Cancer

Abstract

To provide improved techniques for diagnosing esophagogastric cancer in relation to biomarkers and novel biological markers for diagnosing cancer.SOLUTION: Analyzing the level of at least one trace compound in a body sample from a test subject and comparing this level to a reference level, wherein the reference level is the level of the at least one trace compound in an individual or an average level for a population of individuals; Wherein the individual or the population of individuals does not suffer from cancer and wherein (i) an increased level of a particular trace compound in a bodily sample from the test subject as compared to the reference and / or (ii) a decreased level of a particular trace compound in a bodily sample from the test subject as compared to the reference indicates that the subject suffers from or is predisposed to cancer or indicates a negative prognosis for the subject's condition.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to cancer, and in particular, but not exclusively, to the detection of volatile organic compounds (VOCs) for the diagnosis and prognosis of esophagogastric cancer. [Background technology]

[0002] Chemical analysis of human volatile organic compounds (VOCs) is a rapidly evolving field with the potential to contribute to the noninvasive detection of multiple pathologies. A recent systematic review of the diagnostic accuracy of VOC-based breath tests demonstrated their potential for noninvasive cancer detection. Previous studies have reported elevated concentrations of specific VOCs in the exhaled breath, gastric contents, and urine of patients with esophageal and gastric cancer. However, while several studies have suggested a role for these VOCs in key regulatory processes of esophageal and gastric cancer, many of the biochemical pathways associated with their origin in humans remain unknown. Nevertheless, it has been hypothesized that unrestrained production of certain VOCs can occur directly from cancerous tissue, and these VOCs may enter the systemic circulation and then be distributed across the alveolar-capillary barrier. Alternatively, VOCs may be released directly from the mucosa within the aerodigestive tract. Nationwide studies have shown that approximately 9% of gastric and esophageal cancers were missed during prediagnostic endoscopy. Therefore, improved techniques for diagnosing esophageal and gastric cancer are needed.

[0003] The present invention arose from the inventor's research to overcome problems associated with the prior art. Summary of the Invention

[0004] In a first aspect of the present invention, there is provided a method of diagnosing a subject having or predisposed to having cancer, or providing a prognosis of a condition in said subject, said method comprising analyzing the level of at least one trace compound in a body sample from a test subject and comparing said level to a reference level, said reference level being the level of the at least one trace compound in an individual, or an average level for a population, wherein said individual or said population is not affected by cancer, and (i) detecting in a body sample from said test subject the level of at least one trace compound in an individual, or an average level for a population, wherein said individual or said population is not affected by cancer, (ii) detecting in a body sample from said test subject the level of at least one trace compound in an individual, or an average level for a population, wherein said individual or said population is not affected by cancer, (iii) detecting in a body sample from said test subject the level of at least one trace compound in an individual, or an average level for a population, wherein said individual or said population is not affected by cancer, and (iv) detecting in a body sample from said test subject the level of at least one trace compound in an individual, or an average level for a population, wherein said individual or said population is not affected by cancer, Tetradecyl esters;1-Dotriacontanol;1-Chlorotetradecane;4-Methyloctan-1-ol;Carbon monoxide;Nickel;2-Ethylcyclohexylamine;3-Ethyl-1-octene;Ethyl lactate;Tetramethylsuccinimide;6-Methyl-2-heptanone;(E)-Ethene-2-d-ol;4-Anilino-4-keto-2-phenyl-butyric acid;Diisobutyl(oxybis(ethane-2,1-diyl))dicarbonate;Nickel tetracarbonyl;(E)-2-Ethylene-4-methylene-5-hexenal;3-Methyl-1-butyne;(R)-5-Methyl-2-(1-methylethylidene)-cyclohexanone;α-Propyl-benzeneethanol;4-Methyl-1-pentene;1,3,3-Trimethyl-2-oxabicyclo[2.2.(ii) an increase in the level of at least one trace compound selected from the group consisting of 2]oct-5-ene; 2,2'-(ethene-1,2-diylbis(sulfanediyl))diethanol; 3-methyl-thiophene; butanal; tert-butyl alcohol; and 2-methoxysuccinonitrile, or an analog or derivative thereof, compared to the reference, and / or (ii) a decrease in the level of at least one trace compound selected from the group consisting of 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 4,4-dimethyl-octane; benzyl-3-deutero-α-diazopropionate; and formic acid 1-methylethyl ester, or an analog or derivative thereof, in a body sample from the test subject, compared to the reference, indicates that the subject is suffering from or predisposed to cancer, or indicates a negative prognosis for the subject's condition.

