Metabolite Biomarkers for the Detection of Esophageal Cancer Using NMR

Abstract

Methods for the detection and screening of esophageal adenocarcinoma (EAC) patients and for the monitoring of EAC treatment using a panel or panels of small molecule metabolite biomarkers are disclosed. In other aspects, methods for detection and screening for the progression of high-risk conditions (BE and HGD) to EAC and to monitoring treatment using a panel or panels of small molecule metabolite biomarkers are disclosed. The biomarkers are sensitive and specific for the detection of EAC, and can also be used to classify Barrett's esophagus (BE) and high-grade dysplasia (HGD), which are widely regarded as precursors of EAC.

What is claimed is: 
     
         1 . A method of determining a stage in the progression of an esophageal adenocarcinoma in a subject, comprising the steps of
 measuring the concentrations of the at least two components of a panel of a plurality of metabolic biomarkers selected from the group consisting of selected from the group consisting of lactic acid, valine, leucine, methionine, carnitine, tyrosine, tryptophan, 5-hydroxytryptophan, myristic acid, margaric acid, linolenic acid, linoleic acid, pyroglutamic acid, glutamine, β-hydroxybutyrate, citrate, lysine, creatinine, α-glucose, proline, histidine, alanine, glutamate, and mixtures thereof in a test sample of a biofluid from the subject to be tested for a stage in the progression of an esophageal adenocarcinoma, wherein the components of the panel have been selected from a population of metabolic biomarkers that have been shown to significantly distinguish a first stage from a second stage in the progression of an esophageal adenocarcinoma; and   wherein a statistical model has been constructed using the combined measured concentration differences of each component in the panel of a plurality of biomarkers in a first sample from a subject in the first stage and the measured concentration of each component of the panel of a plurality of biomarkers in a second sample from a subject in the second stage, wherein the statistical model distinguishes the first sample from the second sample;   using the statistical model to determine the relationship of the concentrations of the components of the panel of metabolic biomarkers measured in the test sample to the concentrations of the components of the panel of metabolic biomarkers that are characteristic of the first stage and characteristic of the second stage;   thereby determining the stage in the progression of the esophageal adenocarcinoma in a subject.   
     
     
         2 . The method of  claim 1  wherein the step of measuring the concentrations further includes measuring the concentration of at least two components of a panel of a plurality of metabolic biomarkers in a sample of a biofluid from a control source, wherein the metabolic biomarker is a component of a panel of a plurality of biomarkers; and wherein the statistical model is a partial least squares model. 
     
     
         3 . The method of  claim 1  wherein the stages in the progression of esophageal adenocarcinoma being determined are:
 a. the first stage is normal and the second stage is esophageal adenocarcinoma, or 
 b. the first stage is normal and the second stage is high grade dysplasia, or 
 c. the first stage is normal and the second stage is high risk (Barrett's esophagus or high grade dysplasia), or 
 d. the first stage is high risk (Barrett's esophagus or high grade dysplasia) and the second stage is esophageal adenocarcinoma, or 
 e. the first stage is Barrett's esophagus and the second stage is esophageal adenocarcinoma, or 
 f. the first stage is normal and the second stage is late stage esophageal adenocarcinoma, or 
 g. the first stage is normal and the second stage is early stage esophageal adenocarcinoma, or 
 h. the first stage is Barrett's esophagus and the second stage is early stage esophageal adenocarcinoma, or 
 i. the first stage is high grade dysplasia and the second stage is early stage esophageal adenocarcinoma. 
 
     
     
         4 . The method of  claim 1  wherein the panel is selected from the group consisting of
 a. the panel consisting of glutamine, β-hydroxybutyrate, citrate, lysine, creatinine, lactate, and α-glucose; 
 b. the panel consisting of glutamine, β-hydroxybutyrate, citrate, lysine, creatinine, lactate, α-glucose, leucine, valine, methionine, carnitine, tyrosine, tryptophan, 5 hydroxytryptophan, myristic acid, margaric acid, linolenic acid, and linoleic acid; 
 c. the panel consisting of β-hydroxybutyrate, citrate, creatinine, lactate, and α-glucose; 
 d. the panel consisting of glutamine, β-hydroxybutyrate, citrate, lysine, creatinine, lactate, α-glucose, leucine, acetone, acetoacetate and asparagine; 
 e. the panel consisting of glutamine, β-hydroxybutyrate, citrate, lysine, creatinine, lactate, α-glucose, acetoacetate and asparagine; 
 f. the panel consisting of glutamine, lysine, creatinine, acetoacetate and asparagine; 
 g. the panel consisting of lactate, pyroglutamic acid, and proline. 
 
