Biomarker detection

Abstract

The present invention relates to a method for diagnosing a subject suffering from or predisposed to cancer. The method comprises detecting, in a body sample from a test subject, the concentration of trace compounds resulting from the metabolism of at least one sugar and / or at least one amino acid or precursor thereof and / or at least one polyol present in a composition previously administered to the subject. The sugar is present in the composition at a concentration greater than 20,000 mg / 100 ml, the amino acid or precursor thereof is present in the composition at a concentration of at least 500 mg / ml, and the polyol is present in the composition at a concentration greater than 25,000 mg / 100 ml. The method further comprises comparing this concentration with a reference concentration of the trace compounds in individuals not suffering from cancer. In particular, an increase or decrease in the concentration of the trace compounds compared to the reference indicates that the subject suffers from or has a predisposition to cancer, or provides a negative prognosis for the subject's condition.

Description

Technical Field

[0001] The present invention relates to the detection of biomarkers, and in particular, but not limited to, methods, compositions and kits for the detection of biological markers for diagnosing various conditions such as cancer. In particular, the present invention relates to the detection of compounds as diagnostic and prognostic markers for detecting cancers such as esophageal gastric cancer or metastatic cancer.

Background Art

[0002] Esophageal adenocarcinoma is one of the five most common cancers and has the fastest increasing incidence rate among all cancers in Western populations. The United Kingdom has the highest incidence rate of esophageal adenocarcinoma in the world. Stomach cancer is the third leading cause of cancer death worldwide. The 5-year survival rates for esophageal and gastric cancers in the United Kingdom remain extremely low (13% and 18% respectively), ranking among the worst in Europe. An important aspect in improving cancer survival rates is earlier diagnosis. However, the symptoms are non-specific and are commonly shared with benign diseases. By the time the symptoms become cancer-specific, the disease is often in an advanced stage and has a poor prognosis. The costs are high due to the cancer burden and unnecessary investigations of patients with non-specific symptoms. Therefore, there is an urgent need for non-invasive tests for patients with non-specific gastrointestinal symptoms in order to effectively triage patients who should undergo endoscopy and other diagnostic modalities.

[0003] Previous studies have shown an association between esophageal and gastric cancer and volatile organic compounds (VOCs), and the technique for its diagnosis is the breath test. Using gas chromatography-mass spectrometry (GC-MS), researchers have suggested the existence of breath volatile organic compounds (VOCs) profiles specific to certain cancers [4]. GC-MS is a good technique for VOC identification, but it is essentially semi-quantitative unless a robust calibration curve is utilized, limiting the ability to reproduce study findings by different research groups. Furthermore, it requires significant analysis time per sample, so it is not naturally a high-throughput analysis in itself. Direct infusion mass spectrometry, such as selected ion flow tube mass spectrometry (SIFT-MS) and proton transfer reaction time-of-flight mass spectrometry (PTR-ToF-MS), has the advantage of being quantitative and allowing real-time analysis [5, 6].

[0004] There is a need for reliable non-invasive diagnostic tests to identify patients suffering from cancers such as esophagogastric cancer. Diagnostic methods to identify these patients with cancer would be of great benefit to the patients, increasing the chances of early treatment and improved prognosis. Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have previously developed a non-invasive test for cancer based on the detection of trace compounds such as volatile organic compounds (VOCs) in exhaled breath. The present inventors have now developed a novel method and composition that provides improved and faster accuracy of the test, achieved by administering an optimized concentration of oral stimulant (e.g., beverage, capsule or solid food), thereby transiently inducing or "priming" the cancer to produce a large amount of characteristic trace compounds (e.g., VOCs), thereby improving the test performance and diagnostic and / or prognostic accuracy. This allows earlier identification of patients with non-specific symptoms but at high risk of esophagogastric cancer and referral for further investigation and treatment. [Means for solving the problem]

[0006] Thus, in a first aspect of the invention there is provided a method for diagnosing a subject having or predisposition to having cancer, or for providing a prognosis of a condition in a subject, comprising the steps of: (i) detecting in a body sample from a test subject the concentration of trace compounds resulting from the metabolism of at least one sugar and / or at least one amino acid or precursor thereof and / or at least one polyol present in a composition previously administered to the subject, wherein the sugar is present in the composition at a concentration of greater than 20,000 mg / 100 ml, the amino acid or precursor thereof is present in the composition at a concentration of at least 500 mg / ml, and the polyol is present in the composition at a concentration of greater than 25,000 mg / 100 ml; and (ii) comparing this concentration to a standard of the concentration of the trace compound in individuals not suffering from cancer; wherein an increase or decrease in the concentration of the trace compound compared to a reference indicates that the subject is suffering from or predisposed to cancer or provides a negative prognosis of the subject's condition.

[0007] The detection step (i) may comprise detecting the trace compounds up to 30 minutes, up to 25 minutes, up to 20 minutes, up to 15 minutes, up to 10 minutes, or up to 5 minutes after administration of the composition comprising at least one sugar and / or amino acid or precursor thereof and / or at least one polyol. The detection step (i) may comprise detecting the trace compounds within 30 minutes, up to 25 minutes, up to 20 minutes, up to 15 minutes, up to 10 minutes, or up to 5 minutes after administration of the composition comprising at least one sugar and / or amino acid or precursor thereof and / or at least one polyol. Preferably, the detection step is performed when the composition previously administered to the test subject comprises at least one sugar.

[0008] The detection step (i) may further comprise detecting the trace compound between 30 and 60 minutes from administration of the composition comprising at least one sugar and / or amino acid or precursor thereof and / or at least one polyol, more preferably between 30 and 55 minutes, or between 30 and 50 minutes, or between 30 and 45 minutes, or between 30 and 40 minutes, or between 35 and 60 minutes, or between 35 and 55 minutes, or between 35 and 50 minutes, or between 35 and 45 minutes, or between 35 and 40 minutes from administration of the composition comprising at least one sugar and / or amino acid or precursor thereof and / or at least one polyol. Preferably, the detection step (i) further comprises detecting the second trace compound between 35 and 45 minutes from administration of the composition comprising at least one sugar and / or amino acid or precursor thereof and / or at least one polyol. Preferably, such a detection step is performed when the composition comprises at least one amino acid and / or at least one polyol.

[0009] Thus, preferably, the detection step (i) comprises: a) detecting trace compounds within 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes, within 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes, of administration of a composition comprising at least one sugar and / or amino acid or precursor thereof and / or at least one polyol; and b) detecting the trace compounds between 30 and 60 minutes after administration of the composition comprising at least one sugar and / or amino acid or precursor thereof and / or at least one polyol, more preferably between 30 and 55 minutes, or between 30 and 50 minutes, or between 30 and 45 minutes, or between 30 and 40 minutes, or between 35 and 60 minutes, or between 35 and 55 minutes, or between 35 and 50 minutes, or between 35 and 45 minutes, or between 35 and 40 minutes after administration of the composition comprising at least one sugar and / or amino acid or precursor thereof and / or at least one polyol; Includes.

[0010] Preferably, an increase in the concentration of the trace compound compared to the reference indicates that the subject suffers from or has a predisposition to cancer or provides a negative prognosis of the subject's condition. Preferably, the increase in the concentration of the trace compound is at least a 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% increase in the concentration of the trace compound compared to the reference.

[0011] Preferably, the sugar is present in a concentration of at least 20,000mg / 100ml, at least 20,500mg / 100ml, at least 21,000mg / 100ml, at least 25,000mg / 100ml, at least 50,000mg / 100ml or at least 75,000mg / 100ml. Preferably, the sugar is present in a concentration of about 25,000mg / 100ml. Preferably, the sugar is present in a concentration of greater than 20,000mg / 100ml, greater than 20,500mg / 100ml, greater than 21,000mg / 100ml, greater than 25,000mg / 100ml, greater than 50,000mg / 100ml or greater than 75,000mg / 100ml.

[0012] Preferably, the composition contains sugar in a concentration of between about 20,000 mg / 100 mL and 10,000 mg / 100 mL, more preferably between about 25,000 mg / 100 mL and 75,000 mg / 100 mL.

[0013] Those skilled in the art will understand that the term sugar may refer to monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides or sugar alcohols.Sugars may be selected from the group consisting of D-glucose, D-sucrose, D-lactose, D-fructose, D-mannose, D-gulose, D-galactose, D-xylose, D-arabinose, D-lyxose, D-ribose, D-allose, D-altrose, D-talose, D-idose, L-arabinose, L-rhamnose, L-xylulose, disaccharides, trisaccharides, oligosaccharides and polysaccharides, sorbitol, monosaccharides above c4, c7 and c8, sorbitol, mannitol, maltitol, lactitol, erythritol.

[0014] Preferably, the sugar is glucose, sorbitol, mannose or lactose. More preferably, the sugar is glucose, mannose or lactose. Most preferably, the sugar is glucose or lactose.

[0015] Thus, preferably the composition comprises glucose, and preferably the glucose is present in the composition at a concentration of at least 25,000 mg / 100 ml. More preferably, the sugar is glucose and is present in the composition at a concentration of at least 25,000 mg / 100 ml, and the trace compound is detected up to 10 minutes after administration of the glucose-containing composition.

[0016] The composition administered to the subject may contain citric acid. This may be a substitute for or in addition to sugar. Preferably, citric acid is used in combination with sugar. Preferably, sugar is glucose. Therefore, preferably, the composition contains citric acid and glucose.

[0017] Preferably, citric acid is present in the composition at a concentration of at least 1,000 mg / 100 ml, at least 1,100 mg / 100 ml, at least 1,200 mg / 100 ml, at least 1,300 mg / 100 ml, or at least 1,400 mg / 100 ml. Preferably, citric acid is present in a concentration of about 1,400 mg / 100 ml.

[0018] Thus, preferably, the composition comprises glucose and citric acid, preferably, glucose is present in the composition at a concentration of at least 25,000 mg / 100 ml, and citric acid is present in the composition at a concentration of at least 1,400 mg / 100 ml. More preferably, the composition comprises glucose present in the composition at a concentration of at least 25,000 mg / 100 ml, and citric acid present in the composition at a concentration of at least 1,400 mg / ml, and trace compounds are detected up to 10 minutes after administration of the composition comprising glucose and citric acid.

[0019] In another embodiment, the composition preferably comprises amino acids in a concentration of at least 500mg / 100ml, at least 1000mg / 100ml, at least 2000mg / 100ml, at least 3000mg / 100ml, at least 4000mg / 100ml, at least 5000mg / 100ml, or at least 6000mg / 100ml.

[0020] Preferably, the composition comprises the amino acid in a concentration of preferably greater than 500mg / 100ml, greater than 1000mg / 100ml, greater than 2000mg / 100ml, greater than 3000mg / 100ml, greater than 4000mg / 100ml, greater than 5000mg / 100ml or greater than 6000mg / 100ml.

[0021] Preferably, the amino acid is between 500mg / 100ml and 10,000mg / 100ml, between 500mg / 100ml and 6000mg / 100ml, between 500mg / 100ml and 5000mg / 100ml, between 500mg / 100ml and 4000mg / 100ml, between 500mg / 100ml and 3000mg / 100ml, between 500mg / 100ml and 2500mg / 100ml, Between 500mg / 100ml~2000mg / 100ml, Between 1000mg / 100ml~10000mg / 100ml, Between 1500mg / 100ml~10000mg / 100ml, Between 2000mg / 100ml~10000mg / 100ml, Between 2500mg / 100ml~10000mg / 100ml, Between 3000mg / 100ml~10000mg / 100ml, Between 4000 mg / 100ml~10000mg / 100ml, between 5000mg / 100ml~10000mg / 100ml, between 6000mg / 100ml~10000mg / 100ml, between 1000mg / 100ml~5000mg / 100ml, between 1000mg / 100ml~3000mg / 100ml, between 1000mg / 100ml~2500mg / 100ml, between 1000mg / 100ml~3000mg / 100ml, between 1000mg / 100ml~2500mg / 100ml, between 1000mg / 10

[0033] The composition may be present in a concentration of between 0mg / 100ml and 2000mg / 100ml, between 1500mg / 100ml and 10000mg / 100ml, between 1500mg / 100ml and 5000mg / 100ml, between 1500mg / 100ml and 3000mg / 100ml, between 1500mg / 100ml and 2500mg / 100ml, or between 1500mg / 100ml and 2000mg / 100ml.

[0022] Preferably, the amino acid is present in the composition at a concentration of about 2000 mg / ml.

[0023] The amino acid may be selected from the group consisting of tyrosine, glutamic acid, glutamate, phenylalanine, tryptophan, proline and histidine.

