Method of detecting a brain tumour using CSF based metabolites

Abstract

The invention provides a method of detecting a brain tumour in a subject, the method comprising the steps of: (i) measuring the amount of at least one metabolite in a sample of cerebrospinal fluid (CSF) taken from a subject, wherein the at least one metabolite is selected from the group consisting of: propionyl carnitine, butyryl carnitine, creatine, creatinine, valine, glutamine, leucine, betaine, isoleucine, asparagine, pyruvate, acetyl carnitine, carnitine, proline, valine and any combination thereof; and (ii) analysing the amount of the at least one metabolite in the sample of CSF, wherein when the amount of the at least one metabolite in the sample of CSF is above a threshold amount a brain tumour in the subject is detected. The invention further provides a method of detecting a brain tumour in a subject comprising identifying elevated or reduced amounts of metabolites in the CSF of a subject, compared to concentrations of the same metabolites in subjects known not to have a brain tumour.

Description

[0001] METHOD OF DETECTING A BRAIN TUMOUR USING CSF BASED METABOLITES

[0002] Technical Field of the Invention

[0003] The present invention relates to a method of detecting a brain tumour. In particular, the present invention relates to a method of detecting a brain tumour involving measuring and analysing the amount of at least one metabolite in a sample of cerebrospinal fluid taken from a patient.

[0004] Background to the Invention

[0005] Cancers of the brain are acknowledged to have poor outcomes, for example, they have high 5-year mortality rates. One of the key issues is the lack of practical diagnostic tools. Magnetic resonance imaging (MRI) is the gold standard test to visualise solid tumours in the brain. However, MRI requires a tumour to be a certain size (e.g. at least a few millimeters) before it becomes conspicuous and is impractical for frequent screening purposes, owing to the device’s large footprint, labour requirements and costs. Thus, a more practical diagnostic method is needed for the detection of smaller-sized tumours and for more frequent screening.

[0006] In addition, MRI is unable to detect minimum residual disease (MRD) following initial treatment, which is often complete or near-complete tumour removal. Cytology of cerebrospinal fluid (CSF) may be performed to detect MRD, but this is an extremely crude method that is rarely effective at detecting MRD.

[0007] Ependymoma and medulloblastoma are two common types of malignant brain tumours in children. 50% of children with ependymoma and 30% of children with medulloblastoma relapse within 2 years, despite there being no evidence of disease on

[0008] MRI scans at the end of their treatment. This strongly suggests that some cancer cells (or radial glial stem cells that give rise to ependymoma) persist at the end of treatment, remain undetected by MRI and can then re-grow.

[0009] There is currently no practically useful test to detect MRD in brain cancers. Such test would allow for continuation therapy to be provided to MRD-positive patients in order to reduce the risk of recurrence, and could also help to prognose the likelihood of relapse.

[0010] Tumours like ependymoma and medulloblastoma are often bathed in or are in close proximity to CSF. Thus, the inventors have identified that detecting metabolites from aberrant cancer metabolism, from a sample of CSF, may be promising for detecting MRD for brain tumours.

[0011] The aim of the present invention is to develop a more practical and effective method for diagnosing brain cancer.

[0012] It is a further aim of the present invention to develop a method for detecting MRD in brain cancer which would allow for prognosis of brain cancer before, during and after treatment, as well as prognosticating the risk of relapse of brain cancer. It is preferable for the method to be minimally invasive.

[0013] It is also an aim of the present invention to overcome or mitigate at least one problem of the prior art, whether disclosed herein or not.

[0014] Summary of the Invention

[0015] According to a first aspect of the invention there is provided a method of detecting a brain tumour in a subject, the method comprising the steps of: (i) measuring the amount of at least one metabolite in a sample of cerebrospinal fluid (CSF) taken from a subject, wherein the at least one metabolite is selected from the group consisting of: propionyl carnitine, butyryl carnitine, creatine, creatinine, valine, glutamine, leucine, betaine, isoleucine, asparagine, pyruvate, acetyl carnitine, carnitine, proline, valine and any combination thereof;

[0016] (ii) analysing the amount of the at least one metabolite in the sample of CSF, wherein when the amount of the at least one metabolite in the sample of CSF is above a threshold amount a brain tumour in the subject is detected.

[0017] The term “brain tumour” may be used herein to describe a neoplasm derived from cells of the brain or central nervous system, and includes any cancer of the brain, “brain tumour” and “brain cancer” may be used synonymously throughout this specification.

[0018] In some embodiments, the brain tumour is an ependymoma or a medulloblastoma. Brain tumours such as ependymoma or medulloblastoma are often bathed in CSF or are in close proximity. Thus, the detection of metabolites arising from aberrant cancer metabolism in CSF samples have been identified as a good indicator of the presence of such brain tumours in a subject.

[0019] The subject may be a mammal, preferably a human.

[0020] Preferably, the subject is suspected of having a brain cancer, has previously been diagnosed with brain cancer, or is being treated or has received treatment for brain cancer.

[0021] The subject may be an adult or a child. Preferably, the subject is a child. The subject may be a child under the age of 18, 17, 16, 15, 14, 13, 12, 11 or under the age of

[0022] 10. The term “metabolite” used herein may describe a molecule arising from aberrant cancer metabolism. The or each metabolite may be a known biomarker for brain cancer.

[0023] The or each metabolite may have different isomeric forms, such as stereoisomers or enantiomers of the or each metabolite. The amount of the or each metabolite measured in the sample of CSF may be a sum of the different isomers of the or each metabolite, or it may be one or more specific isomer form of the or each metabolite.

[0024] The or each metabolite claimed may be an amino acid and its derivatives or a metabolite associated with one carbon metabolism, the carnitine cycle, aerobic glycolysis, purine catabolism, ascorbate degradation or creatinine synthesis.

[0025] It was surprisingly found that the or each metabolite claimed in the first aspect of the invention is present at elevated levels in samples of CSF taken from subjects with a brain tumour compared to samples of CSF taken from subjects without a brain tumour.

[0026] In preferred embodiments, the at least one metabolite may be selected from the group consisting of: betaine, carnitine, propionyl carnitine, butyryl carnitine, proline, creatine and any combinations thereof. In some embodiments the at least one metabolite is creatine and betaine.

[0027] In some embodiments, only one metabolite is measured in the sample of CSF taken from the subject. In other embodiments, more than one metabolite is measured in the sample of CSF taken from the subject. Two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen metabolites may be measured in the sample of CSF taken from the subject. Preferably, a panel of more than one metabolite is used in the method of the present invention. A panel of metabolites will enhance the accuracy of detection. The sample of CSF may be taken from the subject using a minimally invasive medical procedure, which may be used as a liquid biopsy. This is particularly useful for frequent screening.

[0028] In some embodiments, the sample of CSF is obtained by lumbar puncture.

[0029] The present invention uses samples of CSF, as these samples are more representative of brain tumours than samples of other biological fluids, such as blood.

[0030] In some embodiments, the sample of CSF may be taken from the subject at different time points during the subject’s diagnosis and treatment pathway. For example, in some embodiments, the sample is taken for diagnosis purposes at the start of the subject’s diagnosis, or at different time points during treatment to monitor the disease during treatment. In some embodiments, the sample is taken at one or more time points after treatment to detect the presence of minimal residual disease, and / or to prognose disease recurrence. For example, the sample may be taken at around 14 days after a primary brain tumour has been surgically excised to detect the presence of minimal residual disease.

[0031] In some embodiments, the method comprises a step of sample preparation. This step may precede step (i) and step (ii).

[0032] The sample preparation step may comprise one or more sub-steps.

[0033] The sample preparation step may involve one or more sample preparation techniques known in the art. The sample preparation step may involve a technique known in the art for the precipitation of proteins and / or the removal of blood from the sample of CSF taken from the subject. The sample preparation step may involve agitation techniques, separation techniques and / or incubation periods. The sample preparation step may be performed at room temperature (i.e. 20-25 °C), below room temperature (no more than 20 °C) or above room temperature (at least 25 °C).

