Method for determining possibility of lung cancer

Abstract

To provide a method for determining a possibility of presence of a lung cancer of an object.SOLUTION: A method includes a process of determining the level of biomarker selected from the group of catalase (CAT), C-X-C motif chemokine receptor 4(CXCR4), superoxide dismutase 3(SOD3), and surfactant protein B(SFTPB) from a vesicle population isolated from an object-derived biological sample, variation in level of the biomarker compared with a reference showing a possibility of lung cancer of the object. In a preferable embodiment, the presence of non-small cell lung cancer (NSCLC) is determined on the basis of an increase in level of the marker based upon a plasma-derived exosome, compared with a healthy object. The present disclosure further relates to a process of determining responsiveness to lung cancer development or anticancer therapy on the object suffering from lung cancer on the basis of those biomarkers.SELECTED DRAWING: Figure 1

Description

[Technical field]

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

[0002] 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 both men and women. Lung cancer is primarily diagnosed in older people, with a median age of approximately 70 years.

[0003] Current lung screening options include chest x-ray, sputum cytology, and chest computed tomography (CT), each with their respective weighted advantages and limitations. Among these techniques, sputum cytology is a non-invasive method, but has a very poor detection rate. As for chest x-ray, it also shows low sensitivity and specificity for early detection of lung cancer. Although low-dose CT has high sensitivity, it has been demonstrated to have very poor specificity, resulting in a false positive rate of about 96%. The majority of patients detected by low-dose CT have been demonstrated to have false positive lung cancer by invasive biopsy. Therefore, the clinical utility of these costly invasive tools remains controversial and unsatisfactory in promoting early lung cancer detection and intervention to improve mortality, due in part to the high incidence of benign nodules, making interpretation very difficult.

[0004] It would therefore be generally desirable to overcome or ameliorate one or more of the above-mentioned difficulties. Summary of the Invention [Means for solving the problem]

[0005] Disclosed herein is a method of determining the possible presence of lung cancer in a subject, comprising determining a 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 possible presence of lung cancer in the subject.

[0006] Also disclosed herein is a method of determining the progression of lung cancer in a subject, comprising determining a 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 between the presence of early stage and late stage lung cancer in the subject.

[0007] Also disclosed herein are methods of detecting and treating lung cancer in a subject, comprising: (a) determining a 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 alteration 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.

[0008] Also disclosed herein is a method of monitoring the responsiveness of a subject afflicted with lung cancer to an anti-cancer therapy, comprising determining a 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 (a) an increase or no change in the level of the biomarker compared to a reference indicates that the subject is non-responsive to the anti-cancer therapy, and (b) a decrease in the level of the biomarker compared to the reference indicates that the subject is responsive to the anti-cancer therapy.

[0009] Embodiments of the invention will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a diagram of the exosomal biomarker discovery workflow. [Diagram 2] Characterization of exosomes from human plasma (200 μl) isolated by ultracentrifugation (UC) and total exosome isolation kit (Invitrogen). (A) Electron micrographs of enriched plasma exosomes from pooled healthy donors (n=33) at low magnification (80,000×, scale bar=500 nm). Inserts show enlarged sections of the micrographs (200,000×, scale bar=200 nm). (B) Concentration (particles / ml) and size distribution (nm) profiles by NTA for each exosome-enriched preparation from pooled healthy donors. (C) Immunoblot analysis of pooled plasma exosomes and pooled exosome-depleted plasma against four positive (+) and two negative (-) exosome markers. Transferrin (TF) served as a loading control. Healthy, n=33; early stage NSCLC, n=13; late stage NSCLC, n=60. [Figure 3A] FIG. 1. Assessment of quantitative MS data quality. (A) Venn diagram of overlapping proteins quantified in three TMT technical replicates. [Figure 3B] FIG. 10. Assessment of quantitative MS data quality. (B) Correlation assessment between quantitative data sets. Log2 scatter plot of measured ratios for each protein among triplicates. [Figure 4-1]Figure 4. Immunoblot validation of plasma exosomes. Normalized mean densitometry values ​​(log2) of six candidate exosome biomarkers in early stage NSCLC (n=14); late stage NSCLC (n=14); and healthy individuals (n=14) shown as box plots. Lines represent median values, boxes represent 25-75 percentiles, whiskers represent maximum and minimum ranges, and points represent individual values. One-way ANOVA comparison between control and NSCLC phenotypes. Densitometry values ​​are normalized to transferrin (loading control). Abbreviations: **, p<0.01; ***, p<0.001; ****, p<0.0001. [Figure 4-2] Continued from Figure 4. [Figure 4-3] Continued from Figure 4. [Figure 5A] Receiver operating characteristic curves (ROC). (A) ROC analysis of exosomal protein candidates and clinically used markers for distinguishing NSCLC phenotype from healthy subjects (healthy, n=167; early stage NSCLC, n=64; total NSCLC, n=357). [Figure 5B] Receiver operating characteristic curves (ROC). (B) ROC analysis of exosomal protein candidates and clinically used markers for distinguishing cancer phenotypes from healthy subjects (healthy, n=167; breast, n=113; colorectal, n=144; nasopharyngeal, n=101). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Disclosed herein is a method of determining the likely presence of lung cancer in a subject, comprising determining a 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 indicates the likely presence of lung cancer in the subject. In one embodiment, the method determines the likely presence of both early stage and / or late stage lung cancer in the subject.

[0012] Disclosed herein is a method of determining the possible presence of lung cancer in a subject, comprising determining a 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 an increase in the level of the biomarker compared to a reference indicates the possible presence of lung cancer in the subject.

[0013] Disclosed herein is a method of detecting the presence of lung cancer in a subject, comprising determining a 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 indicates the presence of lung cancer in the subject. In one embodiment, the method detects the presence of early and / or late stage lung cancer in the subject.

[0014] Disclosed herein is a method of detecting the presence of lung cancer in a subject, comprising determining a 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 an increase in the level of the biomarker compared to a reference indicates the presence of lung cancer in the subject.

[0015] Without being bound by theory, the inventors have found that with exosomes, one can see clearly defined entities in the blood with all the advantages of blood samples, but without background (fluctuations of marker proteins due to other diseases or injuries) and interference from plasma proteins. Because tumor cells are known to release much more circulating exosomes than normal proliferating cells, this approach best supports the direct interrogation of NSCLC tumor-derived exosomes in plasma.

[0016] A three or four marker exosome panel can be used, for example, in annual screening and prior to imaging to define high-risk patients. In combination with the panel, those patients with a high-risk clinical profile can proceed to chest computed tomography (CT). Those 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 invention can significantly reduce the false positive cases (approximately 50%) associated with screening CT that may result in unnecessary concerns, biopsies, and / or surgeries; early detection would allow timely tailored therapeutic strategies in management and improved NSCLC prognosis.

[0017] In one embodiment, the method is an in vitro or ex vivo method.

[0018] The phrase "likelihood of the presence of lung cancer" refers to how likely lung cancer is present in a subject. An increase in the level of one or more biomarkers compared to a reference can indicate the likelihood (i.e., probability or risk) of the presence of lung cancer in a subject. This can be, for example, a greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% likelihood of the presence of lung cancer in a subject.

[0019] In one embodiment, there is provided a method of determining the possible presence of lung cancer in a subject, comprising determining a level of a biomarker associated (or bound) to a vesicle 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 indicates the possible presence of lung cancer in the subject.

[0020] The biomarker may be a protein, a peptide. The biomarker may be bound or attached to the surface of the vesicle. Alternatively, it may be contained within the vesicle. In an alternative embodiment, the biomarker is a nucleic acid.

[0021] 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 using an antibody-based technique, such as, for example, an enzyme-linked immunosorbent assay (ELISA), Luminex assay, or Western immunoblotting, which determines the amount of the biomarker associated with, bound to, or contained in the vesicles. The antibody can be an antibody 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 (such as a fluorescent, luminescent, or enzymatic label) that allows detection. Alternatively, the antibody can be detected using a secondary antibody that is conjugated to a label (such as a fluorescent, luminescent, or enzymatic label).

[0022] In one embodiment, the level of the biomarker is determined using a PCR-based technique. Analysis of the vesicles may include RNA sequence analysis by methods known in the art. For example, for RT-PCR analysis, the vesicles may be dissolved and the RNA may be extracted. Methods for determining the mRNA level of a gene in a sample are well known in the art. For example, the mRNA level may be determined by PCR, qPCR, qRT-PCR, RNA sequencing, microarray analysis, SAGE, MassARRAY techniques, next generation sequencing, or FISH. Alternatively, the captured vesicles on or released from the capture surface may be analyzed using immunocytochemistry and other fluorescent imaging techniques. Analysis of the vesicles may also include detecting the presence of DNA molecules using techniques known in the art, such as PCR analysis or genomic sequencing.

[0023] Other techniques such as flow cytometry may also be used. Alternatively, biomarkers may be detected using mass spectrometry. Nucleic acid biomarkers (such as genomic DNA or mRNA) may be detected using PCR-based techniques.

[0024] The terms "peptide," "polypeptide," and "protein" are used interchangeably and include any polymer of amino acids (dipeptides or larger) linked through peptide bonds or modified peptide bonds, whether produced naturally or synthetically. Polypeptides of the invention may contain non-peptidic components, such as carbohydrate groups. Carbohydrate and other non-peptidic substituents may be added to polypeptides by the cell in which the polypeptide is produced and will vary with the type of cell. Polypeptides are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, although may be present.