[0024] Preferably, when the amino acid is glutamic acid, the concentration of the amino acid is at least 5,000mg / 100ml, at least 5,100mg / 100ml, at least 5,200mg / 100ml, at least 5,300mg / 100ml, at least 5,400mg / 100ml, at least 5,500mg / 100ml, at least 6000mg / 100ml, greater than 5,000mg / 100ml, greater than 5,100mg / 100ml, greater than 5,200mg / 100ml, greater than 5,300mg / 100ml, greater than 5,400mg / 100ml, greater than 5,500mg / 100ml, or greater than 6,000mg / 100ml. Preferably, when the amino acid is glutamic acid, the concentration of the amino acid is between 1,800mg / 100ml and 2,200mg / 100ml, between 1,900mg / 100ml and 2,100mg / 100ml. Preferably, when the amino acid is glutamic acid, the concentration of the amino acid is 1,900mg / 100ml, 2,000mg / 100ml, 2,100mg / 100ml, 2,200mg / 100ml or 2,300mg / 100ml. Preferably, when the amino acid is glutamic acid, the concentration of the amino acid is 2,100mg / ml. However, in one embodiment, the amino acid is not glutamic acid.

[0025] Most preferably, the amino acid is tyrosine.

[0026] Thus, preferably the composition comprises tyrosine, and preferably tyrosine is present in the composition at a concentration of at least 2,000 mg / 100 ml. More preferably, the amino acid is tyrosine and is present in the composition at a concentration of at least 2,000 mg / 100 ml, and the trace compound is detected between 35 and 45 minutes after administration of the composition comprising tyrosine.

[0027] The composition administered to the subject may contain an amino acid precursor. This may be in place of or in addition to the amino acid and / or sugar. Preferably, the amino acid precursor is phenylalanine. Preferably, the amino acid precursor is used in combination with its respective amino acid. Thus, preferably, the composition contains tyrosine and phenylalanine.

[0028] Preferably, the amino acid precursors are present in the composition at a concentration of at least 500mg / 100ml, at least 1000mg / 100ml, at least 2000mg / 100ml, at least 3000mg / 100ml, at least 4000mg / 100ml, or at least 5000mg / 100ml. Preferably, the amino acid precursors are present in the composition at a concentration of at least 500mg / 100ml, at least 1000mg / 100ml, at least 2000mg / 100ml, at least 3000mg / 100ml, at least 4000mg / 100ml, or at least 5000mg / 100ml. Preferably, the amino acid precursor is between 500mg / 100ml and 10000mg / 100ml, between 500mg / 100ml and 5000mg / 100ml, between 500mg / 100ml and 4000mg / 100ml, between 500mg / 100ml and 3000mg / 100ml, between 500mg / 100ml and 2500mg / 100ml, between 500mg / 100ml and 2000mg / 100ml, between 1000mg / 100ml and 10000mg / 100ml, between 1500mg / 100ml and 10000mg / 100ml, between 2000mg / 100ml and 10000mg / 100ml, between 2500mg / 100ml and 10000mg / 100ml, between 30 The composition may be present in a concentration between 100mg / 100ml and 10000mg / 100ml, between 1000mg / 100ml and 5000mg / 100ml, between 1000mg / 100ml and 3000mg / 100ml, between 1000mg / 100ml and 2500mg / 100ml, between 1000mg / 100ml and 2000mg / 100ml, between 1500mg / 100ml and 10000mg / 100ml, between 1500mg / 100ml and 5000mg / 100ml, between 1500mg / 100ml and 3000mg / 100ml, between 1500mg / 100ml and 2500mg / 100ml, or between 1500mg / 100ml and 2000mg / 100ml.

[0029] Preferably, the amino acid precursor is phenylalanine. Preferably, the phenylalanine is present in a concentration of 3000mg / 100ml.

[0030] Preferably, the composition comprises phenylalanine and tyrosine.

[0031] In one embodiment, the composition comprises tyrosine, phenylalanine and glutamic acid. Preferably, tyrosine is present in a concentration of at least 2,000 mg / 100 ml, phenylalanine is present in a concentration of at least 3,000 mg / 100 ml, and glutamic acid is present in a concentration of at least 2,100 mg / 100 ml.

[0032] Preferably, the polyol is present in the composition at a concentration of more than 25,000 mg / 100 ml. Preferably, the polyol is present in the composition at a concentration of more than 26,000 mg / 100 ml, more than 27,000 mg / 100 ml, more than 28,000 mg / 100 ml, or more than 29,000 mg / 100 ml. Preferably, the polyol is present in the composition at a concentration of more than 30,000 mg / 100 ml, more than 35,000 mg / 100 ml, more than 40,000 mg / ml, more than 45,000 mg / 100 ml, or more than 50,000 mg / 100 ml. Preferably, the polyol is present in the composition in a concentration of at least 30,000mg / 100ml, at least 35,000mg / 100ml, at least 40,000mg / ml, at least 45,000mg / 100ml, at least 50,000mg / 100ml.

[0033] Preferably, the polyol is present in the composition in a concentration of 50,000 mg / 100 ml. Most preferably, the polyol is present in the composition in a concentration of between 23,000 mg / 100 ml and 27,000 mg / 100 ml, or between 24,000 mg / 100 ml and 26,000 mg / 100 ml.

[0034] Preferably, the polyol is glycerol. Preferably, glycerol is present in the composition at a concentration of more than 30,000mg / ml, more preferably 50,000mg / 100ml. Most preferably, glycerol is present in the composition at a concentration between 23,000mg / 100ml and 27,000mg / 100ml, or between 24,000mg / 100ml and 26,000mg / 100ml.

[0035] In one embodiment, at least one sugar and / or at least one amino acid or precursor thereof and / or at least one polyol is metabolized by a cancer-associated microorganism.

[0036] It is understood that "prognosis" may refer to the determination of the outcome of treatment in a subject diagnosed with cancer.Prognosis may refer to predicting the rate and / or duration of progression or improvement of cancer in a subject, survival probability and / or the effectiveness of various treatment regimes.Thus, poor prognosis may indicate the progression of cancer, low survival probability and reduced effectiveness of treatment regimes.Good prognosis may indicate the improvement of cancer, high survival probability and increased effectiveness of treatment regimes.

[0037] Cancer-associated microorganisms may be bacteria.It is understood that the microorganisms and bacteria present in the intestine form what is called "microbiome".Therefore, the cancer-associated microorganisms that metabolize at least one substrate into trace compounds that are detected and / or analyzed in the method of the present invention for diagnosing cancer preferably form part of the microbiome.

[0038] The cancer-associated microorganism may be Streptococcus, Lactobacillus, Veillonella, Prevotella, Neisseria, Haemophilus, L. coleohominis, Lachnospiraceae, Klebsiella, Clostridiales, Erysipelotrichales, or any combination thereof.

[0039] The cancer-associated microorganism may be S. pyogenes, Klebsiella pneumoniae, Lactobacillus acidophilus, or any combination thereof.

[0040] Cancer-associated microorganisms include E. coli, P. mirabili, B. cepacia, S. pyogenes, Streptococcus salivarius, Actinomyces naeslundii, Lactobacillus fermentum, Streptococcus anginosus, Clostridium bifermentans, Clostridium perfringens, Clostridium septicum, Clostridium sporogenes, Clostridium tertium, Eubacterium lentum, Eubacterium sp., Fusobacterium simiae, Fusobacterium necrophorum, Lactobacillus acidophilus, Peptococcus niger, Peptostreptococcus anaerobius, Peptostreptococcus asaccharolyticus, Peptostreptococcus prevotii, P. aeruginosa, S. aureus, P. mirabilis, E. faecalis, S. pneumoniae, N. meningitides、Acinetobacter baumannii、Bacteroides capillosus、Bacteroides fragilis、Bacteroides pyogenes、Clostridium difficile、Clostridium ramosum、Enterobacter cloacae、Klebsiella pneumoniae、Nocardia sp.、Propionibacterium acnes、Propionibacterium propionicum or any combination of these may be possible. coli、L. fermentation、S. salivarius、S. anginosus or K. pneumoniae.

[0041] In an embodiment, the cancer is esophagogastric junction cancer, gastric cancer, esophageal cancer, esophageal squamous cell carcinoma (ESCC), or esophageal adenocarcinoma (EAC). Thus, in a preferred embodiment, the diagnosis is for diagnosing esophagogastric junction cancer, gastric cancer, esophageal cancer, esophageal squamous cell carcinoma (ESCC), or esophageal adenocarcinoma (EAC). Most preferably, the cancer is esophagogastric cancer, such that this condition can be diagnosed or prognosed. The cancer may be metastatic.

[0042] Preferably, the cancer is gastric cancer, esophageal cancer or metastatic cancer.

[0043] In an embodiment, the cancer is pancreatic cancer or colorectal cancer. Thus, the diagnosis or prognosis may be for diagnosing or prognosing pancreatic cancer or colorectal cancer.

[0044] In a second aspect, there is provided a method for detecting a trace compound in a test subject, comprising the steps of: (i) providing to a subject a composition comprising at least one substrate according to the first aspect that results in a trace compound; and (ii) detecting the concentration of the trace compound in a body sample from the subject. A method is provided, comprising:

[0045] Preferably, the detection step is carried out according to the first aspect.

[0046] In a third aspect of the present invention there is provided a composition comprising at least one sugar and / or at least one amino acid or precursor thereof and / or at least one polyol suitable for metabolism to trace compounds, preferably for use in a method for diagnosis or prognosis of cancer, wherein the sugar is present in the composition at a concentration of more than 20,000mg / 100ml, the amino acid is present in the composition at a concentration of at least 500mg / ml and the polyol is present in the composition at a concentration of more than 25,000mg / 100ml.

[0047] Preferably, the composition and the cancer are as defined in the first aspect.

[0048] In a fourth aspect, there is provided a composition comprising at least one substrate suitable for metabolism by a cancer-associated microorganism into a trace compound, for use in the method of the first or second aspect.

[0049] In a fifth aspect, there is provided a kit for diagnosing a subject having or predisposition to having cancer, or for providing a prognosis of a condition in a subject, comprising: (a) a composition comprising at least one substrate as defined in the first aspect, (b) a means for determining the concentration of the trace compound in a sample from a test subject; and (c) A measure of the concentration of the trace compound in samples from individuals without cancer. wherein the kit identifies an increase or decrease in the concentration of the trace compound in a body sample from a test subject compared to a reference, thereby being used to suggest that the subject suffers from or has a predisposition to cancer, or to provide a negative prognosis of the subject's condition.

[0050] Preferably, the composition and the cancer are as defined in the first aspect.

[0051] The methods of the first and second aspects may include administering or having the subject administered a therapeutic agent that prevents, reduces or delays the progression of the cancer, or having the subject follow a special diet, or administering chemotherapy or chemoradiotherapy.

[0052] Thus, in a sixth aspect, there is provided a method of treating a subject suffering from cancer, comprising: (i) providing to a subject a composition comprising at least one substrate as defined in the first aspect; (ii) analyzing the concentration of a trace compound produced by metabolism of at least one substrate in a body sample from a test subject and comparing said concentration with a standard of the concentration of the trace compound in an individual not suffering from cancer, wherein an increase or decrease in the concentration of the trace compound in the body sample from the test subject compared to the standard is indicative of the subject suffering from or having a predisposition to cancer, or having a negative prognosis; and (iii) administering or having administered to the subject a therapeutic agent, or having the subject follow a special diet, or administering chemotherapy or chemoradiotherapy, wherein the therapeutic agent or special diet, or chemotherapy or chemoradiotherapy, prevents, reduces or delays the progression of the cancer. A method is provided, comprising:

[0053] Preferably, the composition and the cancer are as defined in the first aspect.

[0054] The method of the present invention is useful for monitoring the effectiveness of treatment of cancers of relevance. For example, treatment of resectable esophagogastric cancer may include neoadjuvant chemotherapy, or chemoradiotherapy followed by surgery and adjuvant chemotherapy. Treatment of very early stage esophagogastric cancer may include endoscopic resection. Treatment of advanced esophagogastric cancer may include palliative chemotherapy. Cancer-associated microbiome has recently been shown to enhance metastasis to the liver (Bullman et al., Science, 2017). Thus, the present invention described herein can be used to monitor the response of treatments directed at the cancer-associated microbiome.

[0055] If the cancer is pancreatic cancer, the treatment may include administering chemotherapy, chemoradiotherapy with or without surgery. For example, if the cancer is colorectal cancer, the treatment may include administering chemotherapy, chemoradiotherapy with or without surgery, or endoscopic resection.

[0056] In a seventh aspect, there is provided a method for determining the effectiveness of treatment of a subject with cancer with a therapeutic agent or a special diet, or chemotherapy or chemoradiotherapy, comprising: (i) providing to a subject a composition comprising at least one substrate according to the first aspect; and (ii) analyzing the concentration of a trace compound produced by metabolism of at least one substrate in a body sample from the test subject and comparing this concentration to a standard of the concentration of the trace compound in individuals not suffering from cancer; wherein an increase or decrease in the concentration of the trace compound in a body sample from a test subject compared to a reference indicates that a therapeutic agent or a special diet, or a chemotherapy or chemoradiotherapy treatment regime is or is not effective.

[0057] Preferably, the composition and the cancer are as defined in the first aspect.

[0058] The composition may be an existing composition, food or drink that includes any one of the aforementioned components. Preferably, the composition includes water. The composition of the present invention is ingested by the subject. The composition may be an edible or swallowable solid or liquid. In an embodiment, the composition can be chewed, which results in the release of the substrate, which descends to the intestine. In an embodiment, the composition may be in the form of a capsule designed to break down at a specific location by the digestive tract, thereby resulting in the targeted release of at least one substrate. However, the composition is preferably a liquid (i.e., a drink) that can be swallowed, which may be referred to as an oral stimulation drink (OSD).

