Method for determining the possibility of lung cancer

Abstract

The present disclosure is directed to a method for determining the possible presence of lung cancer in a subject. The method includes 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 possible 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 marker in plasma-derived exosomes compared to a healthy subject. The present disclosure further relates to a method for determining lung cancer progression or responsiveness to anticancer therapy in a subject with lung cancer based on these biomarkers.

Description

Technical Field

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

Background Art

[0002] Lung cancer had approximately 234,030 new cases in the United States in 2018 and is the second most common type of cancer diagnosed in the United States. It is the leading cause of cancer death in both men and women. Lung cancer has an average age of about 70 and is mainly diagnosed in older people.

[0003] Current lung screening options include chest x-ray, sputum cytology, and chest computed tomography (CT), each having their respective weighted advantages and limitations. Among these techniques, sputum cytology is a non-invasive method but has a very poor detection rate. Regarding 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 extremely poor specificity, resulting in a false positive rate of about 96%. The majority of patients detected by low-dose CT have been proven 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, in part due to the high incidence of benign nodules, and requires extremely strict interpretation.

[0004] Therefore, it is generally desirable to overcome or improve one or more of the above difficulties.

Summary of the Invention

Means for Solving the Problems

[0005] A step of 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 derived from a 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, a method for determining the likelihood of the presence of lung cancer in a subject, including this step, is disclosed herein.

[0006] A step of 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 derived from a subject, wherein a change in the level of the biomarker compared to a reference differentiates the presence of early-stage and late-stage lung cancer in the subject, a method for determining the progression of lung cancer in a subject, including this step, is also disclosed herein.

[0007] (a) A step of 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 derived from a 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) a step of administering an anti-cancer therapy to the subject, a method for detecting and treating lung cancer in a subject, including these steps, is also disclosed herein.

[0008] A step of 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 derived from a subject, wherein (a) no increase or change in the level of the biomarker compared to a reference indicates that the subject is non-responsive to anti-cancer therapy, and (b) a decrease in the level of the biomarker compared to a reference indicates that the subject is responsive to anti-cancer therapy, a method for monitoring the responsiveness of a subject with lung cancer to anti-cancer therapy, including this step, is also disclosed herein.

[0009] Embodiments of the present invention will be described below by way of non-limiting example only, with reference to the accompanying drawings.

Brief Description of the Drawings

[0010]

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[0011] A method for 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 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. In one embodiment, the method determines the likelihood of the presence of both early-stage and / or late-stage lung cancer in the subject.

[0012] A method for 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 from a population of vesicles isolated from a biological sample derived from the subject, wherein an increase in the level of the biomarker as compared to a reference indicates the likelihood of the presence of lung cancer in the subject, is disclosed herein.

[0013] A method for 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 from a population of vesicles isolated from a biological sample derived from the subject, wherein a change in the level of the biomarker as compared to a reference indicates the presence of lung cancer in the subject. In one embodiment, the method detects the presence of lung cancer at an early and / or advanced stage in the subject.

[0014] A method for 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 from a population of vesicles isolated from a biological sample derived from the subject, wherein an increase in the level of the biomarker as compared to a reference indicates the presence of lung cancer in the subject, is disclosed herein.

[0015] Without being bound by theory, the inventors have found that using exosomes allows for the identification of a well-defined entity in the blood that has all the advantages of a blood sample but without the background (fluctuations in marker proteins due to other diseases or injuries) and interference from plasma proteins. Since tumor cells are known to release far more circulating exosomes than normal proliferating cells, this approach optimally supports a direct examination of NSCLC tumor-derived exosomes in plasma.

[0016] A 3- or 4-marker exosome panel can be used, for example, in annual check-ups and before imaging to define high-risk patients. In combination with the panel, such patients with a high-risk clinical profile can proceed to a chest computed tomography (CT). Those with test results suggesting a low probability of cancer can be re-evaluated using 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 false-positive cases (about 50%) associated with screening CT that can lead to unnecessary worry, biopsies, and / or surgeries; early detection will enable timely adjusted treatment strategies in management and improvement of 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 it is that lung cancer is present in the 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 the 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 the subject.

[0019] In one embodiment, a method for determining the likelihood of the presence of lung cancer in a subject is provided, which includes the step of determining the level of a biomarker associated with (or bound to) vesicles 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 likelihood of the presence of lung cancer in the subject.

