BIOMARKER DETECTION FOR CANCER DIAGNOSIS AND PROGNOSIS
20220221443 · 2022-07-14
Inventors
- George Hanna (Northwood, GB)
- Bhamini Vadhwana (Hounslow, GB)
- Ilaria Belluomo (London, GB)
- Piers Boshier (London, GB)
Cpc classification
A61B5/082
HUMAN NECESSITIES
International classification
Abstract
The invention relates to a method for diagnosing a subject suffering from cancer, or a pre-disposition thereto. The method comprises detecting, in a bodily sample from a test subject, the concentration of a signature compound resulting from the metabolism of at least one sugar, and/or at least one amino acid or a 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 of more than 20,000 mg/100 ml, the amino acid or a 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 more than 25,000 mg/100 ml. The method further comprises comparing this concentration with a reference for the concentration of the signature compound in an individual who does not suffer from cancer. In particular, an increase or decrease in the concentration of the signature compound compared to the reference, suggests that the subject is suffering from cancer, or has a pre-disposition thereto, or provides a negative prognosis of the subject's condition.
Claims
1. A method for diagnosing a subject suffering from cancer, or a pre-disposition thereto, or for providing a prognosis of the subject's condition, the method comprising: (i) detecting, in a bodily sample from a test subject, the concentration of a signature compound resulting from the metabolism of at least one sugar and/or at least one amino acid or a 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 more than 20,000 mg/100 ml, the amino acid or a 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 more than 25,000 mg/100 ml; and (ii) comparing this concentration with a reference for the concentration of the signature compound in an individual who does not suffer from cancer, wherein an increase or a decrease in the concentration of the signature compound compared to the reference, suggests that the subject is suffering from cancer, or has a pre-disposition thereto, or provides a negative prognosis of the subject's condition.
2. The method according to claim 1, wherein the detection step (i) comprises detecting a signature compound 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 from administration of the composition comprising at least one sugar and/or an amino acid or a precursor thereof and/or at least one polyol.
3. The method according to claim 1 or claim 2, wherein detection step (i) comprises detecting a signature compound at between 30 and 60, or 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 30 minutes from administration of the composition comprising at least one sugar and/or an amino acid or a precursor thereof and/or at least one polyol.
4. The method according to any preceding claim, wherein an increase in the concentration of the signature compound compared to the reference suggests that the subject is suffering from cancer, or has a pre-disposition thereto, or provides a negative prognosis of the subject's condition.
5. The method according to claim 4, wherein the increase in the concentration of the signature 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 signature compound when compared to the reference.
6. The method according to any preceding claim, wherein the sugar is present in the composition previously administered to the subject at a concentration of at least 20,500 mg/100 ml.
7. The method according to any preceding claim, wherein the sugar is glucose, sorbitol, mannose or lactose.
8. The method according to any preceding claim, wherein 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 signature compound is detected up to 10 minutes from administration of the composition comprising glucose.
9. The method according to any preceding claim, wherein the composition administered to the subject comprises citric acid in combination with the sugar, wherein the citric acid is present in the composition at a concentration of at least 1,000 mg/100 ml, optionally wherein the sugar is glucose.
10. The method according to any preceding claim, wherein the amino acid is selected from a group consisting of: tyrosine, glutamic acid, glutamate, phenylalanine, tryptophan, proline and histidine, optionally wherein the composition comprises tyrosine, phenylalanine and glutamic acid.
11. The method according to claim 10, wherein 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, optionally wherein the signature compound is detected between 35 and 45 minutes from administration of the composition comprising tyrosine.
12. The method according to any preceding claim, wherein the amino acid precursor is phenylalanine, optionally present at a concentration of at least 3000 mg/100 ml.
13. The method according to any preceding claim, wherein the polyol is glycerol.
14. The method according to any preceding claim, wherein the polyol is present in the composition at a concentration of more than 30,000 mg/100 ml.
15. The method according to any preceding claim, wherein the cancer is oesophago-gastric junction cancer, gastric cancer, oesophageal cancer, oesophageal squamous-cell carcinoma (ESCC) or oesophageal adenocarcinoma (EAC).
16. The method according to any preceding claim, wherein the cancer is gastric cancer, oesophageal cancer or a metastasised cancer.
17. The method according to any preceding claim, wherein the signature compound is a short chain fatty acid, aldehyde, alcohol or any combination thereof.
18. The method according to claim 17, wherein the signature compound is a C1-C3 aldehyde, a C1-C3 alcohol, a C2-C10 alkane wherein a first carbon atom is substituted with the ═O group and a second carbon atom is substituted with an —OH group, a C1-C20 alkane, a C4-C10 alcohol, a C1-C6 carboxylic acid, a C4-C20 aldehyde, phenol optionally substituted with a C1-C6 alkyl group, a C2 aldehyde, a C3 aldehyde, a C8 aldehyde, a C9 aldehyde, a C10 aldehyde, a C11 aldehyde, an analogue or derivative of any aforementioned species, or any combination thereof.
19. The method according to either claim 17 or claim 18, wherein the signature compound is selected from a 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.
20. The method according to any one of claims 17 to 19, wherein the substrate is a sugar, preferably glucose, and the signature compound is acetic acid, butanoic acid, pentanoic acid, propanoic acid, hexanoic acid, acetaldehyde, propanal, butanal, hexanal, pentanal, decanal, 1-hydoxytheylbenzene and/or P-cresol.
21. The method according to any one of claims 17 to 19, wherein the substrate is an amino acid or precursor thereof, and the signature compound is butanal, decanal, heptanal, hexanal, phenol, decane, P-cresol, 1-hydoxytheylbenzene and/or dodecane.
22. The method according to claim 21, wherein the amino acid or precursor thereof is tyrosine, and the signature compound is decanal and/or dodecane.
23. The method according to any one of claims 17 to 19, wherein the substrate is a polyol, preferably glycerol, and the signature compound is butanoic acid, acetic acid, hexanoic acid, pentanoic acid, propanoic acid, butanal, hexanal, pentanal, and/or propanal.
24. A method for detecting a signature compound in a test subject, the method comprising: (i) providing the subject with a composition comprising at least one substrate according to any preceding claim into a signature compound; and (ii) detecting the concentration of the signature compound in a bodily sample from the subject.
25. The method according to claim 24, wherein the signature compound is as defined in any one of claims 17 to 23.
26. A composition comprising at least one sugar and/or at least one amino acid or a precursor thereof and/or at least one polyol present suitable for metabolism into a signature compound, wherein the sugar is present in the composition at a concentration of more than 20,000 mg/100 ml and 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 25,000 mg/100 ml, for use in a method of diagnosis or prognosis.
27. A composition comprising at least one sugar and/or at least one amino acid or a precursor thereof and/or at least one polyol present suitable for metabolism into a signature compound, wherein the sugar is present in the composition at a concentration of more than 20,000 mg/100 ml and 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 25,000 mg/100 ml for use in a method of diagnosing or prognosing cancer, optionally wherein the cancer is oesophago-gastric junction cancer, gastric cancer, oesophageal cancer, oesophageal squamous-cell carcinoma (ESCC) or oesophageal adenocarcinoma (EAC).
28. A composition comprising at least one substrate according to any one of claims 1 to 14, for use in the method according to any one of claims 1 to 23.
29. A kit for diagnosing a subject suffering from cancer, or a pre-disposition thereto, or for providing a prognosis of the subject's condition, the kit comprising: (a) a composition comprising at least one substrate as defined in any one of claims 1 to 14; (b) means for determining the concentration of a signature compound in a sample from a test subject; and (c) a reference for the concentration of the signature compound in a sample from an individual who does not suffer from cancer, wherein the kit is used to identify an increase or a decrease in the concentration of the signature compound in the bodily sample from the test subject, compared to the reference, thereby suggesting that the subject suffers from cancer, or has a pre-disposition thereto, or provides a negative prognosis of the subject's condition.
30. The kit according to claim 29, wherein the signature compound is as defined in any one of claims 17 to 23.
31. A method for determining the efficacy of treating a subject suffering from cancer with a therapeutic agent or a specialised diet or chemotherapy or chemoradiotherapy, the method comprising: (i) providing the subject with a composition comprising at least one substrate according to any one of claims 1 to 14; and (ii) analysing the concentration of the signature compound resulting from metabolism of the at least one substrate in a bodily sample from a test subject, and comparing this concentration with a reference for the concentration of the signature compound in an individual who does not suffer from cancer, wherein an increase or a decrease in the concentration of the signature compound in the bodily sample from the test subject compared to the reference suggests that the treatment regime with the therapeutic agent or the specialised diet or chemotherapy or chemoradiotherapy is effective or ineffective.
