plasma Proline betaine down 0.97 0.8797 0.15 Serum + Plasma Pyruvate down 0.84 0.199114 1.29 Serum + Plasma Serine down 0.89 0.019434 2.35 Serum + Plasma Sorbitol down 0.84 0.192819 1.31 Serum + Plasma Sphinganine-1-phosphate down 0.78 0.004251 2.88 (d18:0) Serum + Plasma Sphingomyelin (d17:1, C16:0) up 1.27 0.002065 3.11 Serum + Plasma Sphingomyelin (d17:1, C16:0) up 1.14 0.012593 2.51 Serum + Plasma Sphingomyelin (d17:1, C16:0) up 1.19 0.006193 2.76 Serum + Plasma Sphingomyelin (d17:1, C18:0) up 1.32 2.4E05 4.29 Serum + Plasma Sphingomyelin (d17:1, C18:0) up 1.47 0.013879 2.48 Serum + Plasma Sphingomyelin (d17:1, C20:0) up 1.34 1.64E05 4.38 Serum + Plasma Sphingomyelin (d17:1, C24:1) up 1.31 4E05 4.17 Serum + Plasma Sphingomyelin (d18:1, C18:0) up 1.27 0.001083 3.30 Serum + Plasma Sphingomyelin (d18:1, C18:1) up 1.31 0.000203 3.76 Serum + Plasma Sphingomyelin (d18:1, C19:0) up 1.37 7.78E08 5.50 Serum + Plasma Sphingomyelin (d18:1, C20:1) up 1.37 3.12E05 4.23 Serum + Plasma Sphingomyelin (d18:1, C22:1) up 1.45 2.18E05 4.31 Serum + Plasma Sphingomyelin (d18:1, C23:1) up 1.34 3.21E05 4.22 Serum + Plasma Sphingomyelin (d18:1, C24:1) up 1.11 0.026027 2.24 Serum + Plasma Sphingomyelin (d18:1, C24:2) up 1.38 0.00011 3.92 Serum + Plasma Sphingomyelin (d18:2, C17:0) up 1.11 0.046649 2.00 Serum + Plasma Sphingomyelin (d18:2, C19:0) up 1.38 3.21E06 4.74 Serum + Plasma Sphingomyelin (d18:2, C20:1) up 1.33 1.91E05 4.34 Serum + Plasma Sphingomyelin (d18:2, C21:0) up 1.30 0.000168 3.81 Serum + Plasma Sphingomyelin (d18:2, C22:1) up 1.41 7.54E08 5.51 Serum + Plasma Sphingomyelin (d18:2, C24:2) up 1.27 9.47E06 4.50 Serum + Plasma TAG (C16:0, C18:1, C18:3) up 1.36 0.000333 3.63 Serum + Plasma TAG (C18:2, C18:2) up 1.31 0.000433 3.56 Serum + Plasma Threonine down 0.83 0.001967 3.12 Serum + Plasma Trimethylamine-N-oxide down 0.97 0.878188 0.15 (TMAO) Serum + Plasma Uric acid down 0.73 3.87E05 4.18 Serum + Plasma Uridine down 0.86 0.029178 2.19 Serum Kynurenine down 0.96 0.711959 0.37 Plasma Kynurenine up 1.16 0.115984 1.58 Serum + Plasma Kynurenine up 1.08 0.308068 1.02 Serum Sphingosine-1-phosphate down 0.87 0.318297 1.00 (d18:1) Plasma Sphingosine-1-phosphate down 0.93 0.325758 0.99 (d18:1) Serum + Plasma Sphingosine-1-phosphate down 0.91 0.236226 1.19 (d18:1) Serum Tryptophan down 0.88 0.266443 1.12 Plasma Tryptophan down 0.86 0.129383 1.52 Serum + Plasma Tryptophan down 0.87 0.059412 1.89

(41) TABLE-US-00029 TABLE 28 Single metabolite biomarkers for diagnosing pancreatic cancer ANOVA result of pancreatic Direc- cancer relative to pancreatitis Matrix Metabolite tion Ratio p-value t-value Serum Lysophosphatidyl- down 0.83 0.0975655 1.67 ethanolamine (C18:2) Serum Sphingomyelin up 1.79 0.1261030 1.54 (d17:1, C18:0) Serum Sphingomyelin up 1.16 0.1434933 1.48 (d17:1, C16:0) Plasma Lysophosphatidyl- down 0.61 0.0000014 5.02 ethanolamine (C18:2) Plasma Sphingomyelin up 1.28 0.0003381 3.67 (d17:1, C16:0) Plasma Sphingomyelin up 1.21 0.0015925 3.22 (d17:1, C18:0) Plasma Sphingomyelin up 1.20 0.0045531 2.88 (d17:1, C16:0) Plasma Sphingomyelin up 1.15 0.0056123 2.81 (d18:2, C17:0) Plasma Sphingomyelin up 1.19 0.0173175 2.41 (d18:2, C24:0) Plasma Cholesterolester, down 0.99 0.0866887 1.72 total Plasma Lysophosphatidyl- down 0.87 0.0957764 1.68 choline (C18:0) Serum + Lysophosphatidyl- down 0.71 0.0000021 4.85 Plasma ethanolamine (C18:2) Serum + Sphingomyelin up 1.19 0.0061928 2.76 Plasma (d17:1, C16:0) Serum + Sphingomyelin up 1.14 0.0125930 2.51 Plasma (d17:1, C16:0) Serum + Sphingomyelin up 1.47 0.0138791 2.48 Plasma (d17:1, C18:0) Serum + Sphingomyelin up 1.11 0.0260273 2.24 Plasma (d18:2, C24:0) Serum + Lysophosphatidyl- down 0.90 0.0375720 2.09 Plasma choline (C18:0) Serum + Sphingomyelin up 1.11 0.0466493 2.00 Plasma (d18:2, C17:0) Serum + TAG (C18:1, up 1.14 0.1343839 1.50 Plasma C18:2, C18:3)