[0020] The biomarker can be a protein, a peptide. The biomarker can be bound to or associated with the surface of the vesicles. Alternatively, it can be contained within the vesicles. 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 an enzyme-linked immunosorbent assay (ELISA), a Luminex assay, or Western immunoblotting, which determines, for example, the amount of biomarker bound to, associated with, or contained within 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 enzyme label) that enables detection. Alternatively, the antibody can be detected using a secondary antibody conjugated to a label (such as a fluorescent, luminescent, or enzyme label).

[0022] 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, for RT-PCR analysis, the vesicles can be lysed and the RNA can 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 can be determined by PCR, qPCR, qRT-PCR, RNA sequencing, microarray analysis, SAGE, MassARRAY technique, next-generation sequencing, or FISH. Alternatively, captured vesicles on or released from a capture surface can be analyzed using immunocytochemical and other fluorescence imaging techniques. Analysis of the vesicles can 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 can also be used. Alternatively, the biomarker can also be detected using mass spectrometry. Nucleic acid biomarkers (such as genomic DNA or mRNA) can also 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. The polypeptides of the present invention may include non-peptidic components such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to the polypeptide by the cells in which the polypeptide is produced and will vary depending on the cell type. Polypeptides are defined herein in terms of their amino acid backbone structure; substituents such as carbohydrate groups are generally not specified but may nonetheless be present.

[0025] The nucleic acids of the present invention can be in the form of RNA such as mRNA, or in the form of DNA including, for example, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA can be double-stranded or single-stranded. The 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 Fv (scFv), Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv) (including bispecific sdFv), and anti-idiotype (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.

[0027] The term "polymerase chain reaction" or "PCR" means a reaction for the in vitro amplification of a specific nucleic acid sequence by simultaneous primer extension of complementary strands of a nucleic acid molecule. In other words, PCR is a reaction for creating multiple copies or replications of a target nucleic acid flanked by primer sites, and such a reaction involves one or more repetitions of the following steps: (i) a step of denaturing the target nucleic acid, (ii) a step of annealing the primer to the primer site, and (iii) a step of extending the primer by a nucleic acid polymerase in the presence of nucleoside triphosphates. Usually, the reaction is cycled through various temperatures optimized for each step in a thermal cycler device. The specific temperatures, the duration in each step, and the rate 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 includes determining the level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB that is present in, or associated with, bound to, or contained within, a population of vesicles. In one embodiment, the method includes determining the level of CAT, CXCR4, SOD3, or SFTPB. The method can include determining the level of a panel of biomarkers (i.e., two or more biomarkers). In one embodiment, the method includes 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 includes 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 includes determining the level of three biomarkers including CAT, CXCR4, and SFTPB. In one embodiment, the method includes determining the level of four biomarkers including CAT, CXCR4, SOD3, and SFTPB. In one embodiment, the method includes determining the level of four biomarkers consisting of CAT, CXCR4, SOD3, and SFTPB.

[0029] In one embodiment, a method for determining the likelihood of the presence of lung cancer in a subject is provided, the method including determining the level 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 as compared to a reference indicates the likelihood of the presence of lung cancer in the subject. The method can further include determining the level of SOD3.

[0030] In one embodiment, a method for determining the levels of biomarkers including CAT, CXCR4, SOD3, and SFTPB from a population of vesicles isolated from a biological sample derived from a 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, is provided.

[0031] 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.

[0032] The method can include a step of 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.

[0033] In one embodiment, the population of vesicles (or exosome population) can be isolated using differential centrifugation followed by ultracentrifugation (a 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 can include a step of isolating the population of vesicles prior to measuring the levels of the biomarkers. The method can further include a step of lysing the population of vesicles prior to measuring the levels of the biomarkers. Methods for 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 a radioimmunoprecipitation assay (RIPA) buffer.