[0005] In a second aspect, there is provided a method for determining the effectiveness of a treatment for a subject suffering from cancer, said method comprising analyzing the level of at least one trace compound in a body sample from a test subject and comparing said level to a reference level, said reference level being the level of at least one trace compound in a sample previously taken from said subject, and comprising: (i) determining in said body sample from said test subject the level of at least one trace compound selected from the group consisting of methyl-2,3,5,6-tetra-O-methyl-α-D-galactofuranoside; 1-tetradecanol; N-butylbenzenesulfonamide; hexadecanoic acid; tetradec-5-yl ester-3,5,5-trimethyl-hexanoic acid; undec-10-ynoic acid tetradecyl ester; 1-dotriacontanol; 1 -Chlorotetradecane;4-Methyloctan-1-ol;Carbon monoxide;Nickel;2-Ethylcyclohexylamine;3-Ethyl-1-octene;Ethyl lactate;Tetramethylsuccinimide;6-Methyl-2-heptanone;(E)-Ethene-2-d-ol;4-Anilino-4-keto-2-phenyl-butyric acid;Diisobutyl(oxybis(ethane-2,1-diyl))dicarbonate;Nickel tetracarbonyl;(E)-2-Ethylene-4-methylene-5-hexenal;3-Methyl-1-butyne;(R)-5-Methyl-2-(1-methylethylidene)-cyclohexanone;α-Propyl-benzeneethanol;4-Methyl-1-pentene;1,3,3-Trimethyl-2-oxabicyclo[2.2.a decrease in the level of at least one trace compound selected from the group consisting of 2,2'-(ethene-1,2-diylbis(sulfanediyl))diethanol; 3-methyl-thiophene; butanal; tert-butyl alcohol; and 2-methoxysuccinonitrile, or an analog or derivative thereof, in a body sample from the test subject compared to the reference, or (ii) an increase in the level of at least one trace compound selected from the group consisting of 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 4,4-dimethyl-octane; benzyl-3-deutero-α-diazopropionate; and formic acid 1-methylethyl ester, or an analog or derivative thereof, in a body sample from the test subject compared to the reference, indicates that the treatment is effective; or (i) an increase in the level of at least one trace compound selected from the group consisting of methyl-2,3,5,6-tetra-O-methyl-α-D- Galactofuranoside;1-Tetradecanol;N-Butylbenzenesulfonamide;Hexadecanoic acid;Tetradecan-5-yl ester-3,5,5-trimethyl-hexanoic acid;Undec-10-ynoic acid tetradecyl ester;1-Dotriacontanol;1-Chlorotetradecane;4-Methyloctan-1-ol;Carbon monoxide;Nickel;2-Ethyl-cyclohexylamine;3-Ethyl-1-octene;Ethyl lactate;Tetramethylsuccinimide;6-Methyl-2-heptanoic acid (E)-Ethen-2-d-ol; 4-Anilino-4-keto-2-phenyl-butyric acid; Diisobutyl(oxybis(ethane-2,1-diyl))dicarbonate; Nickel tetracarbonyl; (E)-2-Ethylene-4-methylene-5-hexenal; 3-Methyl-1-butyne; (R)-5-Methyl-2-(1-methylethylidene)-cyclohexanone; α-Propyl-benzeneethanol; 4-Methyl-1-pentene; 1,3,3-Trimethyl-2-oxabicyclo[2.2.(ii) an increase in the level of at least one trace compound selected from the group consisting of 2]oct-5-ene; 2,2'-(ethene-1,2-diylbis(sulfanediyl))diethanol; 3-methyl-thiophene; butanal; tert-butyl alcohol; and 2-methoxysuccinonitrile, or an analog or derivative thereof, compared to the reference, or (ii) a decrease in the level of at least one trace compound selected from the group consisting of 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 4,4-dimethyl-octane; benzyl-3-deutero-α-diazopropionate; and formic acid 1-methylethyl ester, or an analog or derivative thereof, in a body sample from the test subject, compared to the reference, indicates that the treatment is ineffective.

[0006] The method of the first aspect may comprise treating said subject, wherein said treating prevents, reduces or delays the progression of cancer and / or treats cancer.

[0007] According to a third aspect of the present invention, there is provided a method of treating an individual suffering from cancer, said method comprising the steps of: (a) determining the level of at least one trace compound in a body sample from a test subject and comparing said level with a reference level, said reference level being an average level of the at least one trace compound in an individual, or a population, wherein said individual or said population is not suffering from cancer; and (i) determining the level of at least one trace compound in a body sample from said test subject, said trace compound being an average level of at least one trace compound in an individual, or a population, wherein said individual or said population is not suffering from cancer, and (ii) determining the level of at least one trace compound in a body sample from said test subject, said trace compound being an average level of at least one trace compound in an individual, or a population, wherein said individual or said population is not suffering from cancer, and (iii) determining the level of at least one trace compound in a body sample from said test subject, said trace compound being an average level of at least one trace compound in an individual, or a population, wherein said individual or said population is not suffering from cancer, and (iv) determining the level of at least one trace compound in a body sample from said test subject, said trace compound being an average level of at least one trace compound in an individual, or a population, wherein said individual or said population is not suffering from cancer, and Dotriacontanol;1-Chlorotetradecane;4-Methyloctan-1-ol;Carbon monoxide;Nickel;2-Ethylcyclohexylamine;3-Ethyl-1-octene;Ethyl lactate;Tetramethylsuccinimide;6-Methyl-2-heptanone;(E)-Ethene-2-d-ol;4-Anilino-4-keto-2-phenyl-butyric acid;Diisobutyl(oxybis(ethane-2,1-diyl))dicarbonate;Nickel tetracarbonyl;(E)-2-Ethylene-4-methylene-5-hexenal;3-Methyl-1-butyne;(R)-5-Methyl-2-(1-methylethylidene)-cyclohexanone;α-Propyl-benzeneethanol;4-Methyl-1-pentene;1,3,3-Trimethyl-2-oxabicyclo[2.2.2]oct-5-ene; 2,2'-(ethene-1,2-diylbis(sulfanediyl))diethanol; 3-methyl-thiophene; butanal; tert-butyl alcohol; and 2-methoxysuccinonitrile, or analogs or derivatives thereof, is increased compared to said reference, or (ii) 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 4,4 a) determining that the level of at least one trace compound selected from the group consisting of: 1-dimethyl-octane; benzyl-3-deutero-α-diazopropionate; and formic acid 1-methylethyl ester, or an analog or derivative thereof, is decreased compared to the reference, indicating that the subject is suffering from or predisposed to cancer, or has a poor prognosis; and b) treating the test subject, wherein the treatment reduces or delays the progression of cancer and / or treats cancer.