32. The method according to claim 31, wherein the signature compound is as defined in any one of claims 17 to 23.
33. The method according to either claim 31 or claim 32, wherein the cancer is oesophago-gastric junction cancer, gastric cancer, oesophageal cancer, oesophageal squamous-cell carcinoma (ESCC) or oesophageal adenocarcinoma (EAC).
Description
[0120] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figure, in which:
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MATERIALS AND METHODS
EXAMPLE 1
Glucose Dose Study
[0157] Subjects
[0158] Four healthy subjects volunteered for participation and informed written consent was obtained.
[0159] Dose Concentrations
[0160] Four doses of the substrate were guided by the (i) daily recommended intake levels by the Food and Nutrition Board and (ii) the already established glucose tolerance test. The glucose tolerance test uses an acceptable 75 g of glucose dissolved in 100 ml water, which is satisfactory for patients. The daily maximum recommended dose is 130 g per day for an adult.[1] Based on these findings, the inventors selected doses of 75 g, 50 g, 25 g, 10 g to compare the dose responses against glucose concentration. All findings were compared with a baseline of 0 g.
[0161] Breath Sampling
[0162] Methods for the detection of short chain fatty acids were established on the 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 fluid diet for a minimum of 6 hours prior to 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 mls of warm water, followed by three oral water rinses to decontaminate the oral cavity. Direct sampling was performed for 3 exhaled breath samples over 60 seconds, consecutively with all four methods at five-minute intervals up to 60 minutes (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mins).
[0163] SIFT-MS
[0164] SIFT-MS allows real-time quantification and identification of VOCs within the exhaled breath using chemical ionisation. Precursor ions (H3O+, NO+ and O2+) are discharged into a quadrupole mass filter and carried by an inert helium gas along a flow tube. Breath is injected into the flow tube to react with the precursor ions to create product ions which are subsequently separated according to mass-to-charge ratio (m/z). The SIFT-MS was subjected to daily automated validation cycles, to operate within temperatures of 10-30° C. Data was obtained in concentrations in parts per billion.
[0165] Sugars
[0166] Comparison of 4 different sugars at a dose of 25 g each. 25 g was chosen after the initial glucose study where similar VOC concentrations were observed between 25 g and 75 g. Glucose, lactose and mannose follow a similar pattern with an increase maximally at 10 minutes after sugar consumption (
[0167] Glucose
[0168] Patient Selection
[0169] All patients were recruited from St Mary's Hospital from February 2019-May 2019. Patients were recruited from three cohorts; oesophagial cancer (n=6), gastric cancer (n=6) and age-matched healthy controls (n=6). Informed written consent was obtained by all participants. Patients diagnosed with oesophagogastric adenocarcinoma ranged from early disease on the curative pathway 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 collated.
[0170] Breath Sampling
[0171] Methods for the detection of 4 classes of volatile compounds; short chain fatty acids, alcohols, aldehydes and phenol-alkanes were established on the 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 fluid diet for a minimum of 6 hours prior to 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 mls of warm water, followed by three oral water rinses to decontaminate the oral cavity. Direct sampling was performed for 3 exhaled breath samples over 60 seconds, consecutively with all four methods at five-minute intervals up to 60 minutes (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mins).
[0172] SIFT-MS
[0173] SIFT-MS allows real-time quantification and identification of VOCs within the exhaled breath using chemical ionisation. Precursor ions (H3O+, NO+ and O2+) are discharged into a quadrupole mass filter and carried by an inert helium gas along a flow tube. Breath is injected into the flow tube to react with the precursor ions to create product ions which are subsequently separated according to mass-to-charge ratio (m/z). The SIFT-MS was subjected to daily automated validation cycles, to operate within temperatures of 10-30° C.
[0174] Statistical Analysis
[0175] Data was obtained in concentrations in parts per billion. Univariate Kruskal Wallis analysis was performed across the three groups using SPSS statistical software (v25, Armonk N.Y.; IBM Corp). Mann Whitney U test was performed to identify differences between oesophageal and gastric cancers compared with controls. P value of <0.05 was considered statistically significant.
EXAMPLE 2
Tyrosine
[0176] Patient Selection
[0177] All patients were recruited from St Mary's Hospital from February 2019-May 2019. Patients were recruited from three cohorts; oesophageal cancer (n=6), gastric cancer (n=6) and age-matched healthy controls (n=6). Informed written consent was obtained by all participants. Patients diagnosed with oesophagogastric adenocarcinoma ranged from early disease on the curative pathway 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 collated.
[0178] Breath Sampling
[0179] Methods for the detection of 4 classes of volatile compounds; short chain fatty acids, alcohols, aldehydes and phenol-alkanes were established on the 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 fluid diet for a minimum of 6 hours prior to 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 dissolved in 100 mls of warm water, followed by three oral water rinses to decontaminate the oral cavity. Direct sampling was performed for 3 exhaled breath samples over 60 seconds, consecutively with all four methods at five-minute intervals up to 60 minutes (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mins).
[0180] SIFT-MS
[0181] SIFT-MS allows real-time quantification and identification of VOCs within the exhaled breath using chemical ionisation. Precursor ions (H3O+, NO+ and O2+) are discharged into a quadrupole mass filter and carried by an inert helium gas along a flow tube. Breath is injected into the flow tube to react with the precursor ions to create product ions which are subsequently separated according to mass-to-charge ratio (m/z). The SIFT-MS was subjected to daily automated validation cycles, to operate within temperatures of 10-30° C.
[0182] Statistical Analysis
[0183] Data was obtained in concentrations in parts per billion. Univariate Kruskal Wallis analysis was performed across the three groups using SPSS statistical software (v25, Armonk N.Y.; IBM Corp). Mann Whitney U test was performed to identify differences between oesophageal and gastric cancers compared with controls. P value of <0.05 was considered statistically significant.
EXAMPLE 3
Phenylalanine
[0184] Subjects: One healthy subject.
[0185] Dose concentrations: The daily recommended intake levels advised by the Food and Nutrition Board is 100 mg/kg daily for an adult, with a maximum dose of 3 g. [1] A single dose of 3 g was selected for this study.
[0186] Breath Sampling
[0187] Methods for the detection of short chain fatty acids, aldehydes and phenol-alkanes were established on the 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 fluid diet for a minimum of 6 hours. Exhalation was performed 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 mls of warm water, followed by three oral water rinses to decontaminate the oral cavity. Direct sampling was performed for 3 exhaled breath samples over 60 seconds, consecutively with all four methods at five-minute intervals up to 60 minutes (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mins).
[0188] SIFT-MS
[0189] SIFT-MS allows real-time quantification and identification of VOCs within the exhaled breath using chemical ionisation. Precursor ions (H3O+, NO+ and O2+) are discharged into a quadrupole mass filter and carried by an inert helium gas along a flow tube. Breath is injected into the flow tube to react with the precursor ions to create product ions which are subsequently separated according to mass-to-charge ratio (m/z). The SIFT-MS was subjected to daily automated validation cycles, to operate within temperatures of 10-30° C. Data was obtained in concentrations in parts per billion.
EXAMPLE 4
Glutamic Acid
[0190] Subjects: One healthy subject.
[0191] Dose Concentrations
[0192] The daily recommended intake levels advised by the Food and Nutrition Board is 30 mg/kg daily for an adult. [1] A single maximum dose of 2.1 g was selected for an average adult of 70 kg.
[0193] Breath Sampling
[0194] Methods for the detection of short chain fatty acids, aldehydes and phenol-alkanes were established on the 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 fluid diet for a minimum of 6 hours. Exhalation was performed 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 mls of warm water, followed by three oral water rinses to decontaminate the oral cavity. Direct sampling was performed for 3 exhaled breath samples over 60 seconds, consecutively with all four methods at five-minute intervals up to 60 minutes (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mins).
[0195] SIFT-MS
[0196] SIFT-MS allows real-time quantification and identification of VOCs within the exhaled breath using chemical ionisation. Precursor ions (H3O+, NO+ and O2+) are discharged into a quadrupole mass filter and carried by an inert helium gas along a flow tube. Breath is injected into the flow tube to react with the precursor ions to create product ions which are subsequently separated according to mass-to-charge ratio (m/z). The SIFT-MS was subjected to daily automated validation cycles, to operate within temperatures of 10-30° C. Data was obtained in concentrations in parts per billion.
EXAMPLE 5
Glycerol Doses
[0197] Subjects
[0198] Two healthy subjects volunteered for participation and informed written consent was obtained.
