Methods for determining the likelihood of lung cancer

Abstract

The present invention relates to a method of determining the likelihood of the presence of lung cancer in a subject. The method comprises determining the level of a biomarker selected from the group consisting of catalase (CAT), C-X-C motif chemokine receptor 4 (CXCR4), superoxide dismutase 3 (SOD3) and surfactant protein B (SFTPB) from a population of vesicles isolated from a biological sample from said subject, wherein a change in the level of the biomarker compared to a reference is indicative of the likelihood of the presence of lung cancer in the subject. In a preferred embodiment, the presence of non-small cell lung cancer (NSCLC) is determined based on an increase in the level of the biomarker based on plasma-derived exosomes compared to a healthy subject. The present disclosure also relates to determining lung cancer progression or responsiveness of a subject with lung cancer to an anti-cancer therapy based on said biomarker.

Description

Field of the invention

[0001] The present invention relates generally to the field of biotechnology. In particular, the present disclosure relates to methods of determining the likelihood of the presence of lung cancer in a subject and methods of treating lung cancer in a subject.

[0002] BACKGROUND

[0003] Lung cancer is the second most common type of cancer diagnosed in the United States, with approximately 234,030 new cases in the United States in 2018. It is the leading cause of cancer death in men and women. Lung cancer is primarily diagnosed in older adults, with an average age of diagnosis around 70 years.

[0004] Current lung screening options include chest X-ray, sputum cytology, and chest computed tomography (CT), each with their own weighted benefits and limitations. Of these methods, sputum cytology is a non-invasive method, but has a very low detection rate. For chest X-ray, it also shows low sensitivity and specificity for early detection of lung cancer. While low-dose CT has a high sensitivity, it has been shown to have very poor specificity, resulting in a false positive rate of approximately 96%. The majority of patients detected by low-dose CT have been confirmed by invasive biopsy to have false positive lung cancer. Thus, the clinical utility of these expensive invasive tools in promoting early lung cancer detection and intervention to improve mortality remains controversial and unsatisfactory, in part due to the high incidence of benign nodules, making interpretation extremely challenging.

[0005] Accordingly, it is generally desirable to overcome or ameliorate one or more of the above difficulties.

[0006] SUMMARY

[0007] Disclosed herein is a method of determining the likelihood of the presence of lung cancer in a subject, the method comprising determining the level of a biomarker selected from the group consisting of catalase (CAT), C-X-C motif chemokine receptor 4 (CXCR4), superoxide dismutase 3 (SOD3), and surfactant protein B (SFTPB) from a population of vesicles isolated from a biological sample from the subject, wherein a change in the level of the biomarker compared to a reference indicates the likelihood of the presence of lung cancer in the subject.

[0008] Also disclosed herein is a method of determining the progression of lung cancer in a subject, the method comprising determining the level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB from a population of vesicles isolated from a biological sample from the subject, wherein a change in the level of the biomarker compared to a reference distinguishes the presence of early stage and late stage lung cancer in the subject.

[0009] Also disclosed herein is a method of detecting and treating lung cancer in a subject, the method comprising: (a) determining the level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB in a population of vesicles isolated from a biological sample of the subject, wherein a change in the level of the biomarker compared to a reference is indicative of the presence of lung cancer in the subject, and (b) administering an anti-cancer therapy to the subject.

[0010] Also disclosed herein is a method of monitoring the responsiveness of a subject having lung cancer to an anti-cancer therapy, the method comprising determining the level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB in a population of vesicles isolated from a biological sample of the subject, wherein (a) an increase or no change in the level of the biomarker compared to a reference is indicative of the subject being unresponsive to the anti-cancer therapy, and wherein (b) a decrease in the level of the biomarker compared to a reference is indicative of the subject being responsive to the anti-cancer therapy. SUMMARY

[0012] Embodiments of the present application are hereinafter described by way of non-limiting example only with reference to the accompanying drawings, in which:

[0013] Figure 1 Exosome biomarker discovery workflow

[0014] Figure 2 Characterization of exosomes from human plasma (200 μΐ) isolated from ultracentrifugation (UC) and total exosome isolation kit (Invitrogen). (A) Respective electron micrographs of plasma exosomes enriched from pooled healthy donors (n=33), low magnification (80000x, scale bar = 500 nm). Inset shows magnified portion of micrograph (200000x, scale bar = 200 nm). (B) Concentration (particles / ml) and size distribution (nm) profile of respective exosome enriched preparations from pooled healthy donors (by NTA). (C) Respective immunoblot analysis of pooled plasma exosomes and pooled exosome-depleted plasma for four positive (+) and two negative (-) exosome markers. Transferrin (TF) as a loading control. Healthy, n=33; early stage NSCLC, n=13; advanced stage NSCLC, n=60.

[0015] Figure 3. Assessment of quality of quantitative MS data. (A) Venn diagram of overlapping proteins quantified in three TMT technical replicates (B) Assessment of correlation between quantitative datasets. Scatter plot of log2 of measured ratios for each protein between triplicates.

[0016] Figure 4Immunoblot validation of plasma exosomes. Normalized mean intensity values (log2) of the six candidate exosomal biomarkers in early NSCLC (n=14); advanced NSCLC (n=14) and healthy individuals (n=14) as shown in box-and-whisker plots. The line represents the median, the box represents the 25th to 75th percentile, the whiskers represent the maximum and minimum range, and the dots represent individual values. One-way ANOVA comparison between controls and NSCLC phenotypes. Intensity values were normalized to transferrin (loading control). Abbreviations: **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0017] Figure 5. Receiver operating characteristic curves (ROC). (A) ROC analysis of exosomal candidate proteins and clinically used markers to distinguish NSCLC phenotypes from healthy subjects. (Healthy, n=167; early NSCLC, n=64; all NSCLC, n=357). (B) ROC analysis of exosomal candidate proteins and clinically used markers to distinguish cancer phenotypes from healthy subjects. (Healthy, n=167; breast, n=113; colorectal, n=144; nasopharyngeal, n=101).