[0059] Preferably, a sample is taken from the subject and then the trace compounds are detected in the body sample, hi some embodiments, the concentration of the trace compounds is measured.

[0060] A trace compound may be any compound that may indicate or correlate with the presence of a microorganism. The trace compounds detected may be volatile organic compounds (VOCs) that result in a fermentation profile, which may be detected in a body sample by various techniques. In an embodiment, these compounds may be detected in a liquid or semi-solid sample in which they are dissolved. However, in a preferred embodiment, the compounds are detected from a gas or vapor. For example, if the trace compounds are VOCs, they may emanate from or form part of the sample, and therefore can be detected in gas or vapor form.

[0061] An increase or decrease in the concentration of these trace compounds compared to the reference standard indicates that the subject suffers from or has a predisposition to cancer, or provides a negative prognosis of the subject's condition. Preferably, an increase in the concentration of these trace compounds compared to the reference standard indicates that the subject suffers from or has a predisposition to cancer, or provides a negative prognosis of the subject's condition.

[0062] The VOCs may be short chain fatty acids, aldehydes, alcohols or any combination thereof.

[0063] VOCs are C 1 ~C 3 Aldehyde, C 1 ~C 3 Alcohol, where the first carbon atom is substituted with an =O group and the second carbon atom is substituted with an -OH group 2 ~C 10 Alkane, C 1 ~C 20 Alkane, C 4 ~C 10 Alcohol, C. 1 ~C 6 Carboxylic acid, C 4 ~C 20 Aldehyde, optionally C 1 ~C 6 Alkyl-substituted phenols, C 2 Aldehyde, C 3 Aldehyde, C 8 Aldehyde, C9 Aldehydes, C 10 Aldehydes, C 11 Aldehydes, analogs or derivatives of any of the aforementioned species, or any combination thereof, may also be used.

[0064] C 1 ~C 6 The carboxylic acid may be selected from the group consisting of formic acid, acetic acid, propanoic acid, butanoic acid, pentanoic acid, and hexanoic acid. C 1 ~C 3 The aldehyde may be selected from the group consisting of formaldehyde, acetaldehyde, and propanal. C 4 ~C 20 The aldehyde is C 4 ~C 10 It may be an aldehyde. C 4 ~C 20 The aldehyde may be selected from the group consisting of butanal, pentanal, hexanal, heptanal, octanal, nonanal, decanal, undecanal, dodecanal, tridecanal, tetradecanal, pentadecanal, hexadecanal, heptadecanal, octadecanal, nonadecanal, and icodanal. C 1 ~C 20 The alkane is preferably C 4 ~C 16 The alkane, more preferably C 8 ~C 14 It is an alkane. C 1 ~C 20 The alkane may be methane, ethane, propane, butane, pentane, hexane, heptane, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, and icodane. The phenol may be unsubstituted. Alternatively, the phenol may be substituted with a C 1 ~C 6 alkyl group at the trans position. The phenol is C 1 ~C 3 It may be substituted with an alkyl group. Optionally C 1 ~C6 The alkyl group substituted phenol may be phenol, 1-hydroxy-4-ethylbenzene or p-cresol.

[0065] Preferably, the volatile organic compound (VOC) is selected from the group consisting of acetic acid, butanoic acid, hexanoic acid, pentanoic acid, propanoic acid, acetaldehyde, decanal, heptanal, hexanal, nonanal, octanal, pentanal, butanal, propanal, 1-hydroxy-4-ethylbenzene, decane, dodecane, p-cresol and phenol or any combination thereof.

[0066] When the substrate is a sugar, preferably glucose, the trace compounds may be acetic acid, butanoic acid, pentanoic acid, propanoic acid, hexanoic acid, acetaldehyde, propanal, butanal, hexanal, pentanal, decanal, 1-hydroxytheylbenzene and / or p-cresol.

[0067] When the substrate is a sugar, preferably glucose, an increase in acetic acid, butanoic acid, pentanoic acid, propanoic acid, acetaldehyde, butanal, hexanal, pentanal, 1-hydroxyethylbenzene and / or p-cresol may be indicative of gastric cancer. Preferably, when the substrate is glucose and the trace compound is butanoic acid, the increase in the concentration of the trace compound is at least a 300% increase in the concentration of the butanoic acid compound compared to the reference, indicative of gastric cancer. Preferably, when the substrate is glucose and the trace compound is propanoic acid, the increase in the concentration of the trace compound is at least a 100% increase in the concentration of the propanoic acid compound compared to the reference, indicative of gastric cancer. Preferably, when the substrate is glucose and the trace compound is acetic acid, the increase in the concentration of the trace compound is at least a 200% increase in the concentration of the acetate compound compared to the reference, indicative of gastric cancer. Preferably, when the substrate is glucose and the trace compound is pentanoic acid, the increase in the concentration of the trace compound is at least a 50% increase in the concentration of the pentanoic acid compound compared to the reference, indicative of gastric cancer.

[0068] When the substrate is a sugar, preferably glucose, an increase in acetic acid, pentanoic acid, propanoic acid, butanal, propanal and / or hexanoic acid may be indicative of esophageal cancer. Preferably, when the substrate is glucose and the trace compounds are butanoic acid, propanoic acid and / or acetic acid, an increase in the concentration of the trace compounds is at least a 50% increase in the concentration of butanoic acid, propanoic acid and / or acetic acid compounds compared to the reference, indicative of esophageal cancer.

[0069] When the substrate is a sugar, preferably glucose, in combination with citric acid, an increase in the trace compounds butanoic acid, propanoic acid and / or propanal may indicate esophageal cancer.

[0070] When the substrate is a sugar, preferably glucose, in combination with citric acid, an increase in the trace compounds butanoic acid, propanoic acid and / or propanal may be indicative of gastric cancer.

[0071] When the substrate is an amino acid or a precursor thereof, the trace compound may be butanal, decanal, heptanal, hexanal, phenol, decane, p-cresol, 1-hydroxyethylbenzene and / or dodecane. Preferably, when the substrate is an amino acid or a precursor thereof, the increase in the concentration of the trace compound is at least a 10%, 20%, 30%, 40% or 50% increase compared to the reference.

[0072] When the substrate is tyrosine, the trace compound may be butanal, decanal, heptanal, hexanal, phenol, decane, p-cresol and / or dodecane. Preferably, the trace compound is decanal and / or dodecane. Preferably, when the substrate is tyrosine, the increase in the concentration of the trace compound is at least a 10%, 20%, 30%, 40% or 50% increase compared to the reference.

[0073] When the substrate is tyrosine, an increase in decanal may indicate esophageal cancer.

[0074] When the substrate is tyrosine, an increase in dodecane may indicate gastric cancer.

[0075] When the substrate is phenylalanine, the trace compounds may be dodecane, decane, phenol, decanal and / or dodecane.

[0076] When the substrate is phenylalanine, increases in the trace compounds decanal, 1-hydroxyethylbenzene, decane, dodecane, p-cresol, and / or phenol may indicate esophageal cancer.

[0077] When the substrate is phenylalanine, increases in the trace compounds hydroxyethylbenzene, decane, dodecane, p-cresol and / or phenol may indicate stomach cancer.

[0078] When the substrate is glutamic acid, the trace compounds may be propanal, dodecane, phenol and / or butanoic acid.

[0079] When the substrate is glutamic acid, increases in the trace compounds propanal, dodecane, phenol, and / or butanoic acid may indicate esophageal cancer.

[0080] When the substrate is glutamic acid, increases in the trace compounds propanal, dodecane, phenol and / or butanoic acid may indicate gastric cancer.

[0081] When the substrate is a polyol, preferably glycerol, the trace compounds may be butanoic acid, acetic acid, hexanoic acid, pentanoic acid, propanoic acid, butanal, hexanal, pentanal and / or propanal.

[0082] When the substrate is a polyol, preferably glycerol, an increase in the trace compounds butanoic acid, acetic acid, hexanoic acid, pentanoic acid, propanoic acid, butanal, hexanal, pentanal and / or propanal may be indicative of esophageal cancer.

[0083] When the substrate is a polyol, preferably glycerol, an increase in the trace compounds butanoic acid, acetic acid, hexanoic acid, pentanoic acid, propanoic acid, butanal, hexanal, pentanal and / or propanal may be indicative of gastric cancer.

[0084] Preferably, the sample is any body sample in which the trace compound is present or secreted. Thus, preferably, the detection or diagnosis method is performed in vitro. However, the prognosis method may be performed in vivo. For example, the sample may include urine, feces, hair, sweat, saliva, blood or tears. In one embodiment, the sample may be immediately assayed for the level of the trace compound. Alternatively, the sample may be stored at low temperature, for example in a freezer, or even frozen, until the concentration of the trace compound is determined. The measurement of the trace compound in the body sample may be made on the whole sample or on a processed sample, for example whole blood or processed blood.

[0085] In an embodiment, the sample may be a urine sample. The concentration of trace compounds in a body sample is preferably measured in vitro from a urine sample collected from a subject. Compounds may be detected from gas or vapor emanating from a urine sample. It is understood that detection of compounds in the gas phase emanating from urine is preferred.

[0086] It is also understood that a "fresh" body sample may be analyzed immediately after it is taken from a subject. Alternatively, the sample may be frozen and stored. The sample may then be thawed and analyzed at a later date.

[0087] However, most preferably, the body sample may be a breath sample from the test subject. The sample may be collected by the subject exhaling air from the mouth, preferably after nasal inhalation. Preferably, the sample includes the subject's alveolar air. Preferably, the alveolar air is collected beyond the dead space air by capturing the end-tidal air. The VOCs from the breath bag are then preconcentrated in the tube, preferably by moving the exhaled air across a thermal desorption tube.

[0088] The difference in the concentration of the trace compound indicative of cancer or a predisposition to cancer in a subject may be an increase or decrease compared to the standard. It is understood that the concentration of the trace compound in a patient suffering from a disease is highly dependent on many factors, such as how advanced the disease is, and the age and sex of the subject. It is also understood that the standard concentration of the trace compound in individuals not suffering from a disease may vary to some extent, but on average, over a given period of time, the concentration tends to be substantially constant. It should further be understood that the concentration of the trace compound in one group of individuals suffering from a disease may differ from the concentration of the compound in another group of individuals not suffering from a disease. However, it is possible to determine the average concentration of the trace compound in individuals not suffering from cancer, which is referred to as the standard or "normal" concentration of the trace compound. The normal concentration corresponds to the reference value described above.

[0089] In one embodiment, the method of the present invention preferably involves determining the ratio of chemicals in the exhaled breath (i.e., using other components therein as a reference) and comparing markers for these diseases to indicate whether they are elevated or decreased.

[0090] Trace compounds are preferably volatile organic compounds (VOCs) that provide a profile and can be detected in or from body samples by various techniques.Accordingly, these compounds can be detected using gas analyzers.Examples of suitable detectors for detecting trace compounds preferably include electrochemical sensors, semiconductor metal oxide sensors, quartz crystal microbalance sensors, optical dye sensors, fluorescent sensors, conductive polymer sensors, composite polymer sensors, or optical spectrometry.

[0091] The inventors have demonstrated that trace compounds can be reliably detected using gas chromatography, mass spectrometry, GCMS or TOF. Dedicated sensors may also be used for the detection step.

[0092] The reference value may be obtained by assaying a statistically significant number of control samples (i.e. samples from subjects not suffering from the disease). Thus, reference (ii) according to the kit of the fifth aspect of the invention may be a control sample (for assay).

[0093] The device preferably includes a positive control (most preferably provided in a container) corresponding to the trace compound. The device preferably includes a negative control (preferably provided in a container). In a preferred embodiment, the kit may include a reference, a positive control, and a negative control. The kit may also include additional controls, such as "spike-in" controls to provide a concentration reference, if desired, and additional positive controls for each of the trace compounds, or their analogs or derivatives.

[0094] Therefore, the inventors have recognized that the difference in concentration of the trace compounds between the reference normal (i.e., control) level and the increased / decreased level can be used as a physiological marker that indicates the presence of disease in the test subject. It is understood that if a subject has an increase / decrease in the concentration of one or more trace compounds that is significantly higher / lower than the control value, which is the "normal" concentration of the compound in the reference, the subject is at higher risk of having a disease or a more advanced condition than if the concentration of the compound is only slightly higher / lower than the "normal" concentration.

[0095] One of ordinary skill in the art will understand how to measure the concentration of a trace compound in a statistically significant number of control individuals and the concentration of the compound in a test subject, and use the respective values ​​to determine whether the test subject has a statistically significant increase / decrease in the concentration of the compound, and therefore infer whether the subject suffers from the disease being screened for.

[0096] The kit of the fifth aspect may include a sample extraction means for obtaining a sample from a test subject. The sample extraction means may include a needle or a syringe, or the like. The kit may include a sample collection vessel for receiving an extracted sample, which may be liquid, gaseous, or semi-solid. The kit may further include instructions for use.