[0199] Dose Concentrations
[0200] Two doses of the substrate were guided by the (i) daily recommended intake levels by the Food and Nutrition Board and (ii) the initial glucose method development study. The daily maximum recommended dose is 276 mg/kg per day for an adult, however, there was no reported harm from higher doses.[1] An average 70 kg individual would be recommended a maximum of 19 g of glycerol. Based on these findings, we selected doses of 50 g, 25 g, 10 g to compare the dose responses against glucose concentration. All findings were compared with a baseline of 0 g.
[0201] Breath Sampling
[0202] Methods for the detection of short chain fatty acids and aldehydes were established on the 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 fluid diet for a minimum of 6 hours prior to 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 mls of warm water, followed by three oral water rinses to decontaminate the oral cavity. Direct sampling was performed for 3 exhaled breath samples over 60 seconds, consecutively with all four methods at five-minute intervals up to 60 minutes (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mins).
[0203] SIFT-MS
[0204] SIFT-MS allows real-time quantification and identification of VOCs within the exhaled breath using chemical ionisation. Precursor ions (H3O+, NO+ and O2+) are discharged into a quadrupole mass filter and carried by an inert helium gas along a flow tube. Breath is injected into the flow tube to react with the precursor ions to create product ions which are subsequently separated according to mass-to-charge ratio (m/z). The SIFT-MS was subjected to daily automated validation cycles, to operate within temperatures of 10-30° C. Data was obtained in concentrations in parts per billion.
EXAMPLE 6
Glycerol Patient Selection
[0205] All patients were recruited from St Mary's Hospital from February 2019-December 2019. Patients were recruited from three cohorts; oesophageal cancer (n=6), gastric cancer (n=6) and age-matched healthy controls (n=6). Informed written consent was obtained by all participants. Patients diagnosed with oesophagogastric adenocarcinoma ranged from early disease on the curative pathway 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 collated.
[0206] Breath Sampling
[0207] Methods for the detection of 4 classes of volatile compounds; short chain fatty acids, alcohols, aldehydes and phenol-alkanes were established on the 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 fluid diet for a minimum of 6 hours prior to 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 glycerol dissolved in 100 mls of warm water, followed by three oral water rinses to decontaminate the oral cavity. Direct sampling was performed for 3 exhaled breath samples over 60 seconds, consecutively with all four methods at five-minute intervals up to 60 minutes (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mins).
[0208] SIFT-MS
[0209] SIFT-MS allows real-time quantification and identification of VOCs within the exhaled breath using chemical ionisation. Precursor ions (H3O+, NO+ and O2+) are discharged into a quadrupole mass filter and carried by an inert helium gas along a flow tube. Breath is injected into the flow tube to react with the precursor ions to create product ions which are subsequently separated according to mass-to-charge ratio (m/z). The SIFT-MS was subjected to daily automated validation cycles, to operate within temperatures of 10-30° C.
[0210] Statistical Analysis
[0211] Data was obtained in concentrations in parts per billion. Univariate Kruskal Wallis analysis was performed across the three groups using SPSS statistical software (v25, Armonk N.Y.; IBM Corp). Mann Whitney U test was performed to identify differences between oesophageal and gastric cancers compared with controls. P value of <0.05 was considered statistically significant.
EXAMPLE 7
Combined Amino Acids (Tyrosine, Phenylalanine, Glutamic Acid)
[0212] Patient Selection
[0213] All patients were recruited from St Mary's Hospital from February 2019-December 2019. Patients were recruited from three cohorts; oesophageal cancer (n=6), gastric cancer (n=1) and age-matched healthy controls (n=6). Informed written consent was obtained by all participants. Patients diagnosed with oesophagogastric adenocarcinoma ranged from early disease on the curative pathway 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 collated.
[0214] Breath Sampling
[0215] Methods for the detection of 4 classes of volatile compounds; short chain fatty acids, alcohols, aldehydes and phenol-alkanes were established on the 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 fluid diet for a minimum of 6 hours prior to 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 2.4 glutamic acid dissolved in 100 mls of warm water, followed by three oral water rinses to decontaminate the oral cavity. Direct sampling was performed for 3 exhaled breath samples over 60 seconds, consecutively with all four methods at five-minute intervals up to 60 minutes (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mins).
[0216] SIFT-MS
[0217] SIFT-MS allows real-time quantification and identification of VOCs within the exhaled is breath using chemical ionisation. Precursor ions (H3O+, NO+ and O2+) are discharged into a quadrupole mass filter and carried by an inert helium gas along a flow tube. Breath is injected into the flow tube to react with the precursor ions to create product ions which are subsequently separated according to mass-to-charge ratio (m/z). The SIFT-MS was subjected to daily automated validation cycles, to operate within temperatures of 10-30° C.
[0218] Statistical Analysis
[0219] Data was obtained in concentrations in parts per billion. Mann Whitney U analysis was performed across cancer and non-cancer using SPSS statistical software (v25, Armonk N.Y.; IBM Corp). P value of <0.05 was considered statistically significant.
EXAMPLE 8
Combined Glucose and Citric Acid
[0220] Patient Selection
[0221] All patients were recruited from St Mary's Hospital from February 2019-December 2019. Twelve healthy controls were recruited to form two cohorts to consume; glucose (n=6), and combined glucose and citric acid (n=6). Informed written consent was obtained by all participants. Age-matched healthy controls included patients with benign upper gastrointestinal disease (reflux, dysmotility) or healthy asymptomatic controls.
[0222] Breath Sampling
[0223] Methods for the detection of 4 classes of volatile compounds; short chain fatty acids, alcohols, aldehydes and phenol-alkanes were established on the 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 fluid diet for a minimum of 6 hours prior to 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 dissolved in 100 mls of warm water, followed by three oral water rinses to decontaminate the oral cavity. Direct sampling was performed for 3 exhaled breath w samples over 60 seconds, consecutively with all four methods at five-minute intervals up to 30 minutes (0, 5, 10, 15, 20, 25, 30 mins).
[0224] SIFT-MS
[0225] SIFT-MS allows real-time quantification and identification of VOCs within the exhaled is breath using chemical ionisation. Precursor ions (H3O+, NO+ and O2+) are discharged into a quadrupole mass filter and carried by an inert helium gas along a flow tube. Breath is injected into the flow tube to react with the precursor ions to create product ions which are subsequently separated according to mass-to-charge ratio (m/z). The SIFT-MS was subjected to daily automated validation cycles, to operate within temperatures of 10-30° C.
[0226] Statistical Analysis
[0227] Data was obtained in concentrations in parts per billion. Mann Whitney U analysis was performed across cancer and non-cancer using SPSS statistical software (v25, Armonk N.Y.; IBM Corp). P value of <0.05 was considered statistically significant.
[0228] Results
EXAMPLE 1
Glucose Dosing Study
[0229] Volatile Organic Compound Analysis
TABLE-US-00001 TABLE 1 Median concentrations of short chain fatty acids detected in the exhaled breath of all subjects at 5-15 minutes. Concentration (ppbv) Fold change Baseline 10 g 25 g 50 g 75 g Baseline 10 g 25 g 50 g 75 g Median Acetic acid 22.1 50.2 35.5 47.3 72.6 0.8 2.3 2.0 2.0 3.0 Butanoic acid 5.2 11.9 11.8 16.8 19.6 0.8 2.6 3.0 3.7 5.3 Hexanoic acid 1.1 1.2 1.0 1.0 1.8 0.8 1.6 1.4 0.9 1.2 Pentanoic acid 5.3 5.4 5.4 6.1 10.2 0.9 1.1 1.0 1.4 1.3 Propanoic acid 11.5 39.6 43.4 82.3 77 0.7 5.3 4.3 6.3 9.9 Average Acetic acid 28.1 57.8 36.5 43.2 68.3 1.3 2.6 1.9 2.5 2.6 Butanoic acid 4.7 20.7 11.8 15.4 28.7 0.9 3.5 2.8 4.4 4.8 Hexanoic acid 1.1 1.5 1.1 1.1 1.5 0.9 1.4 1.2 0.8 1.1 Pentanoic acid 4.5 6.4 5.1 6.2 8.1 0.9 1.2 1.1 1.4 1.4 Propanoic acid 12.2 96.5 46.8 82.2 120.5 1.1 7.1 4.6 7.9 7.8
[0230] Increasing glucose concentrations is positively correlated with increasing concentrations of volatile fatty acids detected within the exhaled breath. Butanoic- and propanoic acid demonstrate a maximal response within 5-15 minutes of glucose consumption with subsequent declining values. Rapid glucose degradation via the glycolytic pathway produces volatile end products detected within exhaled breath. Previous work by the inventors has demonstrated oral water rinsing after glucose consumption eliminates potential VOC response originating from the oral cavity. Butanoic- and pentanoic acid demonstrated a difference of 1-fold increase between 10 g and 50 g of glucose. These compounds were used to guide the recommended glucose dose for preliminary clinical studies involving patients with oesophago-gastric cancer. To obtain a balance between an adequate dose response and a drink acceptable to patients, the inventors selected a dose of 25 g dissolved in 100 ml warm water. The next step of this study will assess the VOC response in patients diagnosed with OG cancer compared to healthy age-matched controls to observe any differences in cellular metabolic activity and VOC response.