(42) TABLE-US-00030 TABLE 29 List of identified biomarkers in serum for pancreatic cancer relative to pancreatitis ANOVA result of pancreatic cancer relative to pancreatitis in serum Estimated Direc- fold Metabolite tion change p-value t-value Citrulline down 0.6659 0.000306024 3.72978 Sphingomyelin (d18:1, C19:0) up 1.3323 0.000408675 3.61444 Sphingomyelin (d18:2, C19:0) up 1.4549 0.000637255 3.487735 Coenzyme Q10 down 0.644 0.001001669 3.35476 Uric acid down 0.6634 0.001043796 3.3425 Sphingomyelin (d18:2, C22:1) up 1.3202 0.001102996 3.326448 O-Acetylcarnitine down 0.64 0.001348925 3.26542 Cholesterylester C16:1 down 0.6485 0.001532724 3.22691 Sphingomyelin (d18:2, C18:1) up 1.4172 0.001651561 3.203999 Ceramide (d18:2, C20:0) up 1.4975 0.0019335 3.155992 Sphingomyelin (d18:2, C20:1) up 1.3823 0.002494254 3.075278 TAG (C16:0, C18:1, C18:3) up 1.5231 0.003143958 3.00091 Sphingomyelin (d17:1, C20:0) up 1.3626 0.003989988 2.923562 Sphingomyelin (d18:2, C24:2) up 1.2165 0.00405684 2.91809 Sphingomyelin (d18:1, C21:0) up 1.3025 0.004503963 2.883483 Proline down 0.748 0.004588723 2.877 Sphingomyelin (d18:2, C20:0) up 1.2522 0.004755925 2.865343 N,N-Dimethylarginine (ADMA) down 0.8133 0.005429519 2.82058 Histidine down 0.7173 0.006031531 2.78489 Sphingomyelin (d18:2, C18:0) up 1.2451 0.006065 2.783001 FFA_Palmitoleic acid down 0.5808 0.006354103 2.76734 (C16:cis[9]1) Pseudouridine down 0.6795 0.006517367 2.75838 Sphingomyelin (d18:2, C21:0) up 1.3436 0.006940038 2.736985 Ceramide (d18:1, C20:0) up 1.4129 0.007134217 2.727937 Ceramide (d18:1, C21:0) up 1.4489 0.007309769 2.719503 Cholesterylester C14:1 down 0.639 0.008019146 2.68671 7-Methylguanosine down 0.7758 0.008427974 2.66903 Sphingomyelin (d17:1, C18:0) up 1.3307 0.008979082 2.646896 Ceramide (d17:1, C16:0) up 1.3949 0.008979607 2.647335 1-Methylhistidine down 0.8072 0.009819775 2.61485 Tetradecanoylcarnitine down 0.7371 0.010786698 2.58114 Pantothenic acid down 0.7347 0.011898307 2.5456 Sphingomyelin (d18:1, C18:1) up 1.3253 0.01264873 2.523445 DAG_Palmitic acid (C16:0) down 0.42 0.013190989 2.50822 Ceramide (d18:2, C18:0) up 1.4046 0.013880851 2.489579 Sphingomyelin (d18:2, C23:1) up 1.2538 0.01514839 2.456672 PS_Palmitic acid (C16:0) down 0.3043 0.016545194 2.42371 3-Indoxylsulfate down 0.5227 0.016706631 2.41986 Campesterol down 0.6904 0.016791704 2.41794 Sphingomyelin (d18:2, C22:0) up 1.304 0.017927017 2.393099 Sphingomyelin (d18:2, C18:0) up 1.3422 0.017980277 2.391968 Ceramide (d18:1, C18:0) up 1.4163 0.018761431 2.376059 Palmitoleic acid (C16:cis[9]1) down 0.6784 0.01999529 2.35122 Sphingomyelin (d18:1, C22:1) up 1.3106 0.020282899 2.345704 Sphingomyelin (d18:1, C20:0) up 1.2418 0.021769114 2.318246 Sphingomyelin (d18:1, C20:1) up 1.2497 0.021853538 2.316737 Phenylalanine down 0.8095 0.022717345 2.30142 Coenzyme Q9 down 0.6369 0.023299502 2.29312 Uridine down 0.7878 0.025020547 2.26335 MAG_Stearic acid (C18:0) down 0.6947 0.025859529 2.25083 Hexadecanoylcarnitine down 0.7697 0.026017925 2.24781 Threonine down 0.8146 0.026945311 2.23382 Mannose up 1.7485 0.028419125 2.212844 Ceramide (d18:2, C16:0) up 1.2947 0.030380468 2.185808 Ceramide (d18:1, C22:1) up 1.4072 0.031629333 2.169372 Sphingomyelin (d17:1, C22:0) up 1.25 0.032365612 2.159677 Ceramide (d18:2, C22:0) up 1.279 0.034401307 2.13481 MAG_Oleic acid (C18:cis[9]1) down 0.5128 0.036301979 2.1126 Sphingomyelin (d18:1, C24:2) up 1.2677 0.036538219 2.10952 Cystine down 0.6627 0.03773506 2.09605 Sphingomyelin (d17:1, C24:1) up 1.1767 0.043724822 2.033665 Sarcosine down 0.783 0.04645201 2.0081 Sphingomyelin (d18:1, C18:0) up 1.278 0.047624028 1.996875 Sphingomyelin (d18:1, C23:1) up 1.18 0.048636538 1.987742 Octadecanoylcarnitine down 0.8093 0.05113107 1.96581 TAG_Linoleic acid up 1.4741 0.053960198 1.942212 (C18:cis[9,12]2) Sphingomyelin (d17:1, C16:0) up 1.2523 0.053967727 1.942151 Erythrol down 0.7977 0.055226592 1.93185 N-Acetylcytidine down 0.784 0.055518717 1.92951 Ceramide (d17:1, C22:0) up 1.2712 0.057891482 1.911159 epsilon-Acetyllysine down 0.856 0.059138083 1.90134 Quinic acid down 0.6024 0.060160786 1.89832 Androstenedione down 0.7708 0.066836588 1.84609 Isocitrate down 0.8568 0.075542806 1.78947 Tyrosine down 0.8428 0.07659407 1.78285 Phosphate (inorganic and from up 0.7772 0.078319502 1.77271 organic phosphates) Sphingomyelin (d18:2, C24:1) up 1.2324 0.080227014 1.761131 Aspartate down 0.7832 0.08101072 1.75646 3-Hydroxyisobutyrate down 0.7953 0.081406107 1.75423 LPC_Arachidonic acid up 0.6423 0.082693197 1.74679 (C20:cis[5,8,11,14]4) 3-Hydroxyisobutyrate down 0.5361 0.085234841 1.7323 Ceramide (d17:1, C24:1) up 1.2125 0.086649647 1.724654 1-Hydroxy-2-amino-(cis,trans)- up 1.2439 0.086787503 1.72367 3,5-octadecadiene (from sphin- golipids) TAG (C16:0, C18:1, C18:2) up 1.2912 0.088582763 1.715218 Glycerol, polar fraction down 0.7693 0.089390729 1.70947 FFA_cis-Vaccenic acid down 0.7432 0.089534331 1.70877 Cholesterylester C18:1 up 0.8639 0.092386252 1.69362 TAG_dihomo-gamma-Linolenic up 1.5331 0.097298227 1.668384 acid (C20:cis[8,11,14]3) Ceramide (d18:1, C23:1) up 1.2439 0.102085315 1.644896 Ribonic acid down 0.8059 0.102980747 1.64038 myo-Inositol down 0.8066 0.109457639 1.61001 Ceramide (d16:1, C18:0) up 1.2392 0.116466943 1.578894 Creatinine down 0.8516 0.118716065 1.56899 Ceramide (d18:1, C22:0) up 1.1531 0.119495116 1.565832 Ceramide (d16:1, C20:0) up 1.2218 0.119885873 1.564166 Ceramide (d18:1, C24:2) up 1.209 0.122327836 1.553849 LPC_cis-Vaccenic acid up 0.6524 0.127022566 1.53435 1,5-Anhydrosorbitol down 0.7163 0.129241821 1.52555 Ornithine down 0.8494 0.132740006 1.51144 Ceramide (d18:1, C16:0) up 1.2022 0.133614664 1.508169 Furoylglycine down 0.5465 0.133825096 1.50966 Sphingomyelin (d16:1, C18:1) up 1.204 0.134773332 1.503545 PI_Linoleic acid (C18:cis[9,12]2) up 0.5443 0.135615536 1.50028 TAG_Arachidonic acid up 1.2706 0.146199585 1.460562 (C20:cis[5,8,11,14]4) PS_Stearic acid (C18:0) down 0.5452 0.146600494 1.45915 MAG_Palmitic acid (C16:0) down 0.6587 0.151089957 1.4431 FFA_Palmitic acid (C16:0) down 0.8293 0.15122502 1.44248 trans-4-Hydroxyproline down 0.8387 0.152106042 1.43931 Sphingomyelin (d18:2, C16:0) up 1.1725 0.15961601 1.413287 TAG_gamma-Linolenic acid up 1.4255 0.162341079 1.404059 (C18:cis[6,9,12]3) Citrate down 0.8468 0.167390859 1.38919 Dehydroepiandrosterone sulfate up 0.7467 0.173750506 1.36666 Glycerol-3-phosphate, polar frac- up 0.804 0.179405454 1.34876 tion Phosphatidylcholine down 0.9044 0.18215668 1.34023 (C18:0, C18:1) Sphingomyelin (d16:1, C22:1) up 1.143 0.199694768 1.288025