[0018] DETAILED DESCRIPTION

[0019] Disclosed herein is a method of determining the likelihood of the presence of lung cancer in a subject, the method comprising determining the level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3 and SFTPB in a population of vesicles isolated from a biological sample from said subject, wherein an increase in the level of the biomarker compared to a reference indicates the likelihood of the presence of lung cancer in the subject. In one embodiment, the method determines the likelihood of the presence of early and / or advanced lung cancer in the subject.

[0020] Disclosed herein is a method of determining the likelihood of the presence of lung cancer in a subject, the method comprising determining the level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3 and SFTPB in a population of vesicles isolated from a biological sample from said subject, wherein an increase in the level of the biomarker compared to a reference indicates the likelihood of the presence of lung cancer in the subject. In one embodiment, the method determines the likelihood of the presence of early and / or advanced lung cancer in the subject.

[0021] Disclosed herein is a method of detecting the presence of lung cancer in a subject, the method comprising determining the level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3 and SFTPB in a population of vesicles isolated from a biological sample from said subject, wherein a change in the level of the biomarker compared to a reference indicates the presence of lung cancer in the subject. In one embodiment, the method detects the presence of early and / or advanced lung cancer in the subject.

[0022] Disclosed herein is a method of detecting the presence of lung cancer in a subject, the method comprising determining the level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB in a population of vesicles isolated from a biological sample from the subject, wherein an increase in the level of the biomarker compared to a reference is indicative of the presence of lung cancer in the subject.

[0023] Without being bound by theory, the inventors have found that using exosomes, a well-defined entity in blood can be seen, with all the advantages of a blood sample but without the interference of background (fluctuations in marker proteins due to other diseases or injuries) and plasma proteins. Since tumor cells are known to release many more circulating exosomes than normal proliferating cells, this approach optimally supports the direct interrogation of NSCLC tumor-derived exosomes in plasma.

[0024] For example, a three- or four-marker exosome panel can be used for annual health screening, as well as for defining high-risk patients prior to imaging. In combination with this panel, those patients with a high-risk clinical feature profile can be subjected to chest computed tomography (CT). Those patients whose test results suggest a low probability of cancer can be re-evaluated with plasma markers during their routine follow-up. Without being bound by theory, the point of care diagnostic panel of the present application can greatly reduce the false-positive cases (~50%) associated with screening CT, which can lead to unnecessary anxiety, biopsies, and / or surgery; and early detection will allow timely tailoring of treatment strategies in management and improve NSCLC prognosis.

[0025] In one embodiment, the method is an in vitro or ex vivo method.

[0026] The phrase "the likelihood of the presence of lung cancer" refers to the likelihood of the presence of lung cancer in a subject. An increase in the level of one or more biomarkers compared to a reference can be indicative of the likelihood (i.e., chance or risk) of the presence of lung cancer in a subject. This can be, for example, a likelihood of more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the presence of lung cancer in a subject.

[0027] In one embodiment, there is provided a method of determining the likelihood of the presence of lung cancer in a subject, the method comprising determining the level of a vesicle-associated (or bound) biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB in a biological sample from the subject, wherein a change (or increase) in the level of the biomarker compared to a reference is indicative of the likelihood of the presence of lung cancer in the subject.

[0028] The biomarker can be a protein, a peptide. The biomarker can be associated with or bound to the surface of the vesicle. Alternatively, it can be contained within the vesicle. In alternative embodiments, the biomarker is a nucleic acid.

[0029] In one embodiment, the level of the biomarker is determined using an antibody-based technique or a PCR-based technique. The biomarker can be detected, for example, using an antibody-based technique such as enzyme-linked immunosorbent assay (ELISA), Luminex assay, or Western blot to determine the amount of biomarker associated with, bound to, or contained within the vesicle. The antibody can be one that specifically binds to a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB. The antibody can be further conjugated to a detectable label (e.g., a fluorescent, luminescent, or enzymatic label) to allow detection. Alternatively, the antibody can be detected using a secondary antibody conjugated to a label (e.g., a fluorescent, luminescent, or enzymatic label).

[0030] In one embodiment, the level of the biomarker is determined using a PCR-based technique. Analysis of the vesicles can include RNA sequence analysis by methods known in the art. For example, the vesicles can be lysed and RNA recovered for RT-PCR analysis. Methods of determining mRNA levels of genes in a sample are well known in the art. For example, mRNA levels can be determined by PCR, qPCR, qRT-PCR, RNA sequencing, microarray analysis, SAGE, MassARRAY technology, next generation sequencing, or FISH. Alternatively, captured vesicles on or released from the capture surface can be analyzed using immuno cytochemistry and other fluorescent imaging techniques. Analysis of the vesicles can also include detection of the presence of DNA molecules using techniques known in the art such as PCR analysis or genomic sequencing.

[0031] Other techniques can also be used, such as flow cytometry. Alternatively, mass spectrometry can also be used to detect the biomarker. PCR-based techniques can also be used to detect nucleic acid biomarkers (e.g., genomic DNA or mRNA).

[0032] The terms "peptide," "polypeptide," and "protein" are used interchangeably and include any polymer of amino acids of two or more amino acids joined to each other by peptide or modified peptide bonds, whether produced naturally or synthetically. The polypeptides of the present application can contain non-peptide components, such as carbohydrate groups. Carbohydrate and other non-peptide substituents can be added to polypeptides by the cells in which they are produced and vary with the type of cell. Polypeptides are defined herein in terms of their amino acid backbone structure; substituents such as carbohydrate groups are typically not specified, but can still be present.

[0033] The nucleic acids of the present application can be in the form of RNA, such as mRNA, or DNA, including cDNA and genomic DNA, obtained by cloning or produced synthetically. The DNA can be double-stranded or single-stranded. Single-stranded DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the anti-sense strand.

[0034] As used herein, the term "antibody" includes, but is not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies, including bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), including bispecific sdFv, and anti-idiotypic (anti-Id) antibodies, as well as epitope-binding fragments of any of the above. The antibodies provided herein can be monospecific, bispecific, trispecific, or of greater multispecificity.

[0035] The term "polymerase chain reaction" or "PCR" refers to a reaction for the in vitro amplification of a specific nucleic acid sequence by the simultaneous primer extension of complementary strands of the nucleic acid molecule. In other words, PCR is a reaction that produces multiple copies or replicates of a target nucleic acid flanked by primer sites, which reaction comprises one or more repetitions of the following steps: (i) denaturation of the target nucleic acid, (ii) annealing of primers to the primer sites, and (iii) extension of the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Typically, the reaction is cycled at different temperatures optimized for each step in a thermocycler instrument. The specific temperatures, duration of each step, and rate of change between steps depend on a number of factors well known to those of ordinary skill in the art. The term "PCR" encompasses derivative forms of the reaction, including but not limited to reverse-transcription-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, and the like.

[0036] In one embodiment, the method comprises determining the level of a biomarker selected from CAT, CXCR4, SOD3, and SFTPB, which biomarker is present in or associated with a vesicle population or contained within a vesicle. In one embodiment, the method comprises determining the level of CAT, CXCR4, SOD3, or SFTPB. The method can comprise determining the level of a panel of biomarkers (i.e., two or more biomarkers). In one embodiment, the method comprises determining the level of two biomarkers, which comprises i) CAT and CXCR4, ii) CAT and SOD3, iii) CAT and SFTPB, iv) CXCR4 and SOD3, v) CXCR4 and SFTPB, or vi) SOD3 and SFTPB. In one embodiment, the method comprises determining the level of three biomarkers, which comprises i) CAT, CXCR4, and SOD3, ii) CAT, CXCR4, and SFTPB, iii) CAT, SOD3, and SFTPB, or iv) CXCR4, SOD3, and SFTPB. In one embodiment, the method comprises determining the level of three biomarkers, which comprises CAT, CXCR4, and SFTPB. In one embodiment, the method comprises determining the level of four biomarkers, which comprises CAT, CXCR4, SOD3, and SFTPB. In one embodiment, the method comprises determining the level of four biomarkers consisting of CAT, CXCR4, SOD3, and SFTPB.

[0037] In one embodiment, a method of determining the likelihood of the presence of lung cancer in a subject is provided, the method comprising determining the level of a biomarker comprising CAT, CXCR4, and SFTPB from a population of vesicles isolated from a biological sample from the subject, wherein a change (or increase) in the level of the biomarker compared to a reference indicates the likelihood of the presence of lung cancer in the subject. The method can further comprise determining the level of SOD3.

[0038] In one embodiment, a method of determining the likelihood of the presence of lung cancer in a subject is provided, the method comprising determining the level of a biomarker comprising CAT, CXCR4, and SFTPB from a population of vesicles isolated from a biological sample from the subject, wherein a change (or increase) in the level of the biomarker compared to a reference indicates the likelihood of the presence of lung cancer in the subject. The method can further comprise determining the level of SOD3.

[0039] The biomarkers referred to herein can be used in combination with other biomarkers known in the art for determining the likelihood of the presence of lung cancer in a subject. These include CA125, CEA, and / or Cyfra-21.

[0040] The method can comprise isolating a population of vesicles from the biological sample. The population of vesicles can be isolated using techniques including ultracentrifugation, size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, affinity selection, microfluidic separation, or a combination thereof.

[0041] In one embodiment, the population of vesicles (or population of exosomes) can be isolated by differential centrifugation followed by ultracentrifugation, which is the gold standard method. Other enrichment methods include density gradient centrifugation, size exclusion chromatography, filtration techniques, polymer-based precipitation, immunoisolation, and sieving separation.

[0042] The method of the application can comprise isolating the population of vesicles prior to measuring the level of the biomarker. The method can further comprise lysing the population of vesicles prior to measuring the level of the biomarker. Methods of lysing the population of vesicles are known in the art. For example, the population of vesicles can be lysed using a lysis buffer such as radioimmunoprecipitation assay (RIPA) buffer.

[0043] In one embodiment, the method comprises detecting the population of vesicles with an antibody. For example, this can comprise detecting a surface marker from the population of exosomes. This will preferably allow isolation, purification and / or enrichment of the population of exosomes. For the purposes of the present application, the terms "isolating" and "isolation" (in all their grammatical forms) relate to the act of separating or recovering exosomes from their environment (e.g. a serum or plasma sample or a tissue biopsy). The terms "purifying" and "purified" (in all their grammatical forms) relate to the act of liberating the desired exosomes from (non-exosomal) contaminants. The terms "enriching" and "enriched" (in all their grammatical forms) mean increasing the proportion of exosomes in their respective solvent. Proteins are particularly envisaged as exosomal surface markers, but other biological molecules such as lipids are also possible. The exosomal surface marker can be recognised by an antibody. In one embodiment, the antibody is selected from an anti-CD9 antibody, an anti-CD63 antibody and an anti-CD81 antibody.

[0044] In one embodiment, the method comprises isolating the population of vesicles with a bead-conjugated antibody (e.g. in an ELISA-based assay). The bead-conjugated antibody allows the isolation of any population of vesicles bound by the antibody from the biological sample using techniques such as centrifugation or magnetic separation (in the case where the beads are magnetic beads) and optionally one or more washing steps. In one embodiment, the antibody is selected from an anti-CD9 antibody, an anti-CD63 antibody and an anti-CD81 antibody.

[0045] A biological sample obtained from a subject can be any bodily fluid. For example, the biological sample can be peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen (including prostatic fluid or pre-ejaculatory fluid), female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymphatic fluid, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluid from sinus cavities, bronchopulmonary aspirate or other lavage fluids. Biological samples can also include blastocyl cavity, umbilical cord blood, or maternal circulation, which can be of fetal or maternal origin. The biological sample can also be a tissue sample or biopsy from which vesicles and other circulating biomarkers can be obtained.

[0046] For many diseases, such as many cancers, invasive tissue biopsy followed by histopathological or molecular analysis is considered the diagnostic gold standard. Whether such procedures are performed as highly invasive surgery or as low-trauma needle biopsies, tissue biopsies carry the risk of infection and cannot be applied repeatedly. Moreover, core and needle biopsies often do not yield sufficient amounts of tissue for in-depth diagnostic analysis and can even miss band-like pathophysiological tissue alterations. Since blood samples can be easily and repeatedly obtained, the concept of "liquid biopsy" holds promise as a less-invasive complement to traditional tissue biopsy. After secretion into a body fluid, vesicles can be isolated from the liquid biopsy by ultracentrifugation.