[0025] The nucleic acid of the present invention can be in the form of RNA, such as mRNA, or in the form of DNA, including cDNA and genomic DNA, obtained, for example, 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 antisense strand.

[0026] 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 (scFvs), Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs) (including bispecific sdFvs), and anti-idiotypic (anti-Id) antibodies, as well as epitope-binding fragments of any of the above. The antibodies provided herein may be monospecific, bispecific, trispecific, or of greater multispecificity.

[0027] The term "polymerase chain reaction" or "PCR" refers to a reaction for the in vitro amplification of specific nucleic acid sequences by simultaneous primer extension of complementary strands of a nucleic acid molecule. In other words, PCR is a reaction for making multiple copies or replicas of a target nucleic acid flanked by primer sites, such a reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Typically, the reaction is cycled through various temperatures optimized for each step in a thermal cycler instrument. The specific temperatures, durations at each step, and rates of change between steps depend on many factors well known to those skilled 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, etc.

[0028] In one embodiment, the method comprises determining the level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB present in the vesicle population or associated with, bound to, or contained within the vesicles. In one embodiment, the method comprises determining the level of CAT, CXCR4, SOD3, or SFTPB. The method may 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, including 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, including 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 levels of three biomarkers including CAT, CXCR4, and SFTPB. In one embodiment, the method comprises determining the levels of four biomarkers including CAT, CXCR4, SOD3, and SFTPB. In one embodiment, the method comprises determining the levels of four biomarkers consisting of CAT, CXCR4, SOD3, and SFTPB.

[0029] In one embodiment, a method is provided for determining the possible presence of lung cancer in a subject, comprising determining levels of biomarkers including 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 biomarkers compared to a reference indicates the possible presence of lung cancer in the subject. The method may further comprise determining the level of SOD3.

[0030] In one embodiment, a method is provided for determining the likely presence of lung cancer in a subject, comprising determining levels of biomarkers including CAT, CXCR4, SOD3, 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 biomarkers compared to a reference indicates the likely presence of lung cancer in the subject.

[0031] The biomarkers referred to herein may be used in combination with other biomarkers known in the art to determine the possible presence of lung cancer in a subject. These include CA125, CEA, and / or Cyfra-21.

[0032] The method can include isolating a vesicle population from a biological sample 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.

[0033] In one embodiment, vesicle populations (or exosome populations) can be isolated using differential centrifugation followed by ultracentrifugation (the gold standard method). Other methods of enrichment include density gradient centrifugation, size exclusion chromatography, filtration techniques, polymer-based precipitation, immunological separation, and isolation by sieving.

[0034] The method of the present invention may include isolating the vesicle population before measuring the level of the biomarker. The method may further include dissolving the vesicle population before measuring the level of the biomarker. Methods for dissolving the vesicle population are known in the art. For example, the vesicle population may be dissolved using a dissolving buffer such as a radioimmunoprecipitation assay (RIPA) buffer.

[0035] In one embodiment, the method comprises detecting the vesicle population with an antibody. For example, this may comprise detecting a surface marker from the exosome population. This would preferably allow the isolation, purification, and / or enrichment of the exosome population. For the purposes of the present invention, the terms "isolate" and "isolating" in all their grammatical forms refer to the act of separating or recovering exosomes from their environment, e.g., serum or plasma samples or tissue biopsies. The terms "purify" and "purification" in all their grammatical forms refer to the act of liberating the desired exosomes from (non-exosomal) contaminants. The terms "enrich" and "enrichment" in all their grammatical forms refer to increasing the proportion of exosomes in their respective solvents. Proteins are particularly envisaged as exosomal surface markers, although other biomolecules such as lipids may also be considered. The exosomal surface marker may be recognised by an antibody. In one embodiment, the antibody is selected from the group consisting of an anti-CD9 antibody, an anti-CD63 antibody, and an anti-CD81 antibody.

[0036] In one embodiment, the method includes isolating the vesicle population using a bead-conjugated antibody (such as in an ELISA-based assay). The bead-conjugated antibody allows any antibody-bound vesicle population to be separated from the biological sample using techniques such as centrifugation or magnetic separation (if the beads are magnetic beads) and optionally one or more washing steps. In one embodiment, the antibody is selected from the group consisting of an anti-CD9 antibody, an anti-CD63 antibody, and an anti-CD81 antibody.

[0037] The biological sample obtained from the 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, earwax, breast milk, bronchoalveolar lavage fluid, semen (including prostatic fluid), Cowper's fluid or pre-ejaculate fluid, female vaginal fluid, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menstrual secretions, pus, sebum, vomit, vaginal secretions, mucosal secretions, stool water, pancreatic juice, lavage fluid from sinus cavities, bronchopulmonary aspirate, or other lavage fluid. Biological samples may also include the blastocyl cavity, which may be of fetal or maternal origin, umbilical cord blood, or maternal circulation. Biological samples may also be tissue samples or biopsies from which vesicles and other circulating biomarkers may be obtained.