[0008] In a fourth aspect, there is provided the use of at least one trace compound selected from the group consisting of: methyl-2,3,5,6-tetra-O-methyl-α-D-galactofuranoside; 1-tetradecanol; N-butylbenzenesulfonamide; hexadecanoic acid; tetradec-5-yl ester-3,5,5-trimethyl-hexanoic acid; undec-10-ynoic acid tetradecyl ester; 1-dotriacontanol; 1-chlorotetradecane; 4-methyloctan-1-ol; carbon monoxide; nickel; 2-ethyl-cyclohexylamine; 3-ethyl-1-octene; ethyl lactate; tetramethylsuccinimide; 6-methyl-2-heptanone; (E)-ethen-2-d-ol; 4-anilino-4-keto-2-phenyl-butyric acid; diisobutanediol; Tyl(oxybis(ethane-2,1-diyl))dicarbonate;Nickel tetracarbonyl;(E)-2-Ethylene-4-methylene-5-hexenal;3-Methyl-1-butyne;(R)-5-Methyl-2-(1-methylethylidene)-cyclohexanone;α-Propyl-benzeneethanol;4-Methyl-1-pentene;1,3,3-Trimethyl-2-oxabicyclo[2.2.2]oct-5-ene;2,2'-(ethene-1 ,2-diylbis(sulfanediyl))diethanol; 3-methyl-thiophene; butanal; tert-butyl alcohol; 2-methoxysuccinonitrile; 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 4,4-dimethyl-octane; benzyl-3-deutero-α-diazopropionate; and formic acid 1-methylethyl ester or analogs or derivatives thereof.

[0009] Preferably, the cancer is esophagogastric cancer.

[0010] As described in Example 2, the present inventors have prepared methyl-2,3,5,6-tetra-O-methyl-α-D-galactofuranoside; 1-tetradecanol; N-butylbenzenesulfonamide; hexadecanoic acid; tetradec-5-yl ester-3,5,5-trimethyl-hexanoic acid; undec-10-ynoic acid tetradecyl ester; 1-dotriacontanol; 1-chlorotetradecane; 4-methyloctan-1-ol; carbon monoxide; nickel; 2-ethyl-cyclohexylamine; 3-ethyl-1-octene; ethyl lactate; tetramethylsuccinimide; 6-methyl-2-heptanone; (E)-ethen-2-d-ol; 4-anilino-4-keto-2-phenyl-butyric acid; diisobutyl(oxybis(ethane-2,1-diyl))dicarbonate; nickel tetracarbonyl; (E)-2-ethyl Increased levels of 4-methyl-5-hexenal; 3-methyl-1-butyne; (R)-5-methyl-2-(1-methylethylidene)-cyclohexanone; α-propyl-benzeneethanol; 4-methyl-1-pentene; 1,3,3-trimethyl-2-oxabicyclo[2.2.2]oct-5-ene; 2,2'-(ethene-1,2-diylbis(sulfanediyl))diethanol; 3-methyl-thiophene; butanal; tert-butyl alcohol; and 2-methoxysuccinonitrile, or decreased levels of 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 4,4-dimethyl-octane; benzyl-3-deuterated-α-diazopropionate; and formic acid 1-methylethyl ester, have been shown to be indicative of esophagogastric cancer. The methods, devices and uses described herein may also include analyzing levels of analogs or derivatives of the trace compounds described herein. Examples of suitable analogs or derivatives of chemical groups that may be assayed include alcohols, ketones, aromatics, organic acids and gases (CO, CO, NO, NO, H2S, SO2, CH4, etc.).

[0011] When the at least one trace compound includes nickel, the at least one trace compound may also include carbon monoxide. Similarly, when the at least one trace compound includes carbon monoxide, the at least one trace compound may also include nickel. Thus, it will be understood that carbon monoxide and nickel, when detected together, are considered to be trace compounds.

[0012] The at least one trace compound may be selected from the group consisting of methyl-2,3,5,6-tetra-O-methyl-α-D-galactofuranoside; 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 1-tetradecanol; N-butylbenzenesulfonamide; hexadecanoic acid; tetradec-5-yl ester-3,5,5-trimethyl-hexanoic acid; 4,4-dimethyl-octane; tetradecyl ester undec-10-ynoic acid; 1-dotriacontanol; benzyl-3-deutero-α-diazopropionate; formic acid 1-methylethyl ester; 1-chlorotetradecane; 4-methyloctan-1-ol; carbon monoxide; nickel; and 2-ethyl-cyclohexylamine, or an analog or derivative thereof. Preferably, the at least one trace compound is selected from the group consisting of methyl-2,3,5,6-tetra-O-methyl-α-D-galactofuranoside; 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 1-tetradecanol; N-butylbenzenesulfonamide; hexadecanoic acid; tetradec-5-yl ester-3,5,5-trimethyl-hexanoic acid; 4,4-dimethyl-octane; tetradecyl ester undec-10-ynoic acid; 1-dotriacontanol; and benzyl-3-deuterio-α-diazopropionate, or an analogue or derivative thereof. Most preferably, the at least one trace compound is selected from the group consisting of methyl-2,3,5,6-tetra-O-methyl-α-D-galactofuranoside; 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 1-tetradecanol; N-butylbenzenesulfonamide; and hexadecanoic acid, or an analogue or derivative thereof.

[0013] The at least one trace compound may be selected from the group consisting of 3-ethyl-1-octene; ethyl lactate; tetramethylsuccinimide; 6-methyl-2-heptanone; (E)-ethen-2-d-ol; 4-anilino-4-keto-2-phenyl-butyric acid; diisobutyl(oxybis(ethane-2,1-diyl))dicarbonate; nickel tetracarbonyl; (E)-2-ethylene-4-methylene-5-hexenal; 3-methyl-1-butyne; (R)-5-methyl-2-(1- 4-methyl-1-pentene; 1,3,3-trimethyl-2-oxabicyclo[2.2.2]oct-5-ene; 2,2'-(ethene-1,2-diylbis(sulfanediyl))diethanol; 3-methyl-thiophene; butanal; tert-butyl alcohol; and 2-methoxysuccinonitrile, or an analog or derivative thereof. Preferably, the at least one trace compound is selected from the group consisting of 3-ethyl-1-octene; ethyl lactate; tetramethylsuccinimide; 6-methyl-2-heptanone; (E)-ethen-2-d-ol; 4-anilino-4-keto-2-phenyl-butyric acid; diisobutyl(oxybis(ethane-2,1-diyl))dicarbonate; nickel tetracarbonyl; (E)-2-ethylene-4-methylene-5-hexenal; 3-methyl-1-butyne; (R)-5-methyl-2-(1-methylethylidene)-cyclohexanone; α-propyl-benzeneethanol; 4-methyl-1-pentene; and 1,3,3-trimethyl-2-oxabicyclo[2.2.2]oct-5-ene, or an analogue or derivative thereof. Most preferably, the at least one trace compound is selected from the group consisting of 3-ethyl-1-octene; ethyl lactate; tetramethylsuccinimide; 6-methyl-2-heptanone; (E)-ethen-2-d-ol; 4-anilino-4-keto-2-phenyl-butyric acid; diisobutyl(oxybis(ethane-2,1-diyl))dicarbonate; and nickel tetracarbonyl, or an analogue or derivative thereof.Most preferably, the at least one trace compound is selected from the group consisting of: 3-ethyl-1-octene; and ethyl lactate, or an analogue or derivative thereof.

[0014] It will be understood that in their most preferred embodiments, these aspects involve detecting increases and / or decreases in trace compounds as defined in the previous paragraph.

[0015] An important feature of a useful biomarker used in the diagnosis and prognosis of disease is that it exhibits high sensitivity and specificity for a particular disease. As described in the Examples, the present inventors have surprisingly demonstrated that many trace compounds found in the breath of test subjects function as powerful biomarkers for cancer and can therefore be used for the detection and prognosis of cancer. Furthermore, the present inventors have shown that the use of such trace compounds as cancer biomarkers involves an assay that is simple, reproducible, non-invasive, inexpensive, and minimally inconvenient for the patient.

[0016] Advantageously, the methods of the present invention provide a non-invasive means for diagnosing cancer.These methods are useful for allowing clinicians to make decisions regarding the best course of treatment for subjects who currently have cancer or may have cancer.Preferably, these methods are useful for allowing clinicians to decide how to treat subjects who currently have cancer.Furthermore, said methods are useful for monitoring the effectiveness of the estimated treatment of related cancer.For example, treatment can include the administration of chemotherapy, chemoradiotherapy and / or radiotherapy with or without surgery.

[0017] In a second aspect, the subject may be treated with a therapeutic agent and / or radiation therapy, may be on a special diet, and / or may be treated with surgery. The therapeutic agent may be a chemotherapeutic agent.

[0018] In a third aspect, treating the subject may include administering or having administered to the subject a therapeutic agent and / or radiation therapy, placing the subject on a special diet, and / or treating the subject with surgery. The therapeutic agent may be a chemotherapeutic agent.

[0019] Therefore, the methods defined herein are useful for providing a prognosis of the subject's condition so that the clinician can administer the treatment. Thus, the methods are very useful for guiding a treatment plan for the clinician and monitoring the effectiveness of such a treatment plan. The clinician can use the methods disclosed herein in conjunction with existing diagnostic tests to improve the accuracy of diagnosis.

[0020] The subject may be any animal of veterinary interest, such as, for example, a cat, dog, horse, etc. However, it is preferred that the subject is a mammal. Preferably, the subject is a human. The subject may be male or female.

[0021] Preferably, a sample is taken from said subject and the level of said trace compound in the body sample is then determined.

[0022] The trace compounds detected are known as volatile organic compounds (VOCs), which lead to fermentation profiles, and can be detected in body samples by various techniques. In one embodiment, these compounds can be detected in liquid or semi-solid samples in which they are dissolved. However, in a preferred embodiment, the compounds are detected from gases or vapors. For example, because the trace compounds are VOCs, they can emanate from or form part of the sample and be detected in gaseous or vapor form.

[0023] The method may include extracting a sample from the test subject. The method may include using a needle, syringe, or the like to extract the sample. The method may include placing the sample in a sample collection container. The sample may be liquid, gas, or semi-solid.

[0024] Preferably, the sample is any body sample in which the trace compounds are present or secreted. For example, the sample may include urine, feces, hair, sweat, saliva, blood, or tears. The inventors believe that VOCs are degradation products of other compounds found in blood. In one embodiment, blood samples can be immediately assayed for the level of the trace compounds. Alternatively, blood can be stored at low temperature, for example, in a refrigerator, or even frozen, before measuring the level of the trace compounds. Measurement of the trace compounds in the body sample can be performed on whole blood or processed blood.

[0025] In another embodiment, the sample may be a urine sample. Preferably, the level of the trace compound in the body sample is measured in vitro from a urine sample collected from the subject. The compound may be detected from gases or vapors emanating from the urine sample. It will be appreciated that detection of the compound in the gas phase emitted from urine is preferred.

[0026] It will also be understood that a "fresh" bodily sample may be analyzed immediately after being taken from a subject. Alternatively, the sample may be frozen and stored. The sample may then be thawed and analyzed at a later date.

[0027] However, most preferably, the body sample can be an exhaled breath sample from the test subject. The sample can be collected by the subject exhaling through the mouth, preferably after nasal inhalation. Preferably, the sample contains the subject's alveolar air. Preferably, the alveolar air is collected through dead space air by capturing end-expiration breath. Then, the VOCs from the exhaled breath bag are preferably pre-concentrated on a thermal desorption tube by moving the breath through the tube.

[0028] The difference in the level of trace compounds can be increased or decreased compared to the reference.As described in the examples, the inventors monitored the levels of the trace compounds in a large number of patients with any esophagogastric cancer, and compared them with the levels of these same compounds in individuals who do not suffer from esophagogastric cancer (i.e., reference or control).They showed that there is a statistically significant increase or decrease in the levels of these compounds in patients with esophagogastric cancer.

[0029] It will be understood that the level of trace compounds in patients with esophagogastric cancer can depend greatly on many factors, such as how advanced the cancer is, and the age and sex of the subject. It will also be understood that while the baseline level of trace compounds in individuals without esophagogastric cancer may vary to some extent, on average over a period of time, the levels tend to be substantially constant.

[0030] Furthermore, it should be understood that the level of trace compounds in one group of individuals suffering from esophagogastric cancer may be different from the level of the compound in another group of individuals who do not suffer from esophagogastric cancer.However, it is possible to determine the average level of trace compounds in individuals who do not suffer from cancer, which is called the reference or "normal" level of said trace compounds.Said normal level corresponds to the reference value described in the first and third aspects.

[0031] In one embodiment, the methods of the present invention preferably involve determining the ratio of chemicals in the exhaled breath (i.e., using other components in the exhaled breath as a reference) and then comparing these markers with the disease to indicate whether they are elevated or decreased.

[0032] The trace compounds are preferably volatile organic compounds (VOCs), which lead to fermentation profiles and can be detected in or from body samples by various techniques.Therefore, these compounds can be detected using gas analyzers.Examples of suitable detectors for detecting the trace compounds preferably include electrochemical sensors, semiconductor metal oxide sensors, quartz crystal microbalance sensors, optical dye sensors, fluorescent sensors, conductive polymer sensors, composite polymer sensors, or optical spectroscopy.

[0033] The inventors have demonstrated that the trace compounds can be reliably detected using gas chromatography, mass spectrometry, GCMS or TOF. The detection step may use dedicated sensors.

[0034] The reference value of the first and third aspects may be obtained by assaying a statistically significant number of control samples (ie samples from subjects not affected by cancer).

[0035] Thus, the inventors have recognized that the difference in the level of the trace compounds between the reference normal (i.e., control) level and the increased / decreased level can be used as a physiological marker indicative of the presence of cancer in the test subject. If a subject has an increased / decreased level of one or more trace compounds that is significantly higher / lower than the "normal" level of the trace compounds in the reference, control value, then the subject is at a higher risk of having cancer or of a more advanced state than if the level of the compound is only slightly higher / lower than the "normal" level.

[0036] One skilled in the art would understand how to measure the level of a trace compound in a statistically significant number of control individuals and the level of the compound in a test subject, and use these respective values ​​to determine whether the test subject has a statistically significant increase / decrease in the level of the compound, and thereby infer whether the subject is afflicted with cancer.

[0037] In a second embodiment, the reference sample for the concentration of at least one trace compound is derived from the sample previously collected from the subject.The reference sample can be collected from the subject before starting treatment.Therefore, the method can show whether the subject has improved since starting treatment.

[0038] Alternatively, or in addition, the reference sample can comprise a sample collected from the subject after treatment is initiated.In some embodiments, the reference sample can comprise a plurality of samples collected from the subject at different time points after treatment is initiated.The plurality of samples can also comprise a sample collected from the subject before treatment is initiated.Therefore, the method of the second aspect can determine whether improvement is ongoing.

[0039] Every feature described in this specification (including any accompanying claims, abstract, and drawings), and / or every step of any method or process so disclosed, may be combined with any of the above-described aspects in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0040] For a better understanding of the present invention, and to show how an embodiment thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: [Brief explanation of the drawings]

[0041] [Figure 1] Figure 1 shows receiver operating characteristic (ROC) curves generated using the VOCs found to be significantly altered in the exhaled breath of cancer patients in Study 1 (blue) and Study 2 (red). DETAILED DESCRIPTION OF THE INVENTION

[0042] Example 1 The present inventors conducted two studies (Study 1: n=631 samples, and Study 2: n=219 samples, respectively) on breath analysis related to esophageal cancer and gastric cancer.

[0043] method Recruiting participants Ethical approval was obtained (REC14 / LO / 1136), and all participants provided written informed consent. Participants for this study were recruited from Imperial College Healthcare NHS Trust. Participants were recruited into two possible groups: those with known esophagogastric cancer (OGC) or a non-cancer control group. Participants in the non-cancer control group were recruited from the endoscopy suite at Imperial College Healthcare NHS Trust and underwent an upper gastrointestinal endoscopy (which was found to be free of esophageal or gastric cancer) on the day they provided a breath sample. Patients who also underwent a concurrent colonoscopy (and were receiving bowel preparation medication) were ineligible to participate in the study. Participants in the OGC group were patients with biopsy-proven invasive gastric cancer or esophageal adenocarcinoma. These patients were recruited from three feasible locations: on the day of a diagnostic upper gastrointestinal endoscopy, on the day of a staging laparoscopy and upper gastrointestinal endoscopy under general anesthesia (part of the OGC's routine staging survey), or on the day of an examination in an outpatient clinic. Patients who had already undergone surgical or oncological (chemotherapy or radiation) treatment for esophageal or gastric cancer were ineligible for participation in this study. Participants were aged 18–90 years and were excluded if they had known liver disease (including esophageal varices and known portal hypertension), acute infection, another type of cancer currently or within the past 5 years, or a known inflammatory condition of the small or large intestine.

[0044] Breath sample collection Breath samples were collected using a standardized breath sampling device, the "Respiration Collector for In Vitro Analysis" (ReCIVA™) (Owlstone Medical, Cambridge, UK), in combination with a dedicated clean air supply, the "Clean Air Supply Pump for ReCIVA" (CASPER) (Owlstone Medical, Cambridge, UK). All participants fasted for a minimum of 4 hours and rested for 20 minutes before breath samples were taken.

[0045] Two studies were conducted. In Study 1, four sampling thermal desorption (TD) tubes (Tenax / Carbograph-5TD, Markes International Ltd, Llantrisant, UK) were used per participant. In Study 2, one sampling TD tube (Tenax / Carbograph-5TD, Markes International Ltd, Llantrisant, UK) was used per participant. Prior to sample collection, all TD tubes were conditioned at 330 °C for 40 min using a TC-20 tube conditioner (Markes International Ltd, Llantrisant, UK). TD tubes were stored at room temperature in airtight polypropylene containers and used for sample collection within 24 h after conditioning.

[0046] Breath collection was performed using a standardized protocol, with participants performing normal tidal breathing while seated. In the study, one breath sample collection using the ReCIVA device was performed using a sample volume of 250 ml per TD tube and a sample flow rate of 400 ml / min. In the study, two breath sample collections using the ReCIVA device were performed using a sample volume of 500 ml per TD tube and a sample flow rate of 200 ml / min. Before analysis, TD tubes were stored at room temperature in airtight polypropylene containers, and all TD tubes were analyzed within 12 hours of breath sample collection. TD tubes that were conditioned in preparation for breath sample collection but subsequently unused (due to the lower-than-expected number of participants recruited) were analyzed simultaneously with the breath samples as "blank" TD tubes.

[0047] Analysis by TD-GC-MS Breath and empty TD tube samples were analyzed using TD-GC-MS. Markes TD-100 thermal desorption unit (Markes International) The TD tube was desorbed using a two-stage desorption program with a constant helium flow of 50 ml / min applied using a TD tube desorber (Markes International Ltd, Llantrisant, UK). In the first desorption stage, the TD tube was dry purged for 3 minutes and heated to 280°C for 10 minutes. In the second desorption stage, a cold trap (U-T12ME-2S, Markes International Ltd, Llantrisant, UK) was rapidly heated (99°C / min) from 10°C to 290°C. VOCs were transported from the TD unit to the GC via a capillary line heated to 140°C. GC-MS analysis was performed using a ZB-642 capillary column (60 m x 0.25 mm ID x 1.40 um df; Phenomenex 5977A MSD (Agilent Technologies, Inc., Torrance, USA) The analysis was performed using an Agilent 7890B GC equipped with a GC-MS / MS Analyzer (Agilent Technologies Ltd, Santa Clara, USA) using helium as the carrier gas (flow rate 1.0 ml / min). The GC column temperature program was set as follows: 40 °C for 4 min, ramp to 110 °C at 5 °C / min and hold for 1 min, ramp to 110 °C at 5 °C / min and hold for 1 min, ramp to 200 °C at 5 °C / min and hold for 1 min, and finally ramp to 240 °C at 10 °C / min and hold for 4 min. The MS transfer line temperature was 240 °C, and the EI source conditions were 230 °C and 70 eV. Mass acquisition was performed at a rate of approximately 6 scans / s in the range of 20–250 m / z.

[0048] result In breath, biological signatures are typically obscured by intra- and inter-subject variability and experimental conditions, such as ambient air quality. In most cases, biologically relevant compounds are present in very small amounts. To mitigate this, large numbers of samples were collected, as outlined above. In both studies, we were able to predict OGCs with high accuracy and identify corresponding metabolomic "fingerprints." The results are shown in Tables 1 and 2 below.

[0049] [Table 1]

[0050] [Table 2]

[0051] A negative log fold change indicates an increase in VOCs in the cancer patient samples, whereas a positive log fold change indicates a decrease in VOCs in the cancer patient samples.

[0052] ROC plots (see Figure 1) were generated using the VOCs from Study 1 and Study 2, respectively. The ROC plot for Study 1 biomarkers produced an AUC of 0.902 for distinguishing cancer from non-cancer, and the AUC for Study 2 biomarkers was 0.740.

Claims

1. 1. A method of diagnosing a subject having or predisposed to cancer, or providing a prognosis of said subject's condition, comprising: analyzing the level of at least one trace compound in a body sample from the test subject and comparing the level with a reference level; said reference level being the level of at least one trace compound in an individual, or an average level for a population, wherein said individual or said population is not affected by cancer; (i) detecting in a body sample from said test subject: methyl-2,3,5,6-tetra-O-methyl-α-D-galactofuranoside; 1-tetradecanol; N-butylbenzenesulfonamide; hexadecanoic acid; tetradec-5-yl ester-3,5,5-trimethyl-hexanoic acid; undec-10-ynoic acid tetradecyl ester; 1-dotriacontanol; 1-chlorotetradecane; 4-methyloctan-1-ol; carbon monoxide; nickel; 2-ethyl-cyclohexylamine; 3-ethyl-1-octene; ethyl lactate; tetramethylsuccinimide; 6-methyl-2-heptanone; (E)-ethen-2-d-ol; 4-anilino-4-keto-2-phenyl-butyric acid; diisobutyl(oxybis(ethane-2,1-diisopropyl)amino)-2-methylpropional; an increased level of at least one trace compound selected from the group consisting of: (E)-2-ethylene-4-methylene-5-hexenal; 3-methyl-1-butyne; (R)-5-methyl-2-(1-methylethylidene)-cyclohexanone; α-propyl-benzeneethanol; 4-methyl-1-pentene; 1,3,3-trimethyl-2-oxabicyclo[2.2.2]oct-5-ene; 2,2'-(ethene-1,2-diylbis(sulfanediyl))diethanol; 3-methyl-thiophene; butanal; tert-butyl alcohol; and 2-methoxysuccinonitrile, or an analogue or derivative thereof, compared to said reference; and / or (ii) a decrease in the level of at least one trace compound selected from the group consisting of 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 4,4-dimethyl-octane; benzyl-3-deutero-α-diazopropionate; and formic acid 1-methylethyl ester, or analogues or derivatives thereof, in a body sample from the test subject compared to the reference is indicative of the subject being affected by or predisposed to cancer, or is indicative of a negative prognosis of the subject's condition.

2. 1. A method for determining the effectiveness of a treatment for a subject suffering from cancer, comprising: analyzing the level of at least one trace compound in a body sample from the test subject and comparing the level with a reference level; the reference level is the level of at least one trace compound in a sample previously taken from the subject; (i) methyl-2,3,5,6-tetra-O-methyl-α-D-galactofuranoside; 1-tetradecanol; in a body sample from said test subject; N-Butylbenzenesulfonamide; Hexadecanoic acid; Tetradec-5-yl ester-3,5,5-trimethyl-hexanoic acid; Undec-10-ynoic acid tetradecyl ester; 1-Dotriacontanol; 1-Chlorotetradecane; 4-Methyloctan-1-ol; Carbon monoxide; Nickel; 2-Ethyl-cyclohexylamine; 3-Ethyl-1-octene; Ethyl lactate; Tetramethylsuccinimide; 6-Methyl-2-heptanone; (E)-Ethene-2-d-ol; 4-Anilino-4-keto-2-phenyl-butyric acid; Diisobutyl(oxybis(ethane-2,1-diyl))dicarbonate; Nickel tetracarbonyl; (E)-2-Ethylene a reduced level of at least one trace compound selected from the group consisting of phenyl-4-methylene-5-hexenal; 3-methyl-1-butyne; (R)-5-methyl-2-(1-methylethylidene)-cyclohexanone; α-propyl-benzeneethanol; 4-methyl-1-pentene; 1,3,3-trimethyl-2-oxabicyclo[2.2.2]oct-5-ene; 2,2'-(ethene-1,2-diylbis(sulfanediyl))diethanol; 3-methyl-thiophene; butanal; tert-butyl alcohol; and 2-methoxysuccinonitrile, or an analog or derivative thereof, compared to said reference; or (ii) an increase in the level of at least one trace compound selected from the group consisting of 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 4,4-dimethyl-octane; benzyl-3-deutero-α-diazopropionate; and formic acid 1-methylethyl ester, or analogs or derivatives thereof, in a body sample from the test subject compared to the reference, indicates that the treatment is effective; or (i) methyl-2,3,5,6-tetra-O-methyl-α-D-galactofuranoside; 1-tetradecanol; in a body sample from said test subject; N-Butylbenzenesulfonamide; Hexadecanoic acid; Tetradec-5-yl ester-3,5,5-trimethyl-hexanoic acid; Undec-10-ynoic acid tetradecyl ester; 1-Dotriacontanol; 1-Chlorotetradecane; 4-Methyloctan-1-ol; Carbon monoxide; Nickel; 2-Ethyl-cyclohexylamine; 3-Ethyl-1-octene; Ethyl lactate; Tetramethylsuccinimide; 6-Methyl-2-heptanone; (E)-Ethene-2-d-ol; 4-Anilino-4-keto-2-phenyl-butyric acid; Diisobutyl(oxybis(ethane-2,1-diyl))dicarbonate; Nickel tetracarbonyl; (E)-2-Ethylene an increased level of at least one trace compound selected from the group consisting of phenyl-4-methylene-5-hexenal; 3-methyl-1-butyne; (R)-5-methyl-2-(1-methylethylidene)-cyclohexanone; α-propyl-benzeneethanol; 4-methyl-1-pentene; 1,3,3-trimethyl-2-oxabicyclo[2.2.2]oct-5-ene; 2,2'-(ethene-1,2-diylbis(sulfanediyl))diethanol; 3-methyl-thiophene; butanal; tert-butyl alcohol; and 2-methoxysuccinonitrile, or an analog or derivative thereof, compared to the reference; or (ii) a decrease in the level of at least one trace compound selected from the group consisting of 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 4,4-dimethyl-octane; benzyl-3-deutero-α-diazopropionate; formic acid 1-methylethyl ester, or analogs or derivatives thereof, in a bodily sample from the test subject compared to the reference indicates that the treatment is ineffective.

3. 3. The method of claim 2, wherein the subject is being treated with a therapeutic agent and / or radiation therapy, is on a special diet, and / or is being treated with surgery.

4. 1. A method of treating an individual suffering from cancer, comprising: (a) determining the level of at least one trace compound in a body sample from a test subject and comparing this level with a reference level; said reference level being an average level for at least one trace compound in an individual, or a population, wherein said individual or said population is not afflicted with cancer; (i) detecting in a body sample from said test subject: methyl-2,3,5,6-tetra-O-methyl-α-D-galactofuranoside; 1-tetradecanol; N-butylbenzenesulfonamide; hexadecanoic acid; tetradec-5-yl ester-3,5,5-trimethyl-hexanoic acid; undec-10-ynoic acid tetradecyl ester; 1-dotriacontanol; 1-chlorotetradecane; 4-methyloctan-1-ol; carbon monoxide; nickel; 2-ethyl-cyclohexylamine; 3-ethyl-1-octene; ethyl lactate; tetramethylsuccinimide; 6-methyl-2-heptanone; (E)-ethen-2-d-ol; 4-anilino-4-keto-2-phenyl-butyric acid; diisobutyl(oxybis(ethane-2 an increase in at least one trace compound selected from the group consisting of: (E)-2-ethylene-4-methylene-5-hexenal; 3-methyl-1-butyne; (R)-5-methyl-2-(1-methylethylidene)-cyclohexanone; α-propyl-benzeneethanol; 4-methyl-1-pentene; 1,3,3-trimethyl-2-oxabicyclo[2.2.2]oct-5-ene; 2,2'-(ethene-1,2-diylbis(sulfanediyl))diethanol; 3-methyl-thiophene; butanal; tert-butyl alcohol; and 2-methoxysuccinonitrile, or an analog or derivative thereof, compared to said reference; or (ii) determining that a decrease in the level of at least one trace compound selected from the group consisting of 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 4,4-dimethyl-octane; benzyl-3-deutero-α-diazopropionate; and formic acid 1-methylethyl ester, or analogs or derivatives thereof, in a body sample from the test subject compared to the reference indicates that the subject is suffering from or predisposed to cancer, or has a poor prognosis; (b) treating said test subject, wherein said treatment reduces or delays the progression of cancer and / or cures cancer.

5. 5. The method of claim 4, wherein treating the subject comprises administering or having administered to the subject a therapeutic agent and / or radiation therapy, feeding the subject a special diet, and / or treating the subject with surgery.

6. The method according to any one of claims 1 to 5, wherein the cancer is esophageal gastric cancer.

7. The at least one trace compound is selected from the group consisting of methyl-2,3,5,6-tetra-O-methyl-α-D-galactofuranoside; 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 1-tetradecanol; N-butylbenzenesulfonamide; hexadecanoic acid; tetradec-5-yl ester-3,5,5-trimethyl-hexanoic acid; 4,4-dimethyl-octane; tetradecyl ester undec-10-ynoic acid; 1-dotriacontanol; 7. The method of any one of claims 1 to 6, wherein the compound is selected from the group consisting of benzyl-3-deutero-α-diazopropionate; formic acid 1-methylethyl ester; 1-chlorotetradecane; 4-methyloctan-1-ol; carbon monoxide; nickel; and 2-ethyl-cyclohexylamine, or an analog or derivative thereof.

8. 8. The method of claim 7, wherein the at least one trace compound is selected from the group consisting of methyl-2,3,5,6-tetra-O-methyl-α-D-galactofuranoside; 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 1-tetradecanol; N-butylbenzenesulfonamide; hexadecanoic acid; tetradec-5-yl ester-3,5,5-trimethyl-hexanoic acid; 4,4-dimethyl-octane; tetradecyl ester undec-10-ynoic acid; 1-dotriacontanol; and benzyl-3-deuterio-α-diazopropionate, or an analog or derivative thereof.

9. 9. The method of claim 8, wherein the at least one trace compound is selected from the group consisting of methyl-2,3,5,6-tetra-O-methyl-α-D-galactofuranoside; 4-aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 1-tetradecanol; N-butylbenzenesulfonamide; and hexadecanoic acid, or an analog or derivative thereof.

10. The at least one trace compound is selected from the group consisting of 3-ethyl-1-octene; ethyl lactate; tetramethylsuccinimide; 6-methyl-2-heptanone; (E)-ethen-2-d-ol; 4-anilino-4-keto-2-phenyl-butyric acid; diisobutyl(oxybis(ethane-2,1-diyl))dicarbonate; nickel tetracarbonyl; (E)-2-ethylene-4-methylene-5-hexenal; 3-methyl-1-butyne; (R)-5-methyl-2-(1-methylethylidene)- 7. The method of any one of claims 1 to 6, wherein the methyl group is selected from the group consisting of cyclohexanone; α-propyl-benzeneethanol; 4-methyl-1-pentene; 1,3,3-trimethyl-2-oxabicyclo[2.2.2]oct-5-ene; 2,2'-(ethene-1,2-diylbis(sulfanediyl))diethanol; 3-methyl-thiophene; butanal; tert-butyl alcohol; and 2-methoxysuccinonitrile, or an analog or derivative thereof.

11. 11. The method of claim 10, wherein the at least one trace compound is selected from the group consisting of 3-ethyl-1-octene; ethyl lactate; tetramethylsuccinimide; 6-methyl-2-heptanone; (E)-ethen-2-d-ol; 4-anilino-4-keto-2-phenyl-butyric acid; diisobutyl(oxybis(ethane-2,1-diyl))dicarbonate; nickel tetracarbonyl; (E)-2-ethylene-4-methylene-5-hexenal; 3-methyl-1-butyne; (R)-5-methyl-2-(1-methylethylidene)-cyclohexanone; α-propyl-benzeneethanol; 4-methyl-1-pentene; and 1,3,3-trimethyl-2-oxabicyclo[2.2.2]oct-5-ene, or an analog or derivative thereof.

12. 12. The method of claim 11, wherein the at least one trace compound is selected from the group consisting of 3-ethyl-1-octene; ethyl lactate; tetramethylsuccinimide; 6-methyl-2-heptanone; (E)-ethen-2-d-ol; 4-anilino-4-keto-2-phenyl-butyric acid; diisobutyl(oxybis(ethane-2,1-diyl))dicarbonate; and nickel tetracarbonyl, or an analog or derivative thereof.

13. 13. The method of claim 12, wherein the at least one trace compound is selected from the group consisting of 3-ethyl-1-octene; and ethyl lactate, or an analog or derivative thereof.

14. The method of any one of claims 1 to 13, wherein the at least one trace compound is detected from a gas or vapor.

15. The method of any one of claims 1 to 14, wherein the body sample is a breath sample from the test subject.

16. The method of any one of claims 1 to 15, wherein the at least one trace compound is detected using gas chromatography, mass spectrometry, GCMS and / or TOF.

17. Use of at least one trace compound selected from the group consisting of: methyl-2,3,5,6-tetra-O-methyl-α-D-galactofuranoside; 1-tetradecanol; as a biomarker for diagnosing a subject having cancer or a predisposition thereto, or for providing a prognosis of the condition in said subject. N-Butylbenzenesulfonamide; Hexadecanoic acid; Tetradec-5-yl ester-3,5,5-trimethyl-hexanoic acid; Undec-10-ynoic acid tetradecyl ester; 1-Dotriacontanol; 1-Chlorotetradecane; 4-Methyloctan-1-ol; Carbon monoxide; Nickel; 2-Ethyl-cyclohexylamine; 3-Ethyl-1-octene; Ethyl lactate; Tetramethylsuccinimide; 6-Methyl-2-heptanone; (E)-Ethene-2-d-ol; 4-Anilino-4-keto-2-phenyl-butyric acid; Diisobutyl(oxybis(ethane-2,1-diyl))dicarbonate; Nickel tetracarbonyl; (E )-2-Ethylene-4-methylene-5-hexenal; 3-Methyl-1-butyne; (R)-5-methyl-2-(1-methylethylidene)-cyclohexanone; α-Propyl-benzeneethanol; 4-Methyl-1-pentene; 1,3,3-trimethyl-2-oxabicyclo[2.2.2]oct-5-ene; 2,2'-(ethene-1,2-diylbis(sulfanediyl))diethanol; 3-Methyl-thiophene; Butanal; tert-Butyl alcohol; 2-Methoxysuccinonitrile; 4-Aminobenzoic acid 4-hydroxyimino-2,2,6,6-tetramethyl-1-piperidinyl ester; 4,4-Dimethyl-octane; benzyl-3-deutero-α-diazopropionate; and formic acid 1-methylethyl ester or analogs or derivatives thereof.

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