[0047] In one embodiment, the biological sample is a body fluid for liquid biopsy. In one embodiment, the biological sample is a blood, serum, or plasma sample. In one embodiment, the biological sample comprises cancer cells or circulating tumor cells (CTCs). In another embodiment, the biological sample comprises vesicles from cancer cells or circulating tumor cells.

[0048] The methods of the present application can comprise evaluating one or more vesicles, including evaluating a population of vesicles. As used herein, a “vesicle” can refer to a naturally occurring or synthetic vesicle comprising an internal cavity. A vesicle can comprise a lipid bilayer membrane that encloses the contents of the internal cavity. A vesicle can comprise a liposome, an exosome, an extracellular vesicle, a microvesicle, an apoptotic vesicle (or apoptotic body), a vacuole, a lysosome, a transport vesicle, a secretory vesicle, a gas vesicle, a matrix vesicle, or a multivesicular body. A vesicle can have a size of about 1000 nm or less, about 900 nm or less, about 800 nm or less, about 700 nm or less, about 600 nm or less, about 500 nm or less, about 450 nm or less, about 400 nm or less, about 350 nm or less, about 300 nm or less, about 250 nm or less, about 240 nm or less, about 230 nm or less, about 220 nm or less, about 210 nm or less, about 200 nm or less, about 190 nm or less, about 180 nm or less, about 170 nm or less, about 160 nm or less, about 150 nm or less, about 140 nm or less, about 130 nm or less, about 120 nm or less, about 110 nm or less, about 100 nm or less, about 90 nm or less, about 80 nm or less, about 70 nm or less, about 60 nm or less, about 50 nm or less, about 40 nm or less, about 30 nm or less, about 20 nm or less, or about 10 nm or less.

[0049] Exosomes are a type of vesicle, also referred to in the art as extracellular vesicles, microvesicles, or microparticles. These vesicles are shed by eukaryotic cells or budded from the plasma membrane to the exterior of the cell. These membrane vesicles vary in size, ranging in diameter from about 10 nm to about 5000 nm. Small vesicles (about 10 to 1000 nm in diameter, preferably 30 to 100 nm) released by exocytosis of intracellular multivesicular bodies are referred to in the art as “exosomes.” The methods and compositions described herein are equally applicable to other vesicles of all sizes.

[0050] Structurally, exosomes can be described as spherical bilayered protein-lipid, carrying a variety of biological molecules, including genetic material, such as mRNA, microRNA (miRNA), and other non-coding RNAs, or even small amounts of DNA, lipids, and proteins, including transcription factors, cytokines, growth factors, and the like.

[0051] In one embodiment, the vesicle is an exosome. In another embodiment, the vesicle is a circulating exosome.

[0052] As described above, the vesicles can be isolated from the sample prior to further analysis (e.g., by utilizing surface markers bound by appropriate antibodies) or analyzed directly from the sample (e.g., by detecting the level of one or more biomarkers described herein). "Analysis" can generally include quantification of the amount of vesicles in the sample and / or evaluation of the level of one or more biomarkers indicative of lung cancer.

[0053] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. As used herein, the term "cancer" refers to both non-metastatic and metastatic cancers, including early and late stage cancers. The term "pre-cancerous" refers to a condition or growth that typically precedes or develops into cancer. "Non-metastatic" refers to a cancer that is benign or remains at the primary site and has not infiltrated the lymphatic or blood vessel system or tissues other than the primary site. Generally, a non-metastatic cancer is any Stage 0, I, or II cancer, occasionally a Stage III cancer. "Early stage cancer" refers to a cancer that is non-invasive or metastatic or classified as a Stage 0, I, or II cancer. The term "late stage cancer" generally refers to a Stage III or IV cancer, but can also refer to a Stage II cancer or a sub-stage of a Stage II cancer. Those skilled in the art will appreciate that the classification of a Stage II cancer as an early stage cancer or a late stage cancer depends on the particular type of cancer. In one embodiment, the cancer is lung cancer. In one embodiment, the cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC).

[0054] In one embodiment, the method comprises treating a subject found to have lung cancer.

[0055] The methods as defined herein can comprise the step of determining the level of a biomarker selected from CAT, CXCR4, SOD3, and SFTPB from a population of vesicles isolated from a biological sample from the subject, wherein a change (increase) in the level of the biomarker compared to a reference is indicative of the presence of lung cancer in the subject.

[0056] A "reference" as referred to herein can be one or more non-cancerous samples taken from the same subject or one or more non-cancerous samples taken from another subject (e.g., a healthy subject not suffering from cancer). The reference can also be a predetermined value or average. In one embodiment, the methods as defined herein comprise the step of comparing the level of one or more biomarkers to a reference.

[0057] The term "increase" or "increased" with respect to a biomarker, as used herein, refers to a statistically significant and measurable increase in the biomarker compared to a reference. The increase can be at least about 10% increase, or at least about 20% increase, or at least about 30% increase, or at least about 40% increase, or at least about 50% increase.

[0058] As used herein, the term "decrease" or "decreased" with respect to a biomarker refers to a statistically significant and measurable decrease in the biomarker as compared to a reference. The decrease can be a decrease of at least about 10%, or a decrease of at least about 20%, or a decrease of at least about 30%, or a decrease of at least about 40%, or a decrease of at least about 50%.

[0059] In one embodiment, the increase in biomarker level as compared to a reference can be an increase of 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, 56-fold, 57-fold, 58-fold, 59-fold, 60-fold, 61-fold, 62-fold, 63-fold, 64-fold, 65-fold, 66-fold, 67-fold, 68-fold, 69-fold, 70-fold, 71-fold, 72-fold, 73-fold, 74-fold, 75-fold, 76-fold, 77-fold, 78-fold, 79-fold, 80-fold, 81-fold, 82-fold, 83-fold, 84-fold, 85-fold, 86-fold, 87-fold, 88-fold, 89-fold, 90-fold, 91-fold, 92-fold, 93-fold, 94-fold, 95-fold, 96-fold, 97-fold, 98-fold, 99-fold, or 100-fold increase, or anywhere in between.

[0060] In one embodiment, the increase in one or more, two or more, three or more, or all four of the biomarkers is indicative of the presence of lung cancer in the subject as compared to a reference.

[0061] In one embodiment, the decrease in biomarker level can mean that the biomarker has 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, 0.1-fold or less, or anywhere in between as compared to a reference level.

[0062] The present invention relates to a method of determining the progression of lung cancer in a subject. Provided herein is a method of determining the progression of lung cancer in a subject, the method comprising determining the level of a biomarker selected from CAT, CXCR4, SOD3, and SFTPB from a population of vesicles isolated from a biological sample from the subject.

[0063] Also provided herein is a method of determining the progression of lung cancer in a subject, the method comprising determining the level of a biomarker selected from CAT, CXCR4, SOD3, and SFTPB from a population of vesicles isolated from a biological sample from the subject, wherein a change in the level of the biomarker compared to a reference distinguishes the presence of early stage lung cancer and late stage lung cancer in the subject. The method can provide an indication of whether the lung cancer is a stage 0, I, II, III, and / or IV cancer. In one embodiment, the method comprises determining the level of SOD3 from a population of vesicles isolated from a biological sample from the subject.

[0064] Provided herein is a method of determining the progression of lung cancer in a subject, the method comprising determining the level of a biomarker selected from CAT, CXCR4, SOD3, and SFTPB from a population of vesicles isolated from a biological sample from the subject, wherein an increase in the level of the biomarker compared to a reference distinguishes the presence of early stage lung cancer and late stage lung cancer in the subject.

[0065] As used herein, the term "determining the progression of lung cancer" can refer to determining whether the lung cancer is an early stage cancer or a late stage cancer. It can also refer to whether the lung cancer is a stage 0, I, II, II, and / or IV cancer.

[0066] Also provided herein is a method of determining the prognosis of lung cancer in a subject following an anti-cancer treatment, the method comprising determining the level of a biomarker selected from CAT, CXCR4, SOD3, and SFTPB from a population of vesicles isolated from a biological sample from the subject.

[0067] Also disclosed herein is a method of detecting and treating lung cancer in a subject, the method comprising: (a) determining the level of a biomarker selected from CAT, CXCR4, SOD3, and SFTPB in a population of vesicles isolated from a biological sample from the subject, wherein a change in the level of the biomarker compared to a reference indicates the presence of lung cancer in the subject, and (b) administering an anti-cancer therapy to the subject.

[0068] In one embodiment, provided is a method of detecting and treating lung cancer in a subject, the method comprising: (a) determining the level of a biomarker selected from CAT, CXCR4, SOD3, and SFTPB in a population of vesicles isolated from a biological sample from the subject, wherein an increase in the level of the biomarker compared to a reference indicates the presence of lung cancer in the subject, and (b) administering an anti-cancer therapy to the subject.

[0069] Also disclosed herein is a method of treating lung cancer in a subject. The method can be based on the results of a test performed by determining the level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB in a population of vesicles isolated from a biological sample of the subject, wherein a change (or increase) in the level of the biomarker compared to a reference indicates the presence of lung cancer in the subject. The method of treating lung cancer can comprise administering an anti-cancer therapy to the subject.

[0070] Also disclosed herein is an anti-cancer therapy for use in treating lung cancer in a subject, wherein the level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB in a population of vesicles isolated from a biological sample of the subject has been determined, and wherein a change (or increase) in the level of the biomarker compared to a reference indicates the presence of lung cancer in the subject.

[0071] Also disclosed herein is the use of an anti-cancer therapy in the manufacture of a medicament for treating lung cancer in a subject, wherein the level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB in a population of vesicles isolated from a biological sample of the subject has been determined, and wherein a change (or increase) in the level of the biomarker compared to a reference indicates the presence of lung cancer in the subject.

[0072] As used herein, the term "treatment" or "treating" can refer to (1) preventing or delaying the appearance of one or more symptoms of a disorder; (2) inhibiting the progress of a disorder or one or more symptoms thereof; (3) relieving a disorder, that is, causing regression of the disorder or at least one or more symptoms thereof; and / or (4) causing a decrease in severity of one or more symptoms of a disorder.

[0073] The term "administering" means contacting, applying or providing an anti-cancer therapy to a subject.

[0074] The term "subject" as used throughout the specification is understood to refer to a human or can be a domesticated or companion animal. While the methods of the application are particularly contemplated for use in treating humans, they are also applicable to veterinary treatments, including the treatment of companion animals such as dogs and cats, and domesticated animals such as horses, cows, and sheep, or zoo animals such as primates, felines, canines, bovines, and ungulates. A "subject" can include a human, patient, or individual, and can be of any age or gender.

[0075] In one embodiment, the method further comprises administering an anti-cancer therapy to the subject found to have lung cancer. The anti-cancer therapy can comprise chemotherapy, radiation therapy, targeted therapy, immunotherapy, or a combination thereof. For example, the chemotherapy can be cisplatin, carboplatin, paclitaxel (Taxol), albumin-bound paclitaxel (nab-paclitaxel, Abraxane), docetaxel (Taxotere), gemcitabine (Gemzar), vinorelbine (Navelbine), irinotecan (Camptosar), etoposide (VP-16), vinblastine, or pemetrexed (Alimta). The method can also comprise treating the subject by surgery.

[0076] Also disclosed herein is a method of monitoring the responsiveness of a subject having lung cancer to an anti-cancer therapy, the method comprising determining the level of a biomarker selected from CAT, CXCR4, SOD3, and SFTPB in a population of vesicles isolated from a biological sample of the subject. A change (such as an increase or decrease) in the level of the biomarker compared to a reference can indicate that the subject is responsive to the anti-cancer therapy. In one embodiment, a decrease in the level of the biomarker compared to a reference indicates that the subject is responsive to the anti-cancer therapy. In one embodiment, an increase or no change in the level of the biomarker compared to a reference indicates that the subject is not responsive to the anti-cancer therapy.