[0034] In a specific embodiment, the sample preparation step may comprise methanol protein precipitation.

[0035] The amount of the at least one metabolite present in the sample of CSF may be measured as a concentration.

[0036] The amount of the at least one metabolite present in the sample of CSF may be measured using techniques known in the art. The amount of the at least one metabolite present in the sample of CSF may be measured using a technique selected from the group consisting of: liquid chromatography-mass spectrometry (LC-MS), liquid chromatography with tandem mass spectrometry (LC-MS / MS), gas chromatographymass spectrometry (GC-MS), gas chromatography with tandem mass spectrometry (GC- MS / MS), an enzyme-linked immunosorbent assays (ELISA) and a lateral flow immunoassay.

[0037] In preferred embodiments, the amount of the at least one metabolite present in the sample of CSF is measured using liquid chromatography-mass spectrometry (LC-MS) or liquid chromatography with tandem mass spectrometry (LC-MS / MS). In such embodiments, the LC-MS or LC-MS / MS technique may involve the use of a calibration curve and comparing the or each metabolite peak area / signal to known standards of the or each metabolite. A data processing step may also be required to determine the amount of the at least one metabolite present in the sample of CSF.

[0038] The method may use a volume of the sample of CSF of no more than 200 μL, 180 μL, 160 μL, 140 μL, 120 μL, 100 μL, 90 μL, 80 μL, 70 μL, 60 μL, 50 μL, 45 μL, 40 μL, 35 μL, 30 μL, 25 μL, 20 μL, 15 μL, 10 μL or no more than 5 μL. The method may use a volume of the sample of CSF of between 1-100 μL, or preferably between 25-50 μL.

[0039] The sample volume used in the method of the present invention is reduced compared to other techniques commonly used for detecting biomarkers from CSF, for example, detection of ctDNA and microRNA from CSF typically require around 2 mL and 200 μL of sample respectively. Thus, the method of the present has improved sensitivity over other commonly used techniques.

[0040] The method of the first aspect of the invention involves comparing the measured amount of the at least one metabolite in the sample of CSF with a threshold amount in order to determine the presence of a brain tumour in the subject.

[0041] The threshold amount may be statistically determined, for example using area under the curve (AUC) analysis in receiver operating characteristics (ROC) curves. The method may further comprise the step of determining the threshold amount using area under the curve (AUC) analysis in receiver operating characteristics (ROC) curves. In preferred embodiments, the threshold amount may be an amount determined by an AUC values of more than 0.5, 0.6, 0.7, 0.8, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98 or 0.99. Preferably, the threshold amount may be an amount determined by an AUC value(s) of more than 0.8, or more preferably 0.9. In such cases, the threshold amount provides a diagnosis with statistical confidence. The threshold amount may be determined based on the degree of sensitivity and specificity. Samples of CSF with an amount of the at least one metabolite above the threshold amount may detect the presence of a brain tumour in a subject with a sensitivity and specificity of greater than 70%, 80% or 90%. Preferably, the sensitivity and specificity are greater than 90%. The threshold amount may be determined by analysing the amount of the or each metabolite in samples of CSF taken from normal or healthy subjects compared with samples of CSF taken from subjects with a brain tumour and then using this data to determine an absolute ‘cut-off’ value for the or each metabolite above which a brain tumour can be detected with high statistical confidence.

[0042] The term “normal or healthy subjects” describes subjects without a brain tumour or central nervous system disease.

[0043] In some embodiments, the threshold amount is a pre-determined concentration.

[0044] The threshold amount may be a concentration of at least 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nm, 800 nM, 900 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 150 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, or at least 1000 μM.

[0045] The threshold amount may depend on the or each metabolite being analysed. There may be more than one threshold amount depending on the number of metabolite(s) being analysed.

[0046] In embodiments where the at least one metabolite is propionyl carnitine, the threshold amount may be a concentration of at least 0.050 μM, 0.055 μM, 0.060 μM, 0.065 μM, 0.070 μM, 0.075 μM, 0.080 μM, 0.085 μM, 0.090 μM, 0.095 μM, or at least 0.1 μM. The threshold amount may be a concentration of at least 0.060 μM, 0.061 μM, 0.062 μM, 0.063 μM, 0.064 μM, 0.065 μM, 0.066 μM, 0.067 μM, 0.068 μM, 0.069 μM, or at least 0.070 μM. Preferably, in such embodiments, the threshold amount may be a concentration of at least 0.060 μM, or more preferably around 0.0633 μM. In such embodiments, if the concentration of propionylcamitine in the sample of CSF is above the threshold concentration, this provides a positive result indicating the detection of a brain tumour in the subject.

[0047] In embodiments where the at least one metabolite is butyryl carnitine, the threshold amount may be a concentration of at least 0.0050 μM, 0.010 μM, 0.015 μM, 0.020 μM, 0.025 μM, 0.030 μM, 0.035 μM, 0.040 μM, 0.045 μM, 0.050 μM, 0.055 μM, 0.060 μM, 0.065 μM, 0.070 μM, 0.075 μM, or at least 0.080 μM. The threshold amount may be a concentration of at least 0.025 μM, 0.026 μM, 0.027 μM, 0.028 μM, 0.029 μM, 0.030 μM, 0.031 μM, 0.032 μM, 0.033 μM, 0.034 μM, or at least 0.035 μM. Preferably, in such embodiments, the threshold amount may be a concentration of at least 0.030 μM, or more preferably around 0.0300 μM. In such embodiments, if the concentration of butyrylcamitine in the sample of CSF is above the threshold concentration, this provides a positive result indicating the detection of a brain tumour in the subject.

[0048] In embodiments where the at least one metabolite is betaine, the threshold amount may be a concentration of at least 1.5 μM, 1.6, μM, 1.7 μM, 1.8 μM, 1.8 μM, 1.9 μM, 2.0 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM or at least 3.0 μM. The threshold amount may be a concentration of at least 2.10 μM , 2.11 μM, 2.12 μM, 2.13 μM, 2.14 μM, 2.15 μM, 2.16 μM, 2.17 μM, 2.18 μM, 2.19 μM or at least 2.20 μM. Preferably, in such embodiments, the threshold amount may be a concentration of at least 2.0 μM, or more preferably around 2.14 μM. In such embodiments, if the concentration of betaine in the sample of CSF is above the threshold concentration, this provides a positive result indicating the detection of a brain tumour in the subject. In embodiments where the at least one metabolite is creatine, the threshold amount may be a concentration of at least 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, or at least 50 μM. The threshold amount may be a concentration of at least 45.0 μM, 45.1 μM, 45.2 μM, 45.3 μM, 45.4 μM, 45.5 μM, 45.6 μM, 45.7 μM, 45.8 μM, 45.9 μM or at least 50.0 μM. Preferably, in such embodiments, the threshold amount may be a concentration of at least 45 μM, or more preferably around 45.5 μM. In such embodiments, if the concentration of creatine in the sample of CSF is above the threshold concentration, this provides a positive result indicating the detection of a brain tumour in the subject.

[0049] In embodiments wherein the at least one metabolite is valine, the threshold amount may be a concentration of at least 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, or at least 30 μM. The threshold amount may be a concentration of at least 20.5 μM, 20.6 μM, 20.7 μM, 20.8 μM, 20.8 μM, 20.9 μM, 21.0 μM, 21.1 μM, 21.2 μM, 21.3 μM, 21.4 μM, 21.5 μM, 21.6 μM, 21.7 μM, 21.8 μM, 21.9 μM, or at least 22.0 μM. Preferably, in such embodiments, the threshold amount may be a concentration of at least 21 μM, or more preferably around 21.4 μM. In such embodiments, if the concentration of valine in the sample of CSF is above the threshold concentration, this provides a positive result indicating the detection of a brain tumour in the subject.