[0035] In one embodiment, the method includes detecting a vesicle population using an antibody. For example, this may include detecting surface markers from an exosome population. This will preferably enable isolation, purification, and / or enrichment of the exosome population. For the purposes of the present invention, the terms "isolate" and "isolating" in all of their grammatical forms relate to the act of separating or recovering exosomes from their environment, such as a serum or plasma sample or a tissue biopsy. The terms "purify" and "purifying" in all of their grammatical forms relate to the act of liberating the desired exosomes from (non-exosomal) contaminants. The terms "enrich" and "enriching" in all of their grammatical forms mean increasing the proportion of exosomes in their respective solvents. Proteins are particularly contemplated as exosome surface markers, although other biomolecules such as lipids are also conceivable. Exosome surface markers can be recognized 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 a vesicle population using a bead-conjugated antibody (such as in an ELISA-based assay). The bead-conjugated antibody enables any antibody-bound vesicle population to be separated from a 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 body 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-ejaculatory fluid, female vaginal fluid, sweat, feces, hair, tear fluid, cyst fluid, pleural fluid and peritoneal fluid, pericardial fluid, lymph fluid, chyle, chylous fluid, bile, interstitial fluid, menstrual secretion, pus, sebum, vomit, vaginal secretion, mucosal secretion, stool water, pancreatic juice, washing fluid from sinus cavities, bronchoalveolar aspirate, or other washing fluids. The biological sample can also include a blastocyst cavity, cord blood, or maternal circulation that 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.

[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 a procedure is performed as an invasive surgery or as a minimally invasive needle biopsy, tissue biopsy is associated with a risk of infection and cannot be applied repeatedly. Furthermore, core and needle biopsies often do not provide a sufficient amount of tissue for thorough diagnostic analysis and may even miss zonal pathophysiological tissue changes. Since blood samples can be easily and repeatedly obtained, the concept of "liquid biopsy" is expected to be a minimally invasive complement to traditional tissue biopsy. When 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 contains cancer cells or circulating tumor cells (CTCs). In another embodiment, the biological sample contains vesicles derived from cancer cells or circulating tumor cells.

[0040] The method of the present invention may include a step of assaying one or more vesicles, including assaying a population of vesicles. As used herein, "vesicle" may refer to a naturally occurring or synthetic vesicle that contains a cavity inside. The vesicle may include a lipid bilayer membrane surrounding the contents of the internal cavity. The vesicle may include liposomes, exosomes, extracellular vesicles, microvesicles, apoptotic vesicles (or apoptotic bodies), vacuoles, lysosomes, transport vesicles, secretory vesicles, gas vesicles, matrix vesicles, or multivesicular bodies. The vesicle may have dimensions 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.

[0041] An exosome is a type of vesicle, also referred to in the art as an extracellular vesicle, microvesicle, or microparticle. These vesicles are released outside the cell by eukaryotic cells or bud off from the plasma membrane. These membrane vesicles have a diameter ranging from about 10 nm to about 5000 nm and are of heterogeneous size. Small vesicles (with a diameter of approximately 10 - 1000 nm, 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, an exosome can be described as a spherical bilayer proteolipid that carries a cargo of various biomolecules, including genetic materials such as mRNA, microRNA (miRNA), and other non-coding RNAs, or even smaller amounts of DNA, lipids, and proteins including transcription factors, cytokines, growth factors, and others.

[0043] In one embodiment, the vesicle is an exosome. In another embodiment, the vesicle is a circulating exosome.

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

[0045] The terms "cancer" and "cancerous" in part refer to or describe a physiological state in mammals typically characterized by unregulated cell growth. As used herein, the term "cancer" refers to non-metastatic and metastatic cancers, including early-stage and late-stage cancers. The term "pre-cancerous" typically refers to a state or growth that precedes or develops into cancer. By "non-metastatic" is meant a cancer that is benign or remains at the primary site and does not penetrate into the lymphatic or vascular system or into tissues other than the primary site. Generally, non-metastatic cancers are cancers that are stage 0, I, or II, and sometimes any cancer that is stage III. By "early-stage cancer" is meant a cancer that is not invasive or metastatic or is classified as a stage 0, I, or II cancer. The term "late-stage cancer" generally refers to stage III or stage IV cancers, but may also refer to stage II cancers or sub-stages of stage II cancers. One of ordinary skill in the art will understand that the classification of a stage II cancer as either 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).

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

[0047] The method as defined herein may include 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 a subject, wherein a change (increase) in the level of the biomarker as compared to a reference indicates the presence of lung cancer in the subject.

[0048] As used herein, a "reference" 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). A reference can also be a predetermined value or an average value. In one embodiment, the methods defined herein include the step of comparing the level of one or more biomarkers to a reference.

[0049] As used herein, the terms "increased" or "increase" with respect to a biomarker refer to a statistically significant and measurable increase in the biomarker as 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.

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

[0051] In one embodiment, the increase in the level of the biomarker compared to the reference can be 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.