[0038] 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 minimally invasive needle puncture, tissue biopsy carries the risk of infection and cannot be applied repeatedly. Furthermore, core and needle biopsies often do not yield sufficient amounts of tissue for thorough diagnostic analysis and may even miss band-like pathophysiological tissue changes. Since blood samples can be easily and repeatedly obtained, the concept of "liquid biopsy" is promising as a minimally invasive complement to traditional tissue biopsy. Once secreted into body fluids, vesicles can be isolated by liquid biopsy via ultracentrifugation.

[0039] 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 derived from cancer cells or circulating tumor cells.

[0040] The method of the present invention may include a step of assessing one or more vesicles, including assessing a vesicle population. As used herein, "vesicle" may refer to a naturally occurring or synthetic vesicle that includes an internal cavity. A vesicle may include a lipid bilayer membrane that surrounds the contents of an internal cavity. A vesicle may include 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. Vesicles may be 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 18 ...50 nm or less, about 260 nm or less, about 270 nm or less, about 280 nm or less, about 290 nm or less, about 300 nm or less, about 310 nm or less, about 320 nm or less, about 330 nm or less, about 340 nm or less, about 350 nm or less, about 360 nm or less, about 370 nm or less, about 380 nm or less, about 390 nm or less, about 400 nm or less, about 400 nm or less, about 450 nm or less, about 400 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, The nanoparticles may have dimensions of about 1.5 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.

[0041] Exosomes are a type of vesicle, also referred to in the art as extracellular vesicles, microvesicles, or microparticles. These vesicles are released by eukaryotic cells to the outside of the cell or budded from the plasma membrane. These membrane vesicles are heterogeneous in size, with diameters ranging from about 10 nm to about 5000 nm. Small vesicles (approximately 10-1000 nm in diameter, preferably 30-100 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.

[0042] Structurally, exosomes can be described as spherical, bilayered proteolipids that carry a cargo of various biomolecules, including genetic material such as mRNA, microRNA (miRNA), and other non-coding RNA, or even small amounts of DNA, lipids, and proteins, including transcription factors, cytokines, growth factors, and others.

[0043] In one embodiment, the vesicles are exosomes. In another embodiment, the vesicles are circulating exosomes.

[0044] As described above, vesicles may be isolated from the sample prior to further analysis (e.g., by utilizing surface markers bound by appropriate antibodies) or may be analyzed directly from the sample (e.g., by detecting the level of one or more biomarkers described herein). "Analysis" may generally include quantifying the amount of vesicles in the sample and / or assessing the level of one or more biomarkers indicative of lung cancer.

[0045] The terms "cancer" and "cancerous" refer to or describe, in part, a physiological condition in a mammal that is typically characterized by unregulated cell growth. As used herein, the term "cancer" refers to 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. By "non-metastatic" we mean a cancer that is benign or that remains at the primary site and has not spread to the lymphatic or vascular system or to tissues other than the primary site. Generally, a non-metastatic cancer is any cancer that is a stage 0, I, or II cancer, and sometimes a stage III cancer. By "early stage cancer" we mean a cancer that is not invasive or metastatic, or that is classified as a stage 0, I, or II cancer. The term "late stage cancer" generally refers to stage III or stage IV cancer, but can also refer to stage II cancer or a cancer substage of stage II. Those skilled in the art will understand that the classification of stage II cancer as either early stage cancer or 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).

[0046] In one embodiment, the method comprises treating a subject found to have lung cancer.

[0047] The methods provided herein may include determining a 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 a subject, wherein an alteration (increase) in the level of the biomarker compared to a reference indicates the presence of lung cancer in the subject.

[0048] A "reference" as referred to herein may 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). A reference may also be a predetermined value or an average value. In one embodiment, the method provided herein comprises comparing the level of one or more biomarkers to a reference.

[0049] As used herein, the term "increase" or "increased" with respect to a biomarker refers to a statistically significant and measurable increase in the biomarker compared to a reference. The increase can be at least about a 10% increase, or at least about a 20% increase, or at least about a 30% increase, or at least about a 40% increase, or at least about a 50% increase.

[0050] As used herein, the term "reduction" or "reduced" with respect to a biomarker refers to a statistically significant and measurable decrease in the biomarker compared to a reference. The decrease can be at least about a 10% decrease, or at least about a 20% decrease, or at least about a 30% decrease, or at least about a 40% decrease, or at least about a 50% decrease.

[0051] In one embodiment, the increase in the level of the biomarker compared to the reference is 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, It may be a 5x, 46x, 47x, 48x, 49x, 50x, 51x, 52x, 53x, 54x, 55x, 56x, 57x, 58x, 59x, 60x, 61x, 62x, 63x, 64x, 65x, 66x, 67x, 68x, 69x, 70x, 71x, 72x, 73x, 74x, 75x, 76x, 77x, 78x, 79x, 80x, 81x, 82x, 83x, 84x, 85x, 86x, 87x, 88x, 89x, 90x, 91x, 92x, 93x, 94x, 95x, 96x, 97x, 98x, 99x, or 100x increase, or anywhere in between.

[0052] In one embodiment, an increase in one or more, two or more, three or more, or all four biomarkers relative to the reference indicates the presence of lung cancer in the subject.

[0053] In one embodiment, a decrease in the level of a biomarker can refer to a biomarker having 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, compared to the reference level.

[0054] The present invention is directed to a method for determining the progression of lung cancer in a subject. Provided herein is a method for determining the progression of lung cancer in a subject, comprising determining the level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB from a vesicle population isolated from a biological sample from the subject.

[0055] Also provided herein is a method of determining progression of lung cancer in a subject, comprising determining a 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 between the presence of early stage and late stage lung cancer in the subject. The method may provide an indication of whether the lung cancer is stage 0, I, II, III, and / or IV cancer. In one embodiment, the method comprises determining a level of SOD3 from a population of vesicles isolated from a biological sample from the subject.

[0056] Provided herein is a method of determining the progression of lung cancer in a subject, comprising determining a 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 an increase in the level of the biomarker compared to a reference distinguishes between the presence of early stage and late stage lung cancer in the subject.

[0057] As used herein, the term "determining the progression of lung cancer" may refer to determining whether the lung cancer is an early stage or late stage cancer. It may also refer to whether the lung cancer is a stage 0, I, II, III, and / or IV cancer.

[0058] Also provided herein is a method for determining the prognosis of lung cancer in a subject following anti-cancer therapy, comprising determining a 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.

[0059] Also disclosed herein are methods of detecting and treating lung cancer in a subject, comprising: (a) determining a 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 alteration 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.

[0060] In one embodiment, a method is provided for detecting and treating lung cancer in a subject, comprising: (a) determining a 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 indicates the presence of lung cancer in the subject; and (b) administering an anti-cancer therapy to the subject.

[0061] Also disclosed herein is a method for treating lung cancer in a subject. The method can be based on the test results performed by determining the level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB in a vesicle population isolated from a biological sample from a subject, and a change (or increase) in the level of the biomarker compared to the reference indicates the presence of lung cancer in the subject. The method for treating lung cancer can include administering an anti-cancer therapy to the subject.

[0062] Also disclosed herein is an anti-cancer therapy for use in treating lung cancer in a subject, wherein a level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB is determined in a population of vesicles isolated from a biological sample from the subject, and a change (or increase) in the level of the biomarker compared to a reference indicates the presence of lung cancer in the subject.

[0063] Also disclosed herein is the use of an anti-cancer therapy in the manufacture of a medicament for the treatment of lung cancer in a subject, wherein a level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB is determined in a population of vesicles isolated from a biological sample from the subject, and a change (or increase) in the level of the biomarker compared to a reference indicates the presence of lung cancer in the subject.

[0064] The term "treat" or "treatment" as used herein may refer to: (1) preventing or delaying the appearance of one or more symptoms of a disorder; (2) inhibiting the development of a disorder or one or more symptoms of a disorder; (3) alleviating the disorder, i.e., causing regression of the disorder or at least one or more symptoms of the disorder; and / or (4) causing a decrease in the severity of one or more symptoms of a disorder.

[0065] The term "administering" refers to contacting, applying, or providing an anti-cancer therapy to a subject.

[0066] The term "subject" as used throughout this specification should be understood to mean a human, or may be a domestic or companion animal. Although the methods of the invention are specifically contemplated for the treatment of humans, they are also applicable to veterinary treatment, including the treatment of companion animals such as dogs and cats, as well as domestic animals such as horses, cows, and sheep, or zoo animals such as primates, felines, canines, bovines, and ungulates. A "subject" may include a person, a patient, or an individual, and may be of any age or sex.

[0067] 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. The chemotherapy can be, for example, 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.

[0068] Also disclosed herein is a method for monitoring the responsiveness of a subject suffering from lung cancer to an anti-cancer therapy, comprising determining the level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB in a vesicle population isolated from a biological sample from the subject. A change (such as an increase or decrease) in the level of the biomarker compared to the reference may indicate that the subject is responsive to the anti-cancer therapy. In one embodiment, a decrease in the level of the biomarker compared to the 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 the reference indicates that the subject is non-responsive to the anti-cancer therapy.