(43) TABLE-US-00031 TABLE 30 List of identified biomarkers in plasma for pancreatic cancer relative to pancreatitis ANOVA result of pancreatic cancer relative to pancreatitis in plasma Estimated Direc- fold Metabolite tion change p-value t-value Sphingomyelin (d18:2, C22:1) up 1.6153 3.12104E06 4.842932 Proline down 0.6895 2.2408E05 4.37545 Sphingomyelin (d17:1, C24:1) up 1.587 4.27653E05 4.214581 Sphingomyelin (d18:1, C23:1) up 1.6747 4.68851E05 4.191339 Sphingomyelin (d17:1, C18:0) up 1.3615 4.85522E05 4.182486 Sphingomyelin (d18:1, C22:1) up 1.767 5.46547E05 4.152388 Sphingomyelin (d18:1, C19:0) up 1.4382 7.71969E05 4.063708 Phosphatidylcholine up 1.2637 8.59408E05 4.035873 (C18:0, C22:6) Sphingomyelin (d18:1, C20:1) up 1.6237 0.000147707 3.893232 Sphingomyelin (d18:0, C18:0) up 1.7822 0.000205464 3.804441 Sphingomyelin (d18:2, C20:1) up 1.3931 0.000228537 3.775489 PC_Docosahexaenoic acid up 1.4333 0.000235934 3.767399 (C22:cis[4,7,10,13,16,19]6) Docosahexaenoic acid up 1.4727 0.000275587 3.724169 (C22:cis[4,7,10,13,16,19]6) Sphingomyelin (d18:1, C24:1) up 1.3496 0.000301678 3.699187 Histidine down 0.7707 0.000324059 3.67934 Nervonic acid (C24:cis[15]1) up 1.3554 0.000382933 3.632725 Sphingomyelin (d18:1, C21:0) up 1.55 0.000666074 3.474936 Sphingomyelin (d18:1, C24:2) up 1.5844 0.000702203 3.459606 Cholesterylester C14:1 down 0.5891 0.000804669 3.41984 Sphingomyelin (d16:1, C22:1) up 1.4853 0.000809878 3.417949 Citrulline down 0.757 0.000855559 3.40183 Sphingomyelin (d18:1, C18:1) up 1.3879 0.00092022 3.380335 Sphingomyelin (d18:2, C19:0) up 1.3682 0.000937129 3.374948 1-Hydroxy-2-amino-(cis,trans-3,5- up 1.4196 0.0010776 3.333412 octadecadiene (from sphin- golipids) Sphingomyelin (d17:1, C20:0) up 1.3428 0.001218963 3.296437 Ceramide (d18:1, C24:0) up 0.7909 0.001508765 3.23293 Sphingomyelin (d18:2, C24:2) up 1.3335 0.001829315 3.172443 Sphingomyelin (d18:1, C18:0) up 1.2858 0.002019719 3.141654 Ceramide (d18:2, C24:0) up 0.7029 0.002259236 3.10761 Sphingomyelin (d18:2, C24:1) up 1.364 0.00231516 3.098834 erythro-Dihydrosphingosine up 1.6091 0.002479426 3.077168 (d18:0) Cholesterol, free up 1.2431 0.002588129 3.063549 Sphingomyelin (d18:2, C18:1) up 1.3772 0.002614513 3.060323 Ceramide (d18:1, C22:1) up 1.5754 0.002820175 3.037171 Sphingomyelin (d18:2, C23:1) up 1.3791 0.003127626 3.002909 Coenzyme Q9 down 0.5759 0.00346341 2.96987 Creatine down 0.7493 0.004048717 2.91875 Sphingomyelin (d17:1, C16:0) up 1.2017 0.004553063 2.879876 FS_Cholesterol, free up 1.3773 0.004696542 2.869538 gamma-Tocopherol down 0.6819 0.004716719 2.86811 3-O-Methylsphingosine (d18:1) up 1.3826 0.006240872 2.773492 Sphingomyelin (d18:2, C21:0) up 1.2945 0.007497189 2.710209 Isocitrate down 1.2572 0.008073829 2.684331 5-O-Methylsphingosine (d18:1) up 1.3027 0.008078261 2.684139 erythro-Sphingosine (d18:1) up 1.2569 0.00826139 2.676274 Ornithine down 0.8191 0.008496731 2.6664 Ceramide (d17:1, C16:0) up 1.3308 0.009659761 2.621604 Sphingomyelin (d18:2, C16:0) up 1.1343 0.010189687 2.601829 Ceramide (d18:1, C18:0) up 1.4369 0.010845552 2.580037 gamma-Linolenic acid up 0.666 0.011356235 2.56275 (C18:cis[6,9,12]3) Phosphatidylcholine down 1.1645 0.012758272 2.520282 (C16:0, C16:0) Cholesterylester C16:3 up 0.7378 0.014873216 2.46352 Glycochenodeoxycholic acid down 2.2659 0.015139856 2.457258 Sphingomyelin (d18:2, C18:0) up 1.2097 0.015665452 2.444098 Heptadecanoic acid (C17:0) up 1.21 0.015686666 2.44359 Cortisol up 1.2751 0.016284634 2.429518 Ceramide (d17:1, C24:0) up 0.7629 0.016965835 2.41458 Sphingomyelin (d17:1, C22:0) up 1.3416 0.019550823 2.359887 2-Hydroxybutyrate up 1.367 0.020060215 2.353764 threo-Sphingosine (d18:1) up 1.2126 0.02071436 2.337555 Sphingomyelin (d18:2, C18:0) up 1.1295 0.021207257 2.328427 epsilon-Acetyllysine down 0.8595 0.021629287 2.32076 Sphingomyelin (d18:2, C20:0) up 1.2212 0.022143876 2.311584 Mannose up 1.3289 0.023498047 2.288459 Phosphatidylcholine up 1.0459 0.026971731 2.233566 (C18:0, C20:4) trans-4-Hydroxyproline down 0.7803 0.027481551 2.22606 Sphingomyelin (d18:1, C20:0) up 1.284 0.027583168 2.224575 Ribonic acid down 0.8229 0.028209262 2.21658 Sphingomyelin (d16:1, C18:1) up 1.2821 0.028763727 2.207696 Androstenedione down 0.7491 0.030930675 2.17863 Ceramide (d18:1, C24:2) up 1.3011 0.03270557 2.155788 Ceramide (d18:2, C23:0) up 0.7619 0.033183627 2.14982 Ceramide (d18:1, C23:1) up 1.2985 0.036735645 2.107649 Uric acid down 0.8122 0.039553284 2.07626 Arginine up 0.8407 0.039715907 2.07453 Sphingomyelin (d16:1, C24:1) up 1.2967 0.040063403 2.070854 Sphingomyelin (d18:1, C16:0) up 1.1089 0.041546222 2.055456 Cholesterylester C16:1 down 0.7801 0.042152212 2.0493 PC_cis-Vaccenic acid up 1.2837 0.043221693 2.038727 Ceramide (d18:1, C16:0) up 1.2534 0.047153541 2.001505 Ceramide (d18:1, C23:0) up 0.8451 0.04882378 1.98639 PC_Arachidonic acid up 1.1952 0.049342791 1.981572 (C20:cis[5,8,11,14]4) 2-Hydroxypalmitic acid (C16:0) down 1.1778 0.050970096 1.96729 Quinic acid down 0.5999 0.053497846 1.94873 PI_dihomo-gamma-Linolenic acid up 0.6492 0.054295028 1.93948 (C20:cis[8,11,14]3) LPC_cis-Vaccenic acid up 0.5475 0.055303605 1.93142 Ceramide (d18:1, C20:0) up 1.2401 0.060429827 1.892009 Sphingomyelin (d16:1, C21:0) up 1.2423 0.061539773 1.883522 Ceramide (d18:1, C21:0) up 1.2235 0.063534014 1.869369 Arachidonic acid up 1.175 0.068087223 1.83751 (C20:cis[5,8,11,14]4) PC_Palmitic acid (C16:0) up 1.181 0.078121305 1.773743 Sphingomyelin (d16:1, C18:0) up 1.1273 0.083566982 1.741797 TAG_Arachidonic acid up 1.2579 0.085000353 1.733693 (C20:cis[5,8,11,14]4) Methionine down 0.9103 0.086723537 1.7241 LPC_Arachidonic acid up 0.6348 0.093194442 1.68946 (C20:cis[5,8,11,14]4) PS_Stearic acid (C18:0) down 0.4987 0.095853935 1.76307 Sphingomyelin (d18:2, C16:0) up 1.1954 0.099579228 1.657014 Ceramide (d18:2, C16:0) up 1.1785 0.102455467 1.643174 Isoleucine up 0.8955 0.102682128 1.64188 Phosphate (from Phospholipids) up 1.1238 0.104160102 1.634801 Threonine down 0.8815 0.105824932 1.62692 Ceramide (d18:2, C18:0) up 1.2113 0.108062148 1.616682 PE_Docosahexaenoic acid up 1.1609 0.109022294 1.612065 (C22:cis[4,7,10,13,16,19]6) 1,5-Anhydrosorbitol down 0.6971 0.112183948 1.5979 Cholesterylester C22:6 up 1.157 0.131073284 1.518073 Ceramide (d18:1, C24:1) up 1.1274 0.131395252 1.516961 Furoylglycine down 0.4928 0.132771551 1.51142 1-Methylhistidine down 0.8842 0.134404228 1.50498 PC_trans-Vaccenic acid up 1.3081 0.148310589 1.452957 (C18:trans[11]1) PE_Arachidonic acid up 0.8713 0.154525702 1.43085 (C20:cis[5,8,11,14]4) Tetradecanoylcarnitine down 1.1734 0.159197359 1.414715 Ceramide (d16:1, C22:0) up 0.8501 0.159388736 1.4142 3-Hydroxybutyrate down 1.5634 0.162013144 1.405164 Coenzyme Q10 down 0.8497 0.165096684 1.39485 PC_conjugated Linoleic acid up 1.3348 0.165412272 1.393845 (C18:cis[9]trans[11]2) Erythrol down 0.8866 0.16720991 1.38786 Ceramide (d17:1, C23:0) up 0.8365 0.172373833 1.3712 Sphingomyelin (d18:1, C22:0) up 1.1189 0.174992719 1.3627 Ceramide (d17:1, C24:1) up 1.132 0.181487495 1.342464 Citrate down 1.1266 0.183236849 1.336949 DAG (C18:1, C18:2) up 1.1226 0.191678456 1.311464 Corticosterone up 1.4104 0.193700627 1.305522 Glucuronic acid down 1.2349 0.193825582 1.305193 Cholesterylester C18:1 up 0.9192 0.198420102 1.2917 Ceramide (d16:1, C18:0) up 1.168 0.198501603 1.291583