     
     
         5 . The method of  claim 1  wherein the panel is selected from the group consisting of
 a. the panel consisting of lactate, valine, leucine, methionine, tyrosine, tryptophan, myristic acid and linoleic acid; 
 b. the panel consisting of glutamine, β-hydroxybutyrate, citrate, and lysine; 
 c. the panel consisting of lactate, valine, leucine, methionine, tyrosine, tryptophan, myristic acid, linoleic acid, glutamine, β-hydroxybutyrate, lysine and citrate; 
 d. the panel consisting of glutamine, β-hydroxybutyrate, citrate, lysine, creatinine, lactate, α-glucose, leucine, acetone, acetoacetate and asparagine; and 
 e. the panel consisting of lactate, valine, leucine, methionine, carnitine, tyrosine, tryptophan, 5-hydroxytryptophan, myristic acid, linolenic acid and linoleic acid. 
 
     
     
         6 . The method of  claim 1  wherein the panel comprises:
 a. at least one compound selected from the group consisting of glutamine, valine, leucine, methionine, lysine, tyrosine, tryptophan, β-hydroxybutyrate, citrate, lysine, creatinine, lactate, and α-glucose; 
 b. at least one compound selected from the group consisting of myristic acid, margaric acid; linolenic acid, linoleic acid and β-hydroxybutyrate; and 
 c. at least one compound selected from the group consisting of citrate, lactic acid, and α-glucose. 
 
     
     
         7 . The method of  claim 1  wherein the panel comprises biomarkers that have been identified by a plurality of methods selected from nuclear magnetic resonance (NMR) spectrometry, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), correlation spectroscopy (COSy), nuclear Overhauser effect spectroscopy (NOESY), rotating frame nuclear Overhauser effect spectroscopy (ROESY), LC-TOF-MS, LC-MS/MS, and capillary electrophoresis-mass spectrometry. 
     
     
         8 . The method of  claim 7  wherein the panel comprises biomarkers that have been identified by nuclear magnetic resonance (NMR) spectrometry and liquid chromatography-mass spectrometry (LC-MS). 
     
     
         9 . A method for detecting the esophageal cancer status within a biological sample, comprising:
 measuring one or more metabolite species within the sample by subjecting the sample to a nuclear magnetic resonance spectrometry analysis, the analysis producing a spectrum containing individual spectral peaks representative of the one or more metabolite species contained within the sample;   identifying the at least one or more metabolite species contained within the sample; and   correlating the measurement of the one or more metabolite species with an esophageal cancer status.   
     
     
         10 . The method of  claim 9 , wherein the one or multiple metabolite species is selected from the group consisting of leucine, β-hydroxybutyrate, lysine, glutamine, acetone, acetoacetate, citrate, unknown compound 1 appearing at 2.63 ppm, asparagine, creatinine, lactate, a-glucose, unsaturated lipids, the following lipid species: C═C—CH2-C═C, CH2-CO, CH2-C═C, CH2-CH2-C═C, CH2-CH2-CO, the lipoproteins VLDL2/LDL2, VLDL1/LDL1; and combinations thereof. 
     
     
         11 . The method of method of  claim 9 , wherein the sample comprises a biofluid. 
     
     
         12 . The method of  claim 11 , wherein the biofluid is blood. 
     