[0069] In one embodiment, a method is provided for monitoring the responsiveness of a subject afflicted with lung cancer to an anti-cancer therapy, comprising determining a 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 a change in the level of the biomarker compared to a reference indicates that the subject is responsive to the anti-cancer therapy.

[0070] In one embodiment, the method includes determining the levels of CAT, CXCR4, and SFTPB.

[0071] In one embodiment, a method is provided for monitoring the responsiveness of a subject afflicted with lung cancer to an anti-cancer therapy, comprising determining a 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 (a) an increase or no change in the level of the biomarker compared to the reference indicates that the subject is non-responsive to the anti-cancer therapy, and (b) a decrease in the level of the biomarker compared to the reference indicates that the subject is responsive to the anti-cancer therapy.

[0072] Also provided herein is a composition for detecting lung cancer in a subject. The composition may comprise an antibody that specifically binds to a protein or peptide biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB. The antibody may optionally be conjugated to a detectable label.

[0073] The compositions described herein may further comprise a population of vesicles isolated from a biological sample from a subject, such as a subject with lung cancer. The population of vesicles may optionally be a lysed population of vesicles.

[0074] Also disclosed herein is the use of a composition as defined herein for detecting lung cancer in a subject.

[0075] Also provided herein is a kit for detecting lung cancer in a subject. The kit may include 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 includes an antibody that specifically binds to CAT, CXCR4, and SFTPB. The kit may include a suitable buffer for detecting lung cancer in a subject. The kit may include components for isolating a vesicle population from a biological sample from a subject. The kit may further include a vesicle population isolated from a biological sample from a subject, such as a subject suffering from lung cancer. The vesicle population may optionally be a lysed vesicle population.

[0076] Throughout this specification and the description that follows, unless the context otherwise requires, the word "comprise", and variations such as "comprises" and "comprising", 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 integers or steps, or group of integers or steps.

[0077] Reference herein to any prior publication (or information derived therefrom) or to any publicly known matter is not, and should not be taken as, an admission, acknowledgment, or any form of suggestion that the prior publication (or information derived therefrom) or publicly known matter forms part of the common general knowledge in the field to which this specification pertains.

[0078] Those skilled in the art will understand that the invention described herein may be subject to variations and modifications other than those specifically described. It should be understood that the invention includes all such variations and modifications within its spirit and scope. The invention also includes all of the steps, features, compositions, and compounds referred to or shown in this specification, individually or collectively, and any and all combinations of any two or more of the steps and features.

[0079] Certain embodiments of the present invention will now be described with reference to the following examples, which are intended for illustrative purposes only and are not intended to limit the scope of the generality described above. EXAMPLES

[0080] Example 1 A tandem mass tag (TMT)-based quantitative workflow for plasma exosome proteome analysis Circulating exosomes are a very important bioentity considering their involvement and involvement in virtually every pathophysiological aspect in humans. In the discovery phase, plasma exosomes were isolated using a prolonged ultracentrifugation (PUC) (1) technique that was reported to be effective in streamlining plasma complexity. TMT (2), a chemical labeling technique that affords both quantitative and multiplexed analysis in a single reagent, was employed to establish differential proteomes for pooled plasma exosomes from early stage NSCLC; late stage NSCLC; and healthy individuals. Briefly, for each sample group, equal concentrations of pooled plasma exosomal proteins were proteolytically digested with trypsin. Tryptic peptides from each respective group were labeled with one of the isobaric tags, followed by first dimensional fractionation using weak anion exchange chromatography. Fractionated labeled peptides are analyzed by LC-MS / MS and the relative abundance of specific peptides between samples is determined by comparing the intensity of TMT reporter fragment ions in the 126-131 m / z region of peptide product ion spectra. Two biological replicates and three technical replicates were performed to increase certainty about differences in quantitative changes in protein expression. Differential quantitative proteomic analysis was performed using open-source public tools and differentially expressed (p<0.05) target proteins of interest were further investigated by extensive literature search and based on their novelty and relevance to cancer progression.

[0081] Exosome isolation was performed using a commercial exosome isolation kit from Invitrogen, given the ease and feasibility of sample preparation during both the verification and validation phases. Validation analysis by Western immunoblotting of the shortlisted target exosomal proteins was performed in a subset of individuals from the orthogonal cohort. Proteins with expression significantly associated with both early and late stage NSCLC, independent of disease stage, were prioritized for validation. Enzyme-linked immunosorbent assay (ELISA) validation of validated candidates in exosomal content and in soluble plasma was performed in parallel with well-validated cancer biomarkers in a larger patient cohort. The diagnostic efficiency of the validated exosomal markers was evaluated based on the validated area under the curve (AUC) obtained from receiver operating characteristic (ROC) curve analysis. Finally, a multivariate statistical algorithm was employed to determine the predictive value of the multiprotein signature panel in discriminating between NSCLC and non-cancer individuals.