(44) TABLE-US-00032 TABLE 30a List of further biomarkers in plasma for pancreatic cancer relative to pancreatitis ANOVA result of pancreatic cancer relative to pancreatitis in plasma Estimated Direc- fold Metabolite tion change p-value t-value Allantoin down 0.91 0.010937785 2.58 Glutamate up 1.19 0.199964208 1.29

(45) TABLE-US-00033 TABLE 31 List of identified biomarkers in plasma combined with serum for pancreatic cancer relative to pancreatitis ANOVA result of pancreatic cancer relative to pancreatitis in plasma combined with serum Estimated Direc- fold Metabolite tion change p-value t-value Sphingomyelin (d18:2, C22:1) up 1.4051 7.54095E08 5.510733 Sphingomyelin (d18:1, C19:0) up 1.3679 7.78447E08 5.504586 Citrulline down 0.7092 1.54646E07 5.39096 Proline down 0.733 1.6096E06 4.89161 Sphingomyelin (d18:2, C19:0) up 1.3843 3.21488E06 4.74344 Histidine down 0.73 5.27549E06 4.63458 Sphingomyelin (d18:1, C21:0) up 1.3976 7.98627E06 4.542348 Sphingomyelin (d18:2, C24:2) up 1.2664 9.46741E06 4.503961 Sphingomyelin (d17:1, C20:0) up 1.3408 1.63736E05 4.378543 Sphingomyelin (d18:2, C20:1) up 1.3346 1.91142E05 4.34259 Cholesterylester C14:1 down 0.6228 1.91298E05 4.3424 Sphingomyelin (d18:1, C22:1) up 1.4467 2.17919E05 4.311944 Sphingomyelin (d18:2, C18:1) up 1.3626 2.35355E05 4.293871 Sphingomyelin (d17:1, C18:0) up 1.3162 2.39718E05 4.289549 Sphingomyelin (d18:1, C20:1) up 1.3684 3.11853E05 4.227262 Sphingomyelin (d18:1, C23:1) up 1.3444 3.21218E05 4.220211 Uric acid down 0.7267 3.86817E05 4.17551 Sphingomyelin (d17:1, C24:1) up 1.3056 3.99813E05 4.16775 Sphingomyelin (d18:2, C20:0) up 1.2479 8.216E05 3.991236 Cholesterylester C16:1 down 0.7126 0.000108103 3.92231 Sphingomyelin (d18:1, C24:2) up 1.3787 0.000109753 3.918481 Sphingomyelin (d18:2, C21:0) up 1.3044 0.000168479 3.808754 Sphingomyelin (d18:1, C18:1) up 1.308 0.000203348 3.759783 Ceramide (d18:1, C18:0) up 1.4324 0.000256282 3.699551 Ceramide (d18:1, C20:0) up 1.3539 0.000266424 3.689242 Coenzyme Q9 down 0.6245 0.000287189 3.66982 TAG (C16:0, C18:1, C18:3) up 1.3569 0.000333495 3.628332 Ceramide (d17:1, C16:0) up 1.3396 0.000361905 3.607059 Sphingomyelin (d18:2, C23:1) up 1.2724 0.000379362 3.593627 Ceramide (d18:1, C21:0) up 1.3549 0.000424963 3.563354 Ceramide (d18:1, C22:1) up 1.4368 0.000572374 3.481166 Coenzyme Q10 down 0.7456 0.000588711 3.4726 Sphingomyelin (d18:1, C20:0) up 1.2642 0.00085526 3.367243 Ceramide (d18:2, C20:0) up 1.3193 0.00091215 3.349325 Sphingomyelin (d17:1, C22:0) up 1.2883 0.000980573 3.327957 Sphingomyelin (d18:1, C18:0) up 1.2683 0.001083125 3.299143 Sphingomyelin (d18:2, C18:0) up 1.2626 0.001263734 3.254065 FFA_Palmitoleic acid (C16:cis[9]1) down 0.6627 0.001417392 3.2202 Sphingomyelin (d18:2, C18:0) up 1.16 0.001537581 3.195909 Ceramide (d18:2, C18:0) up 1.3166 0.00179923 3.149319 Threonine down 0.8345 0.001967265 3.12175 1-Hydroxy-2-amino-(cis,trans-3,5- up 1.2773 0.00232077 3.071216 octadecadiene (from sphingolipids) Quinic acid down 0.5812 0.00248201 3.05666 epsilon-Acetyllysine down 0.8578 0.002493692 3.04904 N,N-Dimethylarginine (ADMA) down 0.8707 0.002703501 3.02395 Ornithine down 0.8249 0.002843004 3.00824 1-Methylhistidine down 0.8479 0.002870091 3.00527 Mannose up 1.5127 0.003417068 2.950549 Sphingomyelin (d18:2, C24:1) up 1.2451 0.003825905 2.914237 Phosphatidylcholine (C18:0, C22:6) up 1.1432 0.00420093 2.88411 Sphingomyelin (d16:1, C22:1) up 1.237 0.004558849 2.857561 O-Acetylcarnitine down 0.7814 0.005213329 2.81351 Ceramide (d18:2, C16:0) up 1.2359 0.005307354 2.808007 trans-4-Hydroxyproline down 0.8022 0.005763094 2.78029 Sarcosine down 0.8308 0.006247795 2.75367 TAG_Linoleic acid (C18:cis[9,12]2) up 1.3779 0.006461217 2.742073 Ceramide (d18:1, C24:2) up 1.2516 0.00679935 2.725164 Palmitoleic acid (C16:cis[9]1) down 0.7481 0.00731918 2.69985 Sphingomyelin (d18:2, C22:0) up 1.2164 0.007829807 2.676796 Androstenedione down 0.7803 0.008343756 2.65515 Pseudouridine down 0.8123 0.008413055 2.65201 LPC_cis-Vaccenic acid up 0.5936 0.008467674 2.64988 Cholesterol, free up 1.1667 0.00852677 2.647368 Ribonic acid down 0.8135 0.00876408 2.63825 Phenylalanine down 0.8676 0.00965423 2.60413 1,5-Anhydrosorbitol down 0.6798 0.010300725 2.58171 3-Indoxylsulfate down 0.6483 0.011122739 2.5542 TAG_Arachidonic acid