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

[0053] In one embodiment, a decrease in the level of the 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 level of the reference.

[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 the step of 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 derived from the subject.

[0055] A method for determining the progression of lung cancer in a subject, comprising the step of 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 derived from the subject, wherein a change in the level of the biomarker compared to a reference differentiates 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 the step of determining the level of SOD3 from a population of vesicles isolated from a biological sample derived from the subject.

[0056] A method for determining the progression of lung cancer in a subject, comprising the step of 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 derived from the subject, wherein an increase in the level of the biomarker compared to a reference differentiates 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 early-stage or late-stage cancer. It may also refer to determining whether the lung cancer is stage 0, I, II, III, and / or IV cancer.

[0058] A method for determining the prognosis of lung cancer in a subject after anti-cancer therapy is also provided herein, which includes the step of 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 derived from the subject.

[0059] (a) A step of 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 derived from a 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) a step of administering an anti-cancer therapy to the subject. A method for detecting and treating lung cancer in a subject is also disclosed herein.

[0060] In one embodiment, (a) a step of 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 derived from a 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) a step of administering an anti-cancer therapy to the subject. A method for detecting and treating lung cancer in a subject is provided.

[0061] A method for treating lung cancer in a subject is also disclosed herein. The method can be based on the test results obtained 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 derived 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. The method for treating lung cancer can include the step of administering an anti-cancer therapy to the subject.

[0062] The level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB has been determined in a population of vesicles isolated from a biological sample derived from a 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. Also disclosed herein is an anti-cancer therapy for use in the treatment of lung cancer in a subject.

[0063] The level of a biomarker selected from the group consisting of CAT, CXCR4, SOD3, and SFTPB has been determined in a population of vesicles isolated from a biological sample derived from a 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. 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.

[0064] As used herein, the terms "treating" or "treatment" can 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) reducing a 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] As used throughout this specification, the term "subject" should be understood to mean a human, or can be a domestic or companion animal. While the methods of the present invention are particularly contemplated for use in the treatment of humans, they are also applicable to veterinary treatment, including the treatment of companion animals such as dogs and cats, domestic animals such as horses, cows, and sheep, or zoo animals such as primates, felines, canines, bovines, and ungulates. A "subject" can include a person, patient, or individual, and can be of any age or gender.

[0067] In one embodiment, the method further comprises administering an anti-cancer therapy to a subject found to have lung cancer. The anti-cancer therapy can include chemotherapy, radiation therapy, targeted therapy, immunotherapy, or a combination thereof. 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), vincblastine, or pemetrexed (Alimta). The method can also include treating the subject by surgery.

[0068] Also disclosed herein is a method of monitoring the responsiveness of a subject with lung cancer to anti-cancer therapy, the method including 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. 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 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] Provided is a method of monitoring the responsiveness of a subject with lung cancer to anti-cancer therapy, the method including 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 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 of monitoring the responsiveness of a subject with 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 derived from the subject, wherein (a) no increase or 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.

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

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

[0074] Also disclosed herein is the use of the compositions 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 can 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 antibodies that specifically bind to CAT, CXCR4, and SFTPB. The kit can include a suitable buffer for detecting lung cancer in a subject. The kit can include components for isolating a population of vesicles from a biological sample derived from the subject. The kit can further include a population of vesicles isolated from a biological sample derived from a subject, such as a subject with lung cancer. The population of vesicles can optionally be a lysed population of vesicles.

[0076] Throughout this specification and the following description, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to 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.

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

[0078] A person skilled in the art will appreciate that the present invention is susceptible to variations and modifications other than those specifically described. The invention includes all such variations and modifications that fall within its spirit and scope. The invention includes, individually or collectively, all of the steps, features, compositions, and compounds referred to or indicated in this specification, as well as any combination of any two or more of the steps and features.

[0079] Reference will now be made to certain specific embodiments of the invention, which are described by way of example only and are not intended to limit the generality of the scope described above.