[0077] In one embodiment, there is provided a method of monitoring the responsiveness of a subject having lung cancer to an anti-cancer therapy, the method comprising determining the level of a biomarker selected from CAT, CXCR4, SOD3, and SFTPB in a population of vesicles isolated from a biological sample of the subject, wherein a change in the level of the biomarker compared to a reference indicates that the subject is responsive to the anti-cancer therapy.

[0078] In one embodiment, the method comprises determining the level of CAT, CXCR4, and SFTPB.

[0079] In one embodiment, there is provided a method of monitoring the responsiveness of a subject having lung cancer to an anti-cancer therapy, the method comprising determining the level of a biomarker selected from CAT, CXCR4, SOD3, and SFTPB in a population of vesicles isolated from a biological sample of the subject, wherein (a) an increase or no change in the level of the biomarker compared to a reference indicates that the subject is not responsive to the anti-cancer therapy, and wherein (b) a decrease in the level of the biomarker compared to a reference indicates that the subject is responsive to the anti-cancer therapy.

[0080] Also provided herein are compositions for detecting lung cancer in a subject. The composition can comprise an antibody that specifically binds to a protein or peptide biomarker selected from CAT, CXCR4, SOD3, and SFTPB. The antibody can optionally be conjugated to a detectable label.

[0081] The composition as described herein can also comprise a population of vesicles isolated from a biological sample from a subject, for example a subject having lung cancer. The population of vesicles can optionally be a population of lysed vesicles.

[0082] Also disclosed herein is the use of a composition as defined herein for detecting lung cancer in a subject.

[0083] Also provided herein is a kit for detecting lung cancer in a subject. The kit can comprise an antibody that specifically binds to a protein or peptide biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB. In one embodiment, the kit comprises an antibody that specifically binds to CAT, CXCR4, and SFTPB. The kit can comprise a suitable buffer for detecting lung cancer in a subject. The kit can comprise components for isolating a population of vesicles from a biological sample of a subject. The kit can also comprise a population of vesicles isolated from a biological sample from a subject, for example a subject having lung cancer. The population of vesicles can optionally be a population of lysed vesicles.

[0084] In the description and statements herein, the word "comprising" and variations such as "comprise" and "comprises" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0085] The reference in this specification to any prior publication (or information derived from it), or to anything otherwise known, is not, and should not be taken as, an acknowledgment or admission that the prior publication (or information derived from it) or anything otherwise known forms part of the common general knowledge in the field of endeavour concerned.

[0086] Those skilled in the art will appreciate that the application described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the application includes all such variations and modifications which fall within the spirit and scope of the application. The application also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0087] Certain embodiments of the application will now be described, for the purposes of illustration only, by reference to the following examples, which are not intended to be limiting in any way. Example

[0088] Example 1

[0089] Quantitative workflow based on tandem mass spectrometry tags (TMT) for plasma exosome proteome analysis.

[0090] Given the involvement and relevance of circulating exosomes in almost all pathophysiological aspects in humans, circulating exosomes are highly sought after. In the discovery phase, plasma exosomes were isolated using the prolonged ultracentrifugation (PUC) (1) method, which was reported to effectively simplify plasma complexity. TMT, a chemical labeling method that provides quantification and multiplexing in a single reagent (2), was used to establish the differential proteome of pooled plasma exosomes from early stage NSCLC; advanced stage NSCLC and healthy individuals. Briefly, for each sample group, equal concentrations of pooled plasma exosome proteins were subjected to proteolytic digestion with trypsin. The trypsin peptides from each respective group were labeled with one of the isobaric element tags, followed by first dimensional fractionation using weak anion exchange chromatography. The fractionally labeled peptides were analyzed by LC-MS / MS and the relative abundance of a particular peptide in a sample was determined by comparing the intensity of the TMT reporter fragment ions in the 126-131 m / z region of the peptide product ion spectra. Two biological replicates and three technical replicates were performed to increase the reliability of the differences in quantitative changes in protein expression. Differential quantitative proteomic analysis was performed using open source public tools and the target proteins of interest that were differentially expressed (p<0.05) were further reviewed through extensive literature mining and based on their novelty and association with cancer progression.

[0091] Given the simplicity and utility of the preparation of the samples in the validation and verification phase, exosome isolation was performed using a commercial exosome isolation kit from Invitrogen. The validation analysis of the shortlisted target exosome proteins by western blotting was performed in a subset of individuals from the orthogonal cohort. Proteins with expression that was significantly associated with early and advanced stage NSCLC (independent of disease stage) were prioritized for validation. Enzyme-linked immunosorbent assay (ELISA) validation of the validated candidates in exosome content and soluble plasma was performed alongside well-established cancer biomarkers in a larger patient cohort. The diagnostic efficiency of the validated exosome markers was assessed based on the established area under the curve (AUC) obtained from receiver operating characteristic (ROC) curve analysis. Finally, multivariate statistical algorithms were employed to determine the predictive value of the multi-protein signature in differentiating NSCLC and non-cancer individuals.

[0092] 2) Characterization of plasma exosomes.

[0093] The plasma exosome-rich preparations obtained from ultracentrifugation (UC) and total exosome isolation kit (Invitrogen) were evaluated by TEM, NTA and immunoblot analysis in accordance with international guidelines for exosome characterization. TEM analysis of both exosome-rich preparations (Figure 2) showed the presence of exosome-like vesicles with a characteristic cup-shaped morphology. NTA analysis of both exosome-rich preparations (Figure 3) showed a unimodal distribution of exosome-like vesicles with a mean size of 100 nm. Immunoblot analysis of both exosome-rich preparations (Figure 4) showed the presence of exosome marker proteins, including CD9, CD63 and CD81. Figure 2A) shows the coexistence of individual and aggregated clusters of membrane-bound spherical vesicles with a size of 50-150 nm in diameter, consistent with the typical characteristics of exosomes. NTA shows the average size distribution of the two exosome-rich preparations Figure 2 B), with a particle size diameter ranging from 40 to 500 nm, which is the expected size range of exosomes (50-150 nm) and small microvesicles (150-1000 nm). The main size (mode) of the particles detected from the UC and Invitrogen kit preparations was 56.6 ± 1.3 nm and 69.6 ± 2.1 nm, respectively, within the acceptable exosome size range. Thus, the concentration of exosomes enriched from the UC and Invitrogen kits was 1.74 x 10 9 ± 3.00 x 10 8 and 2.23 x 10 9 ± 8.00 x 10 7 particles / ml, respectively.

[0094] The successful recovery of exosomes from both methods was determined by immunoblotting of four common exosome-specific markers, including the cytosolic markers Alix and TSG101 and the surface markers CD63 and CD9, and their absence in the exosome-depleted plasma preparations after exosome extraction Figure 2 C). The intracellular proteins GM130 and calnexin were chosen as negative exosome markers for purity assessment, and the absence of each marker in the two exosome-rich isolates indicates a lack of Golgi and endoplasmic reticulum (ER) contamination, while both negative markers were detected in all exosome-depleted plasma preparations as expected. However, contamination from other organelles and vesicles cannot be excluded from the two exosome-rich isolates.

[0095] Overall, these results demonstrate that using the UC and Invitrogen methods, plasma-derived exosomes are highly enriched with low organelle contamination. The UC and Invitrogen exosome isolation methods were used for the discovery phase and the validation / verification phase of exosome enrichment, respectively.

[0096] 3) Assessment of the quality of the quantitative MS data.

[0097] Table I. Summary of proteins, peptide spectrum matches (PSMs) (FDR < 1%) and experimental variations.

[0098]

[0099] Abbreviations: R01, replicate 01; R02, replicate 02; R03, replicate 03; SD, standard deviation; %CV, percent coefficient of variation

[0100] Intra-run technical variation was determined according to the percent coefficient of variation (%CV) comparing the number of proteins and PSMs identified in replicate 01 (R01), replicate 02 (R02), and replicate 03 (R03) as summarized in Table 1. Using a stringent FDR <1%, the overall %CV in the replicate identification of proteins and PSMs observed across all three replicates was <2%, which implies minimal intra-run technical variation and good system reproducibility.

[0101] As Figure 3A shown in Table 2, approximately 77% (625) of the total number of proteins were quantified in at least two of the three replicates, suggesting good protein complementation between the three replicate LC-MS / MS runs, and these proteins were used for further analysis. In Figure 3B Table 3, significant (p<0.05) correlation of the measured pairwise early-stage:control ratios for each protein between the three replicates confirmed the reliability and confidence of the quantitative dataset.

[0102] 4) Candidate biomarker selection criteria and validation analysis.

[0103] Table II. Identified list of currently available cancer biomarkers.

[0104]

[0105] Abbreviations: GS, gene symbol; Cov, protein coverage

[0106] Here, known lung cancer biomarkers identified in the proteomic dataset are listed in Table 3. Although the following markers are clinically available, they are currently used with limited capacity as adjunct blood biomarkers for lung cancer. Based on the fold change reference of these proteins, a 1.2-fold change cutoff was considered as altered expression in this study. Next, to further refine the list of candidate markers, the differentially expressed candidates were selected based on the following criteria: (a) the protein must be identified based on >2 peptides with 95% confidence and quantified in at least two of the three replicates; (b) the protein must show at least a 1.2-fold change; (c) and only proteins with significant (p<0.05) differential expression were considered. Proteins that did not meet these stringent criteria were ignored. As a result, out of the core list of 625 proteins, a total of 56 exosomal proteins were found to exhibit simultaneous differential regulation in both NSCLC phenotypes relative to controls. These proteins were further scrutinized by extensive literature mining and based on their novelty and association with cancer progression, ten markers (Table III) were selected for validation by immunoblotting in a separate set of individuals (early-stage NSCLC, n=14; advanced-NSCLC, n=14, and healthy individuals, n=14) that were not used in the discovery experiment.

[0107] Table III. NSCLC-specific candidate proteins significantly (p<0.05) differentially regulated.

[0108]

[0109] The validation analysis returned six exosomal proteins (Table III) that were statistically consistent with the discovery proteomics dataset and had highly correlated expression with early and late stage NSCLC (p<0.05) that will move into the clinical validation phase. It is important to note that surfactant protein B (SFTPB) is a lung-specific protein that is expressed only in lung tissue. The strategy for selecting NSCLC biomarkers is that the candidate proteins should show significantly increased protein expression (p<0.05) in early and late stage NSCLC, respectively, relative to controls. This will ensure that the candidates can be reliably used to detect early stage NSCLC and have shown increased expression that is not associated with disease progression. Figure 4

[0110] Validation of the six markers was performed using enzyme-linked immunosorbent assay (ELISA) on a total of 306 individuals (early stage NSCLC patients (n=53); late stage NSCLC patients (n=139) and healthy individuals (n=114). Of the six candidates, four markers (CAT, CXCR4, SOD3, SFTPB) showed similar significant (p<0.05) differential expression between healthy subjects and NSCLC phenotypes as reported in the discovery and validation phases and were further evaluated using receiver operating characteristic (ROC) curves. ROC curves were plotted based on ELISA results to compare the diagnostic efficiency of the four candidate markers in exosomal content and soluble plasma, as well as 2 well-studied cancer markers. The discriminatory power of each individual candidate and the 4-marker combination set between healthy controls and early stage NSCLC (p<0.05) / all NSCLC cases was evaluated using the AUC under the ROC curve. The 4-marker combination set showed the highest ROC AUC value of 0.93 in differentiating NSCLC from non-cancer controls. The 4-marker combination and all individual markers had higher efficacy in diagnosing NSCLC compared to well-studied cancer biomarkers (CEA, Cyfra21). This is the first lung cancer study that compares the cargo of interest in exosomes with their respective soluble levels in patient plasma. The four biomarkers are associated with cancer progression, with CAT, SOD3 and SFTPB conferring anti-tumorigenic functions and CXCR4 having pro-tumorigenic functions. Figure 5A

[0111] 5) Clinical Validation

[0112] ​​In the clinical validation, patients were roughly split into two halves and assigned to a training set (n=279) and a validation test set (n=305) for phase I and II validation, respectively (Table IV). In phase I validation, data obtained from the training set were used to train multivariate models to give a combined receiver operating curve (ROC) analysis or predictive values of the final signature for differentiating NSCLC and non-cancer individuals. In phase II validation, data obtained from the test set were used to validate the trained models.

[0113] Table IV. Phase I and II data sets used to derive the exosome multivariate predictive model.

[0114]

[0115] Abbreviations: NSCLC, non-small cell lung cancer; n, sample size.

[0116] The initial 6 candidates selected in the validation phase were evaluated using enzyme-linked immunosorbent assay (ELISA) on training set samples (early stage NSCLC patients (n=32); all NSCLC patients (n=133) and healthy individuals (n=114)). Of the six candidates, four markers (CAT, CXCR4, SOD3, SFTPB) showed similar significant (p<0.05) differential expression between healthy subjects and NSCLC phenotypes as reported in the discovery and validation phases. Phase I validation data obtained from these four exosome targets were subjected to internal machine learning multivariate model training to finally arrive at a three-marker signature (CAT, CXCR4, SFTPB) for early stage NSCLC (AUC=0.96; specificity=0.96; sensitivity=0.91). Phase II validation data obtained from test set samples will be used to cross-evaluate the model's predictions without adjusting the model.

[0117] In Figure 5AFrom the I and II phases, validation results were used to plot ROCs to assess and compare the diagnostic efficiency of the four candidate exosome markers and three well-studied cancer markers (CA125, CEA, Cyfra-21) in matching patients. The AUC under the ROC curve was used to assess the ability of each individual candidate and the three-marker signature to distinguish between healthy controls, early-stage NSCLC, and all NSCLC cases. A total of 167 healthy individuals and 353 NSCLC patients were validated on each of the four exosome markers, of which 64 were early-stage cases, and showed that all markers showed better predictive value in exosome content than in plasma. By including the II phase validation data, the three-marker signature showed the highest ROC AUC value of 0.99 in distinguishing early-stage NSCLC from non-cancer controls (specificity = 0.98; sensitivity = 0.97). All individual markers and the three-marker signature had higher efficacy in diagnosing NSCLC compared to the three well-studied cancer markers (CA125, CEA, and Cyfra-21).

[0118] In addition, ELISA validation of each of the four exosome markers and well-studied cancer markers was performed on breast cancer (n = 113), colorectal cancer (n = 144), and nasopharyngeal carcinoma (NPC) (n = 101) cohorts, covering the top 3 most common global cancer types. Figure 5B ) Compared to the well-studied cancer markers (CA125, CEA, Cyfra-21), the four individual markers showed little discrimination ability, with AUC ranging from 0.5 to 0.6 for breast cancer, colorectal cancer, and NPC, suggesting that the four markers are indeed specific to NSCLC diagnosis. It was also indicated that different cancer phenotypes do indeed as claimed, with AUC scores of approximately 0.5 for Cyfra21-1 (a lung marker) for all three cancer phenotypes, 0.877 for CA15-3 (a breast marker) for breast cancer, 0.671 for colorectal cancer (a colorectal marker), and no discrimination ability for NPC for all well-studied cancer markers, as expected.

[0119] The three exosome targets in the signature are associated with cancer progression, with CAT and SFTPB conferring anti-tumorigenic functions, while CXCR4 has pro-tumorigenic functions. Together, the three-marker exosome signature has great clinical utility in the diagnosis of heterogeneous NSCLC.

Claims

1. Use of a reagent for detecting levels of catalase (CAT), surfactant protein B (SFTPB), and C-X-C motif chemokine receptor 4 (CXCR4) biomarkers in the manufacture of a kit for determining the likelihood of the presence of non-small cell lung cancer (NSCLC) in a subject, wherein the likelihood of the presence of NSCLC in a subject is determined by a method of determining the levels of catalase (CAT), surfactant protein B (SFTPB), and C-X-C motif chemokine receptor 4 (CXCR4) biomarkers in a population of vesicles isolated from a biological sample from the subject with the kit, wherein an increase in the levels of the biomarkers compared to a reference indicates the likelihood of the presence of NSCLC in a subject.

2. The use of claim 1, wherein the biomarkers are nucleic acids, proteins, or peptides.

3. The use of claim 1 or 2, wherein the vesicles are extracellular vesicles.

4. The use of any one of claims 1 to 3, wherein the levels of the biomarkers are determined using an antibody-based technique or a PCR-based technique.

5. The use of any one of claims 1 to 4, wherein the method comprises isolating the population of vesicles from the biological sample.

6. The use of any one of claims 1 to 5, wherein the method comprises detecting the population of vesicles with an antibody.

7. The use of claim 6, wherein the antibody is selected from an anti-CD9 antibody, an anti-CD63 antibody, and an anti-CD81 antibody.

8. The use of any one of claims 1 to 7, wherein the biological sample is a blood, serum, or plasma sample.

9. The use of any one of claims 1 to 8, wherein the method further comprises performing a chest computed tomography (CT) scan on the subject.

10. The use of any one of claims 1 to 9, wherein an anti-cancer therapy is to be administered to a subject found to have NSCLC.

11. Use of a reagent for detecting levels of catalase (CAT), surfactant protein B (SFTPB), and C-X-C motif chemokine receptor 4 (CXCR4) biomarkers in the manufacture of a kit for monitoring the responsiveness of a subject having non-small cell lung cancer (NSCLC) to an anti-cancer therapy, wherein the responsiveness of a subject having NSCLC to an anti-cancer therapy is monitored by a method of determining the levels of catalase (CAT), surfactant protein B (SFTPB), and C-X-C motif chemokine receptor 4 (CXCR4) biomarkers in a population of vesicles isolated from a biological sample from the subject with the kit, wherein (a) an increase or no change in the levels of the biomarkers compared to a reference indicates that the subject is unresponsive to the anti-cancer therapy, and wherein (b) a decrease in the levels of the biomarkers compared to a reference indicates that the subject is responsive to the anti-cancer therapy.

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