up 1.2921 0.011985354 2.527525 (C20:cis[5,8,11,14]4) Sphingomyelin (d17:1, C16:0) up 1.1445 0.012593038 2.513154 Ceramide (d18:1, C16:0) up 1.2193 0.013870643 2.47495 PC_Docosahexaenoic acid up 1.2025 0.01435588 2.462252 (C22:cis[4,7,10,13,16,19]6) Ceramide (d18:1, C23:1) up 1.2355 0.016177455 2.418375 Sphingomyelin (d18:2, C16:0) up 1.1045 0.016312122 2.415036 LPC_Arachidonic acid up 0.6567 0.016469289 2.41156 (C20:cis[5,8,11,14]4) Tyrosine down 0.8753 0.017548104 2.3878 MAG_Stearic acid (C18:0) down 0.8222 0.017821754 2.38217 DAG (C18:1, C18:2) up 1.165 0.018480708 2.368391 Ceramide (d18:1, C24:0) up 0.8905 0.020190923 2.33506 Sphingomyelin (d18:0, C18:0) up 1.2639 0.022606384 2.291511 Cortisol up 1.1875 0.024029553 2.267869 Cholesterylester C16:3 up 0.8322 0.025712947 2.24135 FS_Cholesterol, free up 1.1769 0.026355845 2.231743 Cholesterylester C18:1 up 0.8902 0.02669165 2.22664 Sphingomyelin (d16:1, C18:1) up 1.1946 0.02691608 2.223337 erythro-Sphingosine (d18:1) up 1.1554 0.027447936 2.215561 MAG_Oleic acid (C18:cis[9]1) down 0.6993 0.027614056 2.21331 DAG_Palmitic acid (C16:0) down 0.5831 0.027750028 2.21129 Uridine down 0.8641 0.029177631 2.19181 Furoylglycine down 0.5188 0.029504259 2.18818 3-O-Methylsphingosine (d18:1) up 1.1987 0.030012003 2.179966 Ceramide (d17:1, C22:0) up 1.1831 0.031048649 2.166514 Ceramide (d17:1, C24:1) up 1.1639 0.032866637 2.143495 gamma-Tocopherol down 0.8141 0.033443133 2.1363 TAG_dihomo-gamma-Linolenic acid up 1.3628 0.033669386 2.133521 (C20:cis[8,11,14]3) PS_Palmitic acid (C16:0) down 0.3966 0.033859432 2.13874 Erythrol down 0.8606 0.034747545 2.12062 Docosahexaenoic acid up 1.189 0.035833069 2.108004 (C22:cis[4,7,10,13,16,19]6) Cystine down 0.7942 0.03612422 2.10471 Ceramide (d16:1, C18:0) up 1.1999 0.037901529 2.085021 Sphingomyelin (d18:2, C16:0) up 1.1671 0.038667107 2.076574 5-O-Methylsphingosine (d18:1) up 1.1617 0.039884223 2.063637 Dehydroepiandrosterone sulfate up 0.7436 0.042036959 2.04213 Sphingomyelin (d18:1, C24:1) up 1.1165 0.0430598 2.0315 Campesterol down 0.8026 0.044955946 2.01335 FFA_cis-Vaccenic acid down 0.7776 0.046127998 2.00228 Aspartate down 0.8513 0.046850764 1.99561 Heptadecanoic acid (C17:0) up 1.129 0.048667413 1.979294 TAG (C16:0, C18:1, C18:2) up 1.1894 0.04880215 1.978557 MAG_Palmitic acid (C16:0) down 0.647 0.050530745 1.96318 myo-Inositol down 0.8636 0.05510547 1.92531 Glycerol, polar fraction down 0.8274 0.055474539 1.92238 Nervonic acid (C24:cis[15]1) up 1.1217 0.059026207 1.894973 Sphingomyelin (d18:1, C22:0) up 1.1236 0.061872822 1.874008 Ceramide (d16:1, C20:0) up 1.1676 0.066086059 1.84448 Octadecanoylcarnitine down 0.8846 0.068465313 1.8283 threo-Sphingosine (d18:1) up 1.1184 0.072102549 1.804633 Hexadecanoylcarnitine down 0.8822 0.073707452 1.7945 Phosphate (inorganic and from organic up 0.8717 0.073926374 1.79321 phosphates) 7-Methylguanosine down 0.8895 0.076842023 1.77522 Glycerol-3-phosphate, polar fraction up 0.8272 0.087133931 1.71774 PC_Arachidonic acid up 1.1162 0.099668389 1.651454 (C20:cis[5,8,11,14]4) Creatine down 0.8779 0.107084585 1.61612 Tetradecanoylcarnitine down 0.8816 0.110346804 1.60122 Ceramide (d18:2, C24:0) up 0.885 0.110465605 1.60077 Sphingomyelin (d18:1, C16:0) up 1.0465 0.110682387 1.599721 Sphingomyelin (d16:1, C21:0) up 1.1488 0.114900639 1.581002 PS_Stearic acid (C18:0) down 0.5738 0.116878026 1.57589 Corticosterone up 1.3002 0.124798298 1.539148 FFA_Palmitic acid (C16:0) down 0.8775 0.126394868 1.53261 Ceramide (d18:1, C24:1) up 1.0937 0.126592813 1.531877 Pantothenic acid down 0.8834 0.135644564 1.4961 Ceramide (d18:1, C22:0) up 1.0883 0.140321488 1.478455 Lignoceric acid (C24:0) up 0.9147 0.151000349 1.43959 Ceramide (d18:2, C22:0) up 1.1126 0.156759059 1.419565 FFA_Myristoleic acid (C14:cis[9]1) down 0.7209 0.160847349 1.4056 PI_Linoleic acid (C18:cis[9,12]2) up 0.729 0.161090757 1.40478 FFA_Linolenic acid (C18:cis[9,12,15]3) up 0.8051 0.177342977 1.35207 Glycochenodeoxycholic acid down 1.3563 0.179541868 1.345277 Arachidonic acid (C20:cis[5,8,11,14]4) up 1.0983 0.18049037 1.342271 Sphingomyelin (d16:1, C20:0) up 1.1131 0.18475197 1.329232 Methionine down 0.935 0.190912747 1.31075 Eicosanoic acid (C20:0) up 1.0705 0.192183894 1.306993 Ceramide (d7:1, C24:0) up 0.8982 0.197610342 1.29121