Examples

[0080] (Example 1) Tandem mass tag (TMT)-based quantitative workflow for plasma exosome proteome analysis Circulating exosomes are very important bioentities considering their involvement and relevance to virtually every pathophysiological aspect in humans. In the discovery phase, plasma exosomes were isolated using the prolonged ultracentrifugation (PUC) (1) method, which has been reported to be effective in simplifying plasma complexity. The tandem mass tag (TMT) (2), a chemical labeling method that provides both quantification and multiplexing analysis in a single reagent, was employed to establish the differential proteome for early-stage NSCLC; late-stage NSCLC; and pooled plasma exosomes from healthy individuals. Briefly, for each sample group, equal concentrations of pooled plasma exosome proteins were proteolytically digested with trypsin. Trypsin peptides from each respective group were labeled with one of the isobaric tags, followed by one-dimensional fractionation using weak anion exchange chromatography. The fractionated labeled peptides were analyzed by LC-MS / MS, and the relative abundance of specific peptides between samples was determined by comparing the intensities 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 certainty about the differences in the quantitative changes in protein expression. Differential quantitative proteomics analysis was performed using open-source public tools, and the differentially expressed (p < 0.05) target proteins of interest were further investigated in more detail by extensive literature searches and based on their novelty and relevance to cancer progression.

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

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

[0083] The success of exosome recovery from both methods was confirmed by immunoblot detection of four common 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 assessment, and the absence of each marker 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 cell organelles and vesicles could not 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 variability was determined in terms of the coefficient of variation percentage (%CV), and the number of proteins and PSMs identified in replicates 01 (R01), 02 (R02), and 03 (R03) were compared as summarized in Table 1. Using a stringent FDR < 1%, the overall %CV in the reproducible identification of proteins and PSMs observed across all three replicates was < 2%, which translates to minimal run-to-run technical variability and excellent system reproducibility.

[0088] As depicted in Figure 3A, approximately 77% (625 proteins) of the total number of proteins were quantified in at least two of the three replicates, suggesting excellent protein complementarity between the LC-MS / MS runs of the three replicates, and these proteins were used for further analysis. In Figure 3B, the significant (p < 0.05) correlation of the measured pairs of early:control ratios for each protein between the three replicates supported the certainty and reliability of the quantitative dataset.

[0089] 4) Biomarker Candidate Selection Criteria and Validation Analysis

[0090]

Table 2

[0091] In this specification, known lung cancer biomarkers identified in proteomics datasets are listed in Table 3. Although the following markers are clinically available, they are currently used with limited ability as complementary blood biomarkers for lung cancer. Based on the reference of fold change of these proteins, a cutoff of 1.2-fold change was considered as the change in expression in this study. Next, 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 must be quantified in at least 2 of the triplicates; (b) the protein must exhibit at least a 1.2-fold change; and (c) only proteins that are differentially expressed significantly (p<0.05) were considered. Proteins that did not meet these stringent criteria were ignored. As a result, a total of 56 exosomal proteins were found to exhibit simultaneous differential regulation in both NSCLC phenotypes compared to the control from a core list of 625 proteins. These proteins were further investigated in detail by an extensive literature search 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 experiment (early-stage NSCLC, n = 14; late-stage NSCLC, n = 14; and healthy individuals, n = 14).

[0092]

Table 3

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

[0094] The validity of these six markers was confirmed in a total of 306 individuals (early-stage NSCLC patients (n = 53); late-stage NSCLC patients (n = 139); and healthy individuals (n = 114)) using enzyme-linked immunosorbent assay (ELISA). Among the six candidates, four markers (CAT, CXCR4, SOD3, SFTPB) showed comparable significant (p < 0.05) differential expression between healthy subjects and the NSCLC phenotype as reported in both the discovery and validation phases and were further evaluated using receiver operating characteristic (ROC) curves. ROC curves based on the 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 content and soluble plasma. The discriminative ability of each individual candidate and the four-marker combination panel between healthy controls and early-stage NSCLC (Figure 5A) / all NSCLC cases was evaluated using the area under the ROC curve (AUC). The four-marker combination panel showed the highest ROC AUC value of 0.93 in discriminating NSCLC from non-cancer controls. Compared with the well-investigated cancer biomarkers (CEA, Cyfra21), the four-marker panel and all individual markers had greater power for the diagnosis of NSCLC. This is the first lung cancer study to compare the target payloads within exosomes having their respective soluble levels in the plasma of patients. These four biomarkers are associated with cancer progression, with CAT, SOD3, and SFTPB conferring anti-tumorigenic functions and CXCR4 having pro-tumorigenic functions.