[0097] In a further aspect, there is provided a method for diagnosing a subject having or predisposition to cancer, or for providing a prognosis of a subject's condition, comprising: (i) detecting in a body sample from a test subject the concentration of trace compounds resulting from the metabolism of at least one sugar and / or at least one amino acid or precursor thereof and / or at least one polyol present in a composition previously administered to the subject, wherein the sugar is present in the composition at a concentration of greater than 20,000 mg / 100 ml, the amino acid or precursor thereof is present in the composition at a concentration of at least 500 mg / ml, and the polyol is present in the composition at a concentration of greater than 30,000 mg / 100 ml; and (ii) comparing this concentration to a standard of the concentration of the trace compound in individuals not suffering from cancer; wherein an increase or decrease in the concentration of the trace compound compared to a reference indicates that the subject is suffering from or predisposed to cancer or provides a negative prognosis of the subject's condition.

[0098] In another aspect there is provided a composition comprising at least one sugar and / or at least one amino acid or precursor thereof and / or at least one polyol present, suitable for metabolism to trace compounds, preferably for use in a method for diagnosis or prognosis of cancer, wherein the sugar is present in the composition at a concentration of more than 20,000 mg / 100 ml, the amino acid is present in the composition at a concentration of at least 500 mg / ml and the polyol is present in the composition at a concentration of more than 30,000 mg / 100 ml.

[0099] All of the features described in this specification (including any accompanying claims, abstract and drawings) and / or all of the steps of any method or process so disclosed may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0100] For a better understanding of the present invention, and to show how embodiments of the same may be carried out, reference will now be made, by way of example, to the accompanying drawings in which: [Brief description of the drawings]

[0101] [Figure 1] FIG. 1 illustrates an embodiment of an apparatus and method used to concentrate VOCs from a steel breath bag into a thermal desorption tube. [Diagram 2] Figure 1 shows butanoic acid concentrations detected in breath at varying doses (top panel shows fold change, bottom panel shows concentration (ppbv)). The optimal dose response in subject 1 is between 25-75 g glucose 5-10 min after glucose consumption. [Diagram 3] FIG. 13 shows butanoic acid concentrations detected in breath at varying doses. The optimal dose response in subject 2 is between 25-75 g glucose 5-15 minutes after glucose consumption. [Figure 4] Figure 10. Butanoic acid concentrations detected in breath at varying doses. **Only 2 doses out of 5 completed. A comparable dose response is between 25-50g glucose 5-10 minutes after glucose consumption. 50g glucose demonstrates approximately a 2-fold fold change compared to 25g glucose in subject 3. [Diagram 5] FIG. 10 shows butanoic acid concentrations detected in breath at varying doses. The optimal dose response in subject 4 is between 10-75 g glucose 5-10 minutes after glucose consumption. [Figure 6] FIG. 13 shows subjective comparison between volatile butanoic acid concentrations in breath for 75 g glucose (n=3). [Figure 7] FIG. 13 shows subjective comparison between volatile butanoic acid concentrations in breath for 50 g glucose (n=4). [Figure 8] FIG. 13 shows subjective comparison between volatile butanoic acid concentrations in breath for 25 g glucose (n=4). [Figure 9]FIG. 13 shows subjective comparison between volatile butanoic acid concentrations in breath for 10 g glucose (n=3). [Figure 10A] FIG. 1 shows that multiple volatile short chain fatty acids tested (acetic acid, butanoic acid, hexanoic acid, pentanoic acid and propanoic acid) were maximally increased 5-10 min after glucose consumption. [Figure 10B] FIG. 1 shows that multiple volatile short chain fatty acids tested (acetic acid, butanoic acid, hexanoic acid, pentanoic acid and propanoic acid) were maximally increased 5-10 min after glucose consumption. [Figure 10C] FIG. 1 shows that multiple volatile short chain fatty acids tested (acetic acid, butanoic acid, hexanoic acid, pentanoic acid and propanoic acid) were maximally increased 5-10 min after glucose consumption. [Figure 10D] FIG. 1 shows that multiple volatile short chain fatty acids tested (acetic acid, butanoic acid, hexanoic acid, pentanoic acid and propanoic acid) were maximally increased 5-10 min after glucose consumption. [Figure 10E] FIG. 1 shows that multiple volatile short chain fatty acids tested (acetic acid, butanoic acid, hexanoic acid, pentanoic acid and propanoic acid) were maximally increased 5-10 min after glucose consumption. [Figure 11A] FIG. 1 shows that several volatile aldehydes tested showed maximal increases at 5 min (butanal, decanal, propanal) and 15 min (pentanal). [Figure 11B] FIG. 1 shows that several volatile aldehydes tested showed maximal increases at 5 min (butanal, decanal, propanal) and 15 min (pentanal). [Figure 11C] FIG. 1 shows that several volatile aldehydes tested showed maximal increases at 5 min (butanal, decanal, propanal) and 15 min (pentanal). [Figure 11D] FIG. 1 shows that several volatile aldehydes tested showed maximal increases at 5 min (butanal, decanal, propanal) and 15 min (pentanal). [Figure 11E]FIG. 1 shows that several volatile aldehydes tested showed maximal increases at 5 min (butanal, decanal, propanal) and 15 min (pentanal). [Figure 11F] FIG. 1 shows that several volatile aldehydes tested showed maximal increases at 5 min (butanal, decanal, propanal) and 15 min (pentanal). [Figure 11G] FIG. 1 shows that several volatile aldehydes tested showed maximal increases at 5 min (butanal, decanal, propanal) and 15 min (pentanal). [Figure 11H] FIG. 1 shows that several volatile aldehydes tested showed maximal increases at 5 min (butanal, decanal, propanal) and 15 min (pentanal). [Figure 11I] FIG. 1 shows that several volatile aldehydes tested showed maximal increases at 5 min (butanal, decanal, propanal) and 15 min (pentanal). [Figure 12A] FIG. 1 shows that several volatile phenolics tested demonstrated increased breath concentrations 5 min after glucose consumption (1-hydroxy-4-ethylbenzene, dodecane, p-cresol, phenol). [Figure 12B] FIG. 1 shows that several volatile phenolics tested demonstrated increased breath concentrations 5 min after glucose consumption (1-hydroxy-4-ethylbenzene, dodecane, p-cresol, phenol). [Figure 12C] FIG. 1 shows that several volatile phenolics tested demonstrated increased breath concentrations 5 min after glucose consumption (1-hydroxy-4-ethylbenzene, dodecane, p-cresol, phenol). [Figure 12D] FIG. 1 shows that several volatile phenolics tested demonstrated increased breath concentrations 5 min after glucose consumption (1-hydroxy-4-ethylbenzene, dodecane, p-cresol, phenol). [Figure 12E]FIG. 1 shows that several volatile phenolics tested demonstrated increased breath concentrations 5 min after glucose consumption (1-hydroxy-4-ethylbenzene, dodecane, p-cresol, phenol). [Figure 13A] FIG. 1 shows that the volatile short chain fatty acids tested did not demonstrate significant changes following tyrosine consumption. [Figure 13B] FIG. 1 shows that the volatile short chain fatty acids tested did not demonstrate significant changes following tyrosine consumption. [Figure 13C] FIG. 1 shows that the volatile short chain fatty acids tested did not demonstrate significant changes following tyrosine consumption. [Figure 13D] FIG. 1 shows that the volatile short chain fatty acids tested did not demonstrate significant changes following tyrosine consumption. [Figure 13E] FIG. 1 shows that the volatile short chain fatty acids tested did not demonstrate significant changes following tyrosine consumption. [Figure 14A] FIG. 1 shows that several volatile aldehydes tested (butanal, decanal, heptanal and hexanal) demonstrated a slight increase approximately 30 minutes after tyrosine ingestion. [Figure 14B] FIG. 1 shows that several volatile aldehydes tested (butanal, decanal, heptanal and hexanal) demonstrated a slight increase approximately 30 minutes after tyrosine ingestion. [Figure 14C] FIG. 1 shows that several volatile aldehydes tested (butanal, decanal, heptanal and hexanal) demonstrated a slight increase approximately 30 minutes after tyrosine ingestion. [Figure 14D] FIG. 1 shows that several volatile aldehydes tested (butanal, decanal, heptanal and hexanal) demonstrated a slight increase approximately 30 minutes after tyrosine ingestion. [Figure 14E] FIG. 1 shows that several volatile aldehydes tested (butanal, decanal, heptanal and hexanal) demonstrated a slight increase approximately 30 minutes after tyrosine ingestion. [Figure 14F] FIG. 1 shows that several volatile aldehydes tested (butanal, decanal, heptanal and hexanal) demonstrated a slight increase approximately 30 minutes after tyrosine ingestion. [Figure 14G] FIG. 1 shows that several volatile aldehydes tested (butanal, decanal, heptanal and hexanal) demonstrated a slight increase approximately 30 minutes after tyrosine ingestion. [Figure 14H] FIG. 1 shows that several volatile aldehydes tested (butanal, decanal, heptanal and hexanal) demonstrated a slight increase approximately 30 minutes after tyrosine ingestion. [Figure 14I] FIG. 1 shows that several volatile aldehydes tested (butanal, decanal, heptanal and hexanal) demonstrated a slight increase approximately 30 minutes after tyrosine ingestion. [Figure 15A] Figure 1 shows that volatile phenols demonstrated slight increases in breath concentrations 35-45 min after tyrosine consumption (with the exception of 1-hydroxy-4-ethylbenzene). [Figure 15B] Figure 1 shows that volatile phenols demonstrated slight increases in breath concentrations 35-45 min after tyrosine consumption (with the exception of 1-hydroxy-4-ethylbenzene). [Figure 15C] Figure 1 shows that volatile phenols demonstrated slight increases in breath concentrations 35-45 min after tyrosine consumption (with the exception of 1-hydroxy-4-ethylbenzene). [Figure 15D] Figure 1 shows that volatile phenols demonstrated slight increases in breath concentrations 35-45 min after tyrosine consumption (with the exception of 1-hydroxy-4-ethylbenzene). [Figure 15E] Figure 1 shows that volatile phenols demonstrated slight increases in breath concentrations 35-45 min after tyrosine consumption (with the exception of 1-hydroxy-4-ethylbenzene). [Figure 16] FIG. 1 shows butanoic acid concentrations detected in breath at varying doses of four different sugars at a concentration of 25 g per 100 ml. [Figure 17] 1 shows decanal concentrations detected in breath with 3 g of phenylalanine, with optimal responses observed 15 min after phenylalanine consumption. [Figure 18] 1 shows the concentration of dodecane detected in breath with 3 g of phenylalanine, with the optimal response observed 10 min after phenylalanine consumption. [Figure 19] Figure 1 shows phenol concentrations detected in breath with 3 g phenylalanine, with optimal responses observed 60 min after phenylalanine consumption with 3 g phenylalanine. [Figure 20] 1 shows decane concentrations detected in breath with 3 g of phenylalanine, with optimal responses observed 15 min after phenylalanine consumption. [Figure 21A] FIG. 1 shows a comparison between phenylalanine and tyrosine consumption in the same subjects for (a) decanal, (b) dodecane, (c) phenol, and (d) decane. Elevated VOC responses are demonstrated for phenylalanine compared to tyrosine, and most significantly for decanal and dodecane. [Figure 21B] FIG. 1 shows a comparison between phenylalanine and tyrosine consumption in the same subjects for (a) decanal, (b) dodecane, (c) phenol, and (d) decane. Elevated VOC responses are demonstrated for phenylalanine compared to tyrosine, and most significantly for decanal and dodecane. [Figure 22] 1 shows propanal concentrations detected in exhaled breath, with optimal responses observed 5 min after glutamate consumption. [Figure 23] FIG. 1 shows the concentration of dodecane detected in exhaled breath, with the optimal response observed 20 min after glutamate consumption. [Figure 24] Figure 1 shows phenol concentrations detected in exhaled breath. Optimal responses were observed 35-45 min after phenylalanine consumption. [Diagram 25]Figure 1 shows butanoic acid concentrations detected in exhaled breath. The optimal response was observed 5 min after glutamate consumption. This is likely secondary to the generation of keto acids during transamination of amino acids. Keto acids are used as intermediates in the citric acid cycle of glycolysis. [Figure 26] FIG. 1 shows butanoic acid concentrations detected in breath at varying doses of glycerol for subject 1. The optimal dose response was 45-55 minutes after glycerol consumption at 50 g glycerol. [Figure 27] FIG. 13 shows butanoic acid concentrations detected in breath at varying doses of glycerol in subject 2. The optimal dose response was 45-55 minutes after glycerol consumption with 50 g glycerol. [Figure 28] FIG. 13 shows a subjective comparison between volatile butanoic acid concentrations in breath for 50 g of glycerol. [Figure 29] FIG. 13 shows a subjective comparison between volatile butanoic acid concentrations in breath for 50 g of glycerol. [Figure 30A] FIG. 1 shows that multiple volatile short-chain fatty acids tested (i.e., acetic acid, butanoic acid, and propanoic acid) were maximally increased in the esophageal cancer group 45-60 min after consumption of 25 g of glycerol. [Figure 30B] FIG. 1 shows that multiple volatile short-chain fatty acids tested (i.e., acetic acid, butanoic acid, and propanoic acid) were maximally increased in the esophageal cancer group 45-60 min after consumption of 25 g of glycerol. [Figure 30C] FIG. 1 shows that multiple volatile short-chain fatty acids tested (i.e., acetic acid, butanoic acid, and propanoic acid) were maximally increased in the esophageal cancer group 45-60 min after consumption of 25 g of glycerol. [Figure 30D] FIG. 1 shows that multiple volatile short-chain fatty acids tested (i.e., acetic acid, butanoic acid, and propanoic acid) were maximally increased in the esophageal cancer group 45-60 min after consumption of 25 g of glycerol. [Figure 30E]FIG. 1 shows that multiple volatile short-chain fatty acids tested (i.e., acetic acid, butanoic acid, and propanoic acid) were maximally increased in the esophageal cancer group 45-60 min after consumption of 25 g of glycerol. [Figure 31A] FIG. 1 shows that multiple volatile aldehydes tested (hexanal, propanal, octanal, and pentanal) were maximally increased in the esophageal cancer group between 40 and 55 min after consumption of 25 g of glycerol. [Figure 31B] FIG. 1 shows that multiple volatile aldehydes tested (hexanal, propanal, octanal, and pentanal) were maximally increased in the esophageal cancer group between 40 and 55 min after consumption of 25 g of glycerol. [Figure 31C] FIG. 1 shows that multiple volatile aldehydes tested (hexanal, propanal, octanal, and pentanal) were maximally increased in the esophageal cancer group between 40 and 55 min after consumption of 25 g of glycerol. [Figure 31D] FIG. 1 shows that multiple volatile aldehydes tested (hexanal, propanal, octanal, and pentanal) were maximally increased in the esophageal cancer group between 40 and 55 min after consumption of 25 g of glycerol. [Figure 31E] FIG. 1 shows that multiple volatile aldehydes tested (hexanal, propanal, octanal, and pentanal) were maximally increased in the esophageal cancer group between 40 and 55 min after consumption of 25 g of glycerol. [Fig. 31F] FIG. 1 shows that multiple volatile aldehydes tested (hexanal, propanal, octanal, and pentanal) were maximally increased in the esophageal cancer group between 40 and 55 min after consumption of 25 g of glycerol. [Figure 31G] FIG. 1 shows that multiple volatile aldehydes tested (hexanal, propanal, octanal, and pentanal) were maximally increased in the esophageal cancer group between 40 and 55 min after consumption of 25 g of glycerol. [Fig. 31H]FIG. 1 shows that multiple volatile aldehydes tested (hexanal, propanal, octanal, and pentanal) were maximally increased in the esophageal cancer group between 40 and 55 min after consumption of 25 g of glycerol. [Fig. 31I] FIG. 1 shows that multiple volatile aldehydes tested (hexanal, propanal, octanal, and pentanal) were maximally increased in the esophageal cancer group between 40 and 55 min after consumption of 25 g of glycerol. [Figure 32A] FIG. 1 shows that the volatile phenols tested did not demonstrate changes in breath concentrations among the three patient groups following consumption of 25 g of glycerol. [Figure 32B] FIG. 1 shows that the volatile phenols tested did not demonstrate changes in breath concentrations among the three patient groups following consumption of 25 g of glycerol. [Figure 32C] FIG. 1 shows that the volatile phenols tested did not demonstrate changes in breath concentrations among the three patient groups following consumption of 25 g of glycerol. [Fig. 32D] FIG. 1 shows that the volatile phenols tested did not demonstrate changes in breath concentrations among the three patient groups following consumption of 25 g of glycerol. [Figure 32E] FIG. 1 shows that the volatile phenols tested did not demonstrate changes in breath concentrations among the three patient groups following consumption of 25 g of glycerol. [Diagram 33] 1 shows the concentration of decanal detected in breath, with optimal responses observed in the esophagogastric cancer group 30 minutes after consumption of the complex amino acid drink. [Figure 34A] Figure 1 shows that p-cresol was significantly increased in the esophageal cancer group 40 minutes after consumption of the amino acid drink. Phenol and decane showed an overall increase across both the cancer and non-cancer groups. [Figure 34B] Figure 1 shows that p-cresol was significantly increased in the esophageal cancer group 40 minutes after consumption of the amino acid drink. Phenol and decane showed an overall increase across both the cancer and non-cancer groups. [Figure 34C] Figure 1 shows that p-cresol was significantly increased in the esophageal cancer group 40 minutes after consumption of the amino acid drink. Phenol and decane showed an overall increase across both the cancer and non-cancer groups. [Fig. 34D] Figure 1 shows that p-cresol was significantly increased in the esophageal cancer group 40 minutes after consumption of the amino acid drink. Phenol and decane showed an overall increase across both the cancer and non-cancer groups. [Figure 34E] Figure 1 shows that p-cresol was significantly increased in the esophageal cancer group 40 minutes after consumption of the amino acid drink. Phenol and decane showed an overall increase across both the cancer and non-cancer groups. [Figure 35A] FIG. 1 shows that volatile short chain fatty acids (i.e., butanoic and propanoic acids) had increased concentrations in the control group following consumption of a combination of glucose and citric acid. [Figure 35B] FIG. 1 shows that volatile short chain fatty acids (i.e., butanoic and propanoic acids) had increased concentrations in the control group following consumption of a combination of glucose and citric acid. [Figure 35C] FIG. 1 shows that volatile short chain fatty acids (i.e., butanoic and propanoic acids) had increased concentrations in the control group following consumption of a combination of glucose and citric acid. [Figure 35D] FIG. 1 shows that volatile short chain fatty acids (i.e., butanoic and propanoic acids) had increased concentrations in the control group following consumption of a combination of glucose and citric acid. [Figure 35E] FIG. 1 shows that volatile short chain fatty acids (i.e., butanoic and propanoic acids) had increased concentrations in the control group following consumption of a combination of glucose and citric acid. [Diagram 36] FIG. 1 shows that propanal concentrations increased in the control group after consumption of a combination of glucose and citric acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0102] material and method [Example 1] Glucose Dosage Test subject Four healthy subjects volunteered to participate and gave written informed consent.