TABLE-US-00002 TABLE 2 Demographics and clinical information of participants. Oesophageal Gastric Cancer Cancer Controls (n = 6) (n = 6) (n = 6) Age (years) * 70.5 71.5 69 Male 5 4 3 Ethnicity White 6 5 5 Asian 0 0 1 Black 0 1 0 Metastatic disease 1 1 — Neoadjuvant therapy 3 5 — Co-morbidities Diabetes 1 0 0 Benign UGI disease 0 0 2 Healthy 0 0 4 * median
TABLE-US-00003 TABLE 3 Details of volatile organic compound analysed by selected ion flow tube mass spectrometry Compound Formula precursor Ion Product Ion m/z Acetone C.sub.3H.sub.6O H.sub.3O+ 59 Short Chain Fatty Acids Acetic acid CH.sub.3COOH NO+ 90 Butanoic acid C.sub.4H.sub.8O NO+ 118 Hexanoic acid C.sub.6H.sub.12O.sub.2 NO+ 146 Pentanoic acid C.sub.5H.sub.10O.sub.2 NO+ 85 Propanoic acid CH.sub.3CH.sub.2COOH NO+ 104 Aldehydes Acetaldehyde C.sub.2H.sub.4O H.sub.3O+ 45 Decanal C.sub.10H.sub.20O NO+ 155 Heptanal C.sub.7H.sub.14O NO+ 113 Hexanal C.sub.6H.sub.12O NO+ 99 Nonanal C.sub.9H.sub.18O NO+ 141 Octanal C.sub.8H.sub.16O NO+ 127 Pentanal C.sub.5H.sub.10O NO+ 85 Butanal C.sub.4H.sub.8O NO+ 71 Propanal C.sub.3H.sub.6O NO+ 57 Phenols 1-hydroxy-4- C.sub.8H.sub.10O NO+ 122 ethylbenzene Decane C.sub.10H.sub.22 NO+ 141 Dodecane C.sub.12H.sub.26 H.sub.3O+ 189 P-cresol C.sub.7H.sub.8O NO+ 108 Phenol C.sub.6H.sub.5OH NO+ 94
[0231] Glucose
[0232] Volatile Organic Compound Analysis
[0233] Short Chain Fatty Acids
TABLE-US-00004 TABLE 4 Volatile short chain fatty acids (acetic-, butanoic-, pentanoic-, propanoic acid) increased maximally at 5-10 mins after glucose consumption Median Control Oesophageal Cancer Gastric Cancer Post- Post- Post- Baseline glucose: Baseline glucose: Baseline glucose Concen- Concen- Concen- Concen- Concen- Concen- Time tration tration Fold tration tration Fold tration tration Fold point Increase/ P (ppbv) (ppbv) change (ppbv) (ppbv) change (ppbv) (ppbv) change (mins) decrease value* Acetic acid 38.03 31.37 0.66 19.58 23.63 1.11 16.63 39.0 2.49 5-10 ↑ 0.023 Butanoic acid 5.57 9.64 2.72 4.40 12.34 1.61 4.96 34.18 10.53 5-10 ↑ 0.351 Hexanoic acid 0.64 0.52 0.87 1.00 0.65 0.63 0.76 0.63 0.87 5-10 — 0.081 Pentanoic acid 2.45 2.38 0.94 3.62 1.88 0.67 2.86 2.69 1.16 25-30 ↑ <0.001 Propanoic acid 15.89 17.14 1.17 17.15 30.20 1.33 8.84 38.22 3.52 5-10 ↑ 0.243
TABLE-US-00005 TABLE 5 Mann Whitney U test comparing (i) Control vs. Gastric cancer groups and (ii) Control vs. Oesophageal cancer groups; p < 0.05 is considered statistically significant (highlighted bold) Control vs Gastric Ca Control vs Oesophageal Ca P value P value P value (Fold P value (Fold (ppbv) change) (ppbv) change) Fatty Acids Acetic acid 0.190 <0.001 0.005 0.007 Butanoic acid 0.208 0.038 0.912 1.000 Hexanoic acid 0.041 0.818 0.280 <0.001 Pentanoic acid <0.001 <0.001 0.018 0.011 Propanoic acid 0.984 0.048 0.174 0.407 Aldehydes Acetaldehyde 0.190 <0.001 0.012 0.119 Butanal 0.001 0.021 0.004 <0.001 Decanal <0.001 0.779 0.001 0.646 Heptanal <0.001 0.156 <0.001 0.384 Hexanal <0.001 0.035 0.023 0.522 Nonanal 0.001 0.031* 0.006 0.008* Octanal <0.001 0.001* 0.112 <0.001* Pentanal <0.001 <0.001 0.004 0.818 Propanal 0.174 0.250 <0.001 0.007 Phenol-alkanes 1-hyroxy-4- 0.008 0.019 0.103 0.384 ethylbenzene Decane <0.001 <0.001* <0.001 <0.001* Dodecane 0.503 0.522 0.631 0.711 P-cresol 0.002 <0.001 <0.001 0.313 Phenol <0.001* 0.107 0.046* 0.582
[0234] Discussion
[0235] Three chemical classes of VOCs within exhaled breath have demonstrated a significant difference in patients diagnosed with oesophago-gastric (OG) cancer. A total of 13 compounds from the groups short chain fatty acids (SCFA) (n=4), aldehydes (n=6) and phenols (n=3) have demonstrated increased concentrations after glucose consumption.
[0236] Volatile SCFAs demonstrated the largest alteration in breath concentrations; namely butanoic acid and propanoic acid. Optimal concentrations were reached within 10 minutes of consumption suggesting rapid glucose degradation. Glucose, a monosaccharide, enters the glycolytic pathway producing end products of metabolism detected in the breath. Pentanoic acid was detected in higher concentrations at 30 minutes compared to baseline values. These results suggest breath VOCs can be augmented by oral substrates by manipulating the intrinsic metabolic pathways of known VOCs associated with OG cancer. Gastric cancers demonstrate a stronger response than oesophageal cancers in all significant VOCs detected. The gastric cancer group displayed a significant fold change in SCFA (acetic-, butanoic-, pentanoic-, and propanoic acid), with two overlapping significances with the oesophageal cancer group (acetic- and pentanoic acid).
[0237] Similarly, aldehydes such as pentanal and propanal followed a similar pattern of response to the SCFA with an optimal increase in concentrations at 5-10 minutes. The remainder of the aldehydes show consistently increased levels in the cancer groups, with 4 of the nine with higher baseline values. Acetaldehyde, butanal, hexanal and pentanal demonstrate a significant increase in fold change from the baseline in gastric is cancer patients. Oesophageal cancer patients show this effect with both butanal and propanal only. On the contrary, both groups demonstrate a significant fold increase in the control groups for nonanal and octanal, which needs to be further explored. These results are consistent with previous work published by the inventors associating volatile butanoic acid, butanal and decanal with OG cancer.[1] The remainder of the aldehydes and the phenol-alkanes (except dodecane) have demonstrated consistently increased concentrations in the cancer groups for the duration of the study. Previous work by the inventors group has implicated phenol as a potential breath biomarker in OG cancer.[2]
[0238] Decane, from the phenol family, displays a higher baseline concentration in both cancer groups. A fold increase with the control group after glucose consumption needs to be further explored. A similar pattern of response was observed with P-cresol, but with a fold increase seen only with the gastric cancer group, a new finding. This may be reflective of the transient passage of glucose by the oesophageal tumour compared to pooling of glucose in the stomach.
[0239] Currently, NICE guidelines recommend an upper gastrointestinal endoscopy within 2 weeks for patients presenting with ‘red flag’ symptoms suggestive of OG cancer. [3] However, the insidious nature of the disease means the majority present with non-specific symptoms, delaying diagnosis and translating into poor overall survival outcomes. A non-invasive breath test will act as a triage tool to stratify patients with non-specific upper gastrointestinal symptoms. Identification of breath biomarkers for early detection of OG cancer has the potential to offer patients curative treatment and influence overall survival outcomes. This study assessed patients in early and advanced stages of disease.