(46) TABLE-US-00034 TABLE 32 List of identified biomarkers in serum for pancreatic cancer relative to control (blood donors) ANOVA result of pancreatic cancer relative to control (blood donors) in serum Estimated Direc- fold Metabolite tion change p-value t-value Lysophosphatidyl- down 0.577 0.00156622 3.25511 ethanolamine (C18:2) Sphingomyelin up 1.3587 0.01407457 2.50108847 (d17:1, C16:0) Lysophosphatidyl- up 1.268 0.02237293 2.30771756 choline (C18:0) Sphingomyelin up 2.0372 0.19534056 1.30403267 (d17:1, C16:0)

(47) TABLE-US-00035 TABLE 33 List of identified biomarkers in plasma combined with serum for pancreatic cancer relative to control (blood donors) ANOVA results of pancreatic cancer relative to control (blood donors) in plasma combined with serum Estimated Direc- fold Metabolite tion change p-value t-value Lysophosphatidyl- down 0.5082 1.2246E05 4.50 ethanolamine (C18:2) Sphingomyelin up 1.5107 0.00012772 3.91937588 (d17:1, C16:0) Lysophosphatidyl- up 1.2576 0.02410202 2.27065088 choline (C18:0) Sphingomyelin up 1.1914 0.06618025 1.84605689 (d18:2, C24:0) Sphingomyelin up 1.8984 0.07358616 1.80010609 (d17:1, C16:0) Sphingomyelin up 2.2114 0.10399739 1.63438039 (d18:2, C17:0) Sphingomyelin up 2.0599 0.18632206 1.32680428 (d17:1, C18:0)

(48) TABLE-US-00036 TABLE 33a List of identified biomarkers in plasma for pancreatic cancer relative to non-pancreatic control ANOVA result of pancreatic cancer relative to non-pancreatic control in plasma Estimated Direc- fold Metabolite tion change p-value t-value Glutamate up 1.52 1.11502E05 4.54 Allantoin down 0.93 0.010084541 2.61 Butyrylcarnitine down 0.83 0.027773999 2.22 Sedoheptulose-7- up 1.17 0.05257924 1.97 phosphate Hexanoylcarnitine down 0.85 0.124472537 1.54

(49) TABLE-US-00037 TABLE 34 List of identified biomarkers in serum for pancreatic cancer relative to critical controls (pancreatitis and liver cirrhosis) ANOVA results of pancreatic cancer relative to critical controls (pancreatitis and liver cirrhosis) in serum Estimated Direc- fold Metabolite tion change p-value t-value Sphingomyelin up 1.186 0.02362018 2.28738611 (d17:1, C16:0) Sphingomyelin up 1.0907 0.07053766 1.81676671 (d18:2, C24:0) Sphingomyelin up 1.1699 0.08867187 1.71396562 (d18:2, C17:0) TAG (C18:1, up 1.1762 0.1013912 1.6456486 C18:2, C18:3) Sphingomyelin up 1.3842 0.168661 1.38342438 (d17:1, C16:0)

(50) TABLE-US-00038 TABLE 35 List of identified biomarkers in plasma combined with serum for pancreatic cancer relative to critical controls (pancreatitis and liver cirrhosis) ANOVA results of pancreatic cancer relative to critical controls (pancreatitis and liver cirrhosis) in plasma combined with serum Estimated Direc- fold Metabolite tion change p-value t-value Lysophosphatidyl- down 0.7658 5.0955E05 4.11054 ethanolamine (C18:2) Sphingomyelin up 1.1646 0.00110089 3.29524321 (d17:1, C16:0) Sphingomyelin up 1.1677 0.00232646 3.07122831 (d18:2, C17:0) Sphingomyelin up 1.1038 0.0089445 2.62739206 (d18:2, C24:0) Sphingomyelin up 1.3229 0.02096003 2.32090324 (d17:1, C16:0) TAG (C18:1, up 1.1551 0.0430041 2.03089577 C18:2, C18:3) Sphingomyelin up 1.2607 0.16566411 1.3896436 (d17:1, C18:0)

(51) TABLE-US-00039 TABLE 36 List of identified biomarkers in serum for pancreatic cancer relative to liver cirrhosis ANOVA results of pancreatic cancer relative to liver cirrhosis in serum Estimated Direc- fold Metabolite tion change p-value t-value Sphingomyelin up 1.2627 0.01580708 2.45790679 (d18:2, C17:0) Sphingomyelin up 1.1815 0.02797472 2.2322297 (d17:1, C16:0) Lysophosphatidyl- up 1.136 0.06093446 1.88779624 choline (C18:0) TAG (C18:1, up 1.2232 0.06630851 1.85178193 C18:2, C18:3) Sphingomyelin up 1.5759 0.15737731 1.42531836 (d17:1, C16:0)

(52) TABLE-US-00040 TABLE 37 List of identified biomarkers in plasma combined with serum for pancreatic cancer relative to liver cirrhosis ANOVA results of pancreatic cancer relative to liver cirrhosis in plasma combined with serum Estimated Direc- fold Metabolite tion change p-value t-value Sphingomyelin up 1.3174 0.00025755 3.73327693 (d18:2, C17:0) Sphingomyelin up 1.2193 0.00352438 2.95922082 (d17:1, C16:0) Lysophosphatidyl- up 1.1418 0.04297793 2.0350991 choline (C18:0) Sphingomyelin up 1.5795 0.04479279 2.02153544 (d17:1, C16:0) TAG (C18:1, up 1.2124 0.045452 2.01248629 C18:2, C18:3) Sphingomyelin up 1.5184 0.07057557 1.81962329 (d17:1, C18:0) Sphingomyelin up 1.0908 0.10179664 1.64271456 (d18:2, C24:0)

Example 4: Patients and Sample Preparation

(53) From a retrospective case control study, plasma samples of 79 patients suffering from resectable (i.e. stage T1 or T2) PDAC, plasma samples of 80 patients suffering from chronic pancreatitis, and plasma samples of 80 non-pancreatic controls (hernia repair and thyroid resection) were included. Exclusion criteria were a concomitant malignant disease, curative treatment of malignant disease less than 2 years of recruitment to the trial, concomitant cystic diseases of the pancreas, pregnancy or patients unable to give informed consent. After blood drawing and centrifugation, plasma samples were collected in Eppendorf tubes and stored at 80 C. for further analysis as described in example 2. Statistical analysis was done by ANOVA as described in example 3.

(54) Results are shown in Tables 38 and 39.