[0103] dose concentration The four doses of substrate were guided by (i) the recommended daily intake levels by the Food and Nutrition Board, and (ii) the already established glucose tolerance test. The glucose tolerance test used an acceptable 75g of glucose dissolved in 100ml water, which is good for patients. The maximum recommended daily dose is 130g per day for adults. [1] Based on these findings, we selected doses of 75g, 50g, 25g, and 10g to compare the dose response to glucose concentration. All findings were compared to a baseline of 0g.

[0104] Breath sampling The detection method for short-chain fatty acids was established by selected ion flow tube mass spectrometry (SIFT-MS VoiceUltra 200; Syft Technologies, Anatune, UK). All breath sampling was performed in the morning and subjects maintained a clear liquid diet for a minimum of 6 hours before breath sampling. All subjects exhaled directly into the inlet of the SIFT-MS using a disposable mouthpiece. A baseline breath test was performed for each method, followed by consumption of glucose dissolved in 100 ml of warm water, followed by rinsing the mouth three times with water to decontaminate the oral cavity. Direct sampling of three breath samples over 60 seconds was performed for all four methods consecutively at 5-minute intervals for up to 60 minutes (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes).

[0105] SIFT-MS The SIFT-MS allows for real-time quantification and identification of VOCs in breath using chemical ionization. Precursor ions (H3O+, NO+ and O2+) are emitted into a quadrupole mass filter and transported by inert helium gas along a flow tube. Breath is injected into the flow tube and reacts with the precursor ions to produce product ions, which are then separated according to their mass-to-charge ratio (m / z). The SIFT-MS was subjected to a daily automated validation cycle to operate within temperatures of 10-30 °C. Data were obtained in concentration units of parts per billion.

[0106] sugar Comparison of four different sugars at a dose of 25g each. 25g was chosen after initial glucose testing where similar VOC concentrations were observed between 25g and 75g. Glucose, lactose and mannose followed a similar pattern with the greatest increase occurring 10 minutes after sugar consumption (Figure 16). Lactose is a disaccharide composed of both glucose and galactose and is expected to follow a similar pattern as glucose, without wishing to be bound by any particular theory. Similarly, mannose is a simple sugar that is also known to be an isomer of glucose and is believed to be metabolized via the same glycolytic pathway, without wishing to be bound by any particular theory.

[0107] glucose Patient Selection All patients were recruited from St. Mary's Hospital between February 2019 and May 2019. Patients were recruited from three cohorts: esophageal cancer (n=6), gastric cancer (n=6) and age-matched healthy controls (n=6). Written informed consent was obtained from all participants. Patients diagnosed with esophagogastric adenocarcinoma ranged in the treatment pathway from early stage disease to metastatic palliative disease. Age-matched healthy controls included patients with benign upper gastrointestinal disease (reflux, dysmotility) or healthy asymptomatic controls. Demographic and clinical information was matched.

[0108] Breath sampling Detection methods for four classes of volatile compounds, namely short-chain fatty acids, alcohols, aldehydes and phenol-alkanes, were established by selected ion flow tube mass spectrometry (SIFT-MS VoiceUltra 200; Syft Technologies, Anatune, UK). All breath sampling was performed in the morning and patients maintained a clear liquid diet for a minimum of 6 h before breath sampling. All patients exhaled directly into the inlet of the SIFT-MS using a disposable mouthpiece. A baseline breath test was performed for each method, followed by consumption of 25 g glucose dissolved in 100 ml warm water, followed by rinsing the mouth three times with water to decontaminate the oral cavity. Direct sampling of three breath samples over 60 s was performed for all four methods consecutively at 5 min intervals for up to 60 min (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 min).

[0109] SIFT-MS The SIFT-MS allows for real-time quantification and identification of VOCs in breath using chemical ionization. Precursor ions (H3O+, NO+ and O2+) are emitted into a quadrupole mass filter and transported by inert helium gas along a flow tube. Breath is injected into the flow tube and reacts with the precursor ions to produce product ions, which are then separated according to their mass-to-charge ratio (m / z). The SIFT-MS was subjected to a daily automated validation cycle to operate within temperatures of 10-30 °C.

[0110] statistical analysis Data were obtained in parts per billion concentration units. Univariate analysis by Kruskal-Wallis was performed between the three groups using SPSS statistical software (v25, Armonk NY; IBM Corp). Mann-Whitney U test was performed to identify differences between esophageal and gastric cancer compared with controls. P value <0.05 was considered statistically significant.

[0111] [Example 2] Tyrosine Patient Selection All patients were recruited from St. Mary's Hospital between February 2019 and May 2019. Patients were recruited from three cohorts: esophageal cancer (n=6), gastric cancer (n=6) and age-matched healthy controls (n=6). Written informed consent was obtained from all participants. Patients diagnosed with esophagogastric adenocarcinoma ranged in the treatment pathway from early stage disease to metastatic palliative disease. Age-matched healthy controls included patients with benign upper gastrointestinal disease (reflux, dysmotility) or healthy asymptomatic controls. Demographic and clinical information was matched.

[0112] Breath sampling Detection methods for four classes of volatile compounds, namely short-chain fatty acids, alcohols, aldehydes and phenol-alkanes, were established by selected ion flow tube mass spectrometry (SIFT-MS VoiceUltra 200; Syft Technologies, Anatune, UK). All breath sampling was performed in the morning and patients maintained a clear liquid diet for a minimum of 6 h before breath sampling. All patients exhaled directly into the inlet of the SIFT-MS using a disposable mouthpiece. A baseline breath test was performed for each method, followed by the consumption of 2 g tyrosine dissolved in 100 ml warm water, followed by rinsing the mouth three times with water to decontaminate the oral cavity. Direct sampling of three breath samples over 60 s was performed for all four methods consecutively at 5 min intervals for up to 60 min (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 min).

[0113] SIFT-MS The SIFT-MS allows for real-time quantification and identification of VOCs in breath using chemical ionization. Precursor ions (H3O+, NO+ and O2+) are emitted into a quadrupole mass filter and transported by inert helium gas along a flow tube. Breath is injected into the flow tube and reacts with the precursor ions to produce product ions, which are then separated according to their mass-to-charge ratio (m / z). The SIFT-MS was subjected to a daily automated validation cycle to operate within temperatures of 10-30 °C.

[0114] statistical analysis Data were obtained in parts per billion concentration units. Univariate analysis by Kruskal-Wallis was performed between the three groups using SPSS statistical software (v25, Armonk NY; IBM Corp). Mann-Whitney U test was performed to identify differences between esophageal and gastric cancer compared with controls. P value <0.05 was considered statistically significant.

[0115] [Example 3] Phenylalanine subject One healthy subject.

[0116] Dose level: The recommended daily intake level recommended by the Food and Nutrition Board is 100 mg / kg per day for adults, with a maximum dose of 3 g.[1] A single dose of 3 g was chosen for this study.

