DETECTION OF BIOMARKERS
20210318319 · 2021-10-14
Inventors
Cpc classification
International classification
Abstract
The invention relates to the detection of biomarkers, and methods, compositions and kits for the detection of such biological markers for diagnosing various conditions, such as cancer. In particular, the invention relates to the detection of compounds as diagnostic and prognostic markers for detecting cancer, such as oesophago-gastric cancer.
Claims
1. A method for treating a subject suffering from cancer, the method comprising: (i) detecting, in a bodily sample from a test subject, the concentration of a signature compound resulting from the metabolism, by a cancer-associated microorganism, of at least one substrate in a composition previously administered to the subject; and (ii) comparing the concentration of the signature compound with a reference for the concentration of the signature compound in an individual who does not suffer from the cancer, wherein an increase or a decrease in the concentration of the signature compound compared to the reference, indicates that the subject is suffering from the cancer; and (iii) administering a therapeutic agent capable of treating the cancer to the test subject whose concentration of the signature compound in the bodily sample indicates that the subject is suffering from the cancer.
2. The method according to claim 1, further comprising providing the subject with the composition comprising the at least one substrate which is suitable for metabolism by the cancer-associated microorganism into the signature compound.
3. (canceled)
4. The method according to claim 1, wherein the cancer-associated microorganism is associated with oesophago-gastric junction cancer, gastric cancer, oesophageal cancer, oesophageal squamous-cell carcinoma (ESCC), or oesophageal adenocarcinoma (EAC), and wherein the cancer is oesophago-gastric junction cancer, gastric cancer, oesophageal cancer, oesophageal squamous-cell carcinoma, or oesophageal adenocarcinoma.
5. (canceled)
6. The method according to claim 1, wherein the composition comprising the at least one substrate, which is suitable for metabolism by the cancer-associated microorganism into the signature compound, is ingestable by the subject.
7. The method according to claim 1, wherein the composition comprising the at least one substrate is: (i) in the form of a capsule that is designed to degrade at a certain position with the gastrointestinal tract, thereby offering targeted release of the at least one substrate; or (ii) is a solid, foodstuff, fluid or liquid, which is swallowed.
8. The method according to claim 1, wherein the at least one substrate is selected from a group consisting of: acetic acid, ethanol, lactic acid, lactate, glutamate, glycerol, D-glucose, D-sucrose, D-lactose, D-fructose, D-mannose, D-gulose, D-galactose, D-Xylose, D-arabinose, D-lyxose, D-ribose, L-arabinose, L-rhamnose, L-xylulose, di-, tri-oligo and poly-saccharides, c4, c7 and >c8 monosaccharides, pyruvic acid, ascorbic acid, malic acid, citric acid, succinic acid, fumaric acid, oxalic acid, tannic acid, tartaric acid, sorbitol, mannitol, maltitol, lactitol, erythritol, palmitic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, triglycerides, glycolipids, any or all of the 20 proteinogenic amino acids, 2-amino butyric acid, ornithine, canavanine, homoarginine, artificial sweeteners, stevia, aspartame, sucralose, preservatives, E numbers, nitrate, and small molecule inducers.
9. The method according to claim 1, wherein the composition comprises one or more substrates selected from the group consisting of: glucose, sorbitol, lactose tyrosine, glutamic acid, glycerol, citric acid and acetic acid, or any combination thereof.
10. The method according to claim 1, wherein the cancer-associated microorganism is a bacterium.
11. The method according to claim 1, wherein the cancer-associated microorganism forms part of the microbiome of the test subject.
12. The method according to claim 1, wherein the cancer-associated microorganism is Streptococcus, Lactobacillus, Veillonella, Prevotella, Neisseria, Haemophilus, L. coleohominis, Lachnospiraceae, Klebsiella, Clostridiales, Erysipelotrichales, or any combination thereof.
13. The method according to claim 1, wherein the cancer-associated microorganism is S. pyogenes, Klebsiella pneumoniae, Lactobacillus acidophilus, or any combination thereof.
14. The method according to claim 1, wherein the cancer-associated microorganism is E. coli, P. mirabili, B. cepacia, Streptococcus salivarius, Streptococcus anginosus, S. pyogenes, Actinomyces naeslundii, Lactobacillus fermentum, 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 thereof.
15. The method according to claim 1, wherein the signature compound is a volatile organic compound (VOC).
16. The method according to claim 15, wherein the volatile organic compound (VOC) is selected from a group consisting of: butyric acid, gamma amino butyric acid, caproic acid, hydrogen sulphide, pentanol, propanoic acid, acetic acid, 1,2-propanediol, ethanol, and 3-hydroxypropionic acid, or any combination thereof, optionally acetone, acetic acid, butyric acid, pentanoic acid, hexanoic acid, phenol, ethyl phenol, acetaldehyde, or any combination thereof, or hexanoic acid, pentanoic acid, acetic acid, 2 ethyl phenol, or any combination thereof.
17. The method according to claim 1, wherein the VOC is selected from a group consisting of: aldehydes, fatty acids, and alcohols, or any combination thereof.
18. The method according to claim 1, wherein the cancer is oesophageal squamous-cell carcinoma, wherein the composition comprises a substrate selected from acetic acid and/or ethanol, which is metabolised to a signature compound selected from butyric acid and/or caproic acid, optionally which is analysed to indicate the presence of Clostridium spp.
19. The method according to claim 1, wherein the cancer is gastric cancer, wherein the composition comprises a substrate which is lactic acid, which is metabolised to a signature compound selected from acetic acid, 1,2-propanediol, and/or ethanol, optionally which is analysed to indicate the presence of Lactobacillus spp.
20. The method according to claim 1, wherein the cancer is oesophago-gastric cancer, wherein the composition comprises a substrate which is glutamate, which is metabolised to a signature compound which is gamma amino butyric acid, optionally which is analysed to indicate the presence of Lactococcus spp. or Clostridium spp.
21. The method according to claim 1, wherein the cancer is gastric cancer, wherein the composition comprises a substrate which is glycerol, which is metabolised to a signature compound which is 3-hydroxypropionic acid, optionally which is analysed to indicate the presence of Klebsiella spp.
22. (canceled)
23. (canceled)
24. (canceled)
25. The method according to claim 1, wherein the composition comprises: (i) glucose at a concentration of between about 7000 mg/100 mL and 20000 mg/100 mL, or between about 9000 mg/100 mL and 17000 mg/100 mL, or between about 11000 mg/100 mL and 15000 mg/100 mL; (ii) lactose at a concentration of between about 7000 mg/100 mL and 20000 mg/100 mL, or between about 9000 mg/100 mL and 17000 mg/100 mL, or between about 11000 mg/100 mL and 15000 mg/100 mL; (iii) sorbitol at a concentration of between about 1000 mg/100 mL and 6000 mg/100 mL, or between about 2000 mg/100 mL and 5000 mg/100 mL, or between about 3000 mg/100 mL and 4000 mg/100 mL; (iv) tyrosine at a concentration of between about 25 mg/100 mL and 500 mg/100 mL, or between about 50 mg/100 mL and 400 mg/100 mL, or between about 100 mg/100 mL and 300 mg/100 mL; (v) glutamic acid at a concentration of between about 500 mg/100 mL and 5000 mg/100 mL, or between about 1000 mg/100 mL and 3500 mg/100 mL, or between about 1500mg/100mL and 2500mg/100mL; (vi) glycerol at a concentration of between about 10000 mg/100 mL and 30000 mg/100 mL, or between about 14000 mg/100 mL and 25000 mg/100 mL, or between about 17000 mg/100 mL and 22000 mg/100 mL; (vi) citric acid at a concentration of between about 500 mg/100 mL and 3000 mg/100 mL, or between about 1000 mg/100 mL and 2000 mg/100 mL, or between about 1200 mg/100 mL and 1700 mg/100 mL; and/or (vii) acetic acid at a concentration of between about 200 mg/100 mL and 1500 mg/100 mL, or between about 400 mg/100 mL and 1000 mg/100 mL, or between about 600 mg/100 mL and 800 mg/100 mL.
26. (canceled)
27. (canceled)
Description
EXAMPLES
Example 1
Microbial VOCs Produced in Response to Particular Stimulants found in Food
[0082] Table 1 shows VOCs that can be detected to indicate the presence of specific bacteria, and the studies showing this association.
TABLE-US-00001 TABLE 1 Studies showing the association between selected VOCs and bacteria Selected VOCs and Associated Bacteria VOC Bacteria Identified Identification Methodology Reference Butryic E. coli, P. mirabili, B. cepacia, S. pyogenes SIFT-MS, headspace of monoculture 1 Acid after 5 hours incubation (24 for EF) Actinomyces naeslundii, Clostridium bifermentans, Head-space solid phase microextraction 3 Clostridium perfringens, Clostridium septicum, combined with gas chromatography Clostridium sporogenes, Clostridium tertium, cultivated on Vf medium sampled as 1 ml Eubacterium lentum, Eubacterium sp., Fusobacterium simiae, Fusobacterium necrophorum, Lactobacillus acidophilus, Peptococcus niger, Peptostreptococcus anaerobius, Peptostreptococcus asaccharolyticus, Peptostreptococcus prevotii Hydrogen P. aeruginosa, S. aureus, E. coli, P mirabilis SIFT-MS, headspace of monoculture 1 Sulphide B. cepacia, E. faecalis S. pneumoniae, E. coli, N. meningitidis SIFT-MS, blood infected headspace, 2 anaerobic conditions for 24 hours Pentanol S. aureus, E. coli, P. mirabili, B. cepaci, SIFT-MS, headspace of monoculture 1 S. pyogenes, E. faecalis Propanoic Acinetobacter baumannii, Actinomyces naeslundii Head-space solid phase microextraction 3 Acid Actinomyces naeslundii, Bacteroides capillosus, combined with gas chromatography Bacteroides fragilis, Bacteroides pyogenes, cultivated on Vf medium sampled as 1 ml Clostridium bifermentans, Clostridium difficile, Clostridium perfringens, Clostridium ramosum Clostridium septicum, Clostridium sporogenes, Clostridium tertium, Escherichia coli, Enterobacter cloacae, Eubacterium lentum, Eubacterium sp., Fusobacterium simiae, Fusobacterium necrophorum, Klebsiella pnuemoniae Lactobacillus acidophilus, Nocardia sp. Peptostreptococcus anaerobius, Peptostreptococcus prevotii Propionibacterium acnes, Propionibacterium propionicum
[0083] The following provides examples of bacteria whose presence can be indicated by the detection of signature compounds, and examples of the substrates that can be fed to the bacteria to increase the concentration of the signature compounds.
[0084] Clostridium spp. can be detected by initially feeding the bacteria with substrate compounds, acetic acid and/or ethanol, which are metabolised into signature compounds which are detectable. Excess acetic acid produces butyric acid, and excess ethanol produces caproic acid. These signature compounds can be measured to thereby detect the presence of Clostridium spp.
[0085] Lactobacillus spp. can be similarly detected by feeding it first with the substrate, lactic acid, which is converted into acetic acid, 1,2-propanediol, and ethanol. These signature compounds can be measured to detect the presence of Lactobacillus spp.
[0086] Lactobacillus, Clostridia and other bacteria can be detected by feeding with glutamate. Glutamate is converted to gamma amino butyric acid. This signature compound can be measured to detect the presence of Lactococcus spp., Clostridium spp., and others.
[0087] Klebsiella spp. can be detected by feeding with glycerol. Glycerol is metabolised to 3-hydroxypropionic acid, and this signature compound can be measured to detect the presence of Klebsiella spp.
Example 2
An Augmented Microbiome-Mediated Breath Test for the Earlier Diagnosis of Oesophago-Gastric Cancer (AMBEC)
[0088] The inventors have developed a non-invasive test for oesophago-gastric adenocarcinoma (specificity 81%/sensitivity 80%) based on the detection of volatile organic compounds (VOCs) in exhaled breath. The inventors have improved the accuracy of this test by means of an oral drink which induces the cancer-associated microbiome to produce greater quantities of the distinctive VOCs and thereby allow patients with non-specific symptoms, yet at a high-risk of oesophago-gastric cancer, to be referred faster and earlier for treatment. The oral drink (oral stimulant drink-OSD) selectively ‘feeds’ the cancer-associated microbiome with substances that it will preferentially metabolise to generate quantifiably higher levels of distinctive VOCs. Briefly, the patient is fasted for at least 4 hours and then, while at rest for 20 minutes, breathes into a bag or using breath collection device (such as ReCIVA—see below) that concentrate the volatile compounds into a thermal desorption tube. Breath samples will be analysed.
[0089] The test of the invention could be offered immediately by a medical professional in a similar manner to a routine blood test, thus avoiding the need to “watch-and-wait” to see if a patient's symptoms worsen.
[0090] The test is intended to be performed by a medical professional, who would then send breath samples to a laboratory for analysis. A positive result would warrant immediate referral for endoscopy. A negative result would permit the medical professional to reassure the patient and offer retesting if symptoms persist.
[0091] One example of the invention is referred to as AMBEC (an Augmented Microbiome-mediated Breath Test for the Earlier Diagnosis of Oesophago-gastric Cancer). The target population for testing with AMBEC is patients with upper gastrointestinal symptoms attending GP practices. AMBEC is a highly patient-friendly non-invasive test that will enable both earlier and faster diagnosis, and will substantially mitigate rising pressures on central diagnostic endoscopy.
[0092] Patients are given an oral drink (oral stimulant drink—OSD) to stimulate VOC production by the oesophago-gastric cancer-associated microbiome. The OSD is administered and a breath test is undertaken at 30 minute-intervals for 2 hours.
[0093] Breath is collected by a low-cost device and analysed in regional laboratories using automated standard mass-spectrometry equipment, such as the apparatus shown in
[0094] Referring to
[0095] Breath was collected using 500 ml inert aluminium bags that were washed through with synthetic air prior to sampling. Patients were asked to perform deep nasal inhalation followed by complete exhalation through the mouth into secure GastroCHECK steel breath bag. Alveolar air was preferentially collected over dead space air by capturing end-expiratory breath. VOCs from breath bags were then pre-concentrated (see Figure i) onto thermal desorption tubes by transferring 250 ml of breath at 50 ml sec across the tubes with comm diameter tubing and hand-held air pumps (210-1002 MTX, SKC ltd., Dorset, UK).
[0096] Patients are fasted for a minimum of four hours prior to breath sample collection. All breath samples are collected prior to endoscopy or surgery.
[0097] Exhaled breath analysis can be performed using GC-MS as the standard identification technique, and PTR-TOF-MS as the quantitative technique with a Time-of-Flight analyser to guarantee cutting-edge performance in terms of mass and time resolution.
Example 3
Studies Showing the Association between Cancers and Bacteria
[0098] The studies shown in Table 2 indicate bacteria that have been shown to be associated with particular cancer types. Hence, enabling the diagnosis of these cancers by the detection of these bacteria, for example in a patient's microbiome.
TABLE-US-00002 TABLE 2 Bacteria associated with cancer Author and year Analytical Cancer Cancer Cancer Healthy of Publication Platform site type (N) (N) Bacteria Johan Dicksved 16sRNA Gastric GC 6 15 Streptococcus Microbiology Lactobacillus 2009, DOI: Veillonella 10.1099/ Prevotella jmm.0.007302-0. Neisseria Haemophilus Francisco 16sRNA Gastric GC 5 5 Streptococcus Scientific Reports Lactobacillus 2014, DOI: Veillonella 10.1038/srep04202. L. coleohomini Lachnospiraceae Chang Soo Eun 16sRNA Gastric GC 11 10 Streptococcus Helicobacter Lactobacillus 2014, DOI: Veillonella 10.1111/hel.12145. Prevotella Yalda 16sRNA Gastric GC 8 185 Streptococci Scientific World Lactobacilli Journal Volume Neisseria 2014, DOI: 1421. Klebsiella Nasrollahzadeh 16sRNA Esophagus ESCC 37 17 Clostridiales Scientific Reports Erysipelotrichales 2015, DOI: 10.1038/srep08820.
Example 3
Development of Augmented Microbiome-Mediated Breath Test for the Earlier Diagnosis of Oesophago-Gastric Cancer (AMBEC)
[0099] (i) Production of an Oral Stimulant Drink (OSD)
[0100] The aim was to develop an enhanced OSD formulation that enables the inventors to fully optimise the dose-response, reproducibility and robustness of the new triage test for clinical introduction. The production of the OSD was based on: (i) the dataset of gastric microbiomal bacteria most commonly associated with cancer tissue, (ii) an extensive bioinformatics review of the enzymatic pathway regulation and biochemical io flux in key bacterial species, (iii) the scientific literature describing the conversion of particular primary metabolites to specific VOCs, and (iv) ethical, safety and acceptability considerations of OSD components, such as normal dietary presence, recommended daily allowance (RDA) and palatability.
[0101] Sugars, organic acids and amino acids were identified as priority compounds.
[0102] Accordingly, specific stimuli were selected for the initial OSD. Several fatty acid stimuli were discounted due to insolubility within the aqueous OSD formulation. A suitable commercial kitchen was identified for OSD manufacture. All OSD components and consumables were sourced as either “food” or “medical” grade to ensure no possibility of contamination and that the drink is fit for human consumption.
[0103] Results: Table 3 summarises the composition of one embodiment of the OSD.
TABLE-US-00003 TABLE 3 OSD composition Stimulant Mix RDI mg/mL.sub.water mg/kg.sub.bodyweight* mg/kg.sub.bodyweight/d Tyrosine 1.75 2.5 25 Glutamic Acid 21.0 30 30 Glucose 130.2 186 1857 ** Sorbitol 35.0 50 Lactose 130.2 186 Glycerol 193.2*** 276 Citric Acid 14 20 Acetic Acid 7 10 The OSD was prepared under ISO9001 principles of Quality Management with full traceability of batch records. The feasibility results from the OSD provide the proposed further work with an excellent basis for the critical intervention necessary to elicit an augmented response. RDI—recommended daily intake *Total bodyweight defined here as 70 kg. ** Estimated for a 70 kg healthy adult. ***~29% lower than glycerol content in Covonia cough syrup.
[0104] In one embodiment, the OSD is in the form of a capsule that is designed to degrade at a certain position with the gastrointestinal tract, thereby offering targeted release of the at least one substrate. In another embodiment, the OSD is a liquid drink. Glucose, lactose and sorbitol are believed to be most important for augmenting the microbiome to produce the signature VOCs.
[0105] (ii) VOC Production by Microbiome Associated with Patient Cancer Types
[0106] The aim was to identify dominant microbiome (bacterial species) associated with oesophago-gastric cancer. Dominant microbiomes associated with oesophago-gastric cancer were identified from: (i) a literature search, (ii) 16S analysis of cancer and normal tissue samples, and (iii) microbiome cultures from oesophageal and gastric cancer and non-cancer tissues obtained from oesophagio-gastric cancer and control patients.
[0107] (A) 16S Analysis of Cancer and Normal Tissue Samples Methods: 16S RNA sequencing analysis was undertaken upon gastric and oesophago-gastric tissue samples obtained during surgery. Samples were subjected to metataxonomic analysis on the Illumina MiSeq platform, with the V.sub.3/V.sub.4 region of OG cancer microbiomes being targeted in a high-multiplexing approach, thus leading to a high coverage of the microbial diversity. Taxonomic-dependent analysis of reads from amplicon sequencing was performed using Mothur software. Comparison of dominant bacterial phyla within cancer and non-cancer samples using univariate statistical analysis was performed. Supervised and unsupervised statistical modelling was performed with incorporation of clinical metadata. Results: The inventors have identified the presence of a number of bacteria associated with cancer, as shown in Table 4.
TABLE-US-00004 TABLE 4 Elevated VOC levels in active biotrans verses controls Glycerol media Glucose media E. coli Acetate, propionoic acid, Hexanal, butanoic acid, (NCIMB 9552) butanoic acid pentanoic acid, hexanoic acid L. fermentum Acetaldehyde, Heptanal, Acetone (NCIMB 11840) ethyl phenol S. Salivarius Acetate, pentanoic acid, Insufficient growth (NCIMB 701779) octanal S. Anginosus Acetone, Butnaoic acid, Butanoic acid, Hexanal (NCIMB 702496) Hexanal, Ethyl phenol, Nonanal K. pneumoniae Hexanoic acid Butanoic acid, pentanoic (NCIMB 13281) acid
[0108] (Stimuli cocktail composition, all at 0.1 M concentration: tyrosine, glutamic acid, glucose, lactose, sorbitol, glycerol, ethanol, xylose, phenylalanine)
[0109] As shown in Table .sub.4, higher abundance of Firmicutes (Lactobacillus fermentum, Streptococcus salivarius, Streptococcus anginosus, Klebsiella pneumoniae, Escherichia coli) was found in oesophago-gastric cancer tissue compared to control samples. The identification of dominant oesophago-gastric cancer-associated microbiomes provides target microbiomes to be stimulated by OSD in order to elicit an optimal augmented response. (B) Microbiome Culture from Patient Cancer and Normal Tissue Samples
[0110] The aim was to illustrate a difference in VOCs originate from bacteria associated with either cancer or normal tissues obtained from patients in order to provide support for the overall hypothesis that the gastric microbiome can produce markers of cancer presence.
[0111] Methods: Frozen samples of cancer tissue and non-cancer control tissue in glycerol-freeze media were used for Sequencing (16S/Shotgun) and headspace analysis. Tissue samples were defrosted and re-suspended in 100 μL PBS (sterile pH .sub.5) and vortexed vigorously for 60 seconds. 100 μL of supernatant fluid was then spread on pre-prepared FAA (Fastidious Anaerobe Agar+7% Horse Blood) medium on petri plates and incubated in anaerobic ES-Gas pouches at 37° C. for 24 hours. The following day, the plates were removed from the incubator and treated in two ways: (A) Predominant species: Individual bacterial colonies most frequently occurring on each FAA plate were picked and re-suspended in 100 μL PBS in vials for sequencing and headspace analysis; and (B) Overall species composition: 1.5 ml PBS was used to re-suspended all bacteria from individual plates. The resuspension was then pipetted into an eppend.orf tube and micro-centrifuged at 14800 for 5 mins. Supernatant fluid was removed and the solid pellet re-suspended in 100 μL PBS and split into vials for sequencing and headspace analysis via solid phase micro-extraction (SPME-GC-MS) using a carboxen/polydimethysiloxane SPME fibre. SPME extraction was performed at 60° C. with intermittent agitation at 500 μm. Volatiles were collected in the absence of airflow, after 48 hours of incubation followed by direct release into a heated gas chromatography injector.
[0112] Results: Differing abundances of volatile aldehydes and fatty acids were detected in the headspace from predominant and total culturable bacterial species associated with cancer and non-cancer samples. For the predominant bacteria (A), VOCs including benzaldehyde and methyl phenol were significantly higher in samples from cancer tissue versus normal tissue controls. As for the overall bacteria present in the tissue samples (B), acetic and butanoic acid were significantly higher in cancer tissue versus normal controls (see
[0113] (iii) In vitro VOC Production by different Microbiomes in response to OSD
[0114] A) Standard Bacterial Culture and VOC production in response to RDA of OSD components
[0115] The aim was to examine the feasibility to culture relevant microbiomes and analyse their VOC produced in response to stimuli used in the OSD, at human recommended daily allowance concentrations.
[0116] Methods: Using the above preliminary clinical data provided by VODCA, and literature references describing microbiome-associations with gastric cancer, suitable strains were obtained from culture collections such as NCIMB (Aberdeen) and ATCC (USA).
[0117] See Table 4. All in-vitro culture work was performed under conditions as closely simulating the natural gastric environment as possible (e.g. anaerobiosis, pH.sub.5.5) in Cati or Cate laboratories as appropriate, according to UK microbiological regulatory guidelines using well-established Ingenza protocols. All associated quality control testing was performed throughout. Several study parameters were optimised during the work to enhance cell growth, culture sampling and VOC analysis.
[0118] Results: E. coli culture grew satisfactorily and generated detectable VOCs, but all other cultures either did not achieve satisfactory growth under the initial protocol or did not produce detectable VOCs at the concentrations of stimuli used.
[0119] B) High throughput analysis of VOC production by different human microbiome bacteria
[0120] The aims were to: (i) develop a high throughput system that maximises microbiome culture and VOC production and analysis, and (ii) examine VOCs produced by different known cancer-associated microbiome members.
[0121] The inventors set out to develop a high-throughput system as the platform for efficient testing of microbiome responses to different OSD compositions and concentrations, to inform the design of subsequent patient dosage studies, recognising that many foods and common nutritional supplements (e.g. vitamins, minerals, amino acids) often greatly exceed the RDAs for compounds potentially suitable in the OSD.
[0122] Reasons for revising the initial culture protocol: (i) insufficient microbiome growth under initial protocol in experiment (iii)A, (ii) insufficient VOC levels in response to RDA of OSD compounds in experiment (iii)A, (iii) difficulties found with the use of the OmniLog including slow growth rate of many cancer associated bacteria, and inability to maintain efficient vessel sealing during headspace sampling of highly volatile compounds, and (iv) the inventors' VOC analytical capabilities proving significantly more sensitive than Ingenza's equipment. It was therefore decided to use growth media and conditions that allowed greater culture biomass and increased concentrations of potential VOC stimuli, since in vitro work is not bound by patient safety constraints.
[0123] Methods:
[0124] Growth media: The mechanisms of genetic and biochemical regulation of the gastric microbes under evaluation was considered important in deciding the composition of laboratory growth media and carbon source used to generate biomass. Glucose mediated catabolite repression can inhibit enzymes necessary to catabolise alternate carbon sources. Media rich in supplements such as amino acids or metabolic intermediates also represses bacterial biosynthesis of these compounds by enzymes that are non-essential under these conditions but whose activity may be required for VOC production. Defined minimal salts medium lacking non-essential supplementation was therefore used.
[0125] Stimuli: Concentrations of stimulant constituents were significantly increased, permitting much broader assessment of individual stimulant thresholds, temporal profiles and concerted effects of stimuli upon microbial VOC production. Microbiomes: 5 prioritised bacterial species (Lactobacillus fermentum, Streptococcus salivarius, Streptococcus anginosus, Klebseilla pneumonia, E. coli) were cultured with glucose or glycerol carbon sources.
[0126] Procedure: A protocol was established for biomass generation in shake flask cultures followed by VOC stimulation in 50 ml falcon tubes. Final nitrogen sparging and high-throughput VOC sampling in headspace vials provided the required accuracy and reproducibility with no loss of throughput (see
[0127] VOC analysis: Analysis was conducted at St Mary's VOC laboratory using standard Gas chromatography mass-spectrometry (TD/GC-MS) and Proton transfer reaction time-of-flight mass-spectrometry (TD/PTR-TOF-MS).
[0128] Results: Samples were collected for all cultures and specific VOCs found to be elevated 2-10 fold over controls in particular cultures (see Table 4 and
[0129] (iv) Clinical Study
[0130] Ethical approval: REC Reference (18/LO/0078).
[0131] Hypothesis: Cancer cells and their associated bacteria will be able to utilise administered substrates within defined metabolic pathways responsible for the production of VOC's. By exploiting inherent metabolic pathways in this way we expect to observe a transient elevation in cancer associated VOCs.
[0132] Methods: Patients with cancer of the oesophageal or stomach as well as subjects with a normal upper gastrointestinal tract were recruited at the time of routine outpatient assessment. Patients were required to fast overnight prior to breath sampling. A baseline breath sample was collected at the start of the study period by asking participants to exhale directly into a double thickness Nalophan® bag (Kalle UK Ltd, UK). Participants were then asked to consume the OSD. Following consumption of the OSD, participants were asked to rinse their mouth with water in order to eliminate any oral residue of the OSD. Further breath samples were then collected at 30 and 60 minutes following ingestion of the OSD. Breath samples were transferred from Nalophan® bags on to thermal desorption tubes using a precision handheld pump (SKC Ltd, UK).
[0133] VOC analysis: Breath samples were analysed by PTR-TOF-MS and GC-MS techniques for target quantification of cancer biomarkers: fatty acids (acetic acid, butyric acid, pentanoic acid and hexanoic acid) phenol, ethyl phenol and aldehydes. Exhaled acetone, a marker of ketosis (a state of energy depletion) was assessed in order to verify the administration of a nutritional stimulus. Strict quality control measures were followed.
[0134] Results: 30 patients with gastroesophageal cancer and 30 control subjects were recruited. All participants were able to consume the OSD and there were no observed or reported adverse events. Acetone levels in both cancer and control subjects decreased following ingestion of the OSD confirming nutritional stimulation that occurred. Following ingestion of the OSD target VOCs in cancer patients (pentanoic acid, hexanoic acid, butyric acid, acetic acid, phenol, ethyl phenol) were detected at higher levels as indicated by the average fold change in VOC concentrations at 30 and 60 mins. With the exception of butyric acid (30 mins time point), control subjects exhibited a ≤10% variation in target VOC levels following ingestion of the OSD. Mean fold change variation in exhaled hexanoic acid, and pentanoic acid is presented in (see Table 5 and
TABLE-US-00005 TABLE 5 Mean fold change in select exhaled VOCs following administration of oral stimulant drink Cancer Controls 0 mins 30 mins 60 mins 0 mins 30 mins 60 mins Acetone 1.0 1.0 0.9 1.0 1.0 0.9 Acetic Acid 1.0 1.1 1.2 1.0 1.0 0.9 Butyric Acid 1.0 1.6 1.3 1.0 1.2 1.0 Pentanoic Acid 1.0 1.1 1.2 1.0 0.9 1.0 Hexanoic Acid 1.0 1.1 1.2 1.0 1.0 1.0 Phenol 1.0 1.2 1.3 1.0 1.0 0.9 Ethyl Phenol 1.0 1.2 1.3 1.0 0.9 1.0 Acetaldehyde 1.0 1.1 1.2 1.0 1.1 1.1
[0135] Data is derived from breath samples analysed by PTR-TOF-MS (OSD composition at the concentrations listed in Table 3: tyrosine, glutamic acid, glucose, lactose, sorbitol, glycerol, citric acid, acetic acid.
TABLE-US-00006 TABLE 6 Columns 2-5: Mean differences of the log-transformed data between cancer patients and controls and pooled variances at 30 and 60 minutes. Columns 6-7: Sample size calculation and power calculation in all compounds for the chosen sample size Mean diff Mean diff (cases − (cases − Pooled Pooled Final Power controls) controls) variance variance sample size with Compound time = 30 min time = 60 time = 30 time = 60 (Power 90%) n = 188 Acetic Acid 0.069 0.155 0.155 0.205 188 .sup. 90% Butyric Acid 0.138 0.260 0.310 0.249 81 99.7% Phenol 0.050 0.232 0.329 0.420 171 92.2% Pentanoic Acid 0.139 0.106 0.164 0.221 186 90.2% Hexanoic Acid 0.176 0.203 0.221 0.168 90 99.5% Ethyl Phenol 0.219 0.205 0.113 0.230 52 100.0%
[0136] The final sample size was chosen as the minimum sample size between both time points based on the expected maximum difference between cases and controls. The inventors used the formula (3.Math.31) in Chapter 3 (Julious, S A. Sample sizes for clinical trials. 2010-Chapman and Hall) for comparison of two means in a parallel study adjusting for the imprecision of the population variance estimation and assuming the same number of cases and controls.
[0137] VOCs belonging to fatty acids, phenols and aldehydes were produced by cancer-associated microbiomes cultured from commercial strains and cancer tissues obtained from patients with oesophago-gastric adenocarcinoma. Different microbiomes produced distinct VOC profiles. As shown in Tables 5 and 6, key elevated VOCs include pentanoic acid, hexanoic acid, butyric acid, acetic acid, butanoic acid, acetaldehyde, benzaldehyde, hexanal, octanal, heptanal, phenol, methyl phenol and ethyl phenol.
[0138] Conclusions: Preliminary investigations have demonstrated ‘proof or principle’ that exhaled biomarkers of gastroesophageal cancer could be augmented by an OSD. The findings from in-vitro bacterial culture experiments provide evidence that the cancer associated bacteria are, at least in part, responsible for the observed changes in target VOC. The findings have also indicated that this response may be suppressed by the prior chemoradiotherapy, which is known to modify the intestinal microbiome.
[0139] Knowledge of the VOC profile produced by different cancer-associated microbiomes can then be used to: [0140] Generate VOC calibration curves for GC-MS qualitative analysis, [0141] Test the response of microbiomes to different combinations and concentrations of OSD components in a high-throughput microbiome culture system.
SUMMARY
[0142] The inventors have unequivocally demonstrated an increase in the generation of VOCs in patients with oesophago-gastric cancer in comparison to non-cancer subjects in response to the oral stimulant drink (OSD). A major finding has been to obtain common stimulus-inducible VOCs in both the clinical study and in vitro microbiome culture of known cancer-associated bacteria. The inventors, therefore, have a very high confidence in the results because of the consistency of VOC identification using multiple analytical platforms (i.e. GC-MS and PTR-TOF-MS).
[0143] In addition, VOCs discovered in AMBEC are among volatile biomarkers that were found to differentiate oesophago-gastric cancer patients from control patients in the initial non-augmented breath test clinical studies (Ann Surg. 2015 Dec; 262(6):981-90. JAMA Oncol. 2018 May 17). These findings provide the basis for further work with the primary objective of establishing an AMBEC protocol that achieves a higher diagnostic accuracy than the 85% shown in previous non-augmented breath analysis studies.
[0144] The novelty of the work is using the cancer-associated microbiome to elicit a diagnostic augmented VOC response. In order to realise this novelty, the inventors have achieved: [0145] Production of an oral drink that stimulates the cancer associated-microbiome. [0146] In-vitro demonstration of VOC production in the headspace of microbiome derived cultures in response to OSD as a means to achieve the most significant cancer dependent response in vivo. [0147] Conducting of a clinical trial that showed an increase in the generation of VOCs in patients with oesophago-gastric cancer in comparison to non-cancer subjects in response to OSD. [0148] Development of techniques and detailed protocols for a high throughput system for optimum culturing and testing of the cancer-associated microbiome and capturing generated VOCs, as well as VOC transport and analysis. This high throughput system is transferable to other cancers to explore microbiome augmented diagnostic responses.
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