[0082] 2) Characterization of plasma exosomes Plasma exosome-enriched preparations obtained from both ultracentrifugation (UC) and total exosome isolation kit (Invitrogen) were assessed by TEM, NTA, and immunoblot analysis according to the international guidelines for exosome characterization. TEM analysis for both exosome-enriched preparations (Figure 2A) showed the coexistence of single and aggregated clusters of membrane-bound spherical vesicles with sizes ranging from 50 to 150 nm in diameter, consistent with the typical characteristics of exosomes. NTA shows a mean size distribution for both exosome-enriched preparations (Figure 2B), with particle sizes ranging from 40 to 500 nm in diameter, which is the expected size range for exosomes (50 to 150 nm) and small microvesicles (150 to 1000 nm). The predominant sizes (modes) of particles detected from UC and Invitrogen kit preparations were 56.6 ± 1.3 nm and 69.6 ± 2.1 nm, respectively, which are within the generally accepted size range of exosomes. Correspondingly, the concentration of enriched exosomes from UC and the Invitrogen kit was 1.74 × 10 9 ±3.00×10 8 particles / ml and 2.23×10 9 ±8.00×10 7 The concentration was 10 particles / ml.

[0083] Successful recovery of exosomes from both methods was confirmed by immunoblot detection of four general exosome-specific markers, including the cytoplasmic markers Alix and TSG101 and the surface markers CD63 and CD9, along with their absence in the depleted plasma preparations after exosome extraction (Figure 2C). The intracellular proteins GM130 and calnexin were selected as negative exosome markers for purification evaluation, and the absence of the respective markers in both exosome-enriched isolates indicated the absence of Golgi and endoplasmic reticulum (ER) contamination, while both negative markers were detected in all exosome-depleted plasma preparations as expected. However, contamination by other organelles and vesicles cannot be excluded from both exosome-enriched isolates.

[0084] Collectively, these results confirm that plasma-derived exosomes were highly enriched with low organelle contamination using both the UC and Invitrogen methods. The UC and Invitrogen exosome isolation methods were employed for exosome enrichment in the discovery and verification / validation phases, respectively.

[0085] 3) Assessment of quantitative MS data quality

[0086] [Table 1]

[0087] Run-to-run technical variation was determined in terms of the percentage 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 replicated identification of proteins and PSMs observed across all triplicates was <2%, translating into minimal run-to-run technical variation and excellent system reproducibility.

[0088] As depicted in Figure 3A, approximately 77% of the total number of proteins (625 species) were quantified in at least two of the triplicates, suggesting excellent protein complementation between the triplicate LC-MS / MS runs, and these proteins were used for further analysis. In Figure 3B, significant (p<0.05) correlations of measured pairwise early:control relative ratios for each protein among the triplicates supported the robustness and reliability of the quantitative data set.

[0089] 4) Selection criteria for biomarker candidates and validation analysis

[0090] [Table 2]

[0091] Herein, the known lung cancer biomarkers identified in the proteomics dataset are listed in Table 3. Although the following markers are clinically available, they are currently used in a limited capacity as supplementary blood biomarkers for lung cancer. Based on the reference of the fold change of these proteins, a cutoff of 1.2 fold change was considered as expression change in this study. Then, to further refine the list of candidate markers, 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 triplicates; (b) the protein must exhibit at least 1.2 fold change; and (c) only significantly (p<0.05) differentially expressed proteins are considered. Proteins that do not meet these stringent criteria were ignored. As a result, a total of 56 exosomal proteins from the core list of 625 proteins were found to exhibit simultaneous differential regulation in both NSCLC phenotypes compared to controls. These proteins were investigated in more detail by extensive literature searches and based on their novelty and relevance to cancer progression, and 10 markers (Table III) were shortlisted for validation by immunoblotting in a subset of individuals not used in the discovery experiments (early stage NSCLC, n=14; late stage NSCLC, n=14; and healthy individuals, n=14).

[0092] [Table 3]

[0093] The validation analysis returned six exosomal proteins with expression statistically consistent with the discovery proteomic dataset and highly associated with both early and late stage NSCLC (Figure 4), and these proteins will proceed to the clinical validation phase. It is highlighted that surfactant protein B (SFTPB) is a lung-specific protein that is expressed only in lung tissue. The strategy in the selection of NSCLC biomarkers is that candidate proteins should exhibit a simultaneous significant (p<0.05) increase in protein expression in both early and late stage NSCLC, respectively, compared to controls. This would ensure that the candidate can be reliably used to detect early stage NSCLC, indicating that the increase in expression is independent of disease progression.