(55) TABLE-US-00041 TABLE 38 List of identified biomarkers in plasma for resectable pancreatic cancer relative to pancreatitis ANOVA result of resectable pancreatic cancer relative to pancreatitis in plasma Estimated Direc- fold Metabolite tion change p-value t-value Coenzyme Q9 down 0.48 0.000262364 3.77 Sphingomyelin (d17:1, C18:0) up 1.39 0.000619396 3.52 gamma-Tocopherol down 0.64 0.000811368 3.44 Ceramide (d18:1, C18:0) up 1.58 0.001649611 3.22 Sphingomyelin (d18:2, C18:0) up 1.23 0.001968032 3.17 Sphingomyelin (d18:0, C18:0) up 1.62 0.002649038 3.07 Sphingomyelin (d18:2, C20:1) up 1.28 0.003106771 3.02 Sphingomyelin (d18:2, C18:1) up 1.28 0.003233019 3.01 Citrulline down 0.79 0.004234786 2.92 Ceramide (d18:2, C18:0) up 1.42 0.004602512 2.89 Sphingomyelin (d18:2, C22:1) up 1.26 0.005588704 2.82 Sphingomyelin (d18:1, C20:1) up 1.28 0.006349118 2.78 Threonine down 0.79 0.006433821 2.78 Proline down 0.78 0.006467529 2.77 Sphingomyelin (d18:1, C18:0) up 1.24 0.007565255 2.72 Sphingomyelin (d17:1, C18:0) up 1.21 0.008129047 2.69 Ceramide (d16:1, C18:0) up 1.40 0.010129721 2.62 Sphingomyelin (d18:1, C18:1) up 1.21 0.010386287 2.61 Creatine down 0.76 0.011095461 2.58 Sphingomyelin (d18:2, C19:0) up 1.33 0.013250651 2.52 Sphingomyelin (d18:1, C22:1) up 1.26 0.015581643 2.46 N,N-Dimethylarginine (ADMA) down 0.81 0.016604005 2.43 Lysophosphatidylcholine (C18:0) down 0.82 0.018123417 2.40 Sphingomyelin (d18:1, C23:1) up 1.24 0.018256964 2.39 Sphingomyelin (d18:1, C24:2) up 1.24 0.018768704 2.38 Sphingomyelin (d17:1, C24:1) up 1.21 0.018972758 2.38 Ceramide (d17:1, C16:0) up 1.33 0.019194063 2.38 Histidine down 0.83 0.019390345 2.37 Octadecanoylcarnitine down 0.79 0.020960917 2.34 Coenzyme Q10 down 0.71 0.020994194 2.34 Sphingomyelin (d18:2, C17:0) up 1.17 0.022448489 2.31 Allantoin down 0.90 0.024000876 2.29 Sphingomyelin (d17:1, C20:0) up 1.27 0.024115295 2.29 Ceramide (d18:2, C20:0) up 1.31 0.024965844 2.27 Lysophosphatidylethanolamine (C18:2) down 0.79 0.02591185 2.26 Sphingomyelin (d18:2, C24:2) up 1.19 0.028176764 2.22 trans-4-Hydroxyproline down 0.79 0.031352163 2.18 Sphingomyelin (d18:2, C23:1) up 1.19 0.036399458 2.12 Ceramide (d18:1, C20:0) up 1.27 0.037976539 2.10 Ornithine down 0.85 0.042421242 2.05 Dodecanoylcarnitine down 0.74 0.042954903 2.05 Sphingomyelin (d18:2, C18:0) up 1.08 0.045868882 2.02 Oleoylcarnitine down 0.83 0.048667316 1.99 Hexadecanoylcarnitine down 0.86 0.063680802 1.87 Sphingomyelin (d18:1, C20:0) up 1.13 0.064916271 1.86 Glutamate up 1.31 0.068014282 1.84 Sphingomyelin (d18:1, C21:0) up 1.19 0.075358056 1.79 Sphingomyelin (d18:1, C24:1) up 1.10 0.077758649 1.78 Ceramide (d16:1, C20:0) up 1.26 0.0833007 1.75 Cholesterol, free up 1.14 0.087042975 1.73 TAG (C18:1, C18:2, C18:3) up 1.13 0.097051705 1.67 Dodecanoylcarnitine down 0.77 0.099946654 1.66 Sphingomyelin (d16:1, C18:1) up 1.15 0.100118304 1.66 Sphingomyelin (d17:1, C16:0) up 1.14 0.105283297 1.63 Sphingomyelin (d16:1, C22:1) up 1.17 0.115030296 1.59 Octanoylcarnitine down 0.77 0.118134842 1.57 Ceramide (d18:1, C22:1) up 1.31 0.127530329 1.54 Sphingomyelin (d17:1, C16:0) up 1.07 0.127925459 1.53 Sphingomyelin (d16:1, C18:0) up 1.18 0.141744114 1.48 Sphingomyelin (d18:2, C20:0) up 1.10 0.152012301 1.44 Aspartate down 0.91 0.153850266 1.44 Erythrol down 0.89 0.154985801 1.43 3-Indoxylsulfate down 0.71 0.155611929 1.43 Sphingomyelin (d18:1, C19:0) up 1.24 0.157096387 1.42 Ceramide (d18:1, C21:0) up 1.19 0.157392177 1.42 Ceramide (d18:1, C23:1) up 1.23 0.165132835 1.40 Ceramide (d18:1, C16:0) up 1.17 0.168749843 1.39 Methionine down 0.91 0.175505142 1.36 Ceramide (d18:1, C24:2) up 1.26 0.178243698 1.35 Uric acid down 0.88 0.181936467 1.34 Sphingomyelin (d18:2, C21:0) up 1.13 0.187953909 1.32 Quinic acid down 0.70 0.195229999 1.30 Ceramide (d17:1, C24:1) up 1.17 0.198340202 1.29

(56) TABLE-US-00042 TABLE 39 List of identified biomarkers in plasma for resectable pancreatic cancer relative to non-pancreatic control ANOVA result of resectable pancreatic cancer relative to non-pancreatic control in plasma Estimated Direc- fold Metabolite tion change p-value t-value Glutamate up 1.60 1.12894E05 4.60 Lysophosphatidyl- down 0.75 0.000282104 3.75 ethanolamine (C18:2) Sphingomyelin up 1.13 0.00264958 3.07 (d18:1, C24:1) Sphingomyelin up 1.15 0.010065976 2.62 (d17:1, C18:0) Butyrylcarnitine down 0.78 0.014064671 2.49 Allantoin down 0.92 0.024458386 2.28 Dodecanoylcarnitine down 0.85 0.140987832 1.48 Sedoheptulose-7- up 1.14 0.165293308 1.41 phosphate