[0050] In embodiments wherein the at least one metabolite is leucine, the threshold amount may be a concentration of at least 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or at least 20 μM. The threshold amount may be a concentration of at least 11.5 μM, 11.6 μM, 11.7 μM,

[0051] 11.8 μM, 11.9 μM, 12.0 μM, 12.1 μM, 12.2 μM, 12.3 μM, 12.4 μM, 12.5 μM, 12.6 μM, 12.7 μM, 12.8 μM, 12.9 μM,13.0 μM, 13.1 μM, 13.2 μM, 13.3 μM, 13.4 μM, or at least 13.5 μM. Preferably, in such embodiments, the threshold amount is at least 12 μM, or more preferably around 12.6 μM. In such embodiments, if the concentration of leucine in the sample of CSF is above the threshold concentration, this provides a positive result indicating the detection of a brain tumour in the subject.

[0052] In embodiments wherein the at least one metabolite is carnitine, the threshold amount may be a concentration of at least 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2.0 μM, 3.0 μM, 4.0 μM, or at least 5.0 μM. The threshold amount may be a concentration of at least 0.90 μM, 0.91 μM, 0.92 μM, 0.93 μM, 0.94 μM, 0.95 μM, 0.96 μM, 0.97 μM, 0.98 μM, 0.99 μM, 1.00 μM, 1.01 μM, 1.02 μM, 1.03 μM, 1.04 μM, 1.05 μM, 1.06 μM, 1.07 μM, 1.08 μM, 1.09 μM, or at least 2.00 μM. Preferably, the threshold amount may be a concentration of at least 1.0 μM, or more preferably around 1.03 μM. In such embodiments, if the concentration of carnitine in the sample of CSF is above the threshold concentration, this provides a positive result indicating the detection of a brain tumour in the subject.

[0053] In embodiments wherein the at least one metabolite is acetylcamitine, the threshold amount may be a concentration of at least 0.005 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, or at least 1.5 μM. The threshold amount may be a concentration of at least 0.35 μM, 0.36 μM, 0.37 μM40.38 μM, 0.39 μM, 0.40 μMJ).41 μM, 0.42 μM, 0.43 μM, 0.44 μM, 0.45 μM, 0.46 μM, 0.47 μM, 0.48 μM, 0.49, or at least 0.50 μM. Preferably, in such embodiments, the threshold amount may be a concentration of at least 0.40 μM, or more preferably around 0.420 μM. In such embodiments, if the concentration of acetylcamitine in the sample of CSF is above the threshold concentration, this provides a positive result indicating the detection of a brain tumour in the subject.

[0054] In embodiments wherein the at least one metabolite is proline, the threshold amount may be a concentration of at least 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM or at least 2.0 μM. The threshold amount may be a concentration of at least 0.70 μM, 0.71 μM, 0.72 μM, 0.73 μM, 0.74 μM, 0.75 μM, 0.76 μM, 0.77 μM, 0.78 μM, 0.79 μM, 0.80 μM, 0.81 μM, 0.82 μM, 0.83 μM, 0.84 μM, or at least 0.85 μM. Preferably, the threshold amount may be a concentration of at least 0.77 μM, or more preferably around 0.779 μM. In such embodiments, if the concentration of proline in the sample of CSF is above the threshold concentration, this provides a positive result indicating the detection of a brain tumour in the subject.

[0055] In embodiments wherein the at least one metabolite is isoleucine, the threshold amount may be a concentration of at least 1 μM, 2 μM, 3 μM,4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM,15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or at least 20 μM. The threshold amount may be a concentration of at least 7.5 μM 7.6 μM, 7.7 μM / 7.8 μM, 7.9 μM, 8.0 μM, 8.1 μM, 8.2 μM, 8.3 μM, 8.4 μM, 8.5 μM, 8.6 μM, 8.7 μM, 8.8 μMJJ.9 μM, 9.0 μM, 9.1 μM, 9.2 μM, 9.3 μM, 9.4 μM, or at least 9.5 μM. Preferably, in such embodiments, the threshold amount may be a concentration of at least 8.6 μM, or more preferably around 8.63 μM. In such embodiments, if the concentration of isoleucine in the sample of CSF is above the threshold concentration, this provides a positive result indicating the detection of a brain tumour in the subject. In embodiments wherein the at least one metabolite is asparagine, the threshold amount may be a concentration of at least 1 μM, 2 μM, 3 μM,4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM,15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or at least 20 μM. The threshold amount may be a concentration of at least 5.5 μM, 5.6 μM, 5.7 μM 5.8 μM, 5.9 μM, 6.0 μM, 6.1 μM, 6.2 μM, 6.3 μM, 6.4 μM, 6.5 μM, 6.6 μM, 6.7 μM, 6.8 μM,6.9 μM, 7.0 μM, 7.1 μM, 7.2 μM, 7.3 μM, 7.4 μM, or at least 7.5 μM. Preferably, in such embodiments, the threshold amount may be a concentration of at least 6.8 μM, or more preferably around 6.88 μM. In such embodiments, if the concentration of asparagine in the sample of CSF is above the threshold concentration, this provides a positive result indicating the detection of a brain tumour in the subject.

[0056] In embodiments wherein the at least one metabolite is pyruvate, the threshold amount may be a concentration of at least 150 μM, 160 μM ,170 μM, 180 μM, 190 μM, 200 μM, 210 μM, 220 μM, 230 μM, 240 μM,250 μM, 260 μM,270 μM, 280 μM, 290 μM, or at least 300 μM. The threshold amount may be a concentration of at least 210 μM, 211 μM, 212 μM, 213 μM,214 μM, 215 μM, 216 μM,217 μM, 218 μM, 219 μM, 220 μM, 221 μM, 222 μM, 223 μM, 224 μM, 225 μM, 226 μM, 227 μM, 228 μM, 229 μM, or at least 230 μM. Preferably, in such embodiments, the threshold amount may be a concentration of at least 220 μM, or more preferably around 220 μM. In such embodiments, if the concentration of pyruvate in the sample of CSF is above the threshold concentration, this provides a positive result indicating the detection of a brain tumour in the subject.

[0057] In embodiments wherein the at least one metabolite is glutamine, the threshold amount may be a concentration of at least 450 μM, 460 μM, 470 μM, 480 μM,490 μM,

[0058] 500 μM, 510 μM, 520 μM, 530 μM, 540 μM, 550 μM, 560 μM, 570 μM, 580 μM, 590 μM, or at least 600 μM. The threshold amount may be a concentration of at least 501 μM,502 μM, 503 μM, 504 μM, 505 μM, 506 μM, 507 μM, 508 μM, 509 μM, 510 μM, 511 μM, 512 μM, 513 μM,514 μM, 515 μM, 516 μM, 517 μM,518 μM, 519 μM, or at least 520 μM. Preferably, in such embodiments, the threshold amount may be a concentration of at least 510 μM, or more preferably around 512 μM. In such embodiments, if the concentration of glutamine in the sample of CSF is above the threshold concentration, this provides a positive result indicating the detection of a brain tumour in the subject.

[0059] The threshold amounts described above are values at which there is a statistically high confidence that a brain tumour is present in the subject.

[0060] In some embodiments, the method may further comprise the step of identifying the type of brain tumour detected in the subject, wherein the brain tumour is an ependymoma or a medulloblastoma. This step may involve measuring and analysing at least one additional metabolite in the sample of CSF taken from the subject, wherein the at least one additional metabolite is selected from the group consisting of: malic acid, 2- oxoglutarate, glutarate semialdehyde and any combination thereof.

[0061] It was surprisingly found that the metabolites malic acid, 2-oxoglutarate, and glutarate semialdehyde were present in the samples of CSF to differing levels dependent upon the type of brain tumour (e.g. an ependymoma or a medulloblastoma). Thus, measuring the levels of these metabolites can provide a way of distinguishing the type of brain tumour present in the subject.