[0094] Validation of these 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)). Among the six candidates, four markers (CAT, CXCR4, SOD3, SFTPB) exhibited comparable significant (p<0.05) differential expression between healthy subjects and NSCLC phenotypes as reported in both the discovery and validation phases and were further evaluated using receiver operating characteristic (ROC) curves. ROC curves based on ELISA results were plotted to compare the diagnostic efficiency of the four candidate markers in parallel with two well-investigated cancer markers in both exosomal contents and soluble plasma. The discriminatory ability of each individual candidate and the four-marker combination panel between healthy controls and early stage NSCLC (Figure 5A) / total NSCLC cases was evaluated using the AUC under the ROC curve. The four-marker combination panel exhibited the highest ROC AUC value of 0.93 in discriminating between NSCLC and non-cancer controls. Compared with well-investigated cancer biomarkers (CEA, Cyfra21), the four-marker panel and all single markers possessed greater efficacy for the diagnosis of NSCLC. This is the first lung cancer study to compare the target cargo in exosomes with their respective soluble levels in patient plasma. These four biomarkers have been associated with cancer progression, with CAT, SOD3, and SFTPB conferring anti-tumorigenic functions and CXCR4 having pro-tumorigenic functions.

[0095] 5)Clinical validation In clinical validation, patients were roughly split in half and assigned to a training group (n=279) and a validation test set (n=305) for phase I and phase II validation, respectively (Table IV). In phase I validation, data obtained from the training set are used to train a multivariate model to provide a combined receiver operating characteristic curve (ROC) analysis or predictive value of the final signature panel in discriminating between NSCLC and non-cancer individuals. In phase II validation, data obtained from the test set are used to validate the trained model.

[0096] [Table 4]

[0097] The initial six candidates selected in the validation phase were evaluated against training set samples (early stage NSCLC patients (n=32); total NSCLC patients (n=133); and healthy individuals (n=114)) using enzyme-linked immunosorbent assay (ELISA). Among the six candidates, four markers (CAT, CXCR4, SOD3, SFTPB) exhibited comparable significant (p<0.05) differential expression between healthy subjects and NSCLC phenotypes as reported in both the discovery and validation phases. Phase I validated data obtained from these four exosomal targets were subjected to in-house machine learning multivariate model training to finally derive a three-marker signature panel (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, without any adjustments to the model, will be used to cross-validate the model predictions.

[0098] In Figure 5A, using the validated results obtained from phase I and phase II, ROCs were plotted to evaluate and compare the diagnostic efficiency of the four candidates in parallel with three well-investigated cancer markers (CA125, CEA, Cyfra-21) in both exosomal content and soluble plasma of matched patients. The discriminatory ability of each individual candidate and the three-marker signature panel between healthy controls, early stage NSCLC, and all NSCLC cases was evaluated using the AUC under the ROC curve. A total of 167 healthy individuals and 353 NSCLC patients, of which 64 were early stage cases, were validated for each of the four exosomal markers, showing that all markers show good predictive value in exosomal content but not in plasma. Including phase II validation data, the three-marker signature panel exhibited the highest ROC AUC value of 0.99 (specificity = 0.98; sensitivity = 0.97) in discriminating early stage NSCLC from non-cancer controls. Compared with the three well-investigated cancer markers (CA125, CEA, and Cyfra-21), all single markers and the three-marker signature panel possessed greater efficacy for the diagnosis of NSCLC.

[0099] In addition, ELISA validation for each of the four exosomal markers and well-studied cancer markers was performed on breast cancer (n=113), colorectal cancer (n=144), and nasopharyngeal carcinoma (NPC) (n=101) panels covering the top three common cancer types worldwide (Figure 5B). Compared with the well-studied cancer markers (CA125, CEA, Cyfra-21), the four markers alone showed little discriminatory ability, with AUCs ranging from 0.5 to 0.6 for breast cancer, colorectal cancer, and NPC, suggesting that the four markers are indeed specific for NSCLC diagnosis. It has also been shown that the various cancer phenotypes are quite faithful as claimed, with all three cancer phenotypes scoring an AUC of approximately 0.5 for Cyfra21-1 (lung marker), breast cancer scoring an AUC of 0.877 for CA 15-3 (breast marker), colorectal cancer scoring an AUC of 0.671 (colorectal marker), and NPC showing no discrimination against all well-studied cancer markers as expected.

[0100] Three exosomal targets in the signature panel have been associated with cancer progression, with CAT and SFTPB conferring anti-tumorigenic functions and CXCR4 having a pro-tumorigenic function. In combination, the three-marker exosomal signature has great clinical utility in the diagnosis of heterogeneous NSCLC.

Claims

[Claim 1] 1. A method for determining the possible presence of lung cancer in a subject, comprising: determining a 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 possible presence of lung cancer in the subject.

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