Circulating biomarkers for detecting lung cancer and methods thereof

Abstract

Disclosed herein are biomarkers associated with lung cancer and methods of determining whether a subject has or is at risk of having lung cancer, wherein the methods comprise detecting the expression level of at least one or more miRNAs in a biological sample obtained from the subject.

Description

Field of the Invention

[0001] The present invention generally relates to the field of molecular biology. In particular, the present invention relates to biomarkers associated with lung cancer and methods for using the biomarkers to determine whether a subject has lung cancer or is at risk of developing lung cancer. Background of the Invention

[0003] According to global cancer statistics reported by the International Agency for Research on Cancer, lung cancer is the leading cause of cancer death worldwide, accounting for 18% of all cancer deaths in 2020 (Ferlay et al., 2020). The high mortality rate can be attributed to late-stage cancer detection, as the five-year survival rate for lung cancer patients diagnosed at an early stage (stage I or II) is approximately 70%, but when the cancer is diagnosed at a late stage (stage III and IV), the five-year survival rate drops to 20% or less.

[0004] Chest X-rays and sputum smears are the most common lung cancer screening techniques, but the sensitivity of such tests is low and therefore their utility in diagnosing cancer early is limited. If necessary, fiberoptic bronchoscopy or biopsy can be used to directly examine the lesion and determine the nature of the pathology, but such methods are invasive and difficult to apply to larger-scale testing of high-risk populations. Low-dose spiral CT is currently considered the most effective lung cancer screening technology, which is non-invasive and highly sensitive, but has a false positive rate of up to 96.4% and relatively high screening costs. Therefore, there is an unmet need for effective methods for screening populations to increase the rate of early diagnosis and treatment of lung cancer and thus reduce lung cancer mortality. There is a need to provide alternative methods for diagnosing lung cancer. SUMMARY OF THE INVENTION

[0006] In one aspect, the present disclosure relates to a method for determining whether a subject has lung cancer or is at risk of developing lung cancer, wherein the method comprises detecting / determining the expression level of at least one miRNA from a biological sample obtained from the subject, wherein the at least one miRNA is selected from the group consisting of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p.

[0007] In certain embodiments, the method comprises detecting at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve miRNAs.

[0008] In certain embodiments, the method for determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting / determining the expression levels of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p, and hsa-miR-877-5p in a biological sample.

[0009] In certain embodiments, the expression level of at least one miRNA selected from the group consisting of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p is compared to the expression of the one or more miRNAs in a control, wherein the differential expression level of the one or more miRNAs in a biological sample obtained from the subject as compared to the control indicates that the subject has or is at risk for developing lung cancer.

[0010] In certain embodiments, a method for determining whether a subject has lung cancer or is at risk of developing lung cancer comprises:

[0011] a. Obtaining a biological sample from a subject;

[0012] b. contacting the biological sample with an isolated probe set suitable for detecting one or more miRNAs, wherein the one or more miRNAs are selected from hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p.

[0013] c. comparing the expression level of the one or more miRNAs in the biological sample with the level of the same miRNA in a control;

[0014] d. determining whether the subject has lung cancer or is at risk of developing lung cancer by differential expression of the one or more miRNAs compared to a control.

[0015] In certain embodiments, the biological sample obtained from the subject is a non-cellular biological fluid.

[0016] In certain embodiments, the non-cellular biological fluid is plasma or serum.

[0017] In certain embodiments, the method further comprises detecting the presence of at least one additional biomarker. In certain embodiments, the method further comprises detecting the expression level of at least one additional biomarker from a biological sample obtained from a subject. In some embodiments, the additional biomarker is one or more selected from carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 125 (CA-125), or cytokeratin 19 fragment antigen (CYFRA 21-1). In some other embodiments, the biomarker is CEA.

[0018] In certain embodiments, the method for determining whether a subject has lung cancer or is at risk of developing lung cancer further comprises additional clinical trials. In some embodiments, the additional clinical trials comprise one or more imaging tests, sputum cytology, biopsy, or a combination thereof. In certain embodiments, the imaging tests comprise CT scans, X-rays, MRIs, or PET scans.

[0019] Also provided are methods of treating a subject identified as having lung cancer, comprising:

[0020] a. Determining whether a subject has lung cancer or is at risk of developing lung cancer by detecting the expression level of at least one miRNA from a biological sample obtained from the subject, wherein the at least one miRNA is selected from the group consisting of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p.

[0021] b. comparing the expression level of the one or more miRNAs in the biological sample with the expression level in the control;

[0022] c. treating a subject identified as having lung cancer or identified as being at risk for developing lung cancer with one or more therapeutics suitable for treating lung cancer.

[0023] In some embodiments, the one or more treatments suitable for treating lung cancer include administration of an anti-cancer compound, surgery, and / or radiation therapy.

[0024] In some embodiments, the control may include one or more biological samples obtained from healthy subjects, non-diseased subjects, subjects without cancer, subjects without lung cancer, and / or subjects who do not have lung cancer or are not at risk of developing lung cancer. In some embodiments, the control may include the expression levels of the one or more biomarkers measured in the one or more biological samples obtained from the subject for determining whether the subject has lung cancer or is at risk of lung cancer.

[0025] In certain embodiments, the at least one miRNA can be detected by one or more methods such as, but not limited to, sequencing, nucleic acid hybridization, microarray nucleic acid amplification, and the like.

[0026] Also provided is a kit for determining whether a subject has lung cancer or is at risk of developing lung cancer, the kit comprising an isolated probe set capable of detecting one or more miRNAs selected from the group consisting of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p.

[0027] In some embodiments, the kit includes separate probe sets capable of detecting hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p.

[0028] In some embodiments, the probe is selected from the group consisting of an aptamer, an antibody, an affibody, a peptide, and a nucleic acid.

[0029] In some embodiments, the kit comprises isolated probe sets suitable for determining the expression level of at least one miRNA selected from the group consisting of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p, wherein the at least one miRNA can be detected by one or more methods, such as, but not limited to, sequencing, nucleic acid hybridization, microarray, and nucleic acid amplification. In some examples, nucleic acid amplification may include, but is not limited to, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), reverse transcription-polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), locked nucleic acid PCR, clustered regularly interspaced short palindromic repeats (CRISPR)-based assays, or isothermal amplification assays.

[0030] In certain embodiments, the kit further comprises a probe and / or reagent for detecting at least one additional biomarker. In some embodiments, the additional biomarker is one or more selected from carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 125 (CA-125), or cytokeratin 19 fragment antigen (CYFRA 21-1). In some embodiments, the additional biomarker is CEA.

[0031] In another aspect, the present invention relates to a combination of biomarkers suitable for determining whether a subject has or is at risk of developing lung cancer, wherein the combination comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve biomarkers selected from the group consisting of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p. In some embodiments, the biomarker combination includes hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p, and hsa-miR-877-5p. In some embodiments, the biomarker combination further includes CEA. In some embodiments, the biomarker combination is suitable for detecting biomarkers in a biological sample obtained from a subject, wherein the biological sample is a non-cellular biological fluid, such as a plasma and / or serum sample.

[0032] In another aspect, the present invention also relates to the use of at least one reagent suitable for detecting one or more biomarkers in the manufacture or preparation of a diagnostic agent / kit for use in any of the above methods for determining whether a subject has lung cancer or is at risk of developing lung cancer. In some embodiments, the at least one reagent is used to measure / determine the expression level of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve biomarkers selected from hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p in a biological sample obtained from a subject. In some embodiments, the reagent is used to measure / determine the expression level of biomarkers, wherein the biomarkers include hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p, and hsa-miR-877-5p. In some embodiments, the reagent is used to measure / determine the expression level of at least one additional biomarker. In some embodiments, the additional biomarker is one or more selected from carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 125 (CA-125), or cytokeratin 19 fragment antigen (CYFRA21-1).

[0033] definition

[0034] As used herein, the term "miRNA" refers to microRNA, a small non-coding RNA molecule that comprises approximately 19 to 25 nucleotides in some instances and is present in plants, animals, and some viruses. Known miRNA plays a role in post-transcriptional regulation of RNA silencing and gene expression. These highly conserved RNAs regulate gene expression by binding to the 3'-untranslated region (3'-UTR) of specific mRNA. For example, each miRNA is considered to regulate multiple genes, and since hundreds of miRNA genes are expected to exist in higher eukaryotes, miRNAs are often transcribed from several different loci in the genome. These gene encodings have long RNAs with hairpin structures that, when processed by a series of RNase III (including Drosha and Dicer), form miRNA duplexes that are typically approximately 19-25 nucleotides long, with 2nt overhangs at the 3' end. As will be appreciated by those skilled in the art, miRNA is a type of polynucleotide that has a sequence comprising letters such as "AUGC." It should be understood that nucleotides are arranged from left to right in a 5'>3' order, and unless otherwise indicated, "A" represents adenosine, "U" represents uracil, "G" represents guanosine, and "C" represents cytosine. The letters A, U, G, and C may be used to refer to the bases themselves.

[0035] As used herein, "lung cancer" (abbreviated as "LC" in some cases) refers to a disease in which malignant (cancer) cells form in the lung tissue. The main subtypes of lung cancer are non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), but also include adenoid cystic carcinoma, lymphoma, and sarcoma. The main subtypes of non-small cell lung cancer are adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. These subtypes may arise from different types of lung cells, but are collectively classified as NSCLC because their treatment and prognosis are generally similar.

[0036] As used herein, "biomarker" or "marker" may refer to a gene, protein or miRNA whose expression level or concentration in a sample is changed compared to a control. As used herein, a control refers to the expression level or concentration of a biomarker that indicates a different result than the result of interest or is associated with the different result. For example, a biomarker can be a miRNA whose expression level or concentration is changed (e.g., increased or decreased) compared to a control in a sample of a subject suffering from a disease (i.e., lung cancer). It will be understood by any relevant technician in this field that it is not necessary to obtain a sample from a subject without lung cancer and test the sample simultaneously with the test subject for comparison with the expression level in the control. In some embodiments, the control can be a control sample or a threshold value included in the kit, the threshold being set to represent an expression range of the biomarker, wherein the expression level falling within the range will identify the subject as having lung cancer or at risk of lung cancer.

[0037] As used herein, "biological sample" or "sample" is intended to include any sampling of cells, tissues, or body fluids in which the expression of a biomarker can be detected. Examples of such biological samples include, but are not limited to, biopsies, smears, blood, lymph, urine, saliva, or any other body secretion or derivative thereof. Blood can, for example, include whole blood, plasma, serum, or any derivative of blood. In some embodiments, the biological sample is a liquid biological sample. In some embodiments, the biological sample is a non-cellular biological fluid. In some embodiments, the non-cellular body fluid can include serum and / or plasma. Samples can be obtained from a subject by a variety of techniques known to those skilled in the art.

[0038] As used herein, the term "expression level" or "level" of a biomarker refers to the presence / absence, amount or concentration of a biomarker in a biological sample and can be expressed in any suitable form or unit determined by a person skilled in the art. For example, the expression level of a nucleic acid (e.g., DNA or RNA) can be expressed as, but not limited to, copy number (copy / mL), Ct (cycle threshold), Cq (quantitative cycle), Ct / Cq or log2 scale expression level. In addition, the expression level can be expressed as a score constructed using any form of mathematical model or algorithm.

[0039] As used herein, the term "differential expression" refers to the measurement of a cellular component compared to a control or another sample, thereby determining, for example, a difference in the concentration, presence, or intensity of the cellular component. The results of such a comparison can be given as an absolute value, i.e., the component is present in the sample but not in the control, or as a relative value, i.e., the expression or concentration of the component is increased or decreased compared to the control. In this context, the terms "increase" and "decrease" are interchangeable with the terms "up-regulate" and "down-regulate," which are also used in this disclosure.

[0040] When describing some embodiments, the present disclosure may have disclosed the method as a specific order of steps. However, unless otherwise required, it should be understood that the method should not be limited to the specific order of steps disclosed. Other order of steps are also possible. The specific order of steps disclosed herein should not be interpreted as unduly limiting. Unless otherwise required, the method disclosed herein should not be limited to the steps performed in the order written. The order of the steps can be changed and still remain within the scope of the present disclosure.

[0041] As used herein, "probe" refers to any molecule or reagent that can selectively detect a target biomolecule, for example, by directly or indirectly binding to the desired target biomolecule. The target molecule can be a biomarker, for example, a nucleotide transcript or protein encoded by or corresponding to a biomarker. In view of the present disclosure, probes can be synthesized by those skilled in the art or derived from suitable biological agents. Probes can be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, oligonucleotides, RNA, DNA (e.g., primers), proteins, peptides, antibodies, aptamers, affibodies, and organic molecules. In the case of probes designed to detect nucleic acid biomarkers, such probes can be directed to a target region, a complementary nucleic acid sequence on the reverse strand, or a copy thereof produced by an amplification process.

[0042] As used herein, the term "imaging test" refers to various non-invasive methods used to visualize the inside of a subject's body to diagnose a disease or determine the extent / progression of a disease, and may include computed tomography (CT) (including low-dose CT scans such as low-dose spiral CT or low-dose helical CT), magnetic resonance imaging (MRI), positron emission tomography (PET), or X-rays.

[0043] As used herein, the term "biopsy" relates to the removal of a sample of cells or tissue for examination, such as by a pathologist, to determine the presence or extent of disease.

[0044] As used herein, the term "sputum cytology" refers to the examination of cells present in sputum to detect abnormal cells, such as lung cancer cells.

[0045] As used herein, the term "(statistical) classification" refers to the problem of identifying to which set of classes (or subgroups) a new observation belongs, based on a training dataset containing observations (or instances) whose class membership is known. An example is assigning a diagnosis to a given patient as described by the patient's observed characteristics (gender, blood pressure, presence or absence of certain symptoms, etc.). In machine learning terms, classification is considered an instance of supervised learning, that is, learning when a training set of correctly identified observations is available. The corresponding unsupervised procedure is called clustering and involves grouping data into categories based on some measure of intrinsic similarity or distance. Typically, each observation is analyzed as a set of quantifiable properties, which are variously referred to as explanatory variables or features. These properties can have different categorical values ​​(e.g., "A", "B", "AB", or "O" for blood type), ordinal numbers (e.g., "large", "medium", or "small"), integer values ​​(e.g., the number of times a certain word appears in an email), or real values ​​(e.g., a measurement of blood pressure). Other classifiers work by comparing observations to previous observations using similarity or distance functions. Algorithms that implement classification, especially in specific implementations, are called classifiers. The term "classifier" sometimes also refers to the mathematical function implemented by a classification algorithm, which maps input data to categories.

[0046] As used herein, the term "pre-training" or "supervised (machine) learning" refers to the machine learning task of inferring a function from labeled training data. The training data may consist of a set of training examples. In supervised learning, each example is a pair consisting of an input object (usually a vector) and a desired output value (also called a supervisory signal). The supervised learning algorithm, i.e., the algorithm to be trained, analyzes the training data and produces an inferred function that can be used to map new examples. A preferred approach would allow the algorithm to correctly determine the class labels of unseen examples. This requires the learning algorithm to generalize from the training data to unseen situations in a "reasonable" way.

[0047] As used herein, the term "score" refers to an integer or number that can be mathematically determined, for example, using a computational model known in the art (which may include, but is not limited to, for example, an SVM), and calculated using any of a plurality of mathematical equations and / or algorithms known in the art for statistical classification purposes. This score is used to enumerate an outcome over a range of possible outcomes. The relevance and statistical significance of this score depends on the size and quality of the underlying data set used to establish the outcome spectrum. For example, a blind sample can be input into an algorithm, which in turn calculates a score based on the information provided by the analysis of the blind sample. This results in a score being generated for the blind sample. Based on this score, a decision can be made, for example, as to how likely the patient from whom the blind sample was obtained is to have cancer or not. The ends of the range can be logically defined based on the data provided, or arbitrarily defined according to the experimenter's requirements. In both cases, the range needs to be defined before the blind sample is tested. Thus, a score generated from such a blind sample, such as the number "45," can indicate that the corresponding patient has cancer based on a range defined on a scale from 1 to 50, where "1" is defined as no cancer and "50" is defined as having cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Shown is a receiver operating characteristic curve ("ROC curve") demonstrating the performance of an exemplary embodiment of a miRNA assay for detecting subjects having or at risk for lung cancer. The x-axis represents the specificity of the assay, while the y-axis represents the sensitivity of the assay.

[0050] Figure 2 Shown is a ROC curve demonstrating the performance of an exemplary embodiment of a miRNA assay in combination with CEA for detecting subjects having or at risk for lung cancer. The x-axis represents the specificity of the assay, while the y-axis represents the sensitivity of the assay.

[0051] Figure 3 The performance of the exemplary miRNA panel provided in Table 4 for detecting subjects having or at risk for lung cancer is shown.

[0052] Figure 4 The performance of CEA in combination with the exemplary miRNA panel provided in Table 11 for detecting subjects having or at risk for lung cancer is shown. Detailed Description of the Invention

[0054] miRNAs are evolutionarily conserved single-stranded non-coding RNAs of 19 to 25 nucleotides whose primary function is to mediate degradation or translational repression of mRNA targets. Under normal physiological conditions, miRNAs are key components of feedback mechanisms in a wide range of biological pathways, such as cell proliferation, differentiation, and apoptosis. Conversely, dysregulated miRNAs are implicated in hallmarks of cancer, including supporting tumor growth by inhibiting growth suppression, maintaining proliferative signaling and resisting cell death, activating invasion and metastasis, and promoting angiogenesis. It is now known that miRNAs regulate tumorigenesis through their tumor suppressor or oncogenic activities, and increasing evidence indicates that miRNAs are abnormally expressed in a variety of malignancies.

[0055] The diagnosis of lung cancer is usually carried out by sputum cytology, biopsy or radiographic procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI) scan or positron emission tomography (PET) scan. Sputum cytology is fast and cheap, but suffers from a high false negative rate. The use of radiographic procedures is both expensive and exposes the subject to radiation, and is therefore not suitable for lung cancer screening in the general population. The blood test of protein biomarkers (such as carcinoembryonic antigen (CEA), neuron-specific enolase (NSE) or cytokeratin 19 fragment antigen (CYFRA 21-1)) cannot provide enough accuracy for the diagnosis in clinical settings.

[0056] Because altered miRNA expression profiles in cancer reflect disease progression, and circulating miRNAs are stable and accessible in numerous body fluids, including blood, urine, and saliva, miRNAs are considered suitable as biomarkers. Minimally invasive approaches, such as miRNA-based liquid biopsies, can potentially overcome these disadvantages and improve overall detection accuracy.

[0057] The present invention relates to a method for determining whether a subject has lung cancer or is at risk of developing lung cancer, comprising detecting and / or measuring and / or determining the expression level of one or more biomarkers (particularly the miRNAs listed in Table 1) present in a biological sample obtained from the subject.

[0058] Table 1: Sequences of miRNA biomarkers described herein. Expression levels of miRNAs are shown as downregulated or upregulated relative to controls.

[0059]

[0060]

[0061] In certain embodiments of the present invention, the method for determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression level of one or more miRNAs selected from the group consisting of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p.

[0062] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression level of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven or at least twelve miRNAs selected from hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p.

[0063] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression level of hsa-miR-1280 and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or at least eleven miRNAs selected from the group consisting of hsa-miR-23b-3p, hsa-miR-320a-3p, hsa-miR-877-5p, hsa-miR-181c-5p, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-210-3p, hsa-miR-16-5p, hsa-miR-92a-3p and hsa-miR-342-3p.

[0064] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression level of hsa-miR-23b-3p and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or at least eleven miRNAs selected from the group consisting of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p.

[0065] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression level of hsa-miR-320a-3p and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or at least eleven miRNAs selected from the group consisting of hsa-miR-23b-3p, hsa-miR-877-5p, hsa-miR-181c-5p, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-1280, hsa-miR-205-5p, hsa-miR-210-3p, hsa-miR-16-5p, hsa-miR-92a-3p and hsa-miR-342-3p.

[0066] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression level of hsa-miR-877-5p and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or at least eleven miRNAs selected from the group consisting of hsa-miR-23b-3p, hsa-miR-320a-3p, hsa-miR-181c-5p, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-1280, hsa-miR-205-5p, hsa-miR-210-3p, hsa-miR-16-5p, hsa-miR-92a-3p and hsa-miR-342-3p.

[0067] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression level of hsa-miR-181c-5p and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or at least eleven miRNAs selected from the group consisting of hsa-miR-23b-3p, hsa-miR-320a-3p, hsa-miR-877-5p, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-1280, hsa-miR-205-5p, hsa-miR-210-3p, hsa-miR-16-5p, hsa-miR-92a-3p and hsa-miR-342-3p.

[0068] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression level of hsa-miR-487b-3p and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or at least eleven miRNAs selected from the group consisting of hsa-miR-23b-3p, hsa-miR-320a-3p, hsa-miR-877-5p, hsa-miR-181c-5p, hsa-miR-199b-5p, hsa-miR-1280, hsa-miR-205-5p, hsa-miR-210-3p, hsa-miR-16-5p, hsa-miR-92a-3p and hsa-miR-342-3p.

[0069] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression level of hsa-miR-199b-5p and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or at least eleven miRNAs selected from the group consisting of hsa-miR-23b-3p, hsa-miR-320a-3p, hsa-miR-877-5p, hsa-miR-181c-5p, hsa-miR-487b-3p, hsa-miR-1280, hsa-miR-205-5p, hsa-miR-210-3p, hsa-miR-16-5p, hsa-miR-92a-3p and hsa-miR-342-3p.

[0070] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression level of hsa-miR-205-5p and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or at least eleven miRNAs selected from the group consisting of hsa-miR-23b-3p, hsa-miR-320a-3p, hsa-miR-877-5p, hsa-miR-181c-5p, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-1280, hsa-miR-210-3p, hsa-miR-16-5p, hsa-miR-92a-3p and hsa-miR-342-3p.

[0071] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression level of hsa-miR-210-3p and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or at least eleven miRNAs selected from the group consisting of hsa-miR-23b-3p, hsa-miR-320a-3p, hsa-miR-877-5p, hsa-miR-181c-5p, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-1280, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-92a-3p and hsa-miR-342-3p.

[0072] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression level of hsa-miR-16-5p and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or at least eleven miRNAs selected from the group consisting of hsa-miR-23b-3p, hsa-miR-320a-3p, hsa-miR-877-5p, hsa-miR-181c-5p, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-1280, hsa-miR-205-5p, hsa-miR-210-3p, hsa-miR-92a-3p and hsa-miR-342-3p.

[0073] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression level of hsa-miR-92a-3p and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or at least eleven miRNAs selected from the group consisting of hsa-miR-23b-3p, hsa-miR-320a-3p, hsa-miR-877-5p, hsa-miR-181c-5p, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-1280, hsa-miR-205-5p, hsa-miR-210-3p, hsa-miR-16-5p and hsa-miR-342-3p.

[0074] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression level of hsa-miR-342-3p and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or at least eleven miRNAs selected from the group consisting of hsa-miR-23b-3p, hsa-miR-320a-3p, hsa-miR-877-5p, hsa-miR-181c-5p, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-1280, hsa-miR-205-5p, hsa-miR-210-3p, hsa-miR-16-5p and hsa-miR-92a-3p.

[0075] In certain embodiments, a method of determining whether a subject has or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression levels of hsa-miR-1280 and hsa-miR-487b-3p.

[0076] In certain embodiments, a method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression levels of hsa-miR-1280, hsa-miR-487b-3p, and hsa-miR-199b-5p.

[0077] In certain embodiments, a method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression levels of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, and hsa-miR-205-5p.

[0078] In certain embodiments, a method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression levels of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, and hsa-miR-16-5p.

[0079] In certain embodiments, a method for determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression levels of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, and hsa-miR-320a-3p.

[0080] In certain embodiments, a method for determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression levels of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, and hsa-miR-23b-3p.

[0081] In certain embodiments, a method for determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression levels of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, and hsa-miR-181c-5p.

[0082] In certain embodiments, a method for determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression levels of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, and hsa-miR-92a-3p.

[0083] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression levels of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p and hsa-miR-210-3p.

[0084] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or measuring and / or determining the expression levels of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p and hsa-miR-342-3p.

[0085] In certain embodiments, the method of determining whether a subject has lung cancer or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression levels of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p.

[0086] In certain embodiments, a method for determining whether a subject has or is at risk of developing lung cancer comprises detecting and / or measuring and / or determining the expression level of one or more miRNAs selected from the group consisting of hsa-miR-23b-3p, hsa-miR-320a-3p, hsa-miR-877-5p, hsa-miR-181c-5p, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-1280, hsa-miR-205-5p, hsa-miR-210-3p, hsa-miR-16-5p, hsa-miR-92a-3p and hsa-miR-342-3p in a biological sample obtained from the subject, and comparing the expression of these one or more miRNAs with their expression in a control, wherein the differential expression level of the one or more miRNAs as compared to the control indicates that the subject has or is at risk of developing lung cancer.

[0087] In some embodiments, the control may include one or more biological samples obtained from healthy subjects, non-diseased subjects, cancer-free subjects, lung cancer-free subjects, and / or subjects who do not have lung cancer or are not at risk of developing lung cancer. In some embodiments, the control may include the expression levels of the one or more biomarkers measured in one or more biological samples obtained from the subject for determining whether the subject has lung cancer or is at risk of developing lung cancer. In some embodiments, the control sample may not be obtained simultaneously with the biological sample from the subject to be tested, and may be represented by a control sample provided by a kit, or in some aspects, by a threshold value for miRNA expression in a control population determined in early clinical studies.

[0088] In some embodiments, the expression level of one or more of hsa-miR-23b-3p, hsa-miR-320a-3p, hsa-miR-877-5p, hsa-miR-181c-5p, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-210-3p, hsa-miR-16-5p, and / or hsa-miR-92a-3p in a biological sample is upregulated compared to the expression level in a control. In some embodiments, the expression level of hsa-miR-1280 and / or hsa-miR-342-3p in a biological sample is downregulated compared to the expression level in a control.

[0089] In some embodiments, the differential expression levels of one or more of hsa-miR-23b-3p, hsa-miR-320a-3p, hsa-miR-877-5p, hsa-miR-181c-5p, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-210-3p, hsa-miR-16-5p, and / or hsa-miR-92a-3p are upregulated in a biological sample in a subject having or at risk of developing lung cancer, as compared to the expression levels in a control. In some embodiments, the differential expression levels of hsa-miR-1280 and / or hsa-miR-342-3p are downregulated in a biological sample in a subject having or at risk of developing lung cancer, as compared to the expression levels in a control.

[0090] In certain embodiments, a method for determining whether a subject has lung cancer or is at risk of developing lung cancer comprises:

[0091] a. contacting a biological sample obtained from a subject with an isolated probe set suitable for detecting one or more miRNAs selected from the group consisting of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p, and hsa-miR-877-5p;

[0092] b. comparing the expression level of the one or more miRNAs in the biological sample with the level of the miRNA in the control;

[0093] c. determining whether the subject has lung cancer or is at risk of developing lung cancer by differential expression of the one or more miRNAs compared to a control.

[0094] In certain embodiments, the biological sample comprises a non-cellular biological fluid. In some other embodiments, the non-cellular biological fluid can be plasma or serum.

[0095] In certain embodiments, the method for determining whether a subject has lung cancer or is at risk for developing lung cancer further comprises detecting the expression level of at least one additional biomarker. In some instances, the additional biomarker may include, but is not limited to, carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 125 (CA-125), cytokeratin 19 fragment antigen (CYFRA 21-1), and the like.

[0096] In certain embodiments, methods for determining whether a subject has or is at risk for developing lung cancer further comprise one or more tests, such as, but not limited to, imaging tests, sputum cytology, biopsy, or a combination thereof.

[0097] In certain embodiments, subjects determined to have lung cancer or to be at risk of lung cancer may be further tested using sputum cytology, biopsy, fine needle aspiration, or diagnostic imaging tests including, but not limited to, X-rays, computed tomography (CT) scans, low-dose CT scans, magnetic resonance imaging (MRI) scans, or positron emission tomography (PET) scans. Methods for lung cancer screening are known in the art, including the latest version of the NCCN Clinical Practice Guidelines for Lung Cancer Screening Oncology, which is incorporated herein by reference in its entirety.

[0098] Also provided are methods of treating a subject determined to have lung cancer using the methods disclosed herein. In some embodiments, the methods disclosed herein may further comprise the step of administering treatment to a subject determined to have lung cancer. Such a subject will be referred to a healthcare practitioner and, where determined appropriate, treated with an appropriate treatment method (such as, but not limited to, anticancer compounds, surgery, immunotherapy, or radiation therapy, or a combination of these methods). When a subject is diagnosed with lung cancer, treatment options may include surgery, radiation therapy (including but not limited to external beam radiation therapy, intensity modulated radiation therapy, proton therapy, stereotactic radiosurgery, or brachytherapy), or administration of one or more anticancer compounds, which may include but are not limited to chemotherapy (including but not limited to cisplatin, carboplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine, etoposide, or pemetrexed, either alone or in combination), targeted therapy (such as targeting a specific aspect such as Drugs or monoclonal antibodies for angiogenesis, EGFR, KRAS, ALK, NTRK, BRAF, ROS1, etc., may include but are not limited to osimertinib, erlotinib, gefitinib, afatinib, dacomitinib, crizotinib, ceritinib, dabrafenib, trametinib, bevacizumab, bevacizumab, ervantumab, cetuximab), immunotherapy (e.g., PD-1 and PDL-1 inhibitors such as pembrolizumab or nivolumab) or cell therapy (including chimeric antigen receptor T cells). Methods for treating lung cancer are known in the art and include the latest versions of the NCCN Clinical Practice Guidelines for Non-Small Cell Lung Cancer Oncology and the NCCN Clinical Practice Guidelines for Small Cell Lung Cancer Oncology, which are incorporated herein by reference in their entirety.

[0099] In certain embodiments, a method for treating a subject having lung cancer comprises:

[0100] a. contacting a biological sample obtained from a subject with an isolated probe set suitable for detecting one or more miRNAs selected from the group consisting of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p, and hsa-miR-877-5p;

[0101] b. comparing the expression level of the one or more miRNAs in the biological sample with the level of the miRNA in the control;

[0102] c. determining whether the subject has lung cancer or is at risk of developing lung cancer by differential expression of the one or more miRNAs compared to a control;

[0103] d. treating the subject determined to have or to be at risk for developing lung cancer with one or more therapies selected from administration of an anti-cancer compound, surgery, and / or radiation therapy.

[0104] In certain embodiments, the methods for treating a subject having lung cancer further comprise measuring the level of at least one additional biomarker in the subject.

[0105] In certain embodiments, the additional biomarker is one or more selected from carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 125 (CA-125), and cytokeratin 19 fragment antigen (CYFRA 21-1). In some instances, the additional biomarker is carcinoembryonic antigen (CEA).

[0106] In certain embodiments, the method further comprises imaging testing, sputum cytology, or a biopsy.

[0107] Also provided herein are compositions / agents / reagents for use in the methods disclosed herein. In some embodiments, such compositions / agents / reagents may include, but are not limited to, probes, antibodies, affibodies, nucleic acids, and / or aptamers. In some embodiments, the compositions can detect (or detect) the expression level of a small group of biomarkers (e.g., miRNA) from a biological sample.

[0108] Any composition can be provided in the form of a kit or a reagent mixture. For example, a labeled probe can be provided in a kit for detecting a small group of biomarkers. The kit may include all necessary or sufficient components for determining, which may include but are not limited to target enrichment reagents, detection reagents (e.g., probes and / or fluorescent dyes), buffers, control reagents (e.g., positive and negative controls), amplification reagents, solid supports, labels, instructions, calibrators, and reference materials. In certain embodiments, the kit includes a group of probes for a small group of biomarkers and a solid support for fixing the group of probes. In certain embodiments, the kit includes a group of probes for a small group of biomarkers, a solid support, and a reagent for processing a sample to be tested (e.g., a reagent for separating proteins or nucleic acids from a sample). In certain embodiments, the present invention may include the use of a detection reagent suitable for detecting a small group of biomarkers in the preparation or preparation for (or when used to) determine whether a subject suffers from lung cancer or whether there is a risk of lung cancer in a kit.

[0109] In certain embodiments, detection reagents (e.g., probes) suitable for use in kits include, but are not limited to, oligonucleotides, RNA, DNA (e.g., primers), proteins, peptides, antibodies, aptamers, affibodies, and organic molecules. In the case of probes designed to detect nucleic acid biomarkers, such probes can be directed to the target region, to a complementary nucleic acid sequence on the reverse strand, or to a copy thereof generated by an amplification process.

[0110] In some embodiments, provided herein are DNA-, RNA-, and protein-based detection methods for directly or indirectly detecting the biomarkers described herein. The present invention also provides compositions, reagents, and kits for such diagnostic purposes. The diagnostic methods described herein can be qualitative or quantitative. Quantitative diagnostic methods can be used, for example, by comparing the detected biomarker levels to a cutoff or threshold level. Where applicable, qualitative or quantitative diagnostic methods can also include amplification of the target, signal, or intermediate.

[0111] In some embodiments, biomarkers are detected at the nucleic acid (e.g., DNA or RNA) level. For example, the amount of biomarker RNA (e.g., miRNA) present in a sample is determined (e.g., to determine the level of biomarker expression). Biomarker nucleic acids (e.g., miRNA, amplified cDNA, etc.) can be detected / quantified using a variety of nucleic acid technologies known to those of ordinary skill in the art, including but not limited to sequencing, nucleic acid hybridization (e.g., northern blot), microarrays, and nucleic acid amplification (e.g., quantitative reverse transcription polymerase chain reaction (qRT-PCR), reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), locked nucleic acid (LNA) real-time PCR, CRISPR-based assays, or isothermal amplification assays. Isothermal amplification assays can include, for example, but not limited to, nicking endonucleases. Amplification reaction (NEAR) assay, transcription-mediated amplification (TMA) assay, loop-mediated isothermal amplification (LAMP) assay, helicase-dependent amplification (HDA) assay, clustered regularly interspaced short palindromic repeats (CRISPR) assay or strand displacement amplification (SDA) assay. In some embodiments, the method for detecting miRNA biomarkers may include using the assay method disclosed in WO2011159256A1, and the kit for detecting miRNA may include stem-loop oligonucleotides designed based on the teachings of WO2011159256A1, the disclosure of which is incorporated herein by reference.

[0112] Also provided herein are kits for determining whether a subject has or is at risk of developing lung cancer, the kit comprising isolated probe sets capable of detecting one or more miRNA biomarkers selected from the group consisting of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p, and hsa-miR-877-5p.

[0113] In certain embodiments, the kit includes separate probe sets capable of detecting hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p, and hsa-miR-877-5p.

[0114] In certain embodiments, the kit further comprises reagents for detecting at least one additional biomarker in a biological sample obtained from a subject.

[0115] In certain embodiments, the additional biomarker is carcinoembryonic antigen (CEA).

[0116] The invention illustratively described herein may be suitably implemented in the absence of any one or more elements, one or more limitations specifically disclosed herein. Thus, for example, the terms "comprise," "include," "contain," etc. should be understood broadly and not restrictively. Furthermore, the terms and expressions employed herein are used as descriptive rather than restrictive terms, and when such terms and expressions are used, it is not intended to exclude any equivalents or portions thereof of the features shown and described, but it should be recognized that various modifications are possible within the scope of the claimed invention. Therefore, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the invention specifically implemented herein as disclosed herein may be adopted by those skilled in the art, and such modifications and variations are considered to be within the scope of the present invention.

[0117] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with the proviso or negative limitation that removes any subject matter from that genus, regardless of whether the removed material is specifically recited herein.

[0118] Throughout this disclosure, certain embodiments may be disclosed in the form of ranges. It should be understood that the description of range format is merely for convenience and brevity and should not be construed as an immutable limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within the range. For example, a description such as a range from 1 to 6 should be considered to have specifically disclosed subranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numerals within the range, such as 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, this principle applies.

[0119] Other embodiments are in the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0120] Exemplary embodiments of the present invention are provided in the following examples. Although the exemplary embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, it should be understood that the invention is not limited to these examples.

[0121] Predictive score for lung cancer risk or sample calculation of risk score

[0122] As known in the art, biomarkers (e.g., miRNAs) can be combined to form a biomarker panel to calculate a disease risk score, for example, using a linear model. One example is to calculate this risk score using logistic regression (a form of linear model). Predictive scores can also be calculated using a classification algorithm selected from a group including a support vector machine (SVM) algorithm, a logistic regression algorithm, a multinomial logistic regression algorithm, a Fisher linear discriminant algorithm, a quadratic classifier algorithm, a perceptron algorithm, a k-nearest neighbor algorithm, an artificial neural network algorithm, a random forest algorithm, a decision tree algorithm, a naive Bayes algorithm, an adaptive Bayesian network algorithm, and an ensemble learning method combining multiple learning algorithms.

[0123] The challenge in this area involves identifying relevant biomarkers, such as circulating miRNAs, that can be used to identify individuals at risk for a disease, such as lung cancer. When relevant miRNAs can be identified through exhaustive and well-designed studies, it will be within the skill of one skilled in the art to apply the measured levels of the relevant miRNAs to such statistical models to generate a score for predicting a subject's risk of developing lung cancer.

[0124] Examples of such mathematical methods for performing the calculations disclosed herein (e.g., calculation of prediction scores) can be, but are not limited to, support vector machine algorithms, logistic regression algorithms, multinomial logistic regression algorithms, Fisher linear discriminant algorithms, quadratic classifier algorithms, perceptron algorithms, k-nearest neighbor algorithms, artificial neural network algorithms, random forest algorithms, decision tree algorithms, naive Bayesian algorithms, adaptive Bayesian network algorithms, and ensemble learning methods that combine multiple learning algorithms. In one example, a linear model and a support vector machine algorithm are used to calculate the prediction score.

[0125] As an illustrative example, a control and a subject with lung cancer have different calculated disease risk scores. The fitted probability distribution of the disease risk scores for the control and the subject with lung cancer shows that a separation between the two groups can be found. Based on this prior probability and the previously determined fitted probability distribution, the probability (risk) of the unknown subject having lung cancer can be calculated based on their disease risk score. The higher the score, the higher the risk of the subject having lung cancer. In addition, the disease risk score can, for example, tell the unknown subject the probability (risk) of having lung cancer compared to, for example, the lung cancer rate in a high-risk population.

[0126] The success of such approaches requires the availability of high-quality data. Quantitative data for all detected miRNAs in a large number of well-defined clinical samples, including appropriate controls from symptomatic non-lung cancer patients (disease controls), not only improves the accuracy and precision of the results, but also ensures the consistency of the identified biomarker panels for further clinical applications using, for example, quantitative polymerase chain reaction (qPCR).

[0127] As an example, the following formula 1 illustrates the use of a linear model for lung cancer risk prediction, wherein a disease risk score (unique for each subject) indicates the likelihood that the subject has lung cancer. This is calculated by summing the weighted measurements of, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 miRNAs.

[0128] Formula 1-

[0129] where log2copy_miRNAi is the log-transformed copy number of n individual miRNAs in the test sample volume (e.g., for the example of 12 miRNAs, n=12). Thus, K i - Coefficients and C-constants for weighting multiple miRNA targets can be derived by application of a linear model. Subjects with a disease risk score below 0 will be considered as 0, and subjects with a disease risk score above 100 will be considered as 100. Those skilled in the art will appreciate that, upon knowing the identity of the relevant miRNA biomarkers, relevant disease risk scores and cutoff values ​​can be derived to identify subjects at risk for lung cancer.

[0130] Another example of such a risk score calculation algorithm involves using a logistic regression model:

[0131] Formula 2-

[0132] Where n is the number of individual miRNAs (e.g., for an example of 12 miRNAs, n=12), K i - coefficients used to weight multiple miRNA targets, Ct - cycle threshold (usually defined as the number of cycles in a real-time PCR reaction required for the fluorescence signal to cross the threshold), and constants can be obtained by applying a linear model.

[0133] Yet another example of this algorithm also incorporates the use of a reference sample with known levels of the assayed miRNA to normalize the score for each assay run to account for variations between runs (which may be referred to as a quantitative reference (QR)), and in some embodiments, the average of the QR scores may be used to calculate an expected QR score. Using the examples provided above, an example of how this normalization may be performed is provided below. Knowing the identity of the relevant miRNA biomarkers, it is within the purview of those skilled in the art to derive the relevant disease risk score, QR score, and cutoff value to identify subjects at risk for lung cancer.

[0134] Formula 3-

[0135] Where n is the number of individual miRNAs (e.g., for an example of 12 miRNAs, n=12), K i - coefficient used to weight multiple miRNA targets, Ct - cycle threshold (usually defined as the number of cycles required for the fluorescence signal to cross the threshold in a real-time PCR reaction, sometimes also referred to as Cq), the constant can be obtained by applying a linear model, QR 运行 -QR score obtained from a specific run, QR 预期 is the average of the QR scores obtained from multiple validation runs.

[0136] In some embodiments, the test can be used to stratify patients into low-risk or high-risk categories based on the results derived from the Ct of the target miRNA. A low-risk result indicates that the patient has a low risk of having or developing lung cancer. A high-risk result indicates that a miRNA associated with a high risk of lung cancer is present, and these patients are at risk for developing lung cancer and should therefore be considered for further diagnostic testing according to clinical guidelines. Example

[0137] Methods and Materials

[0138] A reverse transcription-quantitative polymerase chain reaction method was used to detect multiple miRNA biomarkers associated with lung cancer and non-lung cancer groups. The assay consists of five steps:

[0139] Step 1: Isolation of RNA from platelet-poor plasma samples

[0140] Step 2: cDNA synthesis

[0141] Step 3: Pre-amplification

[0142] Step 4: Detect miRNA by quantitative PCR (qPCR)

[0143] Step 5: Convert miRNA Ct (threshold cycle) values ​​(also called Cq or quantification cycle) into risk scores

[0144] RNA was extracted using a semi-automated system. Platelet-poor plasma (PPP) was pretreated by manually adding lysis buffer C and proteinase K, followed by incubation at 37°C for 15 minutes. The pretreated sample was added to the extraction cartridge, which was then loaded onto the CSC 48, CSC16, RSC 48 or The automated portion of the extraction is performed on an RSC 16 instrument using paramagnetic particles that provide the mobile solid phase to optimize sample capture, washing, and nucleic acid purification.

[0145] During the cDNA synthesis step, miRNA targets from each sample are converted into cDNA using miRNA-specific stem-loop-based reverse transcription primers in a single reaction. Compared to linear primers, the stem-loop structure improves hybridization between the primer and the target miRNA, resulting in higher analytical sensitivity. It also minimizes hybridization between the primer and the precursor form of the miRNA, thereby improving the specificity of the assay.

[0146] In the pre-amplification step, cDNA is amplified using sequence-specific PCR primer pairs in a single reaction. Pre-amplification increases the copy number of the miRNA target before the qPCR step while maintaining target amplification specificity. In the qPCR step, each miRNA target is amplified using a sequence-specific forward PCR primer and a semi-nested sequence-specific reverse PCR primer. This combination enables enhanced discrimination of a wide range of highly homologous family members. Amplicons are then detected using SYBR Green I dye in a single-plex reaction under accelerated cycling conditions.

[0147] Development and validation of miRNA biomarkers for lung cancer diagnosis

[0148] Study Design

[0149] The primary objective of this study was to identify and develop a circulating biomarker signature, identified in plasma and / or serum samples, to identify subjects at risk for or with lung cancer. Blood samples were collected from 623 subjects, of whom 525 were ultimately used for development and validation. Reasons for sample exclusion included lack of clinical information, failure to meet inclusion criteria, or severe hemolysis in the sample. Details of the recruