WO2025224437A1

WO2025224437A1 - Biomarkers for lung cancer

Info

Publication number: WO2025224437A1
Application number: PCT/GB2025/050855
Authority: WO
Inventors: Luis Alejandro Jose Mur; Keir LEWIS; David John Rooke
Original assignee: Aberystwyth University
Current assignee: Aberystwyth University
Priority date: 2024-04-24
Filing date: 2025-04-22
Publication date: 2025-10-30
Anticipated expiration: 2026-10-24
Also published as: GB2640556A; GB202405767D0

Abstract

The present invention relates to a method for determining the presence of lung cancer in a subject. The method comprises: (i) determining the level of one or more biomarkers in a sample from the subject; and (ii) comparing the level of said one or more biomarkers with the level of said one or more metabolites in a control sample to determine whether lung cancer is present in the subject. The biomarkers are selected from: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11-dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3-hydroxypentanedioic acid, adenosine phosphosulfate, 7,8-dihydroneopterin 3'-phosphate, cis,cis,cis-10,13,16-Docosatrienoyl-CoA, inosine, N-(2,4-Dinitrophenyl)-2,4-dinitroaniline, prostaglandin M, pentadecenoic acid, PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1(11Z)/22:1(13Z)).

Description

Biomarkers for Lung Cancer

Field of the Invention

The present invention relates to biomarkers for detecting lung cancer. In particular, the present invention relates to a method for determining the presence of lung cancer in a subject. The present invention further relates to a device for detecting lung cancer in a subject and a kit for detecting lung cancer in a subject.

Background to the Invention

Cancer is still the leading cause of death worldwide while lung cancer is the most common cause of cancer death with 1.80 million deaths and 2.21 million diagnosed cases in 2020. As most lung cancer patients are diagnosed late, the five-year survival rate is around 10%, while patients diagnosed with extensive small cell lung cancer have only 5% chance of survival in the next two years. Due to non-elective hospital stays, expensive diagnostic methods, and palliative care, the economic burden of lung cancer in Europe was €18.8 billion in 2013.

Lung cancer is usually diagnosed only when metastatic disease symptoms, are observed. These symptoms include a cough, haemoptysis, chest discomfort and dyspnoea, but can also be more nonspecific and systemic such as weight loss, anorexia, and fatigue. Lung cancer is often recognised in its late stages, because of inadequate diagnostic techniques or the fact that the effects of smoking masks and mimics its early symptoms. As a result, the five-year mortality rate of lung cancer has been reported to be 90%. To diagnose lung cancer, clinicians use bronchoscopy, X-ray imaging and CT (computerised tomography) scans all of which have made it easier to detect lung cancer but have not managed to improve early detection rates.

Even though there are non-invasive methods of diagnosis, such as CT, chest radiography (X-ray), and positron emission tomography (PET) scan, their sensitivity and specificity makes them likely to give false negatives (X-ray) or false positives (CT and PET scans). Therefore, tissue biopsies are usually needed to confirm the diagnosis and these can only be obtained via invasive methods such as mediastinoscopy and bronchoscopy, or more recently, endobronchial ultrasound (EBUS) and endoscopic ultrasound (EUS). These staging and typing tests are highly invasive procedures which increase the burden on the health care provider. This burden is further elevated if patients are diagnosed late and have to undergo various therapies and hospital stays. The Rivera (2003) guidelines to lung cancer diagnosis considers most methods to give only poor to fair levels of accuracy.

Tests with the potential to yield higher accuracies which utilise genetic and molecular markers found in patient biofluids, would allow for a non-invasive diagnostic test. Plasma, serum, and sputum miRNAs, such as hsa-miR-1254 and hsa-miR-574-5p, have been suggested for detection of early-stage non-small cell lung cancer as the tumour appears to change their expression (Foss et al., 2011 ; Xie et al., 2010; Shen et al., 2011). A study by Berker et al. (2019) suggested magnetic resonance spectroscopybased serum biomarkers which can be used as a minimally invasive method of lung cancer classification; however, they caution that further analyses are needed to understand their observations.

It is an object of the present invention to obviate or mitigate one or more of the abovementioned problems.

Summary of the Invention

The present invention relates to a method for determining the presence of lung cancer in a subject and is based, in part, on studies by the inventors in which they have shown that certain metabolites are present at significantly different levels in subjects with lung cancer as compared to control subjects and are therefore suitable as biomarkers for lung cancer.

In a first aspect of the present invention there is provided a method for determining the presence of lung cancer in a subject, the method comprising the steps of:

(i) determining the level of one or more biomarkers in a sample from the subject;

(ii) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether lung cancer is present in the subject; wherein the one or more biomarkers are selected from: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3- hydroxypentanedioic acid, adenosine phosphosulfate, 7,8-dihydroneopterin 3’- phosphate, cis,cis,cis-10,13,16-Docosatrienoyl-CoA, inosine, N-(2,4-Dinitrophenyl)- 2,4-dinitroaniline, prostaglandin M, pentadecenoic acid, PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11 Z)/22: 1 (13Z)).

As discussed in more detail below, the present inventors have found that the level of the abovementioned biomarkers is significantly altered in subjects in which lung cancer is present. As shown below, the inventors have found that these biomarkers have individual diagnostic accuracies (area under the curve; AUC) of greater than 0.70. The inventors have therefore found that by comparing the level of the abovementioned biomarkers in a sample from a subject with the level in a control sample, the presence of lung cancer in a subject can be determined with high sensitivity and specificity. These metabolite biomarkers are therefore useful for determining whether a subject has lung cancer. By quickly and accurately identifying subjects affected by lung cancer, before clinical symptoms become apparent, spread of the disease can be reduced and/or prevented and outcomes improved.

The levels of the metabolite biomarkers of the invention have been shown by the inventors to be consistently and significantly different between subjects with lung cancer as compared to control subjects. These biomarkers can therefore be used to determine the presence of lung cancer in a subject with extremely high sensitivity and specificity.

The method of the present invention comprises determining the level of one or more biomarkers in a sample from the subject. Preferably, the method of the present invention comprises determining the level of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two or twenty-three (or more) biomarkers in a sample from the subject. By determining the level of multiple biomarkers in the sample and comparing multiple biomarkers with the levels in a control sample, the sensitivity and specificity of the determination (of whether a subject has lung cancer) may be improved. The level of one or more biomarkers in the sample will be determined. Together these biomarker levels may be integrated to form a “fingerprint” for the sample, thereby increasing the specificity of the method.

In embodiments of the present invention, the one or more biomarkers are selected from: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a- dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2- hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3- (Carboxymethyl)-3-hydroxypentanedioic acid and galactosylceramide (d18: 1/22:0).

The inventors have found that these biomarkers have individual diagnostic accuracies (area under the curve; AUC) of greater than 0.72. The inventors have therefore found that by comparing the level of the abovementioned biomarkers in a sample from a subject with the level in a control sample, the presence of lung cancer in a subject can be determined with high sensitivity and specificity.

The present invention therefore provides a method for determining the presence of lung cancer in a subject, the method comprising the steps of:

(ii) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether lung cancer is present in the subject; wherein the one or more biomarkers are selected from: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3- hydroxypentanedioic acid and galactosylceramide (d 18: 1/22:0).

In embodiments of the invention, the method may comprise determining the level of two, three, four, five, six, seven, eight, nine, ten, eleven or twelve of the following biomarkers: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L- glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3-hydroxypentanedioic acid and galactosylceramide (d18: 1/22:0).

In embodiments in which the method involves determining the level of twelve biomarkers in a sample from the subject, the biomarkers may consist of 2- methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a- dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2- hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3- (Carboxymethyl)-3-hydroxypentanedioic acid and galactosylceramide (d18: 1/22:0).

In preferred embodiments, the method of the present invention comprises the steps of:

(i) determining the level of twelve biomarkers in a sample from the subject;

(ii) comparing the level of said twelve biomarkers with the level of said twelve biomarkers in a control sample to determine whether lung cancer is present in the subject; wherein the biomarkers consist of 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11- dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3-hydroxypentanedioic acid and galactosylceramide (d18: 1/22:0).

The present inventors have found that these biomarkers, in combination, offer a “signature” or “diagnostic fingerprint” which allows the highly accurate identification of lung cancer. Individually, each biomarker has accuracies of greater than 0.72 (AUC value) and by utilising a combination of these biomarkers a highly accurate diagnosis can be made with average accuracies of greater than 0.93 (mean AUC value).

In embodiments of the present invention, the method may be for distinguishing between subjects with early-stage lung cancer and subjects without lung cancer.

Thus, additionally the present inventors have shown that a number of the metabolite biomarkers identified can distinguish between subjects with early-stage lung cancer and subjects without lung cancer. In such embodiments, the one or more biomarkers are preferably selected from: PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11Z)/22:1 (13Z)).

Therefore, the present invention provides a method for determining whether a subject has early-stage lung cancer, the method comprising the steps of:

(ii) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether the subject has early-stage lung cancer; wherein the one or more biomarkers are selected from: PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11 Z)/22: 1 (13Z)).

When we refer to “early-stage” lung cancer, we refer to stage I and II lung cancer. As will be appreciated by the skilled person, stage I cancer refers to cancers which have not spread to lymph nodes or other organs. Stage I cancer can be divided into stage IA and IB cancers, IA meaning that the tumour is 3 cm or smaller and IB meaning that the tumour is between 3 and 4 cm. Stage II lung cancer can be divided into stage I IA and IIB. Stage HA tumours are between 4 and 5 cm in size with no cancer cells present in the lymph nodes. Stage IIB tumours are up to 5 cm in size with cancer cells present in the lymph nodes adjacent the affected lung. Stage IIB tumours may also include those where: i) cancer has spread to the main bronchus, but not spread to the carina; and/or ii) cancer has spread to the visceral pleura; and/or iii) part of the lung or the whole lung has collapsed or has developed pneumonitis. Stage IIB tumours also include those where cancer has not spread to lymph nodes and one or more of the following characteristics is present: i) the tumour is between 5 and 7 cm; and/or ii) there are one or more separate tumours in the same lobe of the lung as the primary tumour; and/or iii) cancer has spread to any of the following: the parietal pleura, the chest wall, the nerve that controls the diaphragm, and/orthe outer layer of tissue of the pericardial sac. The present inventors have found that the biomarkers listed above are able to differentiate between subjects with early-stage lung cancer and those without lung cancer.

In embodiments of the invention, the method may comprise determining the level of two, three, four or five of the following biomarkers: PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11 Z)/22: 1 (13Z)).

In embodiments in which the method involves determining the level of five biomarkers in a sample from the subject, the biomarkers may consist of PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11 Z)/22: 1 (13Z)).

In preferred embodiments, the method comprises the steps of:

(i) determining the level of five biomarkers in a sample from the subject;

(ii) comparing the level of said five biomarkers with the level of said five biomarkers in a control sample to determine whether the subject has early-stage lung cancer; wherein the biomarkers consist of PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy- 3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE- NMe2(18:1 (11 Z)/22: 1 (13Z)).

The present inventors have found that using these biomarkers, in combination, offers a “signature” or “diagnostic fingerprint” which allows the highly accurate differentiation between subjects with early-stage lung cancer and without lung cancer. Individually, each biomarker has accuracies of greater than 0.76 (AUC value) and by utilising a combination of these biomarkers a highly accurate diagnosis can be made with accuracies of greater than 0.867 (mean AUC value).

Determining the level of biomarkers The method of the present invention involves determining the level of one or more biomarkers in a sample. The skilled person will appreciate that there are a number of ways in which these biomarker levels can be determined.

For example, the biomarker level may be determined using mass spectrometry, for example high resolution mass spectrometry (HR-MS), gas chromatography time-of- flight mass spectrometry (GC-MS), flow infusion electrospray high resolution mass spectrometry (FIE-HRMS) or liquid chromatography-electrospray mass spectrometry (LC-MS).

GC-MS involves linking a gas chromatograph with a mass spectrometer. The gas chromatograph utilizes a capillary column where the chemical properties between the sampled chemicals in a mixture and their relative affinity for the stationary phase of the column will result in their separation along the column. This provides “retention time”, information. The chemicals then enter the mass spectrometer which will show mass-to- charge ratios.

LC-MS links liquid chromatography (LC or High-Performance LC [HPLC]) with a mass spectrometer. The LC part physically separates chemicals between a liquid mixture of two immiscible phases, i.e., stationary and mobile. The chemicals then enter the mass spectrometer which will show mass-to-charge ratios.

In flow infusion, samples are injected directly into a solvent (usually methanol-water) line leading to a mass spectrometer.

Alternatively, the biomarker level could be determined using NMR, enzymatic assays (e.g., enzymatic reaction followed by colorimetric detection) or immunoassays (i.e., antibody binding based assays).

The most well-established immunoassay is the enzyme-linked immunosorbent assay (ELISA). The most used ELISA technique is the sandwich ELISA which measures the antigen using a capture (often associated within the well of a 96 well plate to allow high- throughput screening) and a detection antibody. Monoclonal or polyclonal antibodies can be used in sandwich or competitive ELISA systems. Other immunoassay systems include lateral flow or flow through systems. The use of such immunoassays for detecting the biomarker level would allow the method to be used as a Point of Care (POC) test, for example a POC device, allowing the presence of lung cancer to be determined in locations away from a laboratory.

The step of determining the biomarker level (and comparing the biomarker level with the biomarker level in a control sample) may be performed using a POC test device, for example the device described below. For example, the step of determining the biomarker level (and comparing the biomarker level with the biomarker level in a control sample) may be performed using a flow through device or a lateral flow device. In embodiments, the step of determining the biomarker level (and comparing the biomarker level with the biomarker level in a control sample) may be performed using a lateral flow device, for example the device described below.

The term “biomarker level” as used herein is used to mean the amount of biomarker present in the samples compared to a baseline previously or concurrently established using appropriate controls (e.g., samples from healthy volunteers or patients with other forms of respiratory disease).

Comparing the level of biomarkers with a control sample

Step (ii) of the method of the present invention involves comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether lung cancer is present in the subject.

As will be appreciated by the skilled person, the origin of the control sample will depend upon the particular subject being tested. However, the control sample may be obtained from an age-matched subject and/or a subject of the same sex. Furthermore, since the comparison of biomarker levels may be used to determine whether lung cancer is present in a subject, the control sample may be derived, for example, from a subject in which lung cancer is not present.

In the method of the present invention the biomarker level in a sample from a subject is compared with the biomarker level in a sample from a control. As will be appreciated by the skilled person, in the comparison step, the level of a biomarker in the sample from the subject is compared with a corresponding biomarker level in the control sample (e.g., in embodiments in which the metabolite 2-methoxyestrone 3-sulfate is utilised, the level of 2-methoxyestrone 3-sulfate in the sample is compared with the level of 2- methoxyestrone 3-sulfate in the control).

In embodiments of the invention, a biomarker level in the sample from the subject which is higher or lower than the biomarker level in the control sample indicates that lung cancer is present in the subject.

In embodiments of the invention in which the one or more biomarkers are selected from: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a- dihydrotestosterone sulfate, corticrocin, octanoic acid, 2-hydroxymuconic semialdehyde, PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18: 1/22:0), 7,8-dihydroneopterin 3'-phosphate, cis,cis,cis- 10,13,16-Docosatrienoyl-CoA, N-(2,4-Dinitrophenyl)-2,4-dinitroaniline and PE- NMe2(18:1 (11 Z)/22: 1 (13Z)) a biomarker level in the sample from the subject which is higher than the biomarker level in the control sample indicates that lung cancer is present in the subject.

In embodiments of the invention in which the one or more biomarkers are selected from beta-citryl-L-glutamic acid, 5,8,11 -dodecatriynoic acid, creatine riboside, 3- (Carboxymethyl)-3-hydroxypentanedioic acid, adenosine phosphosulfate, inosine, prostaglandin M and pentadecenoic acid a biomarker level in the sample from the subject which is lower than the biomarker level in the control sample indicates that lung cancer is present in the subject.

As will be appreciated by the skilled person, when referring to a higher or lower biomarker level in a sample from the subject compared to a control sample, we mean a biomarker level which is significantly higher (at least two-fold higher) or significantly lower (at least 0.5-fold lower) than the biomarker level in a control sample.

In embodiments in which the biomarker level in the sample from the subject is higher than the biomarker level in the control sample, preferably the biomarker level in the sample from the subject is at least two-fold higher than in the control sample. More preferably, the biomarker level in the sample from the subject is at least three-fold, fourfold or five-fold higher than in the control sample. More preferably, the biomarker level in the sample from the subject is at least six-fold, seven-fold, eight-fold, nine-fold or tenfold higher than in the control sample. In embodiments in which the biomarker level in the sample from the subject is lower than the biomarker level in the control sample, preferably the biomarker level in the sample from the subject is at least two-fold lower than in the control sample. More preferably, the biomarker level in the sample from the subject is at least three-fold, fourfold or five-fold lower than in the control sample. More preferably, the biomarker level in the sample from the subject is at least six-fold, seven-fold, eight-fold, nine-fold or tenfold lower than in the control sample.

Subject

The present invention provides a method for determining the presence of lung cancer in a subject. Lung cancer is a disease which primarily affects humans but also a variety of animals such as dogs and cats for example. In embodiments of the present invention, the subject is a mammal, preferably a human.

Other features

The method of the present invention may further comprise providing a sample from a subject. In such embodiments the method of the present invention may comprise the steps of:

(i) providing a sample from a subject;

(ii) determining the level of one or more biomarkers in the sample; and

(iii) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether lung cancer is present in the subject; wherein the one or more biomarkers are selected from: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3- hydroxypentanedioic acid, adenosine phosphosulfate, 7,8-dihydroneopterin 3’- phosphate, cis,cis,cis-10,13,16-Docosatrienoyl-CoA, inosine, N-(2,4-Dinitrophenyl)- 2,4-dinitroaniline, prostaglandin M, pentadecenoic acid, PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11 Z)/22: 1 (13Z)).

Feature of the abovementioned methods described herein apply equally to this method.

The present invention comprises determining the level of one or more biomarkers in a sample from the subject. As will be appreciated by the skilled person, the sample may comprise a biological sample from the subject. The biological sample may have been obtained from a bodily fluid of the subject. The biological sample, may include, for example, urine, blood and blood components (e.g., serum), mucus, saliva, milk, vomit, faeces, sweat, semen, vaginal secretion, tears or pus. Preferably, the biological sample is a urine sample. This is particularly advantageous as urine samples are not invasive as with many tests for lung cancer and are therefore well tolerated by subjects. What is more, such samples can be easily obtained. As will be appreciated by the skilled person, the method of the present invention is therefore an in vitro method for determining the presence lung cancer in a subject, the method being carried out on a sample provided from a subject.

In a further aspect of the invention there is provided a method for determining the presence of lung cancer in a subject, the method comprising the steps of:

(i) obtaining a sample from the subject;

(ii) determining the level of one or more biomarkers in the sample;

(iii) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether lung cancer is present in the subject; wherein the one or more biomarkers are selected from: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3- hydroxypentanedioic acid, adenosine phosphosulfate, 7,8-dihydroneopterin 3’- phosphate, cis,cis,cis-10,13,16-Docosatrienoyl-CoA, inosine, N-(2,4-Dinitrophenyl)- 2,4-dinitroaniline, prostaglandin M, pentadecenoic acid, PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11 Z)/22: 1 (13Z)). The method of obtaining the sample from the subject will depend on the sample type and subject. In embodiments in which the sample is a urine sample, the subject may collect the sample in a urine collection container, for example.

There is also provided a method for determining the presence of lung cancer in a subject, determining progression of lung cancer or assessing response to therapy of a subject with lung cancer. The method comprises the steps of:

(ii) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether the subject has lung cancer, has lung cancer which is progressing, or is responding to therapy for lung cancer; wherein the one or more biomarkers are selected from: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3- hydroxypentanedioic acid, adenosine phosphosulfate, 7,8-dihydroneopterin 3’- phosphate, cis,cis,cis-10,13,16-Docosatrienoyl-CoA, inosine, N-(2,4-Dinitrophenyl)- 2,4-dinitroaniline, prostaglandin M, pentadecenoic acid, PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11 Z)/22: 1 (13Z)).

(i) obtaining a sample from the subject;

(ii) determining the level of one or more biomarkers in a sample from the subject; (iii) comparing the level of said one or more biomarkers with the level of said one or more biomarkers in a control sample to determine whether the subject has lung cancer, has lung cancer which is progressing, or is responding to therapy for lung cancer; wherein the one or more biomarkers are selected from: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3- hydroxypentanedioic acid, adenosine phosphosulfate, 7,8-dihydroneopterin 3’- phosphate, cis,cis,cis-10,13,16-Docosatrienoyl-CoA, inosine, N-(2,4-Dinitrophenyl)- 2,4-dinitroaniline, prostaglandin M, pentadecenoic acid, PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11 Z)/22: 1 (13Z)).

In embodiments where the method is being used to determine whether lung cancer is progressing, the method may comprise monitoring over time to determine whether lung cancer has progressed. In such an embodiment, an initial biomarker level (as described above) may be compared with a biomarker level in a sample obtained later in time. The biomarker level(s) obtained may change over time, to be further removed (either higher or lower) from a control biomarker level. Alternatively, in examples where multiple biomarkers are utilised, for example six biomarkers, in an initial analysis only three of the six biomarkers may be indicative of lung cancer. In the later analysis an increased number of biomarkers may be indicative of lung cancer, which may indicate that lung cancer is progressing.

In embodiments where the method is being used to determine whether a subject is responding to therapy, the method may comprise monitoring over time to determine whether a subject is responding to therapy. In such an embodiment, an initial biomarker analysis (which may be before therapy has commenced) may be compared with one or more biomarker analyses undertaken on samples obtained later in time (for example after therapy has commenced). The biomarker levels may change over time, to be further removed (either higher or lower) from a control biomarker level. Alternatively, in examples where multiple biomarkers are utilised, for example six biomarkers, in an initial analysis, six out of six biomarker levels may be indicative of lung cancer. In a later analysis, fewer biomarkers may be indicative of lung cancer, which may indicate that the subject is responding to therapy. Alternatively, in a later analysis, the number of biomarkers indicative of lung cancer may remain the same or increase, which may indicate that a subject is not responding to therapy.

The method of the present invention may also be used to determine whether a drug is effective at treating lung cancer, in a similar manner to that described above in relation to determining whether a subject is responding to therapy. The use of “effective” is used to indicate that a treatment reduces or alleviates signs or symptoms of lung cancer, improves the clinical course of the disease, decreases the number or severity of exacerbations or reduces any other objective or subjective indicia of the disease. The method of the present invention can be used to determine whether drugs used to treat lung cancer, in addition to other drugs developed to treat lung cancer are effective.

Since a diagnosis of a disease is often not based on the results of a single test alone, the method of the present invention may be used to determine whether a subject is more likely than not to have lung cancer based on comparison of one or more biomarker levels with a control biomarker level. Thus, for example, a subject with a putative diagnosis of lung cancer may be diagnosed as being “more likely” or “less likely” to have lung cancer in light of the information provided by the method of the present invention. The present invention may therefore be used to assist a clinician with the diagnosis of lung cancer.

The method of the present invention may, in certain embodiments, comprise detecting other signs or symptoms of lung cancer, conducting clinical tests of lung cancer and/or measuring other lung cancer markers, for example other alternative biomarkers.

As will be appreciated by the skilled person, the above description is not limited to making an initial identification (or diagnosis) of lung cancer in a subject but is also applicable to confirming a provisional diagnosis of lung cancer or “ruling out” such a diagnosis.

The present invention may also be used to determine a suitable treatment for a subject, depending on whether testing indicates that they have lung cancer. The method of the present invention may therefore comprise a step of determining a suitable treatment for the subject, if the subject has lung cancer. Devices

The present invention also provides an immunological capture device for detecting lung cancer in a subject, the device comprising a substrate carrying capture antibodies to one or more of the following biomarkers: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11- dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3-hydroxypentanedioic acid, adenosine phosphosulfate, 7,8-dihydroneopterin 3’-phosphate, cis, cis, cis-10, 13,16- Docosatrienoyl-CoA, inosine, N-(2,4-Dinitrophenyl)-2,4-dinitroaniline, prostaglandin M, pentadecenoic acid, PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3- oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE- NMe2(18:1 (11Z)/22:1 (13Z)).

In embodiments, the substrate carries capture antibodies to one or more of the following biomarkers: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L- glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3-hydroxypentanedioic acid and galactosylceramide (d18: 1/22:0).

In embodiments, the substrate carries capture antibodies to one or more of the following biomarkers: PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11Z)/22:1 (13Z)). Such a device can be useful in identifying subjects with early-stage lung cancer.

In embodiments, the substrate carries capture antibodies to all listed biomarkers.

The term “immunological capture device” as described herein is used to describe an immunoassay device which can be used to measure the presence of a biomarker in a sample through the use of an antibody. The antibody would be specific to the biomarker of interest, such that the antibody could “capture” the biomarker, through binding, if the biomarker is present. The antibodies may therefore be described as “capture antibodies”. The device comprises antibodies to one or more of the following biomarkers: 2- methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a- dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2- hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3- (Carboxymethyl)-3-hydroxypentanedioic acid, adenosine phosphosulfate, 7,8- dihydroneopterin 3’-phosphate, cis,cis,cis-10,13,16-Docosatrienoyl-CoA, inosine, N- (2,4-Dinitrophenyl)-2,4-dinitroaniline, prostaglandin M, pentadecenoic acid, PE(22:0/P- 18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1(11Z)/22:1(13Z)).

In embodiments, the device comprises antibodies to one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two or twenty-three of the following biomarkers: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L- glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3-hydroxypentanedioic acid, adenosine phosphosulfate, 7,8-dihydroneopterin 3’-phosphate, cis,cis,cis-10,13,16-Docosatrienoyl-CoA, inosine, N-(2,4-Dinitrophenyl)-2,4-dinitroaniline, prostaglandin M, pentadecenoic acid, PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11Z)/22:1 (13Z)).

In embodiments, the device comprises antibodies to one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve of the following biomarkers: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3- hydroxypentanedioic acid and galactosylceramide (d 18: 1/22:0).

In embodiments, the device comprises antibodies to one, two, three, four or five of the following biomarkers: PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3- oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE- NMe2(18:1 (11Z)/22:1 (13Z)).

In embodiments, the device comprises antibodies to all listed biomarkers. The substrate of the present invention may be any suitable surface which can carry an antibody. For example, the substrate may be plastic, for example a plate (e.g. a multiwell plate). Alternatively, the substrate may be a porous substrate. The porous substrate may be any material which allows another medium to pass through it. Any suitable porous substrate could be used, for example a woven material, or a cellulosic material.

The substrate carries the capture antibodies. The antibodies may be carried within the substrate or on the surface of the substrate. The antibodies may form a chemical interaction with the surface of the substrate. The antibodies may be bound to the substrate.

In embodiments, the device is a lateral flow device. Alternatively, the device may be a flow through device or an ELISA device.

Advantageously the device is a rapid, simple, non-invasive diagnostic providing quick diagnosis of lung cancer. It is easily accessible and can improve detection and control of lung cancer.

Lateral flow and flow through devices are particularly advantageous in that they can be used remotely to obtain rapid results in a simple manner.

A sample to be tested (i.e. a sample from the subject) may be applied to the immunological capture device, for example to the substrate of the immunological capture device.

A second antibody may also be applied the substrate of the immunological capture device. The second antibody may be specific to the biomarkers or to the capture antibodies (i.e. the antibodies carried on the substrate). The second antibody may have coloured particles attached. The coloured particles may be covalently linked to the second antibody. The coloured particles may be cellulose beads or plastic microparticles, for example. In alternative embodiments the second antibody may be linked to an enzyme, via bio-conjugation, for example. In such embodiments, a composition comprising a substrate which undergoes a colour change upon reaction with the enzyme, indicating the presence of the enzyme, may be added to the device during use. Suitable enzymes and compositions will be well known to the skilled person. In embodiments in which the second antibody is specific to the biomarkers, binding of the second antibody to the biomarkers bound to the capture antibodies results in a colour change. The presence of the biomarkers can therefore be detected by a colour change.

In embodiments in which the second antibody is specific to the capture antibodies, binding of the second antibody to the capture antibody may take place when the biomarker is not present. In such an embodiment, binding of the second antibody to the capture antibodies, results in a colour change. The absence of the biomarkers can therefore be detected by a colour change.

A reader, for example a lateral flow reader may be used to quantify the colour intensity.

In embodiments in which the device is a lateral flow or flow through device, the device may further comprise a control line. Colouring of the control line indicates successful completion of the test.

In embodiments in which the device is a lateral flow or flow through device, the device may further comprise a test line. The test line may comprise capture antibodies to the one or more biomarkers.

Colouring of the test line indicates the presence or absence of the biomarkers in the sample (as discussed above). Comparison of the coloured intensity of the control line and test line can be used to indicate the biomarker level in the sample and therefore whether the sample is from a subject in which lung cancer is present. A reader, for example a lateral flow reader may be used to quantify the coloured intensity of the control and test lines.

In embodiments, the device may further comprise a housing. The substrate may be positioned within the housing.

The device can be used to detect biomarkers in urine, for example.

Preferably the device provides a test result in 60 minutes or less from test initiation. The device may provide a test result in 30 minutes or less, 20 minutes or less or 10 minutes or less from test initiation. The device may be used in the method of the invention.

Kit

The present invention also provides a kit for determining the presence of lung cancer in a subject, the kit comprising:

(i) an immunological capture device comprising a substrate carrying capture antibodies to one or more of the following biomarkers: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a- dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L- glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3-hydroxypentanedioic acid, adenosine phosphosulfate, 7,8-dihydroneopterin 3’-phosphate, cis,cis,cis-10, 13,16-Docosatrienoyl-CoA, inosine, N-(2,4-Dinitrophenyl)- 2,4-dinitroaniline, prostaglandin M, pentadecenoic acid, PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d 18: 1/22:0) and PE-NMe2(18:1(11Z)/22:1(13Z)); and

(ii) a second antibody, wherein the second antibody is specific to the one or more biomarkers or to the capture antibodies.

The present invention also provides a kit for determining the presence of early-stage lung cancer in a subject, the kit comprising:

(i) an immunological capture device comprising a substrate carrying capture antibodies to one or more of the following biomarkers: PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d 18: 1/22:0) and PE-NMe2(18:1(11Z)/22: 1 (13Z)); and

(ii) a second antibody, wherein the second antibody is specific to the one or more biomarkers or to the capture antibodies. The device and second antibody of the kits are described above in relation to the immunological capture device of the present invention. The kits may be used in the methods of the invention.

Uses

The present invention also provides the use of one or more biomarkers for detecting lung cancer in a subject, the one or more biomarkers being selected from: 2- methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a- dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2- hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3- (Carboxymethyl)-3-hydroxypentanedioic acid, adenosine phosphosulfate, 7,8- dihydroneopterin 3’-phosphate, cis,cis,cis-10,13,16-Docosatrienoyl-CoA, inosine, N- (2,4-Dinitrophenyl)-2,4-dinitroaniline, prostaglandin M, pentadecenoic acid, PE(22:0/P- 18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1(11Z)/22:1(13Z)).

The present invention also provides the use of one or more biomarkers for detecting early-stage lung cancer in a subject, the one or more biomarkers being selected from: PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11 Z)/22: 1 (13Z)).

The described and illustrated embodiments are to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the scope of the inventions as defined in the claims are desired to be protected.

The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention as set out herein are also to be read as applicable to any other aspect or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each exemplary embodiment of the invention as interchangeable and combinable between different exemplary embodiments. It should be understood that while the use of words such as “preferable”, “preferably”, “preferred” or “more preferred” in the description suggest that a feature so described may be desirable, it may nevertheless not be necessary and embodiments lacking such a feature may be contemplated as within the scope of the invention as defined in the appended claims. In relation to the claims, it is intended that when words such as “a,” “an,” or “at least one,” are used to preface a feature there is no intention to limit the claim to only one such feature unless specifically stated to the contrary in the claim.

Detailed Description of the Invention

The present invention will now be further described with reference to the following figures which show:

Figure 1 : PCA and PLSDA of the best performing sources of variation in lung cancer (LC) and control (C) (Experiment 1). A. Principal component analysis (PCA) uses the top sources of variation between lung cancer and the control group. It shows good separation of the two groups as well as good clustering of the group. This means the findings show a low variation within the group itself, but a high variation between the two groups. B. Partial Least-Squares Discriminant Analysis (PLS-DA) also plots the top sources of variation between lung cancer and the control group. This shows an increased separation between the two groups and still a good clustering within the group itself. This again shows a low variation within the group and a high variation between the two groups.

Figure 2: ROC for the lung cancer (LC) vs Control (C) groups. This showed an AUC value of 0.924 for the top five best performing biomarkers (sensitivity and specificity being 90%).

Figure 3: Partial Least-Squares Discriminant Analysis (PLS-DA) of positive ionisation of lung cancer (LC) and control (HC) (Experiment 2). PLS-DA plots the top sources of variation between lung cancer and the control group and shows good separation between the groups.

Figure 4: ROC-AUC assessment of the top sources of variation between lung cancer (LC) and controls (HC) urine samples. The AUC for top 15 metabolites is 82.6% with a confidence interval of 72.3-91.4% Figure 5: PCA and PLSDA of the best performing sources of variation in early stage (stage I & II) lung cancer (early) and control (C). A. PCA uses the top sources of variation between the early-stage lung cancer and the control group. It found good separation of the two groups as well as good clustering of the group. This means the findings show a low variation within the group itself, but a high variation between the two groups. B. PLS-DA also plots the top sources of variation between early-stage lung cancer and the control group. This shows an increased separation between the two groups and still a good clustering withing the group itself. This again shows a low variation within the group and a high variation between the two groups.

Figure 6: The ROC curve for early-stage lung cancer (early) v Control (C) comparisons showing an AUC value of 0.867 for the top five best performing metabolic biomarkers, and 0.897 for the best performing ten biomarkers. The sensitivity being 88% and the specificity being 75%.

As discussed above, the method of the present invention allows the presence of lung cancer in a subject to be detected. The present inventors undertook significant investigation to develop the method of the present invention and identified a number of biomarker metabolites, the level of which is significantly altered in subjects in which lung cancer is present. The present inventors have therefore identified a subset of biomarkers which can be used to quickly and accurately identify subjects affected by lung cancer, so that they can be treated accordingly.

Materials and Methods

Patient Recruitment

In experiment 1 , a total 173 patients voluntarily provided samples to the project; 51 were healthy controls and 122 of lung cancer (LC) patients. Out of the 122 lung cancer patients 109 had non-small cell lung cancer (NSCLC), 9 had small cell lung cancer (SCLC) types and 4 had mesothelioma.

In a parallel experiment (experiment 2) the urine of 208 patients was assessed of which 43 were healthy controls and 62 had lung cancer.

All the patients had a unique ID number, allowing forthe records to be kept confidential. The study was undertaken under ethical approval 16/WA/0036, Novel technologies for diagnosing and monitoring pulmonary diseases.

Metabolomic extraction

Urine samples were defrosted in a 4°C fridge overnight. To prepare samples for refraction analyses, they were centrifuged for 5 minutes at 4°C and 3750 rpm after which 1000 pl of each sample was aliquoted into labelled 2 ml microcentrifuge tubes. The samples were normalised based on refractive index using an OPTI Hand Held Refractometer which was calibrated with dH₂O following manufacturer’s instructions. 300 pl from the urine sample was placed on the refractometer and normalised to 1 with H₂O. Then methanol (500 pl, - 20°C) was added, and the samples and vortexed. Following this, 80 pl of methanol/water solution mixed to a 70:30 ratio was added to the pre-marked micro-inserts of HPLC glass vials. Then, 20 pl of urine was put in each glass vial and the HPLC vials were crimped securely.

Mass Spectrometry Data Collection

The samples were profiled using an Exactive Orbitrap (ThermoFinnigan, San Jose CA) mass spectrometer coupled to an Accela (ThermoFinnigan, San Jose CA) ultraperformance liquid chromatography system. Pre-mixed ultra-pure H₂O (18.2 Q) and HPLC grade MeOH (FisherScientific) at a ratio of 7:3 or a flow solvent (mobile phase) were used to deliver 20 pl of the injected sample to the electrospray ionisation (ESI) source. For the first 1.5 min the flow rate was 200 pl min^-1 and 600 pl min^-1 for the subsequent 1 .5 min. Both ionisation modes were acquired at the same time. To acquire the mass spectra for each mode only one scan event was used with a scan rate of 1 .0 Hz. The maximum injection time was 250 ms with a mass resolution of 100 000 and an automatic gain control (AGC) 5 x 10⁵, for both modes of ionisation. Those metabolite masses were then compared to the Human Metabolome Database (HMDB) to identify specific metabolites (Wishart, 2018). The mass spectrometry data was normalised by removing the metabolites below 50 m/z as well as transforming the metabolite intensity value into a percentage of the total ion count for each metabolite.

Statistical analysis

Principal Component Analyses (PCA), partial least squares discriminant analysis (PLS- DA), receiver operating characteristic (ROC) curves, t- tests, and heat maps of Hierarchical Cluster Analyses (HCA) were used based on custom scripts in R and MetaboAnalyst (Xia et al., 2012). To determine whether the metabolites are clinically viable and whether the study approach can be used in a clinical context, the diagnostic accuracy was assessed based on the receiver operating characteristic (ROC)ZArea under the Curve (AUC) were used (Fawcett, 2006). Checking the AUC calculations allows the confirmation of the validity of the fit as the ROC curve analyses plot the true positive rate (sensitivity) in function of the false positive rate (specificity).

Feature identification

Significant m/z were identified based on accurate masses (5 ppm resolution) as detailed in the Kyoto Encyclopaedia of Genes and Genomes (KEGG)(http://www. genome.jp/kegg/) and also the Human metabolite (HMDB), PubChem, and ChEBI databases). Metabolite identifications considered:

1) the following possible adducts: [M+]+, [M+H]+, [M+NH4]+, [M+Na]+, [M+K]+, [M-NH2+H]+, [M-CO2H+H]+, [M-H2O+H]+; [M— ]— , [M-H]-, [M+Na-2H]-, [M+CI]-, [M+K-2H]-; and

2) C13 isotope versus C12 versions of the metabolites (where the metabolites included carbon).

The levels of multiple adducts and C13 for a given metabolite were used in the identification process. The m/z values provided in the Tables were those of the most abundant C12 isotope version of the metabolite.

Results

Metabolomic assessments were undertaken of lung cancer (general) vs control and early stage (I and II) vs control to indicate metabolomic changes between the groups.

Lung cancer vs. control

The PCA and PLS-DA analyses showed a separation between the lung cancer group and the control group (Figure 1). The receiver operating characteristics (ROC) curves together with the AUC values (negative ionisation) that were above 0.70/70% are used to assess the biomarker value. When focusing on the top sources of variation, the AUC values for the top ten metabolites were 0.93/93% (Figure 2). Some of the top performing compounds/metabolites are shown in Table 1 and include 2-M ethoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide and 5a dihydrotestosterone sulfate. Table 1 : Characteristics for nine biomarkers used for detection of lung cancer

In a further experiment (experiment 2), a pairwise comparison was made between the lung cancer and controls groups. PLS-DA showed a clear separation between lung cancer and control groups (Figure 3). The major sources of variation in the PLS-DA were identified using a f-test (P <0.05, correcting for false discovery rates [FDR]). The targeted m/z were identified using the mummichog algorithm on the MetaboAnalyst website. ROC-AUC curve was derived based on these major sources of variation. When considering the top 15 metabolites the AUC value was 82.6% with a confidence interval (Cl) of 72.3-91.4% (Figure 4). Any AUC value over 70% has the potential to be used in a clinical setting. In this experiment 2, the following additional metabolites were identified as potential biomarkers for identifying lung cancer (Table 2):

Table 2: Further biomarkers which can be used for identifying lung cancer

Early-stage lung cancer vs control

The study also assessed whether metabolomics could be used to detect early-stage lung cancer (stage I and II) compared to healthy controls. The PCA analyses and PLS- DA showed that there is a difference between the two groups (Figure 5). ROC curves with AUC values were used and the AUC value for the top five metabolites that differed between the groups was 0.864 (Figure 6). The top five were identical matches to the top five found for the control vs lung cancer. The next five identified were particularly beneficial for the detection of early-stage lung cancer and were PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1Z 22:0) and PE-NMe2(18:1 (11 Z)/22: 1 (13Z)) . The biomarkers particularly beneficial for the detection of early-stage lung cancer from healthy controls are shown in Table 3.

Table 3: Characteristics for the biomarkers used for detection of early-stage lung cancer Discussion

As survival rates and therapy success heavily depend on the stage that the lung cancer has reached at the time of diagnosis, it is very important to make sure that lung cancer is diagnosed as early as possible. Even though methods such as X-ray and CT scans are used for screenings, they are expensive, stationery, and often give false negative and positive results, respectively. Other methods such as bronchoscopies and similar biopsy methods are more accurate but highly invasive. There is, therefore, a need for a non-invasive test which can accurately detect early-stage lung cancer and can be used outside a laboratory environment. The present inventors have shown that metabolomic biomarkers can be used with high specificity and sensitivity to identify subjects with lung cancer. Using such metabolomic biomarkers has great potential in establishing a lateral flow test, or similar, which can much improve the diagnosis of lung cancer with minimal discomfort or inconvenience to the subject.

It will be appreciated that numerous modifications to the above-described method, including smaller and alternative selections of the identified biomarkers, may be made without departing from the scope of the invention as defined in the appended claims.

References

Berker, Y., Vandergrift, L.A., Wagner, I. et a Magnetic Resonance Spectroscopybased Metaboiomic Biomarkers for Typing, Staging, and Survival Estimation of Early- Stage Human Lung Cancer. Sci Rep 9, 10319 (20 9). https://doi.org/10.1038/s41598- 019-46643-5

Fawcett, 2006. An introduction to ROC analysis. Pattern recognition letters, 27(8), pp.861-874.

Foss, K.M., Sima, C., Ugolini, D., Neri, M., Allen, K.E. and Weiss, G.J., 2011 . miR-1254 and miR-574-5p: serum-based microRNA biomarkers for early-stage non-small cell lung cancer. Journal of thoracic oncology, 6(3), pp.482-488.

Shen, J., Todd, N.W., Zhang, H., Yu, L., Lingxiao, X., Mei, Y., Guarnera, M., Liao, J., Chou, A., Lu, C.L. and Jiang, Z., 2011. Plasma microRNAs as potential biomarkers for non-small-cell lung cancer. Laboratory investigation, 91(4), pp.579-587.

Wishart DS, Feunang YD, Marcu A, Guo AC, Liang K, et al., HMDB 4.0 — The Human Metabolome Database for 2018. Nucleic Acids Res. 2018. Jan 4;46(D1): D608-17. 29140435

Xia, J., Mandal, R., Sinelnikov, I.V., Broadhurst, D. and Wishart, D.S., 2012. MetaboAnalyst 2.0 — a comprehensive server for metaboiomic data analysis. Nucleic acids research, 40(W1), pp.W127-W133.

Xie, Y., Todd, N.W., Liu, Z., Zhan, M., Fang, H., Peng, H., Alattar, M., Deepak, J., Stass, S.A. and Jiang, F., 2010. Altered miRNA expression in sputum for diagnosis of non- small cell lung cancer. Lung cancer, 67(2), pp.170-176.

Claims

1. A method for determining the presence of lung cancer in a subject, the method comprising the steps of:

2. The method according to claim 1 wherein when the one or more biomarkers are selected from beta-citryl-L-glutamic acid, 5,8,11 -dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3-hydroxypentanedioic acid, adenosine phosphosulfate, inosine, prostaglandin M and pentadecenoic acid a biomarker level in the sample from the subject which is lower than the biomarker level in the control sample indicates that lung cancer is present in the subject.

3. The method according to claim 1 or 2 wherein when the one or more biomarkers are selected from 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, 2- hydroxymuconic semialdehyde, PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3- oxohexanedioic acid, galactosylceramide (d18: 1/22:0), 7,8-dihydroneopterin 3'- phosphate, cis, cis, cis- 10,13,16-Docosatrienoyl-CoA, N-(2,4-Dinitrophenyl)-2,4- dinitroaniline and PE-NMe2(18:1(11 Z)/22: 1 (13Z)) a biomarker level in the sample from the subject which is higher than the biomarker level in the control sample indicates that lung cancer is present in the subject.

4. The method according to any preceding claim wherein the method is for identifying subjects with early-stage cancer.

5. The method according to claim 4, wherein the one or more biomarkers are selected from PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11 Z)/22: 1 (13Z)).

6. The method according to claim 5, wherein the one or more biomarkers consist of PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11 Z)/22: 1 (13Z)).

7. The method according to claim 1 , wherein the biomarkers consist of 2- methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a- dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2- hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3- (Carboxymethyl)-3-hydroxypentanedioic acid and galactosylceramide (d18: 1/22:0)..

8. The method according to any preceding claim, wherein the subject is a human.

9. The method according to any preceding claim, wherein the method further comprises providing a sample from the subject.

10. The method according to any preceding claim wherein the sample comprises a urine sample.

11 . An immunological capture device for detecting lung cancer in a subject, the device comprising a substrate carrying capture antibodies to one or more of the following biomarkers: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L- glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3-hydroxypentanedioic acid, adenosine phosphosulfate, 7,8-dihydroneopterin 3’-phosphate, cis,cis,cis-10,13,16-Docosatrienoyl-CoA, inosine, N-(2,4-Dinitrophenyl)-2,4-dinitroaniline, prostaglandin M, pentadecenoic acid, PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11Z)/22:1 (13Z)).

12. The immunological capture device according to claim 11 , wherein the substrate carries capture antibodies to one or more of the following biomarkers: 2- methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a- dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2- hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3- (Carboxymethyl)-3-hydroxypentanedioic acid and galactosylceramide (d18: 1/22:0).

13. The immunological capture device according to claim 12, wherein the substrate carries capture antibodies to 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3-hydroxypentanedioic acid and galactosylceramide (d18:1/22:0).

14. The immunological capture device according to claim 11 , wherein the device is for identifying subjects with early-stage lung cancer.

15. The immunological capture device according to claim 14, wherein the substrate carries capture antibodies to one or more of the following biomarkers: PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1(11Z)/22:1(13Z)).

16. The immunological capture device according to claim 11 , wherein the substrate carries capture antibodies to the following biomarkers: PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11 Z)/22: 1 (13Z)).

17. A kit for determining the presence of lung cancer in a subject, the kit comprising:

(i) an immunological capture device comprising a substrate carrying capture antibodies to one or more of the following biomarkers: 2-methoxyestrone 3-sulfate, testosterone glucuronide, androsterone glucuronide, 5a-dihydrotestosterone sulfate, corticrocin, octanoic acid, beta-citryl-L-glutamic acid, 2-hydroxymuconic semialdehyde, 5,6,11 -dodecatriynoic acid, creatine riboside, 3-(Carboxymethyl)-3- hydroxypentanedioic acid, adenosine phosphosulfate, 7,8-dihydroneopterin 3’- phosphate, cis,cis,cis-10,13,16-Docosatrienoyl-CoA, inosine, N-(2,4-Dinitrophenyl)- 2,4-dinitroaniline, prostaglandin M, pentadecenoic acid, PE(22:0/P-18:0), carbamoyl phosphate, 2-hydroxy-3-oxohexanedioic acid, galactosylceramide (d18:1/22:0) and PE-NMe2(18:1 (11 Z)/22: 1 (13Z)); and

18. The kit according to claim 17 wherein the second antibody comprises a coloured particle covalently linked to the second antibody.