[0117] Breath sampling Detection methods for short-chain fatty acids, aldehydes and phenol-alkanes were established by selected ion flow tube mass spectrometry (SIFT-MS VoiceUltra 200; Syft Technologies, Anatune, UK). All breath sampling was performed in the morning after a clear liquid meal for a minimum of 6 h. Breath was taken directly into the inlet of the SIFT-MS using a disposable mouthpiece. A baseline breath test was performed for each method, followed by consumption of phenylalanine dissolved in 100 ml of warm water, followed by rinsing the mouth three times with water to decontaminate the oral cavity. Direct sampling of three breath samples over 60 s was performed consecutively with all four methods at 5 min intervals for up to 60 min (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 min).

[0118] SIFT-MS The SIFT-MS allows for real-time quantification and identification of VOCs in breath using chemical ionization. Precursor ions (H3O+, NO+ and O2+) are emitted into a quadrupole mass filter and transported by inert helium gas along a flow tube. Breath is injected into the flow tube and reacts with the precursor ions to produce product ions, which are then separated according to their mass-to-charge ratio (m / z). The SIFT-MS was subjected to a daily automated validation cycle to operate within temperatures of 10-30 °C. Data were obtained in concentration units of parts per billion.

[0119] [Example 4] Glutamic acid subject One healthy subject.

[0120] dose concentration The recommended daily intake level recommended by the Food and Nutrition Board is 30 mg / kg per day for adults.[1] A maximum single dose of 2.1 g for an average 70 kg adult was chosen.

[0121] Breath sampling Detection methods for short-chain fatty acids, aldehydes and phenol-alkanes were established by selected ion flow tube mass spectrometry (SIFT-MS VoiceUltra 200; Syft Technologies, Anatune, UK). All breath sampling was performed in the morning after a clear liquid meal for a minimum of 6 h. Breath was taken directly into the inlet of the SIFT-MS using a disposable mouthpiece. A baseline breath test was performed for each method, followed by consumption of glutamic acid dissolved in 100 ml of warm water, followed by rinsing the mouth three times with water to decontaminate the oral cavity. Direct sampling of three breath samples over 60 s was performed consecutively with all four methods at 5 min intervals for up to 60 min (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 min).

[0122] SIFT-MS The SIFT-MS allows for real-time quantification and identification of VOCs in breath using chemical ionization. Precursor ions (H3O+, NO+ and O2+) are emitted into a quadrupole mass filter and transported by inert helium gas along a flow tube. Breath is injected into the flow tube and reacts with the precursor ions to produce product ions, which are then separated according to their mass-to-charge ratio (m / z). The SIFT-MS was subjected to a daily automated validation cycle to operate within temperatures of 10-30 °C. Data were obtained in concentration units of parts per billion.

[0123] [Example 5] Glycerol Dosage subject Two healthy subjects volunteered to participate and gave written informed consent.

[0124] dose concentration The two doses of substrate were derived from (i) the recommended daily intake levels by the Food and Nutrition Board and (ii) early glucose method development studies. The maximum recommended daily dose is 276 mg / kg per day for adults, but there are no reports of harm with higher doses.[1] For an average 70 kg individual, a maximum of 19 g of glycerol is recommended. Based on these findings, we selected doses of 50 g, 25 g, and 10 g to compare the dose response on glucose concentrations. All findings were compared to a baseline of 0 g.

[0125] Breath sampling Detection methods for short-chain fatty acids and aldehydes were established by selected ion flow tube mass spectrometry (SIFT-MS VoiceUltra 200; Syft Technologies, Anatune, UK). All breath sampling was performed in the morning and subjects maintained a clear liquid diet for a minimum of 6 hours before breath sampling. All subjects exhaled directly into the inlet of the SIFT-MS using a disposable mouthpiece. A baseline breath test was performed for each method, followed by consumption of glycerol dissolved in 100 ml of warm water, followed by rinsing the mouth three times with water to decontaminate the oral cavity. Direct sampling of three breath samples over 60 seconds was performed for all four methods consecutively at 5-minute intervals for up to 60 minutes (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes).

[0126] SIFT-MS The SIFT-MS allows for real-time quantification and identification of VOCs in breath using chemical ionization. Precursor ions (H3O+, NO+ and O2+) are emitted into a quadrupole mass filter and transported by inert helium gas along a flow tube. Breath is injected into the flow tube and reacts with the precursor ions to produce product ions, which are then separated according to their mass-to-charge ratio (m / z). The SIFT-MS was subjected to a daily automated validation cycle to operate within temperatures of 10-30 °C. Data were obtained in concentration units of parts per billion.

[0127] [Example 6] Glycerol Patient Selection All patients were recruited from St. Mary's Hospital between February 2019 and December 2019. Patients were recruited from three cohorts: esophageal cancer (n=6), gastric cancer (n=6) and age-matched healthy controls (n=6). Written informed consent was obtained from all participants. Patients diagnosed with esophagogastric adenocarcinoma ranged in the treatment pathway from early stage disease to metastatic palliative disease. Age-matched healthy controls included patients with benign upper gastrointestinal disease (reflux, dysmotility) or healthy asymptomatic controls. Demographic and clinical information was matched.

[0128] Breath sampling Detection methods for four classes of volatile compounds, namely short-chain fatty acids, alcohols, aldehydes and phenol-alkanes, were established by selected ion flow tube mass spectrometry (SIFT-MS VoiceUltra 200; Syft Technologies, Anatune, UK). All breath sampling was performed in the morning and patients maintained a clear liquid diet for a minimum of 6 hours before breath sampling. All patients exhaled directly into the inlet of the SIFT-MS using a disposable mouthpiece. A baseline breath test was performed for each method, followed by the consumption of 25 g of glycerol dissolved in 100 ml of warm water, followed by rinsing the mouth three times with water to decontaminate the oral cavity. Direct sampling of three breath samples over 60 seconds was performed for all four methods consecutively at 5-minute intervals for up to 60 minutes (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes).

[0129] SIFT-MS SIFT-MS allows for real-time quantification and identification of VOCs in breath using chemical ionization. Precursor ions (H 3 O + , NO + and O 2 + ) into a quadrupole mass filter and carried along the flow tube by inert helium gas. Breath is injected into the flow tube and reacts with precursor ions to produce product ions, which are then separated according to their mass-to-charge ratio (m / z). The SIFT-MS was subjected to a daily automated validation cycle to operate within temperatures of 10-30 °C.

[0130] statistical analysis Data were obtained in parts per billion concentration units. Univariate analysis by Kruskal-Wallis was performed between the three groups using SPSS statistical software (v25, Armonk NY; IBM Corp). Mann-Whitney U test was performed to identify differences between esophageal and gastric cancer compared with controls. P value <0.05 was considered statistically significant.

[0131] [Example 7] Complex amino acids (tyrosine, phenylalanine, glutamic acid) Patient Selection All patients were recruited from St. Mary's Hospital between February 2019 and December 2019. Patients were recruited from three cohorts: esophageal cancer (n=6), gastric cancer (n=1) and age-matched healthy controls (n=6). Written informed consent was obtained from all participants. Patients diagnosed with esophagogastric adenocarcinoma ranged in the treatment pathway from early stage disease to metastatic palliative disease. Age-matched healthy controls included patients with benign upper gastrointestinal disease (reflux, dysmotility) or healthy asymptomatic controls. Demographic and clinical information was matched.

[0132] Breath sampling Detection methods for four classes of volatile compounds, namely short-chain fatty acids, alcohols, aldehydes and phenol-alkanes, were established by selected ion flow tube mass spectrometry (SIFT-MS VoiceUltra 200; Syft Technologies, Anatune, UK). All breath sampling was performed in the morning and patients maintained a clear liquid diet for a minimum of 6 h before breath sampling. All patients exhaled directly into the inlet of the SIFT-MS using a disposable mouthpiece. A baseline breath test was performed for each method, followed by consumption of 2 g tyrosine, 3 g phenylalanine and 2.1 g glutamic acid dissolved in 100 ml warm water, followed by rinsing the mouth three times with water to decontaminate the oral cavity. Direct sampling of three breath samples over 60 s was performed for all four methods consecutively at 5 min intervals for up to 60 min (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 min).

[0133] SIFT-MS SIFT-MS allows for real-time quantification and identification of VOCs in breath using chemical ionization. Precursor ions (H 3 O + , NO + and O 2 +) is released into a quadrupole mass filter and transported along the flow tube by an inert helium gas. Exhaled breath is injected into the flow tube, reacted with precursor ions to generate product ions, and then separated according to the mass-to-charge ratio (m / z). SIFT-MS was subjected to a daily automated verification cycle to operate within a temperature range of 10 - 30 °C.

[0134] Statistical analysis Data were obtained in parts per billion concentration units. Mann-Whitney U analysis was performed between cancer and non-cancer using SPSS statistical software (v25, Armonk NY; IBM Corp). A P-value < 0.05 was considered statistically significant.

[0135] [Example 8] Combination of glucose and citric acid Patient selection All patients were recruited from St. Mary's Hospital from February 2019 to December 2019. Twelve healthy controls were recruited to form two cohorts that consumed glucose (n = 6) and a combination of glucose and citric acid (n = 6). Written informed consent was obtained from all participants. Healthy controls of the same age included patients with benign upper gastrointestinal diseases (reflux, dysmotility) or healthy asymptomatic controls.

[0136] Exhaled breath sampling Detection methods for four classes of volatile compounds, namely short-chain fatty acids, alcohols, aldehydes and phenol-alkanes, were established by selected ion flow tube mass spectrometry (SIFT-MS VoiceUltra 200; Syft Technologies, Anatune, UK). All breath sampling was performed in the morning and patients maintained a clear liquid diet for a minimum of 6 h before breath sampling. All patients exhaled directly into the inlet of the SIFT-MS using a disposable mouthpiece. A baseline breath test was performed for each method, followed by consumption of 25 g glucose and 1.4 g citric acid dissolved in 100 ml warm water, followed by rinsing the mouth three times with water to decontaminate the oral cavity. Direct sampling of three breath samples over 60 s was performed for all four methods consecutively at 5 min intervals for up to 30 min (0, 5, 10, 15, 20, 25, 30 min). SIFT-MS SIFT-MS allows for real-time quantification and identification of VOCs in breath using chemical ionization. Precursor ions (H 3 O + , NO + and O 2 + ) into a quadrupole mass filter and carried along the flow tube by inert helium gas. Breath is injected into the flow tube and reacts with precursor ions to produce product ions, which are then separated according to their mass-to-charge ratio (m / z). The SIFT-MS was subjected to a daily automated validation cycle to operate within temperatures of 10-30 °C.

[0137] statistical analysis Data were obtained in parts per billion concentration units. Mann-Whitney U analysis was performed between cancer and non-cancer using SPSS statistical software (v25, Armonk NY; IBM Corp). A P value of <0.05 was considered statistically significant.

[0138] result [Example 1] Glucose Medication Test Volatile Organic Compounds Analysis

[0139] [Table 1]

[0140] Increasing glucose concentrations positively correlate with increasing concentrations of volatile fatty acids detected in breath. Butanoic and propanoic acids demonstrated a maximal response within 5-15 minutes of glucose consumption, with values ​​declining thereafter. Rapid glucose breakdown via the glycolytic pathway produces volatile end products that are detected in breath. Previous work by the inventors has demonstrated that rinsing the mouth with water after glucose consumption eliminates potential VOC responses originating from the oral cavity. Butanoic and pentanoic acids demonstrated a 1-fold increase difference between 10g and 50g glucose. These compounds were used to derive a recommended glucose dose for a preliminary clinical trial involving patients with esophagogastric cancer. To obtain a balance between an appropriate dose response and a beverage that is tolerable to patients, the inventors selected a dose of 25g dissolved in 100ml of warm water. The next step in this study is to evaluate the VOC response in patients diagnosed with OG cancer compared to healthy age-matched controls to observe differences in cellular metabolic activity and VOC response.

[0141] [Table 2] JPEG0007682107000002.jpg126160

[0142] [Table 3]

[0143] glucose Volatile Organic Compounds Analysis Short-chain fatty acids

[0144] [Table 4]

[0145] [Table 5]

[0146] Consideration Three chemical classes of VOCs in exhaled breath demonstrated significant differences in patients diagnosed with esophagogastric (OG) cancer: a total of 13 compounds from the group demonstrated increased concentrations after glucose consumption: short chain fatty acids (SCFAs) (n=4), aldehydes (n=6), and phenols (n=3).

[0147] Volatile SCFAs, namely butanoic and propanoic acids, demonstrated the greatest changes in breath concentrations. Optimal concentrations were reached within 10 min of consumption, suggesting rapid glucose breakdown. Glucose, a monosaccharide, enters the glycolytic pathway generating metabolic end products detected in breath. Pentanoic acid was detected at higher concentrations compared to baseline values ​​at 30 min. These results suggest that breath VOCs may be increased by oral substrates by manipulating the intrinsic metabolic pathways of known VOCs associated with OG cancer. Gastric cancer demonstrated a stronger response than esophageal cancer for all significant VOCs detected. The gastric cancer group showed significant fold changes in SCFAs (acetic acid, butanoic acid, pentanoic acid, and propanoic acid), two of which overlapped in significance with the esophageal cancer group (acetic acid and pentanoic acid).