[0240] In clinical practice, exhaled breath could be collected using: [0241] Breath sampling device coupled with thermal desorption tubes to facilitate storage of samples storage and transport. [0242] Direct sampling using mass spectrometry such as SIFT as demonstrated in this study. [0243] Dedicated sensors for those VOC with large response such as acetic-, butanoic-, pentanoic- and propanoic acid.
[0244] Key Points
[0245] Glucose consumption activates the metabolic pathway associated tumour-microbiome is or increased activity of the tumour cell. This is detected with: [0246] A significant fold increase in SCFA (acetic-, butanoic-, pentanoic- and propanoic acid); more so observed in the gastric cancer than oesophageal cancer group. [0247] A significant increase in aldehydes; acetaldehyde, butanal, hexanal and pentanal in the gastric cancer group. An increase in butanal and propanal are observed with oesophageal cancer. [0248] A new finding of decane and P-cresol in increased concentrations in baseline was observed for both cancer groups. P-cresol fold increase was shown with gastric cancer only.
[0249] Exhaled breath samples will be collected at two intervals after glucose ingestion to identify the VOCs at their optimal concentrations; early at 5-10 minutes, and late at 30 minutes.
EXAMPLE 2
Tyrosine
[0250]
TABLE-US-00006 TABLE 6 Demographics and clinical information of participants. Oesophageal Gastric Cancer Cancer Controls (0 = 6) (n = 6) (n = 6) Age (years) * 69.5 65 66.5 Male 6 5 3 Ethnicity White 5 4 5 Asian 1 1 1 Black 0 1 0 Metastatic disease 0 2 — Neoadjuvant therapy 3 5 — Co-morbidities Diabetes 1 1 0 Benign UGI disease 0 0 3 Healthy 0 0 3 * median
TABLE-US-00007 TABLE 7 Details of volatile organic compound analysed by selected ion flow tube mass spectrometry Compound Formula precursor Ion Product Ion m/z Acetone C.sub.3H.sub.6O H.sub.3O+ 59 Short Chain Fatty Acids Acetic acid CH.sub.3COOH NO+ 90 Butanoic acid C.sub.4H.sub.8O NO+ 118 Hexanoic acid C.sub.6H.sub.12O.sub.2 NO+ 146 Pentanoic acid C.sub.5H.sub.10O.sub.2 NO+ 85 Propanoic acid CH.sub.3CH.sub.2COOH NO+ 104 Aldehydes Acetaldehyde C.sub.2H.sub.4O H.sub.3O+ 45 Decanal C.sub.10H.sub.20O NO+ 155 Heptanal C.sub.7H.sub.14O NO+ 113 Hexanal C.sub.6H.sub.12O NO+ 99 Nonanal C.sub.9H.sub.18O NO+ 141 Octanal C.sub.8H.sub.16O NO+ 127 Pentanal C.sub.5H.sub.10O NO+ 85 Butanal C.sub.4H.sub.8O NO+ 71 Propanal C.sub.3H.sub.6O NO+ 57 Phenols 1-hydroxy-4- C.sub.8H.sub.10O NO+ 122 ethylbenzene Decane C.sub.10H.sub.22 NO+ 141 Dodecane C.sub.12H.sub.26 H.sub.3O+ 189 P-cresol C.sub.7H.sub.8O NO+ 108 Phenol C.sub.6H.sub.5OH NO+ 94
[0251] Volatile Organic Compound Analysis
[0252] Short Chain Fatty Acids
TABLE-US-00008 TABLE 8 Volatile short chain fatty acids demonstrated no significant changes after tyrosine consumption. Median Control Oesophageal Cancer Gastric Cancer Post- Post- Post- Baseline tyrosine: Baseline tyrosine: Baseline tyrosine Concen- Concen- Concen- Concen- Concen- Concen- Time tration tration Fold tration tration Fold tration tration Fold point Increase/ P (ppbv) (ppbv) change (ppbv) (ppbv) change (ppbv) (ppbv) change (mins) decrease value* Acetic acid 37.22 34.95 0.91 25.33 13.19 0.62 22.53 24.60 1.29 25-35 — <0.0001 Butanoic acid 7.08 5.85 0.98 8.60 4.56 0.69 9.77 8.61 0.86 25-35 — 0.0068 Hexanoic acid 1.78 1.57 0.86 1.11 0.84 0.65 1.33 0.83 0.92 25-35 — 0.0016 Pentanoic acid 4.86 5.44 1.19 3.78 2.48 0.66 3.76 6.10 0.80 25-35 — <0.0001 Propanoic acid 22.58 16.44 0.84 17.79 8.49 0.60 31.75 25.60 0.58 25-35 — 0.0004 *Kruskal Wallis Analysis p < 0.05 considered statistically significant
[0253] Aldehydes
TABLE-US-00009 TABLE 9 Volatile aldehydes (butanal, decanal, heptanal and hexanal) demonstrated small increases at approximately 30 minutes after tyrosine ingestion. Median Control Oesophageal Cancer Gastric Cancer Post- Post- Post- Baseline tyrosine: Baseline tyrosine: Baseline tyrosine: Concen- Concen- Concen- Concen- Concen- Concen- Time tration tration Fold tration tration Fold tration tration Fold point Increase/ P (ppbv) (ppbv) change (ppbv) (ppbv) change (ppbv) (ppbv) change (mins) decrease value * Acetaldehyde 40.93 40.44 1.04 22.73 22.3 1.02 29.43 23.93 0.82 25-35 — 0.0001 Butanal 5.45 5.16 0.92 4.57 4.03 1.18 5.16 4.22 0.74 25-35 ↑ 0.2016 Decanal 2.04 2.11 0.89 1.08 1.27 1.11 1.14 1.08 0.83 25-35 ↑ 0.0001 Heptanal 1.46 2.04 1.32 0.70 0.99 1.46 0.95 1.26 1.07 25-35 ↑ <0.0001 Hexanal 9.23 11.48 1.12 4.55 6.36 1.25 4.73 5.53 1.26 45 ↑ 0.0002 Nonanal 3.99 4.65 1.26 2.34 2.41 0.95 2.38 2.64 1.16 45 — <0.0001 Octanal 1.38 1.70 1.11 0.91 1.04 0.94 1.08 0.93 0.79 25-35 — 0.0002 Pentanal 2.26 3.21 0.95 1.66 1.26 0.76 2.63 2.91 1.11 25-35 — <0.0001 Propanal 17.98 17.12 1.01 8.52 9.05 0.94 11.70 10.51 0.85 25-35 — <0.0001 * Kruskal Wallis Analysis p < 0.05 considered statistically significant
[0254] Phenols
TABLE-US-00010 TABLE 10 Volatile phenols demonstrated a small increase in exhaled breath concentrations 35-45 minutes after tyrosine consumption (excluding 1-hydroxy-4-ethylbenzene). Median Control Oesophageal Cancer Gastric Cancer Post- Post- Post- Baseline tyrosine: Baseline tyrosine: Baseline tyrosine: Concen- Concen- Concen- Concen- Concen- Concen- Time tration tration Fold tration tration Fold tration tration Fold point Increase/ P (ppbv) (ppbv) change (ppbv) (ppbv) change (ppbv) (ppbv) change (mins) decrease value * 1-hydroxy-4- 0.59 0.58 1.24 0.59 0.48 0.99 0.32 0.61 1.06 35-45 — 0.0008 ethylbenzene Decane 5.59 8.12 1.22 3.27 3.5 1.16 3.3 4.65 1.27 35-45 ↑ <0.0001 Dodecane 1.21 1.21 1.03 0.56 0.63 1.32 0.74 0.93 1.28 35-45 ↑ 0.0001 P-cresol 1.24 1.59 0.90 0.92 1.18 1.12 1.72 2.70 1.10 35-45 ↑ <0.0001 Phenol 9.01 11.0 1.36 4.65 5.71 1.36 5.41 11.03 1.68 10 ↑ <0.0001 * Kruskal Wallis Analysis p < 0.05 considered statistically significant
TABLE-US-00011 TABLE 11 Mann Whitney U test comparing (i) Control vs. Gastric cancer groups and (ii) Control vs. Oesophageal cancer groups; p < 0.05 is considered statistically significant (highlighted bold) Control vs Gastric Ca Control vs Oesophageal Ca P value P value P value (Fold P value (Fold (ppbv) change) (ppbv) change) Fatty Acids Acetic acid 0.057 0.052 <0.001* <0.001* Butanoic acid 0.395 0.001* 0.017* <0.001* Hexanoic acid 0.001* 0.008* <0.001* 0.004* Pentanoic acid 0.041* 0.280 <0.001* <0.001* Propanoic acid 0.131 0.719 0.001* 0.016* Aldehydes Acetaldehyde <0.001* 0.019* <0.001* 0.139 Butanal 0.021* 0.003* 0.430 0.190 Decanal <0.001* 0.952 <0.001* 0.003 Heptanal <0.001* <0.001* <0.001* 0.741 Hexanal <0.001* 0.424 <0.001* 0.441 Nonanal <0.001* 0.056 <0.001* 0.080 Octanal <0.001* 0.049* <0.001* 0.010* Pentanal 0.052 0.042* <0.001* 0.006* Propanal <0.001* 0.358 <0.001* 0.276 Phenol-alkanes 1-hyroxy-4- <0.001* 0.667 0.006* 0.276 ethylbenzene Decane <0.001* 0.197 <0.001* 0.112 Dodecane <0.001* 0.023 <0.001* 0.230 P-cresol 0.006 0.294 0.003* 0.704 Phenol 0.005* 0.535 <0.001* 0.139 *significant increase in control group.