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

[0096]

Table 4

[0097] The first six candidates selected in the validation phase were evaluated using enzyme-linked immunosorbent assay (ELISA) against training set samples (early-stage NSCLC patients (n = 32); all NSCLC patients (n = 133); and healthy individuals (n = 114)). Among the six candidates, four markers (CAT, CXCR4, SOD3, SFTPB) showed comparable significant (p < 0.05) differential expression between healthy subjects and the NSCLC phenotype as reported in both the discovery and validation phases. The Phase I validity confirmation data obtained from these four exosome targets were subjected to in-house machine learning multivariate model training to finally derive a 3-marker signature panel (CAT, CXCR4, SFTPB) for early NSCLC (AUC = 0.96; specificity = 0.96; sensitivity = 0.91). Without adjustment to the model, the Phase II validity confirmation data obtained from the test set samples will be used to cross-evaluate the model's prediction.

[0098] In FIG. 5A, using the validation-confirmed results obtained from Phases I and II, an ROC was plotted to evaluate and compare the diagnostic efficiency of four candidate markers in parallel with three well-investigated cancer markers (CA125, CEA, Cyfra-21) in both the exosome content and soluble plasma of matched patients. The discriminative ability of each individual candidate and the three-marker signature panel among healthy controls, early-stage NSCLC, and all NSCLC cases was evaluated using the AUC under the ROC curve. For each of the four exosomal markers, a total of 167 healthy individuals and 353 NSCLC patients, 64 of whom were early cases, were validation-confirmed, and it was shown that all markers had good predictive values in the exosome content but not in plasma. Including the Phase II validation data, the three-marker signature panel presented the highest ROC AUC value of 0.99 (specificity = 0.98; sensitivity = 0.97) in discriminating early NSCLC from non-cancer controls. Compared with the three well-investigated cancer markers (CA125, CEA, and Cyfra-21), all individual markers and the three-marker signature panel had greater power for the diagnosis of NSCLC.

[0099] In addition, ELISA validation for each of the four exosome markers and well-investigated cancer markers was performed on breast cancer (n = 113), colorectal cancer (n = 144), and nasopharyngeal carcinoma (NPC) (n = 101) groups that cover the top three most common worldwide cancer types (Figure 5B). Compared with the well-investigated 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. The various cancer phenotypes were indeed faithful as claimed, and all three cancer phenotypes scored an AUC of approximately 0.5 for Cyfra21-1 (lung marker), breast cancer scored an AUC of 0.877 for CA 15-3 (breast marker), colorectal cancer scored an AUC of 0.671 (colorectal marker), and NPC was also shown to have no discriminatory ability for all well-investigated cancer markers as expected.

[0100] The three exosome targets in the signature panel are associated with cancer progression, where CAT and SFTPB confer anti-tumorigenic functions and CXCR4 has a tumorigenesis-promoting function. Collectively, the three-marker exosome signature has great clinical utility in the diagnosis of heterogeneous NSCLC.

Claims

**Claim 1** A method for indicating the possibility of the presence of non-small cell lung cancer (NSCLC) in a subject, comprising the step of determining the levels of biomarkers including catalase (CAT), C-X-C motif chemokine receptor 4 (CXCR4), and surfactant protein B (SFTPB) from a population of vesicles isolated from a blood, serum, or plasma sample derived from the subject, wherein a change in the level of the biomarker compared to a reference indicates the possibility of the presence of NSCLC in the subject. **Claim 2** The method according to claim 1, wherein an increase in the level of the biomarker compared to the reference indicates the possibility of the presence of NSCLC in the subject. **Claim 3** The method according to claim 1 or claim 2, wherein the biomarker is a protein or a peptide. **Claim 4** The method according to any one of claims 1 to 3, wherein the vesicles are exosomes. **Claim 5** The method according to any one of claims 1 to 4, wherein the level of the biomarker is determined using an antibody-based technique. **Claim 6** The method according to any one of claims 1 to 5, further comprising the step of isolating the population of vesicles from the blood, serum, or plasma sample. **Claim 7** The method according to any one of claims 1 to 6, further comprising the step of detecting the population of vesicles using an antibody. **Claim 8** The method according to claim 7, wherein the antibody is selected from the group consisting of an anti-CD9 antibody, an anti-CD63 antibody, and an anti-CD81 antibody. **Claim 9** The method according to any one of claims 1 to 8, wherein the NSCLC is early-stage NSCLC. **Claim 10** The method according to any one of claims 1 to 9, further comprising the step of detecting the levels of CAT, CXCR4, and SFTPB.

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