[0148] Similarly, aldehydes such as pentanal and propanal followed a similar pattern of response to SCFAs with an optimal concentration increase at 5-10 minutes. The remaining aldehydes consistently showed increased levels in the cancer group, with four out of nine having higher baseline values. Acetaldehyde, butanal, hexanal and pentanal demonstrate a significant increase in fold change from baseline in gastric cancer patients. Esophageal cancer patients show this effect only with both butanal and propanal. Meanwhile, for nonanal and octanal, both groups demonstrated a significant fold increase over the control group, which needs to be further explored. These results are consistent with previous studies published by the present inventors linking the volatile butanoic acid, butanal and decanal to OG cancer. [1] The remaining aldehydes and phenol-alkanes (except dodecane) consistently demonstrated increased concentrations in the cancer group over the duration of the study. Previous studies by our group have implicated phenols as potential breath biomarkers in OG cancer.[2] Decane, a member of the phenol family, shows higher baseline concentrations in both cancer groups. The fold increase in the control group after glucose consumption needs to be further explored. As a novel finding, a similar response pattern was observed for P-cresol, although the fold increase was only seen in the gastric cancer group. This may reflect a transient passage of glucose by the esophageal tumor compared to its retention in the stomach.

[0149] Currently, NICE guidelines recommend upper gastrointestinal endoscopy within two weeks for patients who present with "red flag" symptoms suggestive of OG cancer. [3] However, the insidious nature of the disease means that the majority present with non-specific symptoms, leading to delayed diagnosis and poor overall survival outcomes. Non-invasive breath testing acts as a triage tool to stratify patients with non-specific upper gastrointestinal symptoms. Identifying breath biomarkers for early detection of OG cancer has the potential to offer patients therapeutic treatment and impact overall survival outcomes. The study evaluated patients at early and advanced stages of the disease.

[0150] In clinical practice, exhaled breath may be collected using: - A breath sampling device coupled with a thermal desorption tube to facilitate sample storage and transport.

[0151] - Direct sampling using mass spectrometry such as SIFT as demonstrated in this study.

[0152] - Dedicated sensors for VOCs with large response such as acetic acid, butanoic acid, pentanoic acid and propanoic acid.

[0153] Main points Glucose consumption activates metabolic pathways associated with increased tumor microbiome or tumor cell activity, as detected by:

[0154] A significant fold increase in SCFAs (acetic acid, butanoic acid, pentanoic acid, and propanoic acid), more observed in the gastric cancer group than in the esophageal cancer group.

[0155] · Significant increase in aldehydes, namely acetaldehyde, butanal, hexanal and pentanal in gastric cancer group. Increase in butanal and propanal is observed in esophageal cancer.

[0156] Novel findings of increased baseline concentrations of decane and p-cresol were observed in both cancer groups. A fold increase in p-cresol was only demonstrated in gastric cancer.

[0157] Breath samples are collected at two intervals after glucose ingestion, first 5-10 min and then 30 min, to identify optimal concentrations of VOCs.

[0158] [Example 2] Tyrosine

[0159] [Table 6]

[0160] [Table 7]

[0161] Volatile Organic Compounds Analysis Short-chain fatty acids

[0162] [Table 8]

[0163] aldehyde

[0164] [Table 9]

[0165] Phenols

[0166] [Table 10]

[0167] [Table 11]

[0168] Consideration Two chemical classes of volatile compounds (phenols and aldehydes) were detected at slightly increased concentrations 30 min after tyrosine consumption. A total of eight compounds demonstrated slightly increased concentrations in the esophageal cancer group. The underlying biological and mechanistic pathways suggest that tyrosine, an aromatic amino acid, is metabolized to phenolic compounds by enzymatic reactions initiated by gastrointestinal bacteria.

[0169] Volatile phenolic compounds were detected at optimal concentrations 35-45 min after tyrosine consumption, although slight increases from baseline values ​​were reported. Phenol and decane showed a similar pattern of increase between groups, while p-cresol and dodecane concentrations were detected at slightly increased concentrations in the esophageal cancer group. Volatile aldehydes, namely butanal, decanal, heptanal and hexanal, demonstrated higher concentrations in the esophageal cancer group compared to controls (fold change 1.46 vs. 1.32). Overall baseline concentrations of all compounds were significantly higher in the control group.

[0170] Decanal demonstrated the only significant fold increase in the esophageal cancer group, which is supported by our previous studies showing significantly higher baseline values ​​of aldehydes (butanal, decanal) and phenols in OG cancer patients.[1,2] The lack of support from our previous findings regarding baseline concentrations may be due to the results being obtained from separate and small numbers of patients from the esophageal and gastric cancer groups, and further exploration is required. However, selected volatile compounds show a response to tyrosine that was overall increased in fold change in cancer, although not significant.

[0171] Short-chain fatty acid concentrations from the cancer cohort were not affected by tyrosine.

[0172] These results suggest the potential for breath VOCs to be increased by oral metabolic substrates acting via the shikimic acid pathway. In the next phase of research, the inventors intend to use tyrosine, as well as phenylalanine, a precursor of tyrosine, as a combination drink. Without wishing to be bound by any particular theory, the inventors aim to measure breath VOC concentrations between 30 and 45 minutes after ingestion to detect potential changes due to the addition of amino acids.

[0173] Main points Decanal, a member of the aldehyde family, demonstrates a significant fold increase following tyrosine consumption in esophageal cancer patients.

[0174] Aldehydes and phenolic compounds show a small, but not significant, increase in fold change from baseline values.

[0175] The significantly higher baseline levels of aldehydes and phenols in the control group require further exploration.

[0176] Volatile phenolic compounds were detected at optimal concentrations 35–45 min after tyrosine consumption.

[0177] [Example 3] Phenylalanine Results and Discussion Phenylalanine is an essential amino acid that is a known precursor of other amino acids such as tyrosine. Metabolism via the shikimate pathway would be expected to produce volatile phenolic compounds. Three compounds in the phenol family (dodecane, decane, and phenol) demonstrated increased concentrations following phenylalanine consumption (Figures 18-20). Dodecane and decane show the greatest increase 10-15 minutes after consumption (3.2- and 1.8-fold increase, respectively). Phenol showed a 2.7-fold increase at 60 minutes. Decane and dodecane show an elevated response to phenylalanine compared to tyrosine, which exerts no significant effect (Figure 21). Phenol produced similar end results, while decane shows slightly increased values ​​following phenylalanine ingestion.

[0178] [Example 4] Glutamic acid Results and Discussion Three compounds from the aldehyde and phenol families demonstrated increased VOC concentrations after glutamic acid consumption (Figures 22-25). Propanal showed maximally elevated concentrations at 5 min, with a fold change of 3.5. Both dodecane and phenol showed a maximum 2-fold increase at 20 and 45 min, respectively. Glutamic acid is a non-essential amino acid that is metabolized via the shikimate pathway to generate volatile compounds of the phenolic family. Glutamic acid participates in the transamination process during its breakdown. The resulting ketoacids are used as key intermediates in the citric acid cycle for further cellular metabolism. This may explain the slight increase observed in butanoic acid within 5 min of glutamic acid consumption.

[0179] Without wishing to be bound by any particular theory, the inventors believe that an increased VOC response may occur with certain compounds already identified across groups, particularly dodecane, phenol, in combination with the other amino acids tested, phenylalanine and tyrosine.

[0180] [Example 5] Glycerol Dosage result subject Two subjects were recruited, one female and one male, with a mean age of 32 years. No significant comorbidities were observed.

[0181] Volatile Organic Compounds Analysis

[0182] [Table 12]

[0183] Consideration Increasing concentrations of glycerol lead to increased production of volatile fatty acids detected in the breath. Concentrations of volatile fatty acids from 25 g of glycerol are comparable to baseline values. Concentrations of butanoic acid increased 30 minutes after glycerol ingestion, with maximum concentrations detected at 45-55 minutes. Glycerol is a polyol compound found in lipids and can be either (i) metabolized via the glycolytic pathway by directly entering the glycolytic pathway or (ii) converted to glucose by gluconeogenesis. Glucose tests demonstrated maximum detection of fatty acids 5-10 minutes after glucose consumption, and thus responses following glycerol ingestion are expected to be delayed as additional enzymatic reactions may be required before entering the circuit. We intend to use a dose of 50 g to elicit fatty acid VOC responses in the breath of patients diagnosed with OG cancer compared to healthy age-matched controls.

[0184] [Example 6] Glycerol result patient Eighteen patients were recruited (n=6 in each group; esophageal cancer, gastric cancer, and healthy controls). All included cancers were histologically confirmed as adenocarcinoma.

[0185] [Table 13]

[0186] Volatile Organic Compounds Analysis Short-chain fatty acids

[0187] [Table 14]

[0188] aldehyde

[0189] [Table 15]

[0190] Phenols

[0191] [Table 16]

[0192] [Table 17]

[0193] Consideration Three chemical classes of VOCs in exhaled breath demonstrated significant increases in patients diagnosed with esophagogastric (OG) cancer. Glycerol is a polyol compound found in lipids and can be either (i) metabolized via the glycolytic pathway by directly entering the pathway or (ii) converted to glucose by gluconeogenesis. Glucose testing demonstrated maximum detection of fatty acids 5-10 min after glucose consumption, and thus it is expected that the elevated response following glycerol ingestion may be delayed further as an enzymatic reaction may be required prior to entry into the circuit.

[0194] Consistent with the hypothesis, we observed an increase in the levels of short chain fatty acids (SCFAs) and aldehydes between 45 and 60 min after glycerol consumption, as shown in Figures 30A-30E. The SCFAs, namely acetic acid, butanoic acid, and propanoic acid, showed a greater increase in the esophageal cancer group (1.5, 2.02, and 1.75-fold increases, respectively) compared to the gastric cancer group (1.09, 1.43, and 1.46-fold increases, respectively). A gradual increase was observed after 45 min, reaching optimal concentrations at 60 min.

[0195] Similarly, selected aldehydes were found to be significantly increased in the esophageal cancer group (Figures 31A-31I). Hexanal and propanal showed the largest increase with a 1.7-fold increase between 40 and 55 min. Octanal increased with a 1.58-fold change, and pentanal increased with a 1.27-fold change. The gastric cancer group showed only a change with pentanal increasing by 1.46-fold at 55 min. The remaining aldehydes were unaffected.

[0196] The multiple volatile phenols tested did not demonstrate significant changes in breath concentrations among the three patient groups following glycerol consumption (Figures 32A-32E).

[0197] Glycerol consumption uniquely increased target VOCs in the esophageal cancer group, which may be due to an increase in the viscosity of the fluid lining the esophagus allowing more than transient passage. Increased contact time between the stroma and tumor may explain the occurrence of increased VOC levels.

[0198] Main points Glycerol consumption activates glycolytic metabolic pathways that are associated with increased activity of the tumor microbiome or tumor cells, as detected by:

[0199] A significant fold increase in SCFAs (acetic acid, butanoic acid, and propanoic acid), more observed in the esophageal cancer group than in the gastric cancer group.

[0200] Significant increase in aldehydes, namely hexanal, octanal, pentanal and propanal in the esophageal cancer group. Increase in pentanal was observed in gastric cancer.

[0201] [Example 7] Complex amino acids (tyrosine, phenylalanine, glutamic acid) result patient Thirteen patients were recruited (esophageal cancer n = 6, gastric cancer n = 1, healthy controls n = 6). All included cancers were histologically confirmed as adenocarcinoma.

[0202] [Table 18]

[0203] Volatile Organic Compounds Analysis Short-chain fatty acids

[0204] [Table 19]

[0205] aldehyde

[0206] [Table 20]

[0207] Phenol-Alkane

[0208] [Table 21]

[0209] Consideration Two chemical classes, aldehydes and phenol-alkanes, demonstrated increased volatile organic compound levels following consumption of the three complex amino acids, as shown in Figures 33 and 34A-34E. In contrast, when tyrosine alone was administered, only decanal was slightly elevated in the esophageal cancer group.

[0210] The aldehyde, decanal, demonstrated a more significant increase in detection levels with this amino acid combination drink (Figure 33). A fold increase of 1.41 was observed in the cancer group compared to a fold increase of 1.05 in the control group (baseline = 0.69 ppbv, 30 min = 0.83 ppbv). Maximum concentrations occurred 30 min after consumption of the nutrient drink.

[0211] Phenol-alkanes are the primary target of this nutrient group. A pathway describing the metabolism of tyrosine by tyrosine phenol lyase to produce phenol has been detailed. More recently, Saito et al. described a pathway involving metabolism by the enzyme tyrosine lyase to produce p-cresol. This metabolic pathway has been demonstrated in bacteria but not in human cells [4]. p-cresol significantly increased from a baseline level of 0.93 ppbv to 1.25 ppbv at 40 min after consumption of the amino acid drink, leading to a 1.37-fold increase. No changes were observed in the control group. Phenol showed an overall increase across both the cancer group (1.79-fold increase) and the non-cancer group (1.83-fold increase), with no significant difference between the two. Decane also increased at 30 min, resulting in a 1.44-fold increase in the cancer group.

[0212] No significant changes occurred in the remaining aldehydes and short-chain fatty acids. Further investigation is needed to explain the increase in decanals.

[0213] Key points: Consumption of complex amino acids potentially activates metabolic pathways associated with bacteria, as detected by:

[0214] · Significant fold increase in decanal (aldehyde).