[0255] Discussion
[0256] Two chemical classes of volatile compounds (phenols and aldehydes) were detected in slightly higher concentrations 30 minutes after tyrosine consumption. A total of 8 compounds demonstrated small increases in concentrations in the oesophageal cancer group. The underlying biological and mechanistic pathway suggests tyrosine, an aromatic amino acid, is metabolised to phenolic compounds by enzymatic reactions initiated by gastrointestinal bacteria.
[0257] Volatile phenol compounds were detected at optimal concentrations at 35-45 minutes after tyrosine consumption, albeit small increases from the baseline values reported. Phenol and decane displayed a similar pattern of increase across groups, whereas P-cresol and dodecane concentrations were detected in slightly higher concentrations in the oesophageal cancer group. Volatile aldehydes, namely butanal, decanal, heptanal and hexanal demonstrated higher concentrations in the oesophageal cancer group is compared with controls (fold change 1.46 vs. 1.32). The overall baseline concentrations of all compounds were notably higher in the control group.
[0258] Decanal demonstrated the only significant fold increase in the oesophageal cancer group, supported by previous work by the inventors showing significantly higher baseline values of aldehydes (butanal, decanal) and phenols in OG cancer patients. [1, 2] Lack of corroboration with the inventor's previous findings of baseline concentrations may be attributed to obtaining the results from oesophageal and gastric cancer groups separately and the small patient numbers which needs to be further explored. However, the selected volatile compounds show overall higher responses to tyrosine by fold change in the cancers, albeit not significant.
[0259] Short chain fatty acids concentrations from the cancer cohort were not affected by tyrosine.
[0260] These results suggest the potential for the augmentation of breath VOCs with oral metabolic substrates acting via the shikimate pathway. In the next phase of the study, the inventors intend to use phenylalanine, a precursor to tyrosine, in addition to tyrosine, as a combination drink. Without wishing to be bound to any particular theory, the inventors propose measuring breath VOC concentrations between 30-45 minutes after ingestion to detect any potential changes with the addition an amino acid.
[0261] Key Points: [0262] Decanal, from the aldehyde family, demonstrates a significant fold increase after tyrosine consumption in the oesophageal cancer group. [0263] Aldehyde and phenol compounds show a slightly higher fold change from baseline values, albeit not significant. [0264] The significantly higher baseline aldehyde and phenol values in the control groups needs to be further explored. [0265] Volatile phenol compounds were detected at optimal concentrations at 35-45 minutes after tyrosine consumption.
EXAMPLE 3
Phenylalanine
[0266] Results and Discussion
[0267] Phenylalanine is an essential amino acid, a known precursor to other amino acids such as tyrosine. Metabolism via the shikimate pathway is expected to produce volatile phenol compounds. Three compounds from the phenol family (dodecane, decane and phenol) demonstrated increasing concentrations after phenylalanine consumption (
EXAMPLE 4
Glutamic Acid
[0268] Results and Discussion
[0269] Three compounds from the aldehyde and phenol family demonstrated an increase in VOC concentrations after glutamic acid consumption (
[0270] Without wishing to be bound to any particular theory, the inventors believe that in combination with other amino acids tested, phenylalanine and tyrosine, an augmented VOC response may be produced, in particular with consistent compounds already identified across the groups: dodecane, phenol.
EXAMPLE 5
Glycerol Doses
[0271] Results
[0272] Subjects
[0273] Two subjects were recruited with an average age of 32 years; 1 female vs 1 male. No significant co-morbidities were noted.
[0274] Volatile Organic Compound Analysis
TABLE-US-00012 TABLE 12 Concentrations of short chain fatty acids detected in the exhaled breath of each subject at 45-55 minutes. Concentration (ppbv) Fold change Baseline 25 g 50 g Baseline 25 g 50 g Subject 1 Acetic acid 37.4 19.3 53.2 1.8 1.2 2.7 Butanoic acid 4.0 4.0 10.2 0.9 1.4 2.5 Hexanoic acid 0.6 0.4 1.3 0.7 0.3 0.7 Pentanoic acid 2.0 0.7 4.9 1.2 0.4 1.9 Propanoic acid 12.6 13.4 38.1 1.8 1.1 4.1 Subject 2 Acetic acid 25.4 37.9 22.2 0.9 0.6 1.1 Butanoic acid 6.6 13.0 7.2 0.9 1.3 1.5 Hexanoic acid 1.7 2.2 2.7 0.9 0.6 0.9 Pentanoic acid 7.3 9.0 4.4 1.1 1.1 1.3 Propanoic acid 14.7 — 16.0 0.7 — 2.0
[0275] Discussion
[0276] Increasing glycerol concentrations translates to increased production of volatile fatty acids detected within the exhaled breath. Volatile fatty acid concentrations from 25 g glycerol are comparable to the baseline values. Butanoic acid concentrations are elevated after 30 minutes of glycerol ingestion, with maximal concentrations detected at 45-55 minutes. Glycerol is a polyol compound found in lipids and is metabolised via the glycolytic pathway by (i) direct entry into the pathway or (ii) be converted to glucose by gluconeogenesis. The glucose study demonstrated maximal fatty acid detection at 5-10 minutes after glucose consumption, and therefore it is expected responses after glycerol ingestion are delayed as it may require further enzymatic reactions before entry into the cycle. The inventors propose using a dose of 50 g to elicit a fatty acid VOC response within the breath of patients diagnosed with OG cancer compared to healthy age-matched controls.
EXAMPLE 6
Glycerol
[0277] Results
[0278] Patients
[0279] Eighteen patients were recruited (n=6 within each group; oesophageal cancer, gastric cancer, healthy controls). All cancers included were histologically confirmed as adenocarcinomas.