     
         13 . A biomarker for detecting esophageal cancer, comprising at least one metabolite species or parts thereof, selected from the group consisting of leucine, β-hydroxybutyrate, lysine, glutamine, acetone, acetoacetate, citrate, unknown compound 1 appearing at 2.63 ppm, asparagine, creatinine, lactate, α-glucose, unsaturated lipids, glyceryl of lipids, NMR signals from the following lipid species: C═C—CH2-C═C, CH2-CO, CH2-C═C, CH2-CH2-C═C, CH2-CH2-CO, and NMR signals from the following lipoprotein signals: VLDL2/LDL2, VLDL1/LDL1; and combinations thereof. 
     
     
         14 . A panel of biomarkers comprising 2 to 18 compounds selected from the group consisting of lactic acid, valine, leucine, methionine, carnitine, tyrosine, tryptophan, 5 hydroxytryptophan, myristic acid, margaric acid, linolenic acid, linoleic acid, pyroglutamic acid, glutamine, β hydroxybutyrate, citrate, lysine, creatinine, α glucose, proline, histidine, alanine, glutamate, and mixtures thereof. 
     
     
         15 . A kit for the analysis of a sample of a biofluid of a subject, comprising:
 a. aliquots of standards of each compound of a panel of metabolic biomarkers;   b. an aliquot of an internal standard; and   c. an aliquot of a control biofluid.   
     
     
         16 . The kit of  claim 15  wherein the biofluid is serum from a control source that is in the same species as the subject. 
     
     
         17 . The kit of  claim 15  wherein the internal standard is selected from the group consisting of trimethylsilylpropionic acid-d4 sodium salt, tridecanoic acid and chlorophenylalanine 
     
     
         18 . The kit of  claim 15  wherein the panel of metabolic biomarkers is 2 to 18 compounds selected from the group consisting of lactic acid, valine, leucine, methionine, carnitine, tyrosine, tryptophan, 5 hydroxytryptophan, myristic acid, margaric acid, linolenic acid, linoleic acid, pyroglutamic acid, glutamine, β hydroxybutyrate, citrate, lysine, creatinine, α glucose, proline, histidine, alanine, glutamate, and mixtures thereof. 
     
     
         19 . The kit of  claim 15  wherein the panel of metabolic biomarkers is selected from the group consisting of:
 a. the panel consisting of glutamine, β-hydroxybutyrate, citrate, lysine, creatinine, lactate, and α-glucose; 
 b. the panel consisting of glutamine, β-hydroxybutyrate, citrate, lysine, creatinine, lactate, α-glucose, leucine, valine, methionine, carnitine, tyrosine, tryptophan, 5 hydroxytryptophan, myristic acid, margaric acid, linolenic acid, and linoleic acid; 
 c. the panel consisting of β-hydroxybutyrate, citrate, creatinine, lactate, and α-glucose; 
 d. the panel consisting of glutamine, β-hydroxybutyrate, citrate, lysine, creatinine, lactate, α-glucose, leucine, acetone, acetoacetate and asparagine; 
 e. the panel consisting of glutamine, β-hydroxybutyrate, citrate, lysine, creatinine, lactate, α-glucose, acetoacetate and asparagine; 
 f. the panel consisting of glutamine, lysine, creatinine, acetoacetate and asparagine; and 
 g. the panel consisting of lactate, pyroglutamic acid, and proline. 
 
     
     
         20 . The kit of  claim 15  wherein the panel of metabolic biomarkers is selected from the group consisting of:
 a. the panel consisting of lactate, valine, leucine, methionine, tyrosine, tryptophan, myristic acid and linoleic acid; 
 b. the panel consisting of glutamine, β-hydroxybutyrate, citrate, and lysine; 
 c. the panel consisting of lactate, valine, leucine, methionine, tyrosine, tryptophan, myristic acid, linoleic acid, glutamine, β-hydroxybutyrate, lysine and citrate; 
 d. the panel consisting of glutamine, β-hydroxybutyrate, citrate, lysine, creatinine, lactate, α-glucose, leucine, acetone, acetoacetate and asparagine; and 
 e. the panel consisting of lactate, valine, leucine, methionine, carnitine, tyrosine, tryptophan, 5-hydroxytryptophan, myristic acid, linolenic acid and linoleic acid. 
 
     
     
         21 . The kit of  claim 15  further comprising instructions for use.