[0215] · An overall increase in phenol (the enzyme tyrosine phenol lyase) with no difference between the cancer and non-cancer groups.

[0216] A new finding of a significant increase in P-cresol, potentially resulting from enzymatic metabolism using tyrosine lyase.

[0217] [Example 8] Glucose and citric acid combination

[0218] [Table 22]

[0219] Volatile Organic Compounds Analysis Short-chain fatty acids

[0220] [Table 23]

[0221] aldehyde

[0222] [Table 24]

[0223] Consideration Two chemical classes, short chain fatty acids (SCFAs) and aldehydes, demonstrated increased volatile organic compound levels following consumption of a combination of glucose and citric acid, as can be seen in Figures 35A-35E and 36. The results of Example 1 demonstrate a significant increase in these groups within 5-10 minutes of glucose consumption alone. The hypothesis is that glucose is metabolized via the glycolytic pathway, which occurs in human and bacterial cells. Since the glycolytic pathway feeds into the citric acid cycle, the objective was to assess further increases in VOCs with the addition of citric acid.

[0224] Butanoic acid demonstrated the greatest increase, from 2.72-fold with glucose alone to 5.36-fold with the addition of citric acid (Figure 35B). Maximal concentrations were achieved within 5-10 days of consumption of the beverage. Propanoic acid also demonstrated a 1.96-fold increase with the addition of citric acid (Figure 35E). No significant changes were observed in the remaining SCFA groups.

[0225] Propanal appeared to be the only aldehyde showing a slight change, a 1.29-fold increase in the citric acid group within 10-15 minutes (Figure 36). No significant changes were observed in the remaining aldehyde groups.

[0226] The two control groups showed distinct changes in VOCs, with citrate as the differentiating factor. We hypothesize that these nutrients may feed into the intrinsic glycolysis and citrate metabolic pathways.

[0227] Main points The combined consumption of glucose and citrate activates known metabolic pathways related to cellular metabolism, as detected by: · Significant fold increase in volatile short chain fatty acids (butanoic and propanoic acids) within 5-10 minutes of consuming the nutrient drink. · Significant fold increase in propanal within 10-15 minutes. Next steps include recruiting patients with esophagogastric cancer and assessing changes in exhaled breath VOCs in response to additional nutritional substrates.

[0228] References 1. Markar, SR, et al., Assessment of a Noninvasive Exhaled Breath Test for the Diagnosis of Oesophagogastric Cancer. JAMA Oncol, 2018. 4(7): p. 970-976. 2. Kumar K, HJ, Abbassi-Ghadi N, Mackenzie HA, Veselkov KA, Hoare JM, Lovat LB, Spanel P, Smith D and Hanna GB, Mass Spectrometric Analysis of Exhaled Breath for the Identification of Volatile Organic Compound Biomarkers in Esophageal and Gastric Adenocarcinoma. Annals of Surgery, 2015. 262(6): p. 981-990. 3. Excellence, N.I.o.C., Gastrointestinal tract (upper) cancers - recognition and referral. 2016. 4. Saito Y, Sato T, Nomoto K, Tsuji H. Identification of phenol- and p-cresol- producing intestinal bacteria by using media supplemented with tyrosine and its metabolites. FEMS Microbiol Ecol. 2018. 94(9)

Claims

1. 1. A method for diagnosing a subject having or predisposition to cancer, or for providing a prognosis of said subject's condition, comprising: (i) detecting in a breath sample from a test subject the concentration of trace compounds resulting from the metabolism of at least one sugar and / or at least one amino acid or precursor thereof and / or at least one polyol present in a composition previously administered to said subject, wherein said sugar is present in said composition at a concentration of greater than 20,000 mg / 100 ml, said amino acid or precursor thereof is present in said composition at a concentration of at least 500 mg / ml, and said polyol is present in said composition at a concentration of greater than 25,000 mg / 100 ml; (a) the sugar is glucose, sorbitol, mannose, or lactose; (b) the amino acids are selected from the group consisting of tyrosine, glutamic acid, glutamate, phenylalanine, tryptophan, proline, and histidine; and / or (c) the polyol is glycerol; Steps, and (ii) comparing this concentration with a standard of the concentration of said trace compound in individuals not suffering from cancer; Including, an increase or decrease in the concentration of the trace compound compared to the reference indicates that the subject suffers from or has a predisposition to cancer or provides a negative prognosis of the subject's condition; the trace compounds are selected from the group consisting of acetic acid, butanoic acid, hexanoic acid, pentanoic acid, propanoic acid, acetaldehyde, decanal, heptanal, hexanal, nonanal, octanal, pentanal, butanal, propanal, 1-hydroxy-4-ethylbenzene, decane, dodecane, p-cresol, and phenol, or combinations thereof; The method (excluding medical procedures on humans), wherein the cancer is esophagogastric junction cancer, gastric cancer, esophageal cancer, esophageal squamous cell carcinoma (ESCC), or esophageal adenocarcinoma (EAC).

2. the detection step (i) comprises detecting the trace compound up to 30 minutes after administration of said composition comprising at least one sugar and / or amino acid or precursor thereof and / or at least one polyol; and / or 2. The method of claim 1, wherein the detection step (i) comprises detecting trace compounds between 30 and 60 minutes after administration of the composition comprising at least one sugar and / or amino acid or precursor thereof and / or at least one polyol.

3. The method of claim 1 or 2, wherein an increase in the concentration of the trace compound compared to the reference indicates that the subject suffers from or has a predisposition to cancer or provides a negative prognosis of the subject's condition.

4. 4. The method of claim 3, wherein the increase in the concentration of the trace compound is at least a 10% increase in the concentration of the trace compound compared to the reference.

5. (i) the sugar is present in the composition previously administered to the subject at a concentration of at least 20,500 mg / 100 ml; (ii) the sugar is glucose and is present in the composition previously administered to the subject at a concentration of at least 25,000 mg / 100 ml, and the trace compound is detected up to 10 minutes after administration of the composition containing glucose; (iii) the composition administered to the subject comprises citric acid in combination with the sugar, and the citric acid is present in the composition at a concentration of at least 1,000 mg / 100 ml; (iv) the amino acid is tyrosine and is present in the composition previously administered to the subject at a concentration of at least 2,000 mg / 100 ml; (v) the amino acid precursor is phenylalanine; and / or (vi) The method of any one of claims 1 to 4, wherein the polyol is present in the composition at a concentration greater than 30,000 mg / 100 ml.

6. 6. The method of claim 5, wherein in (iii) of claim 5, the sugar is glucose.

7. 6. The method of claim 5, wherein the trace compounds are detected between 35 and 45 minutes after administration of the composition comprising tyrosine.

8. 6. The method of claim 5, wherein in (v) of claim 5, the amino acid precursor is present in the composition at a concentration of at least 3000 mg / 100 ml.

9. (i) the composition previously administered to the subject comprises sugar and the trace compound is acetic acid, butanoic acid, pentanoic acid, propanoic acid, hexanoic acid, acetaldehyde, propanal, butanal, hexanal, pentanal, decanal, 1-hydroxyethylbenzene, and / or p-cresol; (ii) the composition previously administered to the subject comprises an amino acid or a precursor thereof, and the trace compound is butanal, decanal, heptanal, hexanal, phenol, decane, p-cresol, 1-hydroxyethylbenzene, and / or dodecane; and / or 3. The method of claim 1, wherein the composition previously administered to the subject comprises a polyol and the trace compound is butanoic acid, acetic acid, hexanoic acid, pentanoic acid, propanoic acid, butanal, hexanal, pentanal and / or propanal.

10. 10. The method of claim 9, wherein in (i) of claim 9, the sugar is glucose.

11. 10. The method according to claim 9, wherein in (ii) of claim 9, the amino acid or precursor thereof is tyrosine and the trace compound is decanal and / or dodecane.

12. 10. The method of claim 9, wherein in (iii) of claim 9, the polyol is glycerol.

13. 1. A method for detecting a trace compound in a test subject, comprising: (i) providing to said subject a composition comprising at least one substrate according to any one of claims 1 to 12 which results in a trace compound; and (ii) detecting the concentration of said trace compound in a breath sample from said subject; wherein the at least one substrate comprises (a) Sugar; (b) an amino acid, and / or (c) a polyol, and wherein the trace compound is selected from the group consisting of acetic acid, butanoic acid, hexanoic acid, pentanoic acid, propanoic acid, acetaldehyde, decanal, heptanal, hexanal, nonanal, octanal, pentanal, butanal, propanal, 1-hydroxy-4-ethylbenzene, decane, dodecane, p-cresol, and phenol, or combinations thereof, excluding medical procedures on humans.

14. 14. The method of claim 13, wherein the sugar is glucose, sorbitol, mannose, or lactose.

15. 14. The method of claim 13, wherein the amino acid is selected from the group consisting of tyrosine, glutamic acid, glutamate, phenylalanine, tryptophan, proline, and histidine.

16. The method of claim 13 wherein the polyol is glycerol.

17. The method according to any one of claims 13 to 16, wherein said trace compounds are as defined in any one of claims 10 to 12.

18. 1. A composition comprising at least one sugar and / or at least one amino acid or precursor thereof and / or at least one polyol present suitable for metabolism to trace compounds for use in a method for diagnosing or prognosing cancer, wherein said sugar is present in said composition at a concentration of more than 20,000 mg / 100 ml, said amino acid is present in said composition at a concentration of at least 500 mg / ml and said polyol is present in said composition at a concentration of more than 25,000 mg / 100 ml, (a) the sugar is glucose, sorbitol, mannose, or lactose; (b) the amino acids are selected from the group consisting of tyrosine, glutamic acid, glutamate, phenylalanine, tryptophan, proline, and histidine; and / or (c) the polyol is glycerol; the trace compounds are selected from the group consisting of acetic acid, butanoic acid, hexanoic acid, pentanoic acid, propanoic acid, acetaldehyde, decanal, heptanal, hexanal, nonanal, octanal, pentanal, butanal, propanal, 1-hydroxy-4-ethylbenzene, decane, dodecane, p-cresol, and phenol, or combinations thereof; the cancer is esophagogastric junction cancer, gastric cancer, esophageal cancer, esophageal squamous cell carcinoma (ESCC), or esophageal adenocarcinoma (EAC); composition.

19. A composition comprising at least one sugar, and / or at least one amino acid or precursor thereof, and / or at least one polyol according to any one of claims 1 to 8 for use in the method according to any one of claims 1 to 12.

20. 1. A kit for diagnosing a subject having or predisposition to cancer, or for providing a prognosis of said subject's condition, comprising: (a) a composition comprising at least one sugar as defined in any one of claims 1 to 8, and / or at least one amino acid or a precursor thereof, and / or at least one polyol, (b) an apparatus for determining the concentration of a trace compound in a breath sample from a test subject; and (c) a standard of concentrations of said trace compounds in breath samples from individuals not suffering from cancer; Including, identifying an increase or decrease in the concentration of said trace compounds in breath samples from said test subject compared to said standard, thereby being used to indicate that said subject suffers from or has a predisposition to cancer or to provide a negative prognosis of said subject's condition; the trace compounds are selected from the group consisting of acetic acid, butanoic acid, hexanoic acid, pentanoic acid, propanoic acid, acetaldehyde, decanal, heptanal, hexanal, nonanal, octanal, pentanal, butanal, propanal, 1-hydroxy-4-ethylbenzene, decane, dodecane, p-cresol, and phenol, or combinations thereof; The kit, wherein the cancer is esophagogastric junction cancer, gastric cancer, esophageal cancer, esophageal squamous cell carcinoma (ESCC), or esophageal adenocarcinoma (EAC).

21. The kit according to claim 20, wherein the trace compound is as defined in any one of claims 10 to 12.

22. 1. A method for determining the effectiveness of treatment of a subject with cancer with a therapeutic agent or a special diet, or chemotherapy or chemoradiotherapy, comprising: (i) providing to said subject a composition comprising at least one sugar according to any one of claims 1 to 8, and / or at least one amino acid or a precursor thereof, and / or at least one polyol, and (ii) analyzing the concentration of said trace compounds resulting from the metabolism of said at least one sugar, and / or at least one amino acid or precursor thereof, and / or at least one polyol in a breath sample from a test subject and comparing said concentration with a standard of the concentration of said trace compounds in individuals not suffering from cancer; Including, an increase or decrease in the concentration of said trace compound in said breath sample from said test subject compared to said reference indicates that said therapeutic agent or said special diet, or chemotherapy or chemoradiotherapy treatment regime is effective or ineffective; the trace compounds are selected from the group consisting of acetic acid, butanoic acid, hexanoic acid, pentanoic acid, propanoic acid, acetaldehyde, decanal, heptanal, hexanal, nonanal, octanal, pentanal, butanal, propanal, 1-hydroxy-4-ethylbenzene, decane, dodecane, p-cresol, and phenol, or combinations thereof; The method (excluding medical procedures on humans), wherein the cancer is esophagogastric junction cancer, gastric cancer, esophageal cancer, esophageal squamous cell carcinoma (ESCC), or esophageal adenocarcinoma (EAC).

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