TABLE-US-00013 TABLE 13 Demographics and clinical information of participants. Oesophageal Gastric Cancer Cancer Controls (n = 6) (n = 6) (n = 6) Age (years) * 72.5 60 64.5 Male 6 4 1 Ethnicity White 6 2 4 Asian 0 1 1 Black 0 0 0 Arabic 0 3 1 Metastatic disease 1 2 — Neoadjuvant therapy 6 2 — Co-morbidities Diabetes 0 Benign UGI disease 2 Healthy 4 * median
[0280] Volatile Organic Compound Analysis
[0281] Short Chain Fatty Acids
TABLE-US-00014 TABLE 14 Volatile short chain fatty acids (acetic-, butanoic-, propanoic acid) increased maximally at 60 mins after glycerol consumption. Median Control Oesophageal Cancer Gastric Cancer Post- Post- Post- Baseline glycerol: Baseline glycerol: Baseline glycerol Concen- Concen- Concen- Concen- Concen- Concen- Time tration tration Fold tration tration Fold tration tration Fold point Increase/ P (ppbv) (ppbv) change (ppbv) (ppbv) change (ppbv) (ppbv) change (mins) decrease value* Acetic acid 35.68 26.90 0.62 54.77 101.35 1.50 20.99 33.60 1.09 60 ↑ 0.007 Butanoic acid 7.46 8.59 1.12 10.55 18.93 2.02 4.99 8.67 1.43 60 ↑ <0.001 Hexanoic acid 1.56 1.13 0.52 2.04 1.43 0.49 1.95 0.98 0.70 60 — 0.002 Pentanoic acid 3.53 3.75 0.83 5.49 7.87 1.05 2.86 2.49 0.86 60 — <0.001 Propanoic acid 24.70 26.95 0.72 45.95 107.93 1.75 10.19 19.07 1.46 60 ↑ <0.001 *Kruskai Wallis Analysis p < 0.05 considered statistically significant
[0282] Aldehydes
TABLE-US-00015 TABLE 15 Volatile aldehydes were maximally increased for the oesophageal cancer group between 40-55 minutes (hexanal, octanal, pentanal, propanal). Median Control Oesophageal Cancer Gastric Cancer Post- Post- Post- Baseline glycerol: Baseline glycerol: Baseline glycerol Concen- Concen- Concen- Concen- Concen- Concen- Time tration tration Fold tration tration Fold tration tration Fold point Increase/ P (ppbv) (ppbv) change (ppbv) (ppbv) change (ppbv) (ppbv) change (mins) decrease value * Acetaldehyde 25.47 32.05 0.88 51.00 19.30 0.80 23.72 17.85 0.83 60 — <0.001 Butanal 3.39 4.44 0.87 9.02 6.49 1.00 2.78 2.21 1.05 60 — <0.001 Decanal 1.31 1.18 0.97 2.53 1.46 0.77 1.17 0.95 1.05 60 — <0.001 Heptanal 2.28 1.33 0.58 2.48 1.40 0.76 1.60 1.20 0.65 60 — <0.001 Hexanal 6.28 6.93 1.17 7.77 11.56 1.72 4.42 5.52 1.09 40 ↑ <0.001 Nonanal 3.07 2.97 0.98 3.62 3.71 1.09 3.36 2.93 1.09 60 — <0.001 Octanal 1.49 1.35 0.95 1.91 1.68 1.58 1.25 1.39 0.87 40 ↑ <0.001 Pentanal 2.62 1.39 0.63 2.37 3.71 1.27 1.30 1.63 1.46 55 ↑ <0.001 Propanal 15.77 12.51 0.77 16.93 17.83 1.71 7.38 5.67 0.98 55 ↑ <0.001 * Kruskal Wallis Analysis p < 0.05 considered statistically significant
[0283] Phenols
TABLE-US-00016 TABLE 16 Volatile phenols demonstrated no alterations in exhaled breath concentrations between the three patient groups. Median Control Oesophageal Cancer Gastric Cancer Post- Post- Post- Baseline glycerol: Baseline glycerol: Baseline glycerol Concen- Concen- Concen- Concen- Concen- Concen- Time tration tration Fold tration tration Fold tration tration Fold point Increase/ P (ppbv) (ppbv) change (ppbv) (ppbv) change (ppbv) (ppbv) change (mins) decrease value * 1-hydroxy-4- 0.72 1.09 1.47 1.14 0.88 0.99 0.56 0.56 1.47 60 — — ethylbenzene Decane 5.33 4.61 0.92 6.54 7.33 1.13 5.83 4.89 0.81 60 — — Dodecane 1.03 0.75 0.86 1.02 1.30 1.09 0.84 1.05 0.95 60 — — P-cresol 1.71 1.46 0.71 2.23 2.41 1.04 0.96 0.83 0.98 60 — — Phenol 4.21 2.64 1.43 4.73 7.71 1.36 4.18 3.68 0.98 60 — — * Kruskal Wallis Analysis p < 0.05 considered statistically significant
TABLE-US-00017 TABLE 17 Mann Whitney U test comparing (i) Control vs Gastric cancer groups and (ii) Control vs Oesophageal cancer groups; p < 0.05 is considered statistically significant Control vs Gastric Ca Control vs Oesophageal Ca P value P value P value (Fold P value (Fold (ppbv) change) (ppbv) change) Fatty Acids Acetic acid 0.103 <0.001 0.005 0.070 Butanoic acid 0.258 0.012 <0.001 0.008 Hexanoic acid 0.147 0.711 <0.001 0.435 Pentanoic acid <0.001 0.779 <0.001 0.029 Propanoic acid 0.242 <0.001 <0.001 0.005 Aldehydes Acetaldehyde <0.001 0.112 0.056 0.009 Butanal <0.001 <0.001 <0.001 0.053 Decanal 0.048 <0.001 <0.001 0.803 Heptanal <0.001 0.271 0.503 0.459 Hexanal 0.005 0.833 <0.001 0.582 Nonanal 0.107 0.060 <0.001 0.322 Octanal 0.043 0.008 <0.001 <0.001 Pentanal 0.001 <0.001 <0.001 <0.001 Propanal <0.001 <0.001 <0.001 <0.001 Phenol-alkanes 1-hyroxy-4- 0.008 0.503 0.009 0.569 ethylbenzene Decane 1.000 0.944 <0.001 0.056 Dodecane 0.056 0.003 <0.001 <0.001 P-cresol 0.003 0.002 <0.001 0.002 Phenol 0.222 0.764 0.014 0.682
[0284] Discussion
[0285] Three chemical classes of VOCs within exhaled breath have demonstrated a significant increase in patients diagnosed with oesophago-gastric (OG) cancer. Glycerol is a polyol compound found in lipids and is metabolised via the glycolytic pathway by (i) direct entry into the pathway or (ii) converted to glucose by gluconeogenesis. The glucose study demonstrated maximal fatty acid detection at 5-10 minutes after glucose consumption, and therefore it is expected elevated responses after glycerol ingestion may be delayed further as enzymatic reactions may be required before entry into the cycle.
[0286] In keeping with the hypothesis, we observed elevation in short chain fatty acids (SCFA) and aldehydes levels between 45-60 minutes after glycerol consumption, as illustrated in
[0287] Similarly, select aldehydes were found to increase largely in the oesophageal cancer group (
[0288] A number of the volatile phenols tested demonstrated no significant alterations in exhaled breath concentrations between the three patient groups after glycerol consumption (
[0289] Glycerol consumption has uniquely increased target VOCs in the oesophageal cancer group. Potentially this may be due to the higher viscosity of the fluid coating the oesophagus allowing more than a transient passage. Increased contact time between the substrate and the tumour may explain the higher VOC levels produced.
[0290] Key Points:
[0291] Glycerol consumption activates the glycolytic metabolic pathway associated tumour-microbiome or increased activity of the tumour cell. This is detected by: [0292] A significant fold increase in SCFA (acetic-, butanoic-, and propanoic acid); more so observed in the oesophageal cancer group than the gastric cancer group. [0293] A significant increase in aldehydes; hexanal, octanal, pentanal and propanal in the oesophageal cancer. An increase in pentanal was observed with gastric cancer.
EXAMPLE 7
Combined Amino Acids (Tyrosine, Phenylalanine, Glutamic Acid)
[0294] Results
[0295] Patients
[0296] Thirteen patients were recruited (oesophageal cancer n=6, gastric cancer n=1, healthy controls n=6). All cancers included were histologically confirmed as adenocarcinomas.