[0062] This step of identifying the type of brain tumour may comprise the sub-steps of: (a) measuring the amount of at least one additional metabolite in the sample of CSF taken from the subject, wherein the at least one additional metabolite is selected from a group consisting of: malic acid, 2-oxoglutarate, glutarate semialdehyde and any combination thereof;

[0063] (b) analysing the amount of the at least one additional metabolite in the sample of CSF, wherein when the sample of CSF has an elevated amount of 2-oxoglutarate and / or malic acid above a threshold amount a medulloblastoma is identified, and wherein when the sample of CSF has an elevated amount of glutarate semialdehyde above a threshold amount an ependymoma is identified.

[0064] According to a second aspect of the invention there is provided a method of detecting a brain tumour in a subject, the method comprising the steps of:

[0065] (i) measuring the amount of at least one metabolite in a sample of cerebrospinal fluid (CSF) taken from a subject, wherein the at least one metabolite is selected from the group consisting of: N-acetyl histidine, hypoxanthine, threonic acid or threonate, creatinine, glutamine, creatine, arginine, pyruvate, lactate, leucine, lysine, betaine, isoleucine, asparagine, urate, O-acetyl carnitine, carnitine, proline, xylonate, ketoglutarate, and any combination thereof; and

[0066] (ii) analysing the amount of the at least one metabolite present in the sample of CSF by comparing the amount of the at least one metabolite in the sample of CSF with an amount of the at least one metabolite in a control sample of CSF from a subject without a brain tumour, and wherein when the amount of the at least one metabolite in the sample of CSF is elevated or reduced in comparison to the amount of the at least one metabolite in the control sample of CSF a brain tumour in the subject is detected.

[0067] The method of the second aspect of the invention and its components may be as described for the first aspect of the invention and may include any optional feature or combination of optional features described for the first aspect.

[0068] The control sample is a sample of CSF taken from a subject without brain cancer or a central nervous system disease.

[0069] The control sample may be taken from a subject that is aged-matched with the subject from which the sample of CSF taken from the subject.

[0070] The amount of the at least one metabolite in the sample of CSF taken from the subject is compared to the amount of the same metabolite(s) in the control sample of CSF.

[0071] It was surprisingly found that the or each metabolite claimed in the second aspect of the invention is present at elevated or reduced levels in samples of CSF taken from subjects with brain cancer compared to control samples of CSF. Thus, a sample of CSF having a difference in abundance of any one (or more) of the claimed metabolites compared to a control sample, whether an increase or a decrease, may be indicative of the presence of a brain tumour.

[0072] The term “elevated” describes any statistically significant increase in the amount of the or each metabolite in the sample of CSF taken from the subject compared to the control sample of CSF. In some embodiments, the amount of the or each metabolite in the sample of CSF taken from the subject compared to the control sample of CSF is calculated as a fold change. The fold change may be a fold change in the concentration of the or each metabolite between the samples of CSF.

[0073] The amount of the or each metabolite in the sample of CSF may be at least 1.1,

[0074] 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1,

[0075] 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, or at least 21.0 times the amount of the or each metabolite in the control sample of CSF.

[0076] The amount of glutamine in the sample of CSF may be at least 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, or at least 2.00 times the amount of glutamine in the control sample of CSF.

[0077] The amount of creatine in the sample of CSF may be at least 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, or at least 2.00 times the amount of creatine in the control sample of CSF.

[0078] The amount of arginine in the sample of CSF may be at least 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, 2.00, 2.05, 2.10, 2.15, 2.20, 2.25, 2.30, 2.35, 2.40, 2.45, or at least 2.50 times the amount of arginine in the control sample of CSF.

[0079] The amount of pyruvate in the sample of CSF may be at least 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, 2.00, 2.05, 2.10, 2.15, 2.20, 2.25, 2.30, 2.35, 2.40, 2.45, 2.50, 2.55, 2.60, 2.65, 2.70, 2.75, 2.80, 2.85, 2.90, 2.95, or at least 3.00 times the amount of pyruvate in the control sample of CSF. The amount of lactate in the sample of CSF may be at least 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, 2.00, 2.05, 2.10, 2.15, 2.20, 2.25, 2.30, 2.35, 2.40, 2.45,

[0080] 2.50. 2.55. 2.60. 2.65. 2.70. 2.75. 2.80. 2.85. 2.90. 2.95, or at least 3.00 times the amount of lactate in the control sample of CSF.

[0081] The amount of leucine in the sample of CSF may be at least 1.75, 1.80, 1.85, 1.90,

[0082] 1.95, 2.00, 2.05, 2.10, 2.15, 2.20, 2.25, 2.30, 2.35, 2.40, 2.45, 2.50, 2.55, 2.60, 2.65, 2.70, 2.75, 2.80, 2.85, 2.90, 2.95, or at least 3.0 times the amount of leucine in the control sample of CSF.

[0083] The amount of lysine in the sample of CSF may be at leastl .05, 1.10, 1.15, 1.20,

[0084] I I.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, 2.00, 2.05, 2.10, 2.15, 2.20, 2.25, 2.30, 2.35, 2.40, 2.45, 2.50, 2.55, 2.60, 2.65, 2.70, 2.75, 2.80, 2.85, 2.90, 2.95, 3.00, 3.05, 3.10, 3.15, 3.20, 3.25, 3.30, 3.35, 3.40, 3.45, 3.50, 3.55, 3.60, 3.65, 3.70, 3.75, 3.80 3.85, 3.90, 3.95, or at least 4.00 times the amount of lysine in the control sample of CSF.

[0085] The amount of betaine in the sample of CSF may be at least 2.00, 2.05, 2.10, 2.15,

[0086] 2.20. 2.25. 2.30. 2.35. 2.40. 2.45. 2.50. 2.55. 2.60. 2.65. 2.70. 2.75. 2.80. 2.85. 2.90. 2.95, 3.00, 3.05, 3.10, 3.15, 3.20, 3.25, 3.30, 3.35, 3.40, 3.45, or at least 3.50 times the amount of betaine in the control sample of CSF.

[0087] The amount of isoleucine in the sample of CSF may be at least 1.75, 1.80, 1.85, 1.90, 1.95, 2.00, 2.05, 2.10, 2.15, 2.20, 2.25, 2.30, 2.35, 2.40, 2.45, 2.50, 2.55, 2.60, 2.65, 2.70, 2.75, 2.80, 2.85, 2.90, 2.95, 3.00, 3.05, 3.10, 3.15, 3.20, 3.25, 3.30, 3.35, 3.40, 3.45, 3.50, 3.55, 3.60, 3.65, 3.70, 3.75, 3.80, 3.85, 3.90, 3.95, or at least 4.00 times the amount of isoleucine in the control sample of CSF. The amount of asparagine in the sample of CSF may be at least 2.50, 2.55, 2.60, 2.65, 2.70, 2.75, 2.80, 2.85, 2.90, 2.95, 3.00, 3.05, 3.10, 3.15, 3.20, 3.25, 3.30, 3.35, 3.40, 3.45, or at least 3.50 times the amount of asparagine in the control sample of CSF.

[0088] The amount of urate in the sample of CSF may be at least 3.00, 3.05, 3.10, 3.15, 3.20, 3.25, 3.30, 3.35, 3.40, 3.45, 3.50, 3.55, 3.60, 3.65, 3.70, 3.80, 3.90, 4.00, 4.10, 4.20, 4.30, 4.40, 4.50, 4.60, 4.70, 4.80, 4.90, 5.00, 5.10, 5.20, 5.30, 5.40, 5.50, 5.60, 5.70, 5.80, 5.90, or at least 6.00 times the amount of urate in the control sample of CSF.

[0089] The amount of O-acetylcamitine in the sample of CSF may be at least 4.00, 4.10, 4.20, 4.30, 4.40, 4.50, 4.60, 4.70, 4.80, 4.90, 5.00, 5.10, 5.20, 5.30, 5.40, 5.50, 5.60, 5.70, 5.80, 5.90, 6.00, 6.10, 6.20, 6.30, 6.40, or at least 6.50 times the amount of O- acetylcamitine in the control sample of CSF.

[0090] The amount of carnitine in the sample of CSF may be at least 4.80, 4.90, 5.00, 5.10, 5.20, 5.30, 5.40, 5.50, 5.60, 5.70, 5.80, 5.90, 6.00, 6.10, 6.20, 6.30, 6.40, 6.50, 6.60, 6.70, 6.80, 6.90, 7.00, 7.10, 7.20, 7.30, 7.40, 7.50, 7.60, 7.70, 7.80, 7.90, 8.00, 8.10, 8.20, 8.30, 8.40, or at least 8.50 times the amount of carnitine in the control sample of CSF.

[0091] The amount of proline in the sample of CSF may be at least 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, or at least 22.0 times the amount of proline in the control sample of CSF.

[0092] The term “reduced” describes any statistically significant decrease in the amount of the or each metabolite in the sample of CSF taken from the subject compared to the control sample of CSF.

[0093] In some embodiments, the amount of the or each metabolite in the sample of CSF taken from the subject compared to the control sample of CSF is calculated as a fold change. The fold change may be a fold change in the concentration of the or each metabolite between the samples of CSF.

[0094] The amount of the or each metabolite in the sample of CSF may be no more than 0.01, 0.10, 0.20, 0.30, 0.40, 0.41, 0.42, 0.43, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.690.70, 0.71, 0.72, 0.73 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or no more than 0.99 times the amount of the or each metabolite in the control sample of CSF.

[0095] The amount of N-acetyl histidine in the sample of CSF may be no more than 0.20, 0.25, 0.30, 0.35, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, or no more than 0.55 times the amount of N-acetylhistidine in the control sample of CSF.

[0096] The amount of hypoxanthine in the sample of CSF may be no more than 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, or no more than 0.70 times the amount of hypoxanthine in the control sample of CSF.

[0097] The amount of threonic acid in the sample of CSF may be no more than 0.45, 0.50, 0.55, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, or no more than 0.75 times the amount of threonic acid in the control sample of CSF.

[0098] The amount of creatinine in the sample of CSF may be no more than 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, or no more than 0.90 times the amount of creatinine in the control sample of CSF. In particular, the following metabolites were found to have reduced levels reduced in samples taken from subjects with brain cancer compared to control samples: N-acetyl histidine, hypoxanthine, threonic acid and creatinine.

[0099] In particular, the following metabolites were found to have elevated levels in samples taken from subjects with brain cancer compared to control samples: glutamine, creatine, arginine, pyruvate, lactate, leucine, lysine, betaine, isoleucine, asparagine, urate, O-acetyl carnitine, carnitine and proline.

[0100] In preferred embodiments, the at least one metabolite may be selected from the group consisting of: betaine, carnitine, propionyl carnitine, butyryl carnitine, proline, creatine and any combinations thereof. In some embodiments the at least one metabolite is betaine and creatine.

[0101] In some embodiments two metabolites may be selected. The two metabolites may be selected from a pair of metabolites selected from the group consisting of: betaine and threonic acid; betaine and hypoxanthine; L-camitine and acetyl-L-histidine; L-arginine and creatinine; L-camitine and L-threonic acid; betaine and creatinine; malate and hypoxanthine; sulphate and L-threonic acid; a-ketoglutarate and hypoxanthine; betaine and xylonate; a-ketoglutarate and hypoxanthine; sn-glycero-3-phosphocholine and xylonate; 3-methyl-2-oxobutanoate and malate; and 3-methyl-2-oxobutanoate and a- ketoglutarate.

[0102] The or each metabolite may have different isomeric forms, such as stereoisomers or enantiomers of the or each metabolite. The amount of the or each metabolite measured in the sample of CSF may be a sum of the different isomers of the or each metabolite, or it may be one or more specific isomeric form of the or each metabolite. In some embodiments, the at least one metabolite measured from the sample of CSF has the following isomeric form: N-Acetyl-L-histidine, L-threonic acid, L- Glutamine, L- Arginine, (R)-Lactate, L-Leucine, L-Lysine, L-Isoleucine, L- Asparagine, L-Camitine, propionyl-L-camitine, butyryl-L-camitine or L-Proline.

[0103] The or each metabolite may be measured using any technique as described for the first aspect of the present invention.

[0104] The sample of CSF may be as described for the first aspect of the invention and may be prepared as described for the first aspect of the invention.

[0105] In some embodiments, the method may further comprise the step of identifying the type of brain tumour present in the subject. The brain tumour identified may be an ependymoma or a medulloblastoma. This step may involve measuring and analysing at least one additional metabolite in the sample of CSF taken from the subject, wherein the at least one additional metabolite is selected from the group consisting of: malic acid (malate), 2-oxoglutarate, glutarate semialdehyde and any combination thereof.

[0106] It was surprisingly found that the metabolites malic acid (malate), 2-oxoglutarate, and glutarate semialdehyde were present in the samples of CSF to differing levels dependent upon the type of brain tumour (e.g. an ependymoma or a medulloblastomoa). Thus, measuring the levels of these metabolites can provide a way of distinguishing the type of brain tumour present in the subject.

[0107] This step of identifying the type of brain tumour may comprise the sub-steps of: a) measuring the amount of at least one additional metabolite in the sample of CSF taken from the subject, wherein the at least one additional metabolite is selected from a group consisting of: malic acid (malate), 2-oxoglutarate, glutarate semialdehyde and any combination thereof; and b) analysing the amount of the at least one additional metabolite present in the sample of CSF by comparing the amount of the at least one additional metabolite with an amount of the at least one additional metabolite in the control sample of CSF, and wherein when the sample of CSF has an elevated amount of 2-oxoglutarate and / or malic acid compared with the control sample of CSF a medulloblastoma is identified, and wherein when the sample of CSF has an elevated amount of glutarate semialdehyde compared with the control sample of CSF an ependymoma is identified.

[0108] According to a third aspect of the invention there is provided a method for detecting a brain tumour in a subject, the method comprising the steps of:

[0109] (i) measuring the amount of at least one test metabolite in a sample of CSF taken from a subject, wherein the amount of at least one test metabolite is known to be elevated in the CSF of a subject with a brain tumour;

[0110] (ii) measuring the amount of at least one control metabolite in the sample of CSF taken from a subject, wherein the amount of the control metabolite is known not to be elevated in the CSF of a subject with a brain tumour;

[0111] (iii) standardising the amount of the at least one test metabolite based on the amount of the at least one control metabolite;

[0112] (iv) comparing the standardised amount of the at least one test metabolite with a threshold amount, and wherein when the standardised amount is above the threshold amount a brain tumour in the subject is detected. The method of the third aspect of the invention and its components may be as described for the first or second aspect of the invention and may include any optional feature or combination of optional features described for the first or second aspect.

[0113] The or each test metabolite may be selected from the group consisting of: lactate, pyruvate, betaine, carnitine, propionyl carnitine, butyryl carnitine, valine, o- acetylcamitine, proline, leucine, isoleucine, urate, lysine, arginine, asparagine, glutamine and any combination thereof. It was surprisingly found that the amounts of the aforementioned metabolites are elevated in samples of CSF taken from a subject with brain a brain tumour compared with samples of CSF taken from a subject without a brain tumour.

[0114] Preferably, the or each test metabolite may be selected from the group consisting of: betaine, carnitine, O-acetyl carnitine, proline and any combination thereof.

[0115] More preferably, the or each test metabolite may be betaine and / or carnitine.

[0116] The or each control metabolite may be selected from the group consisting of: creatinine, threonic acid or threonate, hypoxanthine, N-acetyl histidine and any combination thereof. It was surprisingly found that the amounts of the aforementioned metabolites are reduced or remained relatively constant in samples of CSF taken from a subject with brain cancer compared with samples of CSF taken from a subject without brain cancer.

[0117] Preferably, the or each control metabolite may be creatinine and / or threonic acid or threonate.

[0118] In some embodiments, the at least one test metabolite is betaine and the at least one control metabolite is creatinine. In some embodiments, the at least one test metabolite is carnitine and the at least one control metabolite is threonic acid or threonate.

[0119] In some embodiments, the at least one test metabolite is betaine and the at least one control metabolite is threonic acid or threonate.

[0120] In some embodiments, the at least one test metabolite is carnitine and the at least one control metabolite is creatinine.

[0121] The or each test metabolite and control metabolite may be measured using any technique as described for the first aspect of the present invention.

[0122] The sample of CSF may be as described for the first aspect or the invention and may be prepared as described for the first aspect of the invention.

[0123] In some embodiments, the amount of the at least one test metabolite is standardised against the amount of the at least one control metabolite in the sample of CSF. This may be calculated as a ratio of the amount of the at least one test metabolite to the amount of the at least one control metabolite. In some embodiments, the following ratios of metabolites may be calculated: betaine / creatinine, betaine / threonate or threonic acid, or camitine / creatinine.

[0124] In some embodiments, the threshold amount is a ratio of the amount of the at least one test metabolite to the amount of the at least one control metabolite at which a brain tumour can be detected from a sample of CSF with a high degree of statistical confidence.

[0125] The threshold amount may be statistically determined, for example using area under the curve (AUC) analysis in receiver operating characteristics (ROC) curves. In preferred embodiments, the threshold amount may be an amount determined by an AUC analysis of more than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, or 0.99. Preferably, the threshold amount may be an amount determined by an AUC analysis of more than 0.8, more preferably 0.9. In such cases, the threshold amount has statistical confidence. In such cases, the threshold amount provides a diagnosis with statistical confidence. The threshold amount may be determined based on the degree of sensitivity and specificity. Samples of CSF with an amount of the at least one metabolite above the threshold amount may detect the presence of a brain tumour in a subject with a sensitivity and specificity of greater than 70%, 80% or 90%. Preferably, the sensitivity and specificity are greater than 90%.

[0126] The threshold amount may be a ratio of at least 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 950, or at least 1000.

[0127] The threshold amount may depend on the or each metabolite being analysed. There may be more than one threshold amount depending on the number of metabolite(s) being analysed.

[0128] In embodiments wherein the at least one test metabolite is betaine and the at least one control metabolite is creatinine, the threshold amount may be a ratio of betaine / creatinine of at least 0.01, 0.05, 0.1, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, or at least 0.50. The threshold amount may be a ratio of betaine / creatinine of 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.30. Preferably, in such embodiments, the threshold amount may be a ratio of betaine / creatinine of 0.178. In such embodiments, if the ratio (i.e. the standardised amount of the or each test metabolite) in the sample of CSF is above this threshold ratio, this provides a positive result indicating the detection of a brain tumour in the subject.

[0129] In embodiments wherein the at least one test metabolite is betaine and the at least one control metabolite is threonate, the threshold amount may be a ratio of betaine / threonate of at least 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or at least 3.0. The threshold amount may be a ratio of betaine / threonate of 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 3.10, 3.11, 3.12, 3.13, 3.14, or 3.15. Preferably, in such embodiments, the threshold amount may be a ratio of betaine / threonate of 2.16. In such embodiments, if the ratio (i.e. the standardised amount of the or each test metabolite) in the sample of CSF is above this threshold ratio, this provides a positive result indicating the detection of a brain tumour in the subject.

[0130] In embodiments wherein the at least one metabolite is carnitine and the at least one control metabolite is creatinine, the threshold amount may be a ratio of camitine / creatinine of at least 0.005, 0.010, 0.015, 0.020, 0.025, 0.030, 0.035, 0.040, 0.045, 0.050, 0.055, 0.060, 0.065, 0.070, 0.075, 0.080, 0.085, 0.090, 0.095, 0.100, 0.105, 0.110, 0.115, 0.120, or at least 0.125. The threshold amount may be a ratio of camitine / creatinine of 0.061, 0.062, 0.063, 0.064, 0.065, 0.066, 0.067, 0.068, 0.069, 0.070, 0.071, 0.072, 0.073, 0.074, 0.075, 0.076, 0.078, 0.079, or 0.080. Preferably, in such embodiments, the threshold amount may be a ratio of camitine / creatinine of 0.0680. In such embodiments, if the ratio (i.e. the standardised amount of the or each test metabolite) in the sample of CSF is above this threshold ratio, this provides a positive result indicating the detection of a brain tumour in the subject.

[0131] The threshold amounts described above are values at which there is a statistically high confidence that a brain tumour is present in the subject.

[0132] In some embodiments, the method may further comprise the step of identifying the type of brain tumour present in the subject. The brain tumour identified may be an ependymoma or a medulloblastoma. This step may involve measuring and analysing at least one additional metabolite in the sample of CSF taken from the subject, wherein when the at least one additional metabolite is selected from the group consisting of: malic acid, 2-oxoglutarate, glutarate semialdehyde and any combination thereof.

[0133] This step of identifying the type of brain tumour may comprise the sub-steps of: a) measuring the amount of malic acid and / or 2-oxoglutarate in the sample of CSF taken from the subject; b) measuring the amount of glutarate semialdehyde in the sample of CSF taken from the subject; c) standardising the amount of glutarate semialdehyde based on the amount of malic acid and / or 2-oxoglutarate; d) comparing the standardised amount of glutamate semialdehyde with a secondary threshold amount, and wherein when the standardised amount is above the threshold amount this indicates the presence of as ependymoma in the subject, and wherein when the standardised amount is below a threshold amount a medulloblastoma is detected in the subject.

[0134] It was surprisingly found that the metabolites malic acid, 2-oxoglutarate, and glutarate semialdehyde were present in the samples of CSF to differing levels dependent upon the type of brain tumour (e.g. an ependymoma or a medulloblastoma). Thus, measuring the levels of these metabolites can provide a way of distinguishing the type of brain tumour present.

[0135] In some embodiments, the amount of the glutamate semialdehyde is standardised against the amount of malic acid and / or 2-oxoglutarate in the sample of CSF. This may be calculated as a ratio of the amount of glutarate semialdehyde to the amount of malic acid and / or 2-oxoglutarate.

[0136] In some embodiments, the threshold amount is a ratio of the amount of the glutamate semialdehyde to the amount of malic acid and / or 2-oxoglutarate at which the type of brain tumour can be distinguished from a sample of CSF with a high degree of statistical confidence. The threshold amount may be determined as described above.

[0137] The method of the first, second or third aspect of the invention may comprise measuring the total amount of, or ratio of the total amount of at least 2 metabolites (or 3 or more metabolites) in the CSF samples, in order to determine the increase / decrease or ratios of the increase or decrease of said metabolites between controls (not having brain cancer) and patient samples or to distinguish between different types of brain cancer. In some embodiments measurement of the total amount of 2 or more metabolites in a sample is compared to the total amount of the 2 or more metabolites in a control sample

[0138] (from a patient not having brain cancer) and the ratios compared, to identify whether a brain cancer is present in the sample. In other embodiments the total amount of 2 or more metabolites from a sample is measured and the increase or decrease in total amount determined, wherein different cut-off values are indicative of particular types of brain cancer. For example, a cut-off value above a first value but below a second value may indicate ependymoma, whereas a cut-off value above the second value may indicated medulloblastoma, or vice versa. Table A below provides embodiments of pairs of metabolites and the cut-off ratio (value) which indicates the presence of brain cancer compared to a control (EPN vs Control), which indicates medulloblastoma rather than ependymoma (MB vs. EPN) or which indicates medulloblastoma compared to a control (MB vs CON) . For example if the ratio of the total amount of betaine and threonic acid in in a sample is at least 2.09 times but lower than 2.97 times the amount of the same metabolites in a control sample, ependymoma is indicated, whereas if it is at least 2.97 times, then medulloblastoma is indicated; and if the ratio of the total amount of 3- Methyl-2-oxobutanoate and malate in a known brain cancer sample (e.g., determined by the methods of the first, second or third aspects of the invention) is no more than 0.0726 times the amount of the same metabolites in a known medulloblastoma sample, this indicates that the brain cancer in the sample is ependymoma.

[0139] Table A: (EPN = ependymoma; MB = medulloblastoma; CON = control)

[0140] According to a further aspect of the invention, the method according to the first, second and / or third aspect is used in the detection of a minimal residual disease in brain cancer after treatment. The method according to the first, second and / or third aspect of the invention may be used in the prognosis of the brain cancer before, during or after treatment.

[0141] The method according to the first, second and / or third aspect of the invention may be used in the prognosis of brain cancer recurrence after treatment.

[0142] The method according to the first, second or third aspect of the invention may be used in the diagnosis of brain cancer. The method may be used to diagnose a subject previously undiagnosed with brain cancer. The method according to the first, second or third aspect of the invention may be used to determine the type of brain cancer present in a subject. The method may be used to distinguish between an ependymoma or a medulloblastoma.

[0143] The method according to the first, second or third aspect of the invention may be used to determine the characteristics of a brain cancer in a subject.

[0144] Detailed Description of the Invention

[0145] In order that the invention may be more clearly understood embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which:

[0146] Figure 1 shows a principal component analysis (PCA) plot showing the metabolic profiles of CSF samples of ependymoma (circular data points, n=32), medulloblastoma (triangular data points, n=14) and a control of end of treatment leukaemia (square data points, n=49). Each data point is one CSF sample and the position in space denotes the effect of the sum total of all metabolites identified in each CSF sample.

[0147] Figure 2 shows the concentration of betaine (Fig. 2A), proline (Fig. 2B), creatine (Fig. 2C) and carnitine (Fig. 2D) in samples of CSF taken from subjects with a brain tumour at post-surgery and relapse, compared with a control sample of CSF (taken from a child who had no brain cancer or neurological condition).

[0148] Figure 3 shows the AUC analysis for proline (Fig. 3A) and valine (Fig. 3B), according to an embodiment of the first aspect of the invention. Figure 4 shows the AUC analysis for betaine / creatinine ratios (Fig. 4A), betaine / threonate ratios (Fig. 4B), and camitine / creatinine ratios (Fig. 4C), according to embodiments of the third aspect of the invention.

[0149] Metabolites as biomarkers for brain tumours

[0150] Figure 1 is a principal component analysis (PCA) plot which demonstrates the total metabolic profiles of ependymoma and medulloblastoma are broadly similar to one another and are different to that of the control. 18 metabolites were identified as differentially abundant between brain tumour samples (ependymoma and medulloblastoma) and a control samples (end of treatment leukaemia). 14 of the metabolites identified were shown to be increased in abundance in brain tumour samples, and 4 of the metabolites identified were shown to be decreased in abundance in brain tumour samples (as shown in Table 2). The metabolites identified included amino acids and derivatives (n=8), metabolites in one carbon metabolism (n=l), the carnitine cycle (n=2), aerobic glycolysis (n=2), purine catabolism (n=2), ascorbate degradation (n=l) and creatinine synthesis (n=2).

[0151] Figure 2 demonstrates that betaine (Fig. 2A), proline (Fig. 2B), creatine (Fig. 2C) and carnitine (Fig. 2D) are present at a higher concentration in samples of CSF taken from subjects with a brain tumour (ependymoma or medulloblastoma) at post-surgery and relapse compared with control samples.

[0152] Preparation of CSF samples

[0153] The CSF samples were prepared as follows: i. Take 25 μL of CSF ii. Add 75 μL of MeOH at 4 °C iii. Vortex for 10 minutes at 2000 rpm at 4 °C iv. Incubate at -80 °C overnight (14 hours) to precipitate proteins (and remove blood, so blood-stained samples can be used) v. Centrifuge for 10 minutes at 13000 G at 4 °C (to remove cells)

[0154] Example Method 1:

[0155] Example method 1 describes an embodiment of a method according to the first aspect of the invention.

[0156] Following sample preparation, metabolites present in CSF samples were identified and their concentrations measured using liquid chromatography with tandem mass spectrometry (LC-MS / MS) using a ZIC-pHILIC column.

[0157] Standards were used to determine the concentration of the metabolite in the samples of CSF. Standards were added to the MeOH in step ii.

[0158] Data processing of the LC-MS / MS data was done through Xcalibur software and anaylsed in Excel.

[0159] In Example Method 1, the presence of a brain tumour in a subject is detected when the concentration of the or each metabolite in the sample of CSF is above a threshold amount. The threshold amount for each metabolite was determined using AUC analysis of a receiver operating characteristic (ROC) curve. As an example, Figure 3 shows the AUC analysis for proline (Fig. 3A) and creatine (Fig. 3B). The threshold amount is determined based on an absolute concentration of a given metabolite which has a high sensitivity and specificity (preferably above around 90% for both) for detecting a brain tumour based on AUC analysis of a receiver operating characteristic (ROC) curve.

[0160] Table 1 below shows the threshold amounts (given as concentrations) for each metabolite calculated based on the AUC analysis as described above. This data demonstrates how the presence of a brain tumour can be detected based on the absolute concentration of a given metabolite in a sample of CSF.

[0161] Table 1:

[0162] Example Method 2:

[0163] Example Method 2 demonstrates an embodiment according to a second aspect of the invention. Following sample preparation, metabolites present in the CSF samples were identified and their concentrations measured using liquid chromatography with tandem mass spectrometry (LC-MS / MS) using a ZIC-pHILIC column.

[0164] Standards were used to determine the concentration of the metabolite in the samples of CSF. 250 authentic standards were selected for identification to create a library of metabolites values which were matched to the CSF results.

[0165] Data processing of the LC-MS / MS data was done through Compound Discoverer (multivariate analysis in SIMCA) and ROC (receiver operator curve) in Matboanalyst.

[0166] In example method 2, the presence of a brain tumour in a subject is detected when the concentration of the or each metabolite in the sample of CSF is increased or decreased compared to a control sample of CSF (end of treatment leukaemia).

[0167] From the PCA analysis, it was revealed that that the 18 metabolites listed in Table 2 are differentially abundant between brain tumour samples and control samples.

[0168] Table 2 below shows the ratio of the concentration of each metabolite in a CSF sample taken from subjects with ependymoma or medulloblastoma compared with the concentration of each metabolite in control CSF samples (end of treatment leukaemia).

[0169] Referring to Table 2, N-acetyl-L-histidine, hypoxanthine, L-threonic acid and creatinine have a ratio of less than 1 and are therefore decreased in abundance in CSF samples taken from subjects with a brain tumour (both ependymoma and medulloblastoma) compared to control samples.

[0170] Referring to Table 2, L-glutamine, creatine, L-arginine, pyruvate, (R)-lactate, L- leucine, L-lysine, betaine, L-isoleucine, L-asparagine, urate, O-acetylcamitine, L- carnitine and L-proline have a ratio of greater than 1 and are therefore increased in abundance in CSF samples taken from subjects with a brain tumour (both ependymoma and medulloblastoma) compared to control samples.

[0171] The data in Table 2 demonstrates that by measuring an increase or decrease in concentration of these metabolites in CSF samples, we can test for both ependymoma and medulloblastoma as they have similar metabolic profiles.

[0172] Table 2: Example Method 3:

[0173] Example Method 3 demonstrates an embodiment according to a third aspect of the invention.

[0174] Following sample preparation, metabolites present in the CSF samples were identified and their concentrations measured using liquid chromatography with tandem mass spectrometry (LC-MS / MS) using a ZIC-pHILIC column.

[0175] Standards were used to determine the concentration of the metabolite in the samples of CSF.

[0176] Data processing of the LC-MS / MS data was done through Xcalibur software.

[0177] In example method 3, the presence of a brain tumour in a subject is detected by comparing the concentrations of a test metabolite with the concentration of a control metabolite.

[0178] The test metabolite is any metabolite that increases in abundance in CSF samples taken from subjects with a brain tumour compared to a control sample of CSF (end of treatment leukaemia). The test metabolite may be any metabolite listed in Table 2 with a ratio of greater than one.

[0179] The control metabolite is any metabolite that decreases in abundance in CSF samples taken from subjects with a brain tumour compared to a control sample of CSF (end of treatment leukaemia). The control metabolite may be any metabolite listed in Table 2 with a ratio less than one.

[0180] The ratio of the concentration of the test metabolite / control metabolite was calculated. This ratio was then compared to a threshold amount which was determined using AUC analysis of a receiver operating characteristic (ROC) curve. As an example, Figure 4 shows the AUC analysis for the following ratios of metabolites: betaine / creatinine (Fig. 4A), betaine / threonate (Fig. 4B) and camitine / creatinine (Fig. 4C). The threshold amount is determined based on a ratio of the concentration of test metabolite / control metabolite which has high sensitivity and specificity (preferably above around 90% for both) for detecting a brain tumour based on AUC analysis of a receiver operating characteristic (ROC) curve.

[0181] As shown in Figure 4, the threshold amount for the betaine / creatine ratio is 0.177, the threshold amount for the betaine / threonate ratio is 2.16, and the threshold amount for the carnitine to creatine is 0.0681. This data demonstrates how the presence of a brain tumour can be detected based on the ratios of the concentrations of two metabolites in a sample of CSF.

[0182] Identifying the type of brain cancer:

[0183] PCA analysis also revealed that the abundance of malic acid, 2-oxoglutate and glutarate semialdehyde in CSF samples differs between ependymoma and medulloblastoma.

[0184] Table 3 shows the difference in abundance of these three metabolites between ependymoma and medulloblastoma. This data demonstrates that these three metabolites can be used to distinguish between ependymoma and medulloblastoma.

[0185] Table 3:

[0186] Metabolites in CSF arise from tumour tissue:

[0187] Table 4 shows the metabolites identified in ependymoma tissue samples using secondary ion mass spectrometry (OrbiSIMS) surface analysis metabolomics. FFPE ependymoma tissue samples were used (n=19) and were matched to the CSF samples used above. The metabolites detected in CSF samples are also detected in ependymoma tissues samples. This demonstrates that metabolites in CSF samples are a good indicator of the presence of a brain tumour.

[0188] Table 4:

[0189] Further investigations were undertaken to determine the origin of metabolites in CSF. Method Secondary ion mass spectrometry (OrbiSIMS) surface analysis metabolomics was performed on FFPE tissue samples of epndymomas. The results showed that the metabolites found in the CSF samples of the above methods matched those found in epndymoma tissue samples. The above embodiments are described by way of example only. Many variations are possible without departing from the scope of the invention.

Claims

CLAIMS1. A method of detecting a brain tumour in a subject, the method comprising the steps of:(i) measuring the amount of at least one metabolite in a sample of cerebrospinal fluid (CSF) taken from a subject, wherein the at least one metabolite is selected from the group consisting of: propionyl carnitine, butyryl carnitine, creatine, creatinine, valine, glutamine, leucine, betaine, isoleucine, asparagine, pyruvate, acetyl carnitine, carnitine, proline, valine and any combination thereof; and(ii) analysing the amount of the at least one metabolite in the sample of CSF, wherein when the amount of the at least one metabolite in the sample of CSF is above a threshold amount a brain tumour in the subject is detected.

2. A method of detecting a brain tumour in a subject, the method comprising the steps of:(i) measuring the amount of at least one metabolite in a sample of cerebrospinal fluid (CSF) taken from a subject, wherein the at least one metabolite is selected from the group consisting of: N-acetylhistidine, hypoxanthine, threonic acid or threonate, creatinine, glutamine, creatine, arginine, pyruvate, lactate, leucine, lysine, betaine, isoleucine, asparagine, urate, acetylcamitine, carnitine, proline and any combination thereof; and(ii) analysing the amount of the at least one metabolite present in the sample of CSF by comparing the amount of the at least one metabolite in the sample of CSF with an amount of the at least one metabolite in a control sample of CSF taken from a subject without a brain tumour, and wherein when the amount of the at least one metabolite in the sample of CSF is elevated or reduced in comparison to the amount of the at least one metabolite in the control sample of CSF a brain tumour in the subject is detected.

3. The method according to claim 1 or 2, wherein the brain tumour is an ependymoma or a medulloblastoma.

4. The method according to any preceding claim, wherein the at least one metabolite is selected from the group consisting of: betaine, carnitine, propionyl carnitine, butyryl carnitine, proline, creatine and any combination thereof.

5. The method according to claim 2, wherein a brain tumour is detected when the amount of at least one of the following metabolites is elevated in the sample of CSF compared with the amount of the at least one metabolite in the control sample: glutamine, creatine, arginine, pyruvate, lactate, leucine, lysine, betaine, isoleucine, asparagine, urate, O-acetyl carnitine, carnitine and proline.

6. The method according to claim 2, wherein a brain tumour is detected when the amount of at least one of the following metabolites is reduced in the sample of CSF compared with the amount of the at least one metabolite in the control sample: N-acetyl histidine, hypoxanthine, threonic acid and creatinine.

7. The method according to any preceding claim, wherein the method comprises a panel of more than one metabolite.

8. The method according to any preceding claim, wherein the amount of the or each metabolite is measured as a concentration in the sample of CSF.

9. The method according to claim 8, wherein the threshold amount for the or each metabolite is a concentration of at least: 0.060 μM for propionyl carnitine, at least 0.030 μM for butyryl carnitine, at least 2.0 μM for betaine, at least 45 μM for creatine, at least 21 μM for valine, at least 12 μM for leucine, at least 1.0 μM for carnitine, at least 0.40 μM for acetyl carnitine, at least 0.70 μM for proline, at least 8.6 μM for isoleucine, at least 8.8 μM for asparagine, at least 220 μM for pyruvate and at least 510 μM for glutamine.

10. The method according to any preceding claim, wherein the amount of the or each metabolite in the sample of CSF is measured using a technique selected from the group consisting of: liquid chromatography-mass spectrometry (LC- MS), liquid chromatography with tandem mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry (GC-MS), gas chromatography with tandem mass spectrometry (GC-MS / MS), an enzyme-linked immunosorbent assay (ELISA) and a lateral flow assay.

11. The method according to any preceding claim, wherein the method further comprises the step of identifying the type of brain tumour detected in the subject, the step comprising:(c) measuring the amount of at least one additional metabolite in the sample ofCSF taken from the subject, wherein the at least one additional metabolite isselected from a group consisting of: malic acid, 2-oxoglutarate, glutarate semialdehyde and any combination thereof; and(d) analysing the amount of the at least one additional metabolite in the sample of CSF, wherein when the sample of CSF has an elevated amount of 2- oxoglutarate and / or malic acid above a threshold amount a medulloblastoma is identified, and wherein when the sample of CSF has an elevated amount of glutarate semialdehyde above a threshold amount an ependymoma is identified.

12. A method according to claim 2 or any claim dependent on claim 2, wherein the method further comprises the step of identifying the type of brain tumour detected in the subject, the step comprising:(c) measuring the amount of at least one additional metabolite in the sample of CSF taken from the subject, wherein the at least one additional metabolite is selected from a group consisting of: malic acid, 2-oxoglutarate, glutarate semialdehyde and any combination thereof;(d) analysing the amount of the at least one additional metabolite present in the sample of CSF by comparing the amount of the at least one additional metabolite with an amount of the at least one additional metabolite in the control sample of CSF, and wherein when the sample of CSF has an elevated amount of 2-oxoglutarate and / or malic acid compared with the control sample of CSF a medulloblastoma is identified, and wherein when the sample of CSF has an elevated amount of glutarate semialdehyde compared with the control sample of CSF an ependymoma is identified.

13. The method according to any preceding claim, wherein the method is used in the detection of a minimal residual disease in brain cancer after treatment.

14. The method according to any preceding claim, wherein the method is used in the prognosis of brain cancer before, during or after treatment, or the prognosis of brain cancer recurrence after treatment.

15. The method according to any preceding claim, wherein the method is used in the diagnosis of a brain cancer.

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