GB2630774A - Lung cancer biomarkers - Google Patents

Abstract

A method for diagnosing lung cancer in an individual comprising or consisting of the step of: (a) providing a test sample from the individual; and (b) measuring the expression in the test sample of one or more biomarkers selected from KCNAB2, SLC15A1, GPR123 and KNDC1; wherein the expression in the test sample of the one or more biomarkers is indicative of lung cancer in the individual. The gene SLC15A1 was significantly upregulated in a lung adenocarcinoma cell line whilst KCNAB2, GPR123 and KNDC1 showed significant downregulation. Uses and methods of diagnosing lung cancer in an individual, and methods of treating lung cancer in an individual, together with arrays and kits for use in the same.

Description

LUNG CANCER BIOMARKERS

Field of Invention

The present invention provides in vitro methods for diagnosing lung cancer in an individual, as well as arrays and kits for use in such methods.

Background

Lung adenocarcinomas (LAUD) are regarded as the leading cause of cancer-related deaths worldwide amongst men and women. The majority of cases are diagnosed when the cancer has already metastasized and surgical resection is no longer an option, resulting in a dismal overall 5-year survival rate for NSCLC of 24% and only 6% in stage 4 disease (seer.cancer.gov).

Despite improvements in the management of the neoplasm, LAUD is associated with low 5-year overall survival rate of 18% (Haghjoo et al., 2020). Generally, the symptoms of lung cancer do not appear until the disease is at an advanced stage, which therefore limits treatment options and leads to poor long-term survival.

Next generation sequencing (NGS) utilizes parallel sequencing strategies to produce millions of short read sequences. Together with bioinformatics tools, they have revolutionised research and have provided unprecedented prospects for the analysis of large data sets rapidly and cost-effectively. Specifically, they enable the detection of a plethora of biomarkers in numerous genes with high diagnostic sensitivity. The inventors used these approaches to identify novel lung cancer diagnostic biomarkers, after identifying a need for the identification of biomarkers associated with lung cancer that could allow earlier and more accurate diagnosis, and therefore improved treatment.

Herein, the inventors have used machine learning tools in order to identify and validate novel lung cancer diagnostic biomarkers that can be utilised alone, or in combination with other known lung cancer biomarkers.

Summary of the Invention

Accordingly, a first aspect of the invention provides a method for diagnosing lung cancer in an individual, the method comprising or consisting of the steps of: (a) providing a test sample from the individual; (b) measuring the expression in the test sample of one or more biomarkers selected from KCNAB2, SLC15A1, GPR123 and KNDC1; wherein the expression in the test sample of the one or more biomarkers is indicative of lung cancer in the individual.

In a preferred embodiment, step (b) comprises or consists of measuring the expression of two or more biomarkers selected from KCNAB2, SLC15A1, GPR123 and KNDC1, wherein the expression in the test sample of the one or more biomarkers is indicative of lung cancer in the individual.

Thus, in one embodiment, the method comprises determining a biomarker signature of the test sample, which enables a diagnosis to be reached in respect of the individual from which the sample is obtained.

By "biomarker" we include biological molecules (or components or fragments thereof) that provide information that is useful in the diagnosis of lung cancer. The biomarker may be a nucleic acid molecule, for example an mRNA or cDNA molecule. The biomarker may also be a protein or polypeptide.

By "biomarker signature" we mean the combination of biomarkers that are measured in the sample that are useful in the diagnosis of lung cancer.

By "measuring the expression" we include measuring the presence and/or amount of a particular biomarker. We also include measuring whether a particular biomarker is up-or down-regulated compared to an expected expression level or a control expression level.

In the methods of the invention, step (b) comprises or consists of measuring the expression of one or more biomarkers, for example at least two, three or all four of the biomarkers selected from KCNAB2, SLC15A1, GPR123 and KNDC1.

In an additional or alternative embodiment, step (b) comprises or consists of measuring the expression of two or more biomarkers, for example at least three or four of the biomarkers selected from KCNAB2, SLC15A1, GPR123 and KNDC1. The skilled person will appreciate that measurement of any combination of these four biomarkers may be measured within the scope of the claimed invention.

In an additional or alternative embodiment, step (b) comprises or consists of measuring the expression of KCNAB2 and SLC15A1. In some embodiments, step (b) comprises or consists of measuring the expression of GPR123 and KNDC1.

In an additional or alternative embodiment, step (b) comprises or consists of measuring the expression of KCNAB2 and GPR123. In some embodiments, step (b) comprises or consists of measuring the expression of SLC15A1 and KNDC1.

In an additional or alternative embodiment, step (b) comprises or consists of measuring the expression of KCNAB2 and KNDC1. In some embodiments, step (b) comprises or consists of measuring the expression of GPR123 and SLC15A1.

In an additional or alternative embodiment, step (b) comprises or consists of measuring the expression of each of KCNAB2, SLC15A1, GPR123 and KNDC1.

In some preferred embodiments, step (b) comprises or consists of measuring the 20 expression of KCNAB2.

In some preferred embodiments, step (b) comprises or consists of measuring the expression of SLC15A1.

In some preferred embodiments, step (b) comprises or consists of measuring the expression of GPR123.

In some preferred embodiments, step (b) comprises or consists of measuring the expression of KNDC1.

In some embodiments, step (b) comprises or consists of measuring the expression of SLC15A1. In some embodiments, step (b) comprises or consists of measuring the expression of GPR123. In some embodiments, step (b) comprises or consists of measuring the expression of KNDC1.

In some preferred embodiments, step (b) comprises or consists of measuring the expression of: - KCNAB2 and SLC15A1; - KCNAB2 and GPR123; - KCNAB2 and KNDC1; - KCNAB2 and SLC15A1 and GPR123; -KCNAB2 and SLD15A1 and KNDC1; or - KCNAB2 and GPR123 and KNDC1.

In an additional or alternative embodiment, step (b) further comprises measuring the expression of one or more biomarkers selected from TP53, KRAS, EGFR, TOP2A, PSAT1 and SNIP1, for example two, three, four, five, or six of these biomarkers.

In an additional or alternative embodiment, step (b) further comprises measuring the expression of two or more biomarkers selected from TP53, KRAS, EGFR, TOP2A, PSAT1 and SNIP1, for example three, four, five, or six of these biomarkers.

In an additional or alternative embodiment, step (b) comprises measuring the expression of TP53 and KRAS. In some embodiments, step (b) comprises measuring the expression of EGFR and TOP2A. In some embodiments, step (b) comprises measuring the expression of PSAT1 and SNIP1.

In an additional or alternative embodiment, step (b) comprises measuring the expression of TP53 and KRAS. In some embodiments, step (b) comprises measuring the expression of EGFR and PSAT1. In some embodiments, step (b) comprises measuring the expression of TOP2A and SNIP1.

In an additional or alternative embodiment, step (b) comprises measuring the expression of TP53 and KRAS. In some embodiments, step (b) comprises measuring the expression of EGFR and SNIP1. In some embodiments, step (b) comprises measuring the expression of PSAT1 and TOP2A.

In an additional or alternative embodiment, step (b) comprises measuring the expression of EGFR and TOP2A. In some embodiments, step (b) comprises measuring the expression of TP53 and SNIP1. In some embodiments, step (b) comprises measuring the expression of PSAT1 and KRAS.

In an additional or alternative embodiment, step (b) comprises measuring the expression of EGFR and TOP2A. In some embodiments, step (b) comprises measuring 20 25 30 35 the expression of TP53 and PSAT1. In some embodiments, step (b) comprises measuring the expression of SNIP1 and KRAS.

In an additional or alternative embodiment, step (b) comprises or consists of measuring the expression of PSAT1 and SNIP1. In some embodiments, step (b) comprises measuring the expression of TP53 and EGFR. In some embodiments, step (b) comprises measuring the expression of TOP2A and KRAS.

In an additional or alternative embodiment, step (b) comprises measuring the expression of PSAT1 and SNIP1. In some embodiments, step (b) comprises measuring the expression of TP53 and TOP2A. In some embodiments, step (b) comprises measuring the expression of (iii) EGFR and KRAS.

In some preferred embodiments, step (b) comprises measuring the expression of TP53.

In some preferred embodiments, step (b) comprises measuring the expression of KRAS.

In some preferred embodiments, step (b) comprises measuring the expression of 20 MFR.

In some preferred embodiments, step (b) comprises measuring the expression of TOP2A.

In some preferred embodiments, step (b) comprises measuring the expression of PSAT1.

In some preferred embodiments, step (b) comprises measuring the expression of SNIP1.

In an additional or alternative embodiment, step (b) comprises or consists of measuring the expression of each of KCNAB2, SLC15A1, GPR123, KNDC1, TP53, KRAS, EGFR, TOP2A, PSAT1 and SNIP1.

In some embodiments, step (b) comprises or consists of measuring the expression of any of the combinations of biomarkers listed in Table 2.

By "lung cancer" we include any type of cancer that forms in tissues in the lungs, usually in the cells lining air passages. The two main types are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).

In specific embodiments the methods of the invention permit the diagnosis of lung cancer and/or early lung cancer.

By "diagnosis" we include determining the presence or absence of a disease state in an individual (e.g., determining whether an individual is or is not suffering from lung cancer).

In some preferred embodiments, the lung cancer is Non-Small Cell Lung Cancer (NSCLC). Therefore, in some embodiments, the method is for the diagnosis of NSCLC in an individual.

By "Non-Small Cell Lung Cancer (NSCLC)" we include any type of lung cancer that is not Small Cell Lung Cancer (SCLC).

In an additional or alternative embodiments, the NSCLC is selected from the group comprising: adenocarcinoma; squamous cell carcinoma; adenosquamous carcinoma; large cell carcinoma; or large cell neuroendocrine cancer. In some preferred embodiments, the NSCLC is adenocarcinoma.

In additional or alternative embodiments, the NSCLC in the individual is early-stage NSCLC (i.e., Stage 0, I or Stage II) or is late-stage NSCLC (i.e. Stage III or Stage IV).

In some embodiments, the lung cancer is Small Cell Lung Cancer (SCLC). Therefore, in some embodiments, the method is for the diagnosis of SCLC in an individual.

By "Small Cell Lung Cancer (SCLC)" we include any type of lung cancer that is not Non-Small Cell Lung Cancer (NSCLC).

In some embodiments, the methods described herein are able to diagnose lung cancer at an early stage, as the methods can be carried out on a sample from an individual either when they are showing no symptoms (i.e., asymptomatic) or when they are displaying minor symptoms. Therefore, one advantage of the invention described herein is that it can allow diagnosis of lung cancer at a much earlier stage than traditional diagnostic methods, allowing patients to seek prompt treatment that is key to long term survival.

By "early lung cancer" (or "early stage lung cancer") we include or mean lung cancer comprising or consisting of stage I and/or stage II non-small lung cancer, for example as determined by the American Joint Committee on Cancer (AJCC) TNM system (e.g., see: Milos, isi):::niwhich is incorporated by reference herein).

It will be appreciated by persons skilled in the art that, in addition to measuring the biomarkers in a sample from an individual to be tested, the methods of the invention may also comprise measuring those same biomarkers in one or more control samples.

Thus, in one embodiment, the method further comprises or consists of the steps of: (c) providing one or more (negative) control samples from an individual not afflicted with lung cancer; and (d) determining a biomarker signature of the one or more control samples by measuring the expression in the control sample of the one or more biomarkers measured in step (b); wherein the lung cancer is identified in the event that the presence and/or amount in the test sample of the one or more biomarkers measured in step (b) is different from the expression in the control sample of the one or more biomarkers measured in step (d).

Preferably the negative control sample is a sample derived from normal lung tissue from the individual; or from a healthy individual; or a pool of healthy individuals.

By "is different from the expression in the negative control sample" we include the situation where the biomarker is detected in the test sample but is not detected in the negative control sample(s), and vice versa. We also include the situation where the biomarker in question is upregulated or downregulated in the test sample compared to the same biomarker in the control sample. By "upregulated or downregulated" we include where the amount of the biomarker in the test sample differs from the amount of the biomarker in the control sample by at least ±5%, for example, at least ±6%, ±7%, ±8%, ±9%, ±10°/0, ±11%, ±12%, ±13%, ±14%, ±15%, ±16%, ±17%, ±18%, ±19%, ±20%, ±21%, ±22%, ±23%, ±24%, ±25%, ±26%, ±27%, ±28%, ±29%, ±30%, ±31%, ±32%, ±33%, ±34%, ±35%, ±36%, ±37%, ±38%, ±39%, ±40%, ±41%, ±42%, ±43%, ±44%, ±45%, ±41%, ±42%, ±43%, ±44%, ±55%, ±60%, ±65%, ±66%, ±67%, ±68%, ±69%, ±70%, ±71%, ±72%, ±73%, ±74%, ±75%, ±76%, ±77%, ±78%, ±79%, ±80%, ±81%, ±82%, ±83%, ±84%, ±85%, ±86%, ±87%, ±88%, ±89%, ±90%, ±91%, ±92%, ±93%, ±94%, ±95%, ±96%, ±97%, ±98%, ±99%, ±100%, ±125%, ±150%, ±175%, ±200%, ±225%, ±250%, ±275%, ±300%, ±350%, ±400%, ±500% or at least ±1000% of the one or more control sample(s) (e.g., the negative control sample).

Alternatively or additionally, the expression in the test sample differs from the mean expression in the control samples by at least >1 standard deviation from the mean presence or amount in the control samples, for example, a1.5, a2, a3, a4, a5, a6, ?7, a8, a9, a10, all, a12, a13, a14 or a15 standard deviations from the mean presence or amount in the control samples. Any suitable means may be used for determining standard deviation, however, in one embodiment, standard deviation is determined using the direct method (i.e., the square root of [the sum the squares of the samples minus the mean, divided by the number of samples]). In additional or alternative embodiments, other statistical methods that are well known in the art can be used to determine whether there is a difference between the expression of a biomarker in the test sample compared to a control sample. Such methods may include but are not limited to the following: Student t-test, Mann-Whitney U test, one-way analysis of variance (ANOVA), Kruskal-Wallis test, Limma test.

In an additional or alternative embodiment, a decrease in the amount of KCNAB2 measured in step (b) as compared to a negative control sample is indicative of lung cancer in the individual.

In an additional or alternative embodiment, an increase in the amount of SLC15A1 measured in step (b) as compared to a negative control sample is indicative of lung cancer in the individual.

In an additional or alternative embodiment, a decrease in the amount of GPR123 measured in step (b) as compared to a negative control sample is indicative of lung cancer in the individual.

In an additional or alternative embodiment, a decrease in the amount of KNDC1 measured in step (b) as compared to a negative control sample is indicative of lung cancer in the individual.

In an additional or alternative embodiment, an increase in the amount of TP53, KRAS, EGFR, TOP2A, PSAT1 and a decrease in the amount of SNIP1 measured in step (b) as compared to a negative control sample is indicative of lung cancer in the individual.

In an additional or alternative embodiment, a decrease in the amount of KCNAB2 measured in step (b) is indicative of lung cancer in the individual.

In an additional or alternative embodiment, an increase in the amount of SLC15A1 measured in step (b) is indicative of lung cancer in the individual.

In an additional or alternative embodiment, a decrease in the amount of GPR123 measured in step (b) is indicative of lung cancer in the individual.

In an additional or alternative embodiment, a decrease in the amount of KNDC1 measured in step (b) is indicative of lung cancer in the individual.

In an additional or alternative embodiment, an increase in the amount of TP53, KRAS, EGFR, TOP2A, PSAT1 and a decrease in the amount of SNIP1 measured in step (b) is indicative of lung cancer in the individual.

In one embodiment, the method of the invention may further comprise or consist of the steps of: (e) providing one or more (positive) control samples from an individual afflicted with lung cancer; and (f) determining a biomarker signature of the control sample by measuring the expression in the control sample of the one or more biomarkers measured in step (b); wherein the lung cancer is identified in the event that the expression in the test sample of the one or more biomarkers measured in step (b) corresponds to the expression in the control sample of the one or more biomarkers measured in step (f).

Preferably the control sample is a sample derived from an individual having NSCLC.

By "corresponds to the expression in the control sample" we include that the presence and/or amount is identical to that of a positive control sample; or closer to that of one or more positive control sample than to one or more negative control sample (or to predefined reference values representing the same). Preferably the presence and/or amount is within ±40% of that of the one or more control sample (or mean of the control samples), for example, within ±39%, ±38%, ±37%, ±36%, ±35%, ±34%, ±33%, ±32%, ±31%, ±30%, ±29%, ±28%, ±27%, ±26%, ±25%, ±24%, ±23%, ±22%, ±21%, ±20%, ±19%, ±18%, ±17%, ±16%, ±15%, ±14%, ±13%, ±12%, ±11°/0, ±10°/0, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.05% or within 0% of the one or more control sample (e.g., the positive control sample).

Alternatively or additionally, the difference in the expression in the test sample is S5 standard deviation from the mean presence or amount in the control samples, for example, 5_4.5, 5_4, 5_3.5, S3, 52.5, 5_2, 5_1.5, 5_1.4, 5_1.3, 5_1.2, 5_1.1, Si, 5_0.9, 5_0.8, s0.7, 5_0.6, 5_0.5, 5_0.4, 550.3, s0.2, 5_0.1 or 0 standard deviations from the from the mean presence or amount in the control samples, provided that the standard deviation ranges for differing and corresponding biomarker expressions do not overlap (e.g., abut, but no not overlap).

Alternatively or additionally, by "corresponds to the expression in the control sample" we include that the presence or amount in the test sample correlates with the amount in the control sample in a statistically significant manner. By "correlates with the amount in the control sample in a statistically significant manner" we mean or include that the presence or amount in the test sample correlates with the that of the control sample with a p-value of 5_0.05, for example, 50.04, 5_0.03, 5_0.02, 5_0.01, 5_0.005, 50.004, 50.003, 50.002, 50.001, 50.0005 or 50.0001.

By "positive control sample" we include samples derived from an individual with confirmed lung cancer or a pool of lung cancer samples. In the case where the positive control is a pool of lung cancer samples, the amount of the biomarker may be an average value of the amount of the biomarker measured in each of the lung cancer samples.

Differential expression (up-regulation or down-regulation) of biomarkers, or lack thereof, can be determined by any suitable means known to a skilled person. Differential expression is determined to a p value of a least less than 0.05 (p = < 0.05), for example, at least <0.04, <0.03, <0.02, <0.01, <0.009, <0.005, <0.001, <0.0001, <0.00001 or at least <0.000001.

It will be appreciated by persons skilled in the art that differential expression may relate to a single biomarker or to multiple biomarkers considered in combination (i.e., as a biomarker signature). Thus, a p value may be associated with a single biomarker or with a group of biomarkers. Indeed, proteins having a differential expression p value of greater than 0.05 when considered individually may nevertheless still be useful as biomarkers in accordance with the invention when their expression levels are considered in combination with one or more other biomarkers.

Therefore, in some embodiments the classification of step (b) may be achieved by comparing the expression of biomarkers in the test sample to those in the one or more positive and/or negative control sample(s).

In an alternative or additional embodiment, the presence and/or amount in the test sample of the one or more biomarkers measured in step (b) may be compared against predetermined reference values representative of the measurements in steps (e) and/or (g), i.e., reference negative and/or positive control values.

As detailed above, the methods of the invention may also comprise measuring, in one or more negative or positive control samples, the presence and/or amount of the one or more biomarkers measured in the test sample in step (b).

In one preferred embodiment of the first aspect of the invention, the method is repeated on the individual. Thus, steps (a) and (b) may be repeated using a sample from the same individual taken at different time to the original sample tested (or the previous method repetition). Such repeated testing may enable disease progression to be assessed, for example to determine the efficacy of a selected treatment regime and (if appropriate) to select an alternative regime to be adopted.

Thus, in one embodiment, the method is repeated using a test sample taken between 1 day to 100 weeks to the previous test sample(s) used, for example, between 1 week to 100 weeks, 1 week to 90 weeks, 1 week to 80 weeks, 1 week to 70 weeks, 1 week to 60 weeks, 1 week to 50 weeks, 1 week to 40 weeks, 1 week to 30 weeks, 1 week to 20 weeks, 1 week to 10 weeks, 1 week to 9 weeks, 1 week to 8 weeks, 1 week to 7 weeks, 1 week to 6 weeks, 1 week to 5 weeks, 1 week to 4 weeks, 1 week to 3 weeks, or 1 week to 2 weeks.

Alternatively or additionally, the method may be repeated using a test sample taken every period from the group consisting of: 1 day, 2 days, 3 day, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 35 weeks, 40 weeks, 45 weeks, 50 weeks, 55 weeks, 60 weeks, 65 weeks, 70 weeks, 75 weeks, 80 weeks, 85 weeks, 90 weeks, 95 weeks, 100 weeks, 104, weeks, 105 weeks, 110 weeks, 115 weeks, 120 weeks, 125 weeks and 130 weeks.

Alternatively or additionally, the method may be repeated at least once, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times or 25 times.

Alternatively or additionally, the method is repeated continuously.

In one embodiment, the method is repeated during therapy, immediately following the completion of therapy, and twice per year for the first five years that the patients are in remission.

In one embodiment, the method is repeated until lung cancer is diagnosed in the individual using the methods of the present invention and/or conventional clinical methods (i.e., until confirmation of the diagnosis is made).

As exemplified in the accompanying Example, the profiles of certain genes in a cell test sample may be indicative of lung cancer in an individual. For example, the relative expression of certain genetic biomarkers in a single test sample may be indicative of the presence of lung cancer in an individual.

In some embodiments, step (b), (d) and/or step (f) is performed using one or more first binding agent capable of binding to a biomarker.

In one preferred embodiment of the method of the invention, step (b), (d) and/or (f) comprises measuring the expression of a nucleic acid molecule encoding the one or more biomarkers.

The nucleic acid molecule may be a gene expression intermediate or derivative thereof, such as an mRNA molecule or a cDNA molecule. In some preferred embodiments, the nucleic acid molecule is an mRNA molecule. By "mRNA molecule" we include premRNA and mature mRNA.

In one embodiment, step (b), (d) and/or (0 is be performed using one or more binding agents, each individually capable of binding selectively to a nucleic acid molecule encoding one of the biomarkers.

Conveniently, the one or more binding moieties may each comprise or consist of a nucleic acid molecule, such as DNA, RNA, peptide nucleic acid (PNA), locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA) or phosphorodiamidate morpholino oligomer (PMO).

Advantageously, the one or more binding moieties may be 5 to 100 nucleotides in length. For example, they may be 15 to 35 nucleotides in length.

It will be appreciated that the nucleic acid-based binding moieties may comprise a

detectable moiety.

The detectable moiety may be selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety (for example, a radioactive atom); or an enzymatic moiety.

Alternatively or additionally, the detectable moiety may comprise or consist of a radioactive atom, for example selected from the group consisting of technetium-99m, iodine-123, iodine-125, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, phosphorus-32, sulphur-35, deuterium, tritium, rhenium-186, rhenium- 188 and yttrium-90.

Alternatively or additionally, the detectable moiety of the binding moiety may be a fluorescent moiety.

In some embodiments, measuring the expression of the one or more biomarker(s) in step (b), (e) and/or (g) may be performed using a method selected from the group consisting of Southern hybridisation, Northern hybridisation, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative reverse transcriptase PCR (RT-qPCR), nanoarray, microarray, autoradiography and in situ hybridisation. In addition, measurement of mRNA may be useful for particular sample types that are more difficult to extract proteins from for analysis.

Preferably, the expression of the biomarkers in the test sample is determined using RT-PCR. In some embodiments, the expression of the biomarkers in the test sample is determined using RT-qPCR. RT-qPCR is used to determine the amount of a particular mRNA in a test sample, and utilises the enzyme reverse transcriptase to convert mRNA in the sample to cDNA, followed by amplification and detection using traditional PCR techniques.

In some embodiments when nucleic acid biomarkers are detected, measurement of the nucleic acid is carried out using another transcriptomics-based technique. These include techniques generally known in the art for detecting nucleic acids (e.g., mRNA) in a sample. They may include, but are not limited to, the following: - Chromogenic in situ hybridization (CISH); -Fluorescence in situ hybridization (FISH); - RNA sequencing; - RNA microarrays; - Digital RT-PCR; - Northern blot; -Digital colour-coded nucleic acid barcode (nCounter®) technology; - RT-PCR-ELISA.

In one embodiment, expression of the one or more biomarker(s) is determined using an RNA or DNA microarray.

In another embodiment of the methods of the invention, step (a) comprises providing a sample from an individual to be tested and step (b) comprises measuring in the sample the expression of the protein or polypeptide of the one or more biomarker(s). Thus, a biomarker signature for the sample may be determined at the protein level.

Therefore, in an alternative or additional embodiment, step (b), (d), and/or (f) comprises measuring the expression of the protein or polypeptide of the one or more biomarkers.

In one preferred embodiment, step (b), (d) and/or step (f) is performed using one or more first binding agent capable of binding to a protein or polypeptide biomarker.

Suitable binding agents (also referred to as binding molecules) can be selected from a library, based on their ability to bind a given target molecule, as discussed below.

In one preferred embodiment, at least one type of the binding agents, and more typically all of the types, may comprise or consist of an antibody or antigen-binding fragment of the same, or a variant thereof.

Methods for the production and use of antibodies are well known in the art, for example see Antibodies: A Laboratory Manual, 1988, Harlow & Lane, Cold Spring Harbor Press, ISBN-13: 978-0879693145, Using Antibodies: A Laboratory Manual, 1998, Harlow & Lane, Cold Spring Harbor Press, ISBN-13: 978-0879695446 and Making and Using Antibodies: A Practical Handbook, 2006, Howard & Kaser, CRC Press, ISBN-13: 9780849335280 (the disclosures of which are incorporated herein by reference).

Thus, a fragment may contain one or more of the variable heavy (VH) or variable light (VL) domains. For example, the term antibody fragment includes Fab-like molecules (Better et al., (1988) Science 240, 1041); Fv molecules (Skerra et al., (1988) Science 240, 1038); single-chain Fv (scFv) molecules where the VH and VL partner domains are linked via a flexible oligopeptide (Bird et al., (1988) Science 242, 423; and single domain antibodies (dAbs) comprising isolated V domains (Ward et al., (1989) Nature 341, 544).

For example, the binding agent(s) may be whole antibodies or scFv molecules.

The term "antibody variant" includes any synthetic antibodies, recombinant antibodies or antibody hybrids, such as but not limited to, a single-chain antibody molecule produced by phage-display of immunoglobulin light and/or heavy chain variable and/or constant regions, or other immuno-interactive molecule capable of binding to an antigen in an immunoassay format that is known to those skilled in the art.

A general review of the techniques involved in the synthesis of antibody fragments which retain their specific binding sites is to be found in Winter & Milstein (1.991) Nature 349, 293-299.

Molecular libraries such as antibody libraries (Clackson et al, 1991, Nature 352, 624- 628; Marks et al., 1991, J Mol Biol 222(3): 581-97), peptide libraries (Smith, 1985, Science 228(4705): 1315-7), expressed cDNA libraries (Santi et al., (2000) J Mol Biol 296(2): 497-508), libraries on other scaffolds than the antibody framework such as affibodies (Gunneriusson et al., 1999, Appl Environ Microbiol 65(9): 4134-40) or libraries based on aptamers (Kenan et al., 1999, Methods Mol Biol 118, 217-31) may be used as a source from which binding molecules that are specific for a given motif are selected for use in the methods of the invention.

Conveniently, the binding agent(s) may be immobilised on a surface (e.g., on a multiwell plate or array).

In one embodiment of the methods of the invention, determining the expression of the protein/polypeptide biomarkers is performed using an assay comprising a second binding agent capable of binding to the one or more biomarkers, the second binding agent comprising a detectable moiety. For example, an immobilised (first) binding agent may initially be used to 'trap' the protein biomarker on to the surface of a microarray, and then a second binding agent may be used to detect the 'trapped' protein.

The second binding agent may be as described above in relation to the (first) binding agent, such as an antibody or antigen-binding fragment thereof.

It will be appreciated by skilled person that the one or more biomarkers (e.g., proteins) in the test sample may be labelled with a detectable moiety, prior to performing step (b). Likewise, the one or more biomarkers in the control sample(s) may be labelled with a detectable moiety.

Alternatively, or in addition, the first and/or second binding agents may be labelled with a detectable moiety.

In an alternative or additional embodiment, the one or more biomarkers in the test and/or control sample(s) are labelled either directly or indirectly with a detectable moiety.

By "directly labelled" we include that the biomarkers are bound by a binding agent that is itself or is coupled to a detectable moiety (e.g., as used during a direct ELISA). By "indirectly labelled" we include that the biomarkers are bound by a first binding agent that is not directly labelled, and a second binding agent is used that is itself, or is coupled to, a detectable moiety (e.g., as used during an indirect ELISA) Suitable detectable moieties are well known in the art. For example, the detectable moiety may be selected from the group comprising: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety; an enzymatic moiety.

Thus, the detectable moiety may be a fluorescent and/or luminescent and/or chemiluminescent moiety which, when exposed to specific conditions, may be detected. For example, a fluorescent moiety may need to be exposed to radiation (i.e., light) at a specific wavelength and intensity to cause excitation of the fluorescent moiety, thereby enabling it to emit detectable fluorescence at a specific wavelength that may be detected.

Alternatively, the detectable moiety may be an enzyme which is capable of converting a (preferably undetectable) substrate into a detectable product that can be visualised and/or detected. Examples of suitable enzymes are discussed in more detail below in relation to, for example, ELISA assays.

In a further alternative, the detectable moiety may be a radioactive atom which is useful in imaging. Suitable radioactive atoms include 99mTc and 1231 for scintigraphic studies. Other readily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRI) such as 1231 again, 1311, 111In, 19F, 13C, 15N, 170, gadolinium, manganese or iron. Clearly, the agent to be detected (such as, for example, the one or more biomarkers in the test sample and/or control sample described herein and/or an antibody molecule for use in detecting a selected protein) must have sufficient of the appropriate atomic isotopes in order for the detectable moiety to be readily detectable.

Preferred assays for detecting proteins include immunohistochemistry (IHC), enzyme linked immunosorbent assays (ELISA), radioimmunoassay (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies. Exemplary sandwich assays are described by David et al., in US Patent Nos. 4,376,110 and 4,486,530, hereby incorporated by reference.

In some embodiments, the assay is an ELISA (Enzyme Linked Immunosorbent Assay) which typically involves the use of enzymes giving a coloured reaction product, usually in solid phase assays. Enzymes such as horseradish peroxidase and phosphatase have been widely employed. A way of amplifying the phosphatase reaction is to use NADP as a substrate to generate NAD which now acts as a coenzyme for a second enzyme system. Pyrophosphatase from Escherichia coil provides a good conjugate because the enzyme is not present in tissues, is stable and gives a good reaction colour.

Chemiluminescent systems based on enzymes such as luciferase can also be used.

ELISA methods are well known in the art, for example see The ELISA Guidebook (Methods in Molecular Biology), 2000, Crowther, Humana Press, ISBN-13: 9780896037281 (the disclosures of which are incorporated by reference).

In one embodiment, the detectable moiety is fluorescent moiety (for example an Alexa Fluor dye, e.g., Alexa647).

By "test sample" (or sample to be tested) we include a sample to be tested in the invention, such as a sample taken or derived from an individual to be tested, wherein the sample comprises endogenous proteins and/or nucleic acid molecules. Preferably, the sample to be tested is provided from an individual that is a mammal, preferably a human.

In some embodiments, the individual is selected from the group comprising: a primate (for example, a human; a monkey; an ape); a rodent (for example, a mouse, a rat, a hamster, a guinea pig, a gerbil, a rabbit); a canine (for example, a dog); a feline (for example, a cat); an equine (for example, a horse); a bovine (for example, a cow); or a porcine (for example, a pig).

In some preferred embodiments, the individual is a human. In some embodiments, the individual is a human who is suspected of having lung cancer.

In one preferred embodiment, the test sample comprises one or more lung cancer cell.

In some embodiments, the sample is selected from the group comprising: a biopsy (such as a core needle biopsy; fine needle biopsy; bronchoscopy sample); a tissue sample; an organ sample; and a bodily fluid sample (such as blood or pleural fluid). It will be appreciated that the test and any control samples should be from the same species. In some preferred embodiments, the sample is a tissue sample. In some preferred embodiments, the sample is a sample of lung tissue.

In some embodiments, the biopsy can be analysed using the methods of the present invention either with or without purification of cells from the biopsy sample. The test sample can be taken specifically for the purpose of performing the methods of the present invention, or, in alternative embodiments, the methods of the invention can be carried out on historical samples that have been appropriately stored.

In one particularly preferred embodiment, the sample is a lung tissue sample. In an alternative or additional embodiment, the sample is a sample comprising or consisting of lung cells, for example epithelial cells or alveolar cells or pleural cells. In a preferred embodiment, the sample comprises one or more lung cancer cells.

In some other embodiments, the sample may be a sample of bodily fluid. For example, the sample may be pleural fluid or blood (i.e., unfractionated blood that can be separated into serum or plasma for testing).

The methods of this invention are suitable for testing a sample from any individual who has, or is suspected of having, lung cancer. For example, the individual may be from one of the following groups: Individuals with previously diagnosed lung cancer (of any type or stage); - Individuals with suspected lung cancer; Individuals with symptoms suggestive of or consistent with lung cancer (e.g., persistent coughing, coughing up blood, chest pain or pain when breathing, shortness of breath, fatigue, unintentional or unexplained weight loss, wheezing, hoarseness).

In an alternative or additional embodiment, the expression of the biomarkers in the test sample is determined using mass spectrometry, an affinity-based method, a transcriptomics-based method, ELISA or flow cytometry.

In some preferred embodiments, the expression of the biomarkers in the test and/or control samples is determined by ELISA.

In some embodiments, the affinity-based method is an array. Once suitable binding molecules (discussed above) have been identified and isolated, the skilled person can manufacture an array using methods well known in the art of molecular biology.

In one preferred embodiment, an array is provided for diagnosing or determining the presence of lung cancer in an individual comprising an agent or agents for detecting the presence and/or amount of one or more of the biomarkers selected from KCNAB2, SLC15A1, GPR123 and KNDC1. In some embodiments, the array further comprises an agent or agents for detecting the expression of one or more of the biomarkers selected from TP53, KRAS, EGFR, TOP2A, PSAT1 and SNIP1.

Arrays per se are well known in the art. Typically, they are formed of a linear or two-dimensional structure having spaced apart (i.e., discrete) regions ("spots"), each having a finite area, formed on the surface of a solid support. An array can also be a bead structure where each bead can be identified by a molecular code or colour code or identified in a continuous flow. Analysis can also be performed sequentially where the sample is passed over a series of spots each adsorbing the class of molecules from the solution. The solid support is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs, silicon chips, microplates, polyvinylidene difluoride (PVDF) membrane, nitrocellulose membrane, nylon membrane, other porous membrane, non-porous membrane (e.g. plastic, polymer, perspex, silicon, amongst others), a plurality of polymeric pins, or a plurality of microtitre wells, or any other surface suitable for immobilising proteins, polynucleotides and other suitable molecules and/or conducting an immunoassay. The binding processes are well known in the art and generally consist of cross-linking covalently binding or physically adsorbing a protein molecule, polynucleotide or the like to the solid support. By using well-known techniques, such as contact or non-contact printing, masking or photolithography, the location of each spot can be defined. For reviews see Jenkins, R.E., Pennington, S.R. (2001, Proteomics, 2,13-29) and Lal et al., (2002, Drug Discov Today 15;7(18 Suppl):5143-9).

Typically, the array is a microarray. By "microarray" we include the meaning of an array of regions having a density of discrete regions of at least about 100/cm2, and preferably at least about 1000/cm2. The regions in a microarray have typical dimensions, e.g., diameters, in the range of between about 10-250 pm, and are separated from other regions in the array by about the same distance. The array may also be a nanoarray.

Once suitable binding molecules (discussed above) have been identified and isolated, the skilled person can manufacture an array using methods well known in the art of molecular biology.

In another aspect, the invention provides use of two or more biomarkers selected from the group comprising KCNAB2, SLC15A1, GPR123 and KNDC1 as biomarkers for diagnosing or determining the presence of lung cancer in an individual.

In some embodiments, the invention provides the use of KCNAB2, SLC15A1, GPR123 and KNDC1 as biomarkers for diagnosing or determining the presence of lung cancer in an individual.

One preferred embodiment of the first aspect of the invention includes wherein in the event that the individual is diagnosed with lung cancer, the method further comprises a step of providing the individual with a lung cancer therapy.

Thus, a related aspect of the invention provides a method of treating lung cancer in an individual comprising the steps of: (i) diagnosing lung cancer according to the method described in the first aspect of the invention; and (ii) providing the individual with lung cancer therapy.

The lung cancer therapy may be selected from the group comprising: surgery, chemotherapy, radiotherapy, immunotherapy, chemoimmunotherapy, and combinations thereof, adoptive cell therapies, gene therapies, cancer vaccines, and oncolytic virus therapies. The skilled person will be aware of the most appropriate treatments in order to provide an individual with lung cancer treatment.

A further aspect of the invention provides a kit for diagnosing or determining the presence of lung cancer, the kit comprising: (i) an array according to the invention, or components for making the same; and (ii) instructions for performing the method as defined above (e.g., in the first or subsequent aspects of the invention).

A further aspect of the invention provides a use of one or more binding agents to a biomarker as described herein in the preparation of a kit for diagnosing or determining a lung cancer-associated disease state in an individual. Thus, multiple different binding agents may be used, each targeted to a different biomarker, in the preparation of such as kit. In one embodiment, the binding agent is an antibody or antigen-binding fragment thereof (e.g., scFv) or a nucleic acid binding molecule, as described herein.

Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures: Figure 1: Confluency of Calu-3 and MRC-S cells A) A 175 flask for growing Calu-3 cells at 90% confluency (X40 magnification), B) a T75 flask for growing MRC-5 cells at 70% confluency (X40 magnification).

Figure 2: Fold gene expression obtained via RT-qPCR A) SLC15A1 was significantly upregulated (p<0.0032), whereas B) GPR123 (p<0.003), C) KNDC1 (p<0.0002), and D) KCNA82 (p<0.0023) were significantly downregulated. There was no significant difference in fold gene expression of E) GPR183 (p<0.4711).

EXAMPLE 1 -Identification of possible lung cancer biomarkers In the current study, the inventors used machine learning tools to identify novel lung cancer diagnostic biomarkers. Specifically, Gene Expression Omnibus (GEO) datasets for SCLC (GSE15240, GSE99316) and NSCLC (GSE40791, GSE30219, GSE43580, GSE18842, GSE37745) were downloaded, including data on cancer tissue samples from 222 patients with SCLC, 786 patients with NSCLC and adjacent normal tissue samples from 145 patients with lung cancer (GSE40791, GSE18842). Least absolute shrinkage and selection operator (LASSO) regression were used to screen potential diagnostic biomarkers for SCLC and NSCLC. Some of the most highly upregulated and downregulated genes were selected, and their potential as diagnostic biomarkers was verified.

EXAMPLE 2 -Validation of lung cancer biomarkers

Background

The biomarkers with the highest potential identified following the methods of Example 1 were taken forward for further analysis and validation.

Materials and Methods Cell Culture The Calu-3 epithelial cell line derived from lung adenocarcinoma and the MRC-5 cell line derived from fibroblasts isolated from the lung tissue (ATCC, Virginia, USA) were used for the purposes of total RNA isolation and DNA profiling. The Calu-3 cells were cultured in complete Dulbecco's Modified Eagle Medium, DMEM (Gibco, Bleiswijk, Netherlands) supplemented with 10°/0 FBS (Gibco, Bleiswijk, Netherlands) and 1°/0 Pen/Strep (Gibco, Bleiswijk, Netherlands) and were incubated at 37°C and 5% CO2 to grow. MRC-5 cells were cultured in complete Minimum Essential Media, MEM (Gibco, Bleiswijk, Netherlands) supplemented with 10% foetal bovine serum, FBS and 1% penicillin G/streptomycin, Pen/Strep and were incubated under the same conditions as Calu-3 cells. The confluency of each T75 flask was assessed before total RNA extraction, and only flasks with confluency above 90% were used for the purpose of total RNA isolation.

Total RNA isolation In brief, 400pL of Trizol reagent (Ambion Life Technology, Auckland, New Zealand) was used to homogenise the pallets of MRC-5/Calu-3 cells, using a 5 mL syringe. Each extraction was performed in triplicate from three different flasks for each cell line. Post homogenisation, the cell suspensions were incubated in TRIzolTm for 5 minutes at room temperature. Post this period, the TRIzolTm cell homogenate was transferred to a 1.5 mL RNAse free Eppendorf tube and 80 pL of chloroform was added to the homogenate. The Eppendorf contents were mixed well by vortexing at medium speed and left to incubate at room temperature for 2-3 min. The Eppendorf tubes containing the samples were then centrifuged (Biofuge Fresco, Germany) for 15 min, at 12,000 G at 4°C. The upper layer containing RNA was carefully pipetted out into a new RNAse free 1.5 mL Eppendorf tube and 200 pL of Isopropanol was added to it. The samples were then incubated for 10 min at 4°C. Once the incubation was complete, the samples were centrifuged for 10 min, at 12,000 G at 4°C. Post centrifugation, a clear palette was visible at the bottom of the Eppendorf tubes. The Isopropanol was withdrawn carefully and 2 wash steps with 400 pL of 75% ethanol were performed. Each wash step involved re-suspension of the clear palette with 75°/s ethanol by reverse pipetting and vortexing, and centrifugation at 7,500 G at 4°C. Once the two wash steps were complete, the ethanol was withdrawn from the Eppendorf tubes carefully and the tubes were left open to air dry for 10 min. After the air-dry step, 20 pL of RNAse free water were added to the Eppendorf tubes and the samples were incubated on a heat block at 58°C for 15 min. The quantity and quality of the sample was then assessed by using Nanodrop (Nanodrop ND1000 Spectrophotometer, USA). All samples were made up to a total volume of 87.5 pL by adding RNAse-free water. DNAse treatment was performed post the first quantification by using the QIAGEN RNase-Free DNase Set (Qiagen). In brief, 10 pL of RNase-free Buffer RDD and 2.5 pL RNase-free DNase I were added to the reaction tubes containing samples and incubated for 10 min at room temperature. Post incubation, further quantification by using the Nanodrop ND1000 was performed.

cDNA synthesis All cDNA synthesis reactions were performed by using a Thermal cycler (Eppendorf, Mastercycler nexus gradient). All the cDNA experiments were performed in triplicate using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems ThermoFisher, Pleasanton, CA) with three independent biologic samples for each cell line. Each sample contained 20 ng/ pL of RNA content. The contents in each PCR tube consisted of 2.00 pL RT Buffer (10X), 0.8 pL dNTP Mix (100 mM) (25X), 2.00 pL RT Random Primers, MultiScribeml Reverse Transcriptase, 4.2 pl Nuclease-free Water, and 10 pL of RNA sample. The thermal cycler ran at 25°C for 10 min, followed by 37°C for 120 min, and 85°C for 5 min. Samples were kept in a -20°C freezer until RT-qPCR was performed.

Reverse transcription-quantitative polvmerase chain reaction (RT-ciPCR) All RT-qPCR experiments were performed by using QuantStudion^ Real-Time PCR (Quant Studio 7 flex). The primer sequences used for the RT-qPCR reactions are shown in Table 1. All the RT-qPCR experiments were performed in triplicate using TaqManTm Fast Advanced Master mix (Applied Biosystems ThermoFisher, Pleasanton, CA) with three independent biologic samples for each cell line. GAPDH was used an internal control. The PCR contents in each well consisted of 1.00 pL TaqManTm Small RNA Assay (20X) (Applied Biosystems ThermoFisher, Pleasanton, CA), 10.00 pL PCR Fast Advanced Master Mix, 7.67 pL Nuclease-free Water, and 1.33 pL of cDNA template. A no template control and no Master mix control were run for each plate. The PCR cycle ran at 95°C for 20 sec (enzyme activation), followed by 40 cycles of 95°C for 1 sec (denture) and 60°C for 20 sec (anneal). Analysis of the Ct values for each well were exported to Microsoft Excel for further analysis. 2-4,O,Ct values of fold change gene expression were used to compare the relative differences between the Calu-3 cancerous cell line and control lung fibroblast MRC-5 cell line.

Statistical analysis All sample data were analysed on GraphPad Prism 9.5.0 software via incorporating an unpaired t-test in order to check the difference in fold change of gene expression between the cancerous and control cell lines. P values of <0.05 were deemed significantly different from the controls and denoted with asterisks where appropriate.

Results There were five gene profiles analysed via RT-qPCR throughout this in vitro study. Table 1 below shows their names and assay IDs.

Table 1: Gene names and assay IDs analysed via RT-qPCR Gene name Assay ID SLC15A1 Hs00192639_ml GPR123 Hs00287219_ml GPR183 Hs00270639 sl KCNAB2 Hs00186308 ml KNDC1 Hs00287000 ml Figure 1A shows growing Calu-3 cells at 900/0 confluency. Figure 1B illustrates growing MRC-5 cells at 70% confluency.

Results of gene fold expression obtained via RT-qPCR are illustrated in Figure 2. The inventor's results showed that one gene, SLC15A1, was significantly upregulated (p<0.0032) within the Calu-3 cell line when compared to the MRC-5 cell line. Three of the genes analysed throughout this study showed significant downregulation. These included, GPR123 (p<0.003), KNDC1 (p<0.0002), and KCNAB2 (p<0.0023). One of the genes analysed, GPR183, showed no significant difference in gene fold expression when compared between the Calu-3 and MRC-5 cell lines (p<0.4711).

Discussion Throughout this in vitro analysis, the inventors carried out a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of five potential biomarker genes for NSCLC/LAUD within cancerous and non-cancerous cell lines.

In silico analysis revealed that KCNAB2 was downregulated in both SCLC and NSCLC when compared to the control suggesting that it could act as a tumour suppressor gene. This is in agreement with the results obtained by RT-qPCR fold gene expression analysis, which also revealed that the voltage-gated potassium channel subunit beta- 2 (KCNAB2) was downregulated in Calu-3 cancerous cells when compared to the non-cancerous MRC-5 cell line. Localised within 1p36 region, KCNAB2 encodes the auxiliary protein KV[32. This protein product then modifies the functional properties and negatively regulates other members of the potassium voltage-gated family, alongside the potassium voltage-gated alpha subunit. Potassium channels have shown to be involved in the processes of cancer developments and patient prognosis, which agrees with the results herein.

The SLC15A1 gene is a downstream target for the leptin signalling pathway. The gene encodes a proton-coupled oligopeptide transporter 1 (PEPT1), which is found to be expressed on small intestine and kidney epithelial cells in mammals. The significantly elevated expression of SLC15A1 depicted from our RT-qPCR experiment suggested that the gene possibly acts as an oncogene. Those results are also in line with the inventor's bioinformatic analysis in which the SLC15A1 was found upregulated in both SCLC and NSCLC cohorts.

GPR123 (ADGRA1) is part of the G protein-coupled receptors (GPCRs) superfamily.

GPCRs are involved in several biological processes, including cellular adhesion, angiogenesis, development, and hormonal regulation. The inventor's results indicated that GPR123 has a significantly downregulated profiles within the cancerous Calu-3 cell line when compared to the non-cancerous MRC-5 cell line, which was also in line with our bioinformatic analysis. GPR123 was noted to be involved in tumour-associated inflammation and immune cell infiltration within endometrial cancers. Of note, no significant difference was obtained between GPR183 expression profiles obtained from Calu-3 and MRC-5 cell lines.

The brain-specific Ras guanine nucleotide, KNDC1 (v-KIND) is localized at chromosome 10826.3. Two isoforms of this gene, KIND1 and KIND2.22. The role of KNDC1 in several signalling pathways is related to the mechanisms of development, regulation, and restriction of cell growth. The results obtained via the RT-qPCR and in silico analyses suggested that the significantly downregulated KNDC1 might potentially act as a tumour suppressor within the LAUD cell line. No previous studies have focused on the role of KNDC1 in LAUD tumours.

G-protein coupled receptors afford the largest family of membrane proteins in the human genome. GPR183 has been widely studied in immune regulation and yet its role in tumour immunity remains scarce. Herein, both bioinformatics and in vitro analyses indicated that GPR183 acts as a tumour suppressor gene. Nevertheless, this did not reach significance in our in vitro studies.

Herein, following in silico and in vitro analyses, as well as extensive literature review, the inventors identified a promising gene panel which could easily discriminate between the SCLC or the NSCLC and the control cohort. Specifically, in silico and in vitro analyses showed that that SLC15A1 seemed to contribute to the development of lung adenocarcinoma when upregulated, whilst KCNAB2, GPR123 and KNDC1 possessed tumour suppressor properties and hence lead to this type of lung cancer when downregulated. The role of GPR1S3 remain unclear, even though GPR183 possibly acts a tumour suppressor gene.

The inventors then also identified additional biomarkers which, in combination with those identified above, can be used as part of a wider panel for lung cancer diagnosis.

Collectively with the literature review analysis, the inventors ended up with a panel including the following genes and expression profiles: TP53, KRAS, EGFR, TOP2A, PSAT1, SLC15A1 when upregulated and GPR123, KNOC1, KCNAB2, SNIP1 when downregulated. The expression profiles of GPR123, SLC15A1, KCNAB2 and KNDC1 were verified by RT-qPCR using the Calu-3 epithelial cell line derived from lung adenocarcinoma and the MRC-5 cell line derived from fibroblasts isolated from the lung tissue.

In summary, the inventors have identified and validated four novel lung cancer biomarkers: SLC15A1; GPR123, KCNAB2; and KNDC1, that can be used individually or in various combinations as a biomarker signature. In combination with other identified biomarkers, the inventors have therefore also identified a wider biomarker signature: TP53, KRAS, EGFR, TOP2A, PSAT1, GPR123, KNDC1, KCNAB2, SLC15A1, and SNIP1.

The novel biomarker signatures identified can be used for the early diagnosis of lung cancer.

References Bird, R. E., et al., (1988) Single-chain antigen-binding proteins. Science. 242(4877): 423-26.

Better, M., et al., (1988). Escherichia coli secretion of an active chimeric antibody fragment. Science. 240(4855): 1041-143.

Clackson, T. et at, (1991). Making antibody fragments using phage display libraries.

Nature. 352(6336): 624-28.

Gunneriusson, E., et al., (1999) Staphylococcal Surface Display of Immunoglobulin A (IgA)-and IgE-Specific In Vitro-Selected Binding Proteins (Affibodies) Based on Staphylococcus aureus Protein A. App! Environ Microbiol. 65(9): 4134-40.

Haghjoo, N., Moeini, A., & Masoudi-Nejad, A. (2020). Introducing a panel for early detection of lung adenocarcinoma by using data integration of genomics, epigenomics, transcriptomics and proteomics. Exp Mol Pathol. 112, 104360.

Harlow & Lane (1988). Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY, ISBN-13: 978-0879693145.

Harlow & Lane (1998) Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY, ISBN-13: 978-0879695446.

Howard & Kaser (2006) Making and Using Antibodies: A Practical Handbook, 2006, CRC Press, ISBN-13: 978-0849335280.

Jenkins, R. E., Pennington, S. R., (2001) Arrays for protein expression profiling: towards a viable alternative to two-dimensional gel electrophoresis? Proteomics. 1:1329.

Kenan, D. J., & Keene, J. D., (1999) Methods Mol Biol. 118, 217-31.

Lal, S. P., Christopherson, R. I., & dos Remedios, C. G., (2002) Antibody arrays: an embryonic but rapidly growing technology. Drug Discov Today. 7(18 Suppl): S143-49).

Santi, E., et al., (2000) Bacteriophage lambda display of complex cDNA libraries: a new approach to functional genomics. J Mol Biol. 296(2): 497-508.

Skerra, A., & PlOckthorn, A., (1988) Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science. 240(4855), 1038-41.

Smith, G. P., (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface, Science. 228(4705): 1315-17.

Ward, E. S. et al., (1989) Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coll. Nature. 341(6242): 544-46.

Winter, G., & Milstein, C., (1991) Man-made antibodies. Nature. 349, 293-99.

Claims (31)

1. CLAIMS1. A method for diagnosing lung cancer in an individual, the method comprising or consisting of the steps of: (a) providing a test sample from the individual; (b) measuring the expression in the test sample of one or more biomarkers selected from KCNAB2, SLC15A1, GPR123 and KNDC1; wherein the expression in the test sample of the one or more biomarkers is indicative of lung cancer in the individual.
2. 2. The method according to Claim 1, wherein step (b) comprises or consists of measuring the expression of 2 or more biomarkers, for example 3 or 4 of the 15 biomarkers selected from KCNAB2, SLC15A1, GPR123 and KNDC1.
3. 3. The method according to Claim 1 or 2, wherein step (b) comprises or consists of measuring the expression of each of KCNAB2, SLC15A1, GPR123 and KNDC1.
4. 4. The method according to any one of Claims 1-3, wherein step (b) further comprises measuring the expression of one or more biomarkers selected from TP53, KRAS, EGFR, TOP2A, PSAT1 and SNIP1, for example 2, 3, 4, 5, or 6 of these biomarkers.
5. S. The method according to any one of Claims 1-4, wherein step (b) comprises or consists of measuring the expression of KCNAB2.
6. 6. The method according to any one of Claims 1-5, wherein step (b) comprises or consists of measuring the expression of each of KCNAB2, SLC15A1, GPR123, KNDC1, 30 TP53, KRAS, EGFR, TOP2A, PSAT1 and SNIP1.
7. 7. The method according to any one of Claims 1-6, wherein the method is for diagnosis of early lung cancer.
8. 8. The method according to any one of Claims 1-7, wherein the lung cancer is Non-Small Cell Lung Cancer (NSCLC).
9. 9. The method according to Claim 8, wherein the NSCLC is selected from the group comprising: adenocarcinoma; squamous cell carcinoma; adenosquamous carcinoma; large cell carcinoma; or large cell neuroendocrine cancer, preferably wherein the NSCLC is adenocarcinoma.
10. 10. The method according to Claim 8 or 9, wherein the NSCLC in the individual is early-stage NSCLC (i.e. Stage 0, I or Stage II) or is late-stage NSCLC (i.e. Stage III or Stage IV).
11. 11. The method according to any one of Claims 1-10, further comprising or consisting of the steps of: (c) providing one or more control samples from an individual not afflicted with lung cancer; and (d) determining a biomarker signature of the one or more control samples by measuring the expression in the control sample of the one or more biomarkers measured in step (b); wherein the lung cancer is identified in the event that the expression in the test sample of the one or more biomarkers measured in step (b) is different from the expression in the control sample of the one or more biomarkers measured in step (d).
12. 12. The method according to Claim 11, wherein the control sample is a negative control sample, for example a sample derived from normal lung tissue from the individual; or from a healthy individual; or a pool of healthy individuals.
13. 13. The method according to any one of Claims 1-12, further comprising or consisting of the steps of: (e) providing one or more control samples from an individual afflicted with lung cancer; and (f) determining a biomarker signature of the control sample by measuring the expression in the control sample of the one or more biomarkers measured in step (b); wherein the lung cancer is identified in the event that the expression and/or amount in the test sample of the one or more biomarkers measured in step (b) corresponds to the expression in the control sample of the one or more biomarkers measured in step (f).
14. 14. The method according to any one of Claims 1 to 13, wherein step (b), (d) and/or (f) comprises measuring the expression of a nucleic acid molecule encoding the one or more biomarkers.
15. 15. The method according to Claim 16, wherein the nucleic acid molecule is an mRNA molecule or a cDNA molecule.
16. 16. The method according to any one of Claims 1-13, wherein step (b), (d), and/or (f) comprises measuring the expression of the protein or polypeptide of the one or more biomarkers.
17. 17. The method according to any one of Claims 1-16, wherein step (b), (d) and/or step (f) is performed using one or more first binding agent capable of binding to a bio marker.
18. 18. The method according to one of Claims 1-17, wherein the one or more biomarkers in the test and/or control sample(s) are labelled either directly or indirectly with a detectable moiety.
19. 19. The method according to Claim 18, wherein the detectable moiety is selected from the group comprising: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety; an enzymatic moiety.
20. 20. The method according to any of Claims 1-19, wherein the test sample comprises one or more lung cancer cell, and is optionally selected from the group comprising: a biopsy (such as a core needle biopsy; fine needle biopsy; bronchoscopy sample); a tissue sample; an organ sample; and a bodily fluid sample (such as blood or pleural fluid).
21. 21. The method according to Claims 1-20, wherein the expression of the biomarkers in the test sample is determined using RT-qPCR.
22. 22. The method according to any of Claims 1-20, wherein the expression of the biomarkers in the test sample is determined using mass spectrometry, an affinity-based method, a transcriptomics-based method, ELISA or flow cytometry.
23. 23. An array for diagnosing or determining the presence of lung cancer in an individual comprising an agent or agents for detecting the expression of one or more of the biomarkers selected from KCNAB2, SLC15A1, GPR123 and KNDC1, optionally wherein the array further comprises an agent or agents for detecting the expression of one or more of the biomarkers selected from TP53, KRAS, EGFR, TOP2A, PSAT1 and SNIP1.
24. 24. Use of two or more biomarkers selected from the group comprising KCNAB2, SLC15A1, GPR123 and KNDC1 as biomarkers for diagnosing or determining the presence of lung cancer in an individual.
25. 25. Use of the biomarkers KCNAB2, SLC15A1, GPR123 and KNDC1 as biomarkers for diagnosing or determining the presence of lung cancer in an individual.
26. 26. The method according to Claims 1-25, wherein in the event that the individual is diagnosed with lung cancer, the method further comprises a step of providing the individual with a lung cancer therapy.
27. 27. The method according to Claim 26, wherein the lung cancer therapy is selected from the group comprising: surgery, chemotherapy, radiotherapy, immunotherapy, chemoimmunotherapy, thermochemotherapy and combinations thereof, adoptive cell therapies, gene therapies, cancer vaccines, and oncolytic virus therapies.
28. 28. A kit for diagnosing or determining the presence of lung cancer, the kit comprising: (i) the array according to Claim 23, or components for making the same; and (ii) instructions for performing the method as defined in any one of Claims 1 to 27.
29. 29. A method of treating lung cancer in an individual comprising the steps of: (i) diagnosing lung cancer according to the method defined in any one of Claims 1-28; and (ii) providing the individual with lung cancer therapy.
30. 30. The method according to any of Claims 1-29, or the use according to any of Claims 25 or 26, wherein the individual is selected from the group comprising: a primate (for example, a human; a monkey; an ape); a rodent (for example, a mouse, a rat, a hamster, a guinea pig, a gerbil, a rabbit); a canine (for example, a dog); a feline (for example, a cat); an equine (for example, a horse); a bovine (for example, a cow); or a porcine (for example, a pig), preferably wherein the individual is a human.
31. 31. A method, use, array, or kit for determining the presence of lung cancer in an individual substantially as described herein.