TABLE-US-00018 TABLE 18 Demographics and clinical information of participants. Oesophagogastric Cancer Controls (n = 7) (n = 6) Age (years) * 65 70 Male 6 3 Ethnicity White 6 6 Asian 1 0 Black 0 0 Arabic 0 0 Metastatic disease 4 — Neoadjuvant therapy 5 — Co-morbidities Diabetes 2 0 Benign UGI disease — 2 Healthy — 0 * median
[0297] Volatile Organic Compound Analysis
[0298] Short Chain Fatty Acids
TABLE-US-00019 TABLE 19 Volatile short chain fatty acids were not altered after amino acid consumption. Median Control Oesophagogastric Cancer Post-amino Post-amino Baseline acids: Baseline acids: Time Concentration Concentration Fold Concentration Concentration Fold point Increase/ P value* P value (ppbv) (ppbv) change (ppbv) (ppbv) change (mins) decrease ppbv Fold change Acetic acid 25.65 24.74 0.67 24.57 29.15 0.99 30 — — — Butanoic acid 5.10 3.74 0.88 8.13 7.76 0.15 30 — — — Hexanoic acid 0.71 0.65 0.94 1.10 0.89 0.80 30 — — — Pentanoic acid 2.08 1.74 1.01 2.24 1.53 0.84 30 — — — Propanoic acid 13.77 9.95 1.10 11.70 11.46 0.87 30 — — — *Mann Whitney U analysis p < 0.05 considered statistically significant
[0299] Aldehydes
TABLE-US-00020 TABLE 20 Volatile decanal demonstrated an increase in the oesophagogastric cancer group at 30 minutes after consumption of the combined amino acid drink. Control Oesophagogastric Cancer Post-amino Post-amino Baseline acids: Baseline acids: Time Concentration Concentration Fold Concentration Concentration Fold point Increase/ P value* P value* (ppbv) (ppbv) change (ppbv) (ppbv) change (mins) decrease ppbv Fold change Acetaldehyde 16.95 16.08 0.93 30.05 19.80 0.68 30 — — — Butanal 2.39 1.73 1.01 2.48 2.42 1.08 30 — — — Decanal 0.64 0.59 1.05 0.69 0.83 1.41 30 ↑ <0.001 <0.001 Heptanal 1.23 0.75 1.10 1.20 0.94 0.81 30 — — — Hexanal 2.73 3.16 1.31 2.52 3.42 1.24 30 — — — Nonanal 1.68 1.88 0.98 2.21 2.55 1.16 30 — — — Octanal 0.82 0.56 0.90 0.88 0.59 0.78 30 — — — Pentanal 1.06 0.72 1.00 0.98 1.14 0.97 30 — — — Propanal 12.15 9.54 0.95 8.55 8.78 1.06 30 — — — *Mann Whitney U analysis p < 0.05 considered statistically significant
[0300] Phenol-Alkanes
TABLE-US-00021 TABLE 21 Volatile phenols demonstrated an increase of p-cresol in exhaled breath concentrations between the cancer and non-cancer groups. The increase in phenol concentrations were comparable between both groups. Control Oesophagogastric Cancer Post-amino Post-amino Baseline acids: Baseline acids: Time Concentration Concentration Fold Concentration Concentration Fold point Increase/ P value * P value * (ppbv) (ppbv) change (ppbv) (ppbv) change (mins) decrease ppbv Fold change 1-hydroxy-4- 0.64 0.44 0.98 0.65 0.45 0.87 30 — 0.289 0.003 ethylbenzene Decane 3.59 4.45 1.25 3.87 4.78 1.44 30 ↑ 0.303 0.066 Dodecane 0.52 0.92 1.38 0.76 0.77 1.09 30 — 0.147 0.089 P-cresol 1.26 1.22 0.84 0.93 1.25 1.37 40 ↑ 0.073 <0.001 Phenol 3.15 4.82 1.79 1.59 2.27 1.83 30 ↑ <0.001 0.051 * Mann Whitney U analysis p < 0.05 considered statistically significant
[0301] Discussion
[0302] Two chemical classes, aldehydes and phenol-alkanes, demonstrated an increase in volatile organic compound levels after the consumption of three combined amino acids, as illustrated in
[0303] Decanal, an aldehyde, demonstrated a more significant increase in detected levels with this combination amino acid drink (
[0304] Phenol-alkanes are the primary target of this nutrient group. Pathways have been detailed describing the metabolism of tyrosine by tyrosine phenol lyase to produce phenols. More recently, Saito et al have delineated a pathway involving metabolism by the enzyme tyrosine lyase to produce p-cresol. This metabolic pathway has been proven within bacteria, not the human cells [4]. P-cresol was significantly increased at 40 minutes after consumption of the amino acid drink from baseline levels of 0.93 ppbv to 1.25 ppbv translating to a 1.37-fold increase. No change was observed in the control group. Phenol showed a global increase across both cancer (1.79-fold increase) and non-cancer groups (1.83-fold increase), with no significant differences between the two. Decane also increased at 30 minutes with a 1.44-fold increase in the cancer group.
[0305] There were no significant alterations in the remainder of the aldehydes and short chain fatty acid groups. Further work needs to be done to explain the increase in decanal.
[0306] Key Points:
[0307] Consumption of combined amino acids potentially activates a metabolic pathway associated with bacteria. This is detected with: [0308] A significant fold increase in decanal (aldehyde). [0309] A global increase in phenol (enzyme tyrosine phenol lyase), with no differences between cancer and non-cancer groups. [0310] A new finding of a significant increase in P-cresol, potentially produced by the enzymatic metabolism using tyrosine lyase.
EXAMPLE 8
Combined Glucose and Citric Acid
[0311]
TABLE-US-00022 TABLE 22 Demographics and clinical information of participants. Control group 1 Control group 2 (glucose only) (glucose + citric acid) (n = 6) (n = 6) Age (years) * 69 67.5 Male 3 2 Ethnicity White 5 5 Asian 1 0 Black 0 1 Arabic 0 0 Co-morbidities Diabetes 0 0 Benign UGI disease 2 3 Healthy 4 3 * median
[0312] Volatile Organic Compound Analysis
[0313] Short Chain Fatty Acids
TABLE-US-00023 TABLE 23 Volatile short chain fatty acids (butanoic- and propanoic acid) had increased concentrations detected in control group 2 (glucose + citric acid). Median Control 1 (glucose only) Control 2 (glucose + citric acid) Baseline Post-drink: Baseline Post-drink: Time Concentration Concentration Fold Concentration Concentration Fold point Increase/ P value* P value (ppbv) (ppbv) change (ppbv) (ppbv) change (mins) decrease ppbv Fold change Acetic acid 38.03 31.37 0.66 19.02 17.93 0.94 5 — <0.001 0.032 Butanoic acid 5.57 9.64 2.72 2.88 14.90 5.36 5 ↑ 0.960 0.093 Hexanoic acid 0.64 0.52 0.87 1.06 0.63 0.62 5 — 0.008 0.246 Pentanoic acid 2.45 2.38 0.73 3.11 2.65 0.99 5 — 0.003 0.142 Propanoic acid 15.89 17.14 1.17 8.19 15.33 1.96 5-10 ↑ 0.024 0.093 *Mann Whitney U analysis p < 0.05 considered statistically significant
[0314] Aldehydes
TABLE-US-00024 TABLE 24 Volatile decanal demonstrated an increase in the oesophagogastric cancer group at 30 minutes after consumption of the combined amino acid drink. Median Control 1 (glucose only) Control 2 (glucose + citric acid) Baseline Post-drink: Baseline Post-drink: Time Concentration Concentration Fold Concentration Concentration Fold point Increase/ P value* P value* (ppbv) (ppbv) change (ppbv) (ppbv) change (mins) decrease ppbv Fold change Acetaldehyde 16.02 15.25 1.04 19.72 21.09 0.94 5 — — — Butanal 2.46 2.21 1.03 2.82 2.37 0.87 5 — — — Decanal 0.66 0.59 0.97 0.87 0.77 1.13 5 — — — Heptanal 0.60 0.53 0.86 1.80 1.22 0.77 5 — — — Hexanal 2.71 3.09 1.06 4.04 4.86 1.07 5 — — — Nonanal 1.07 1.56 1.23 2.30 2.49 1.08 5 — — — Octanal 0.43 0.58 1.40 1.43 1.12 0.81 5 — — — Pentanal 1.17 1.05 1.00 1.32 1.43 0.89 5 — — — Propanal 9.67 9.29 1.06 6.96 9.41 1.29 10-15 ↑ 0.001 0.912 *Mann Whitney U analysis p < 0.05 considered statistically significant
[0315] Discussion
[0316] Two chemical classes, short chain fatty acids (SCFA) and aldehydes, demonstrated an increase in volatile organic compound levels after the consumption of glucose and citric acid combined, as can be seen in
[0317] Butanoic acid demonstrated the largest increase from 2.72-fold with glucose alone to 5.36-fold with the addition of citric acid (
[0318] Propanal appeared to be the only aldehyde to show an increase of 1.29-fold in the citric acid group within 10-15 minutes, albeit a small change (
[0319] Clear alterations in VOCs have been shown in two control groups, with citric acid as the differentiating factor. We hypothesis these nutrients may feed into the glycolytic and citric acid intrinsic metabolic pathways.
[0320] Key Points:
[0321] Consumption of combined glucose and citric acid activates known metabolic pathways associated with cell metabolism. This is detected by: [0322] A significant fold increase in volatile short chain fatty acids (butanoic- and propanoic acid) within 5-10 minutes of consumption of the nutrient drink. [0323] A significant fold increase in propanal within 10-15 minutes.
[0324] The next steps will involve recruiting patients with oesophago-gastric cancer to assess breath VOC alterations in response to additional nutritional substrates.
REFERENCES
[0325] 1. Markar, S. R., et al., Assessment of a Noninvasive Exhaled Breath Test for the Diagnosis of Oesophagogastric Cancer. JAMA Oncol, 2018. 4(7): p. 970-976.
[0326] 2. Kumar K, H. J., Abbassi-Ghadi N, Mackenzie H A, Veselkov K A, Hoare J M, Lovat L B, Spanel P, Smith D and Hanna G B, 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.
[0327] 3. Excellence, N. I. o. C., Gastrointestinal tract (upper) cancers—recognition and referral. 2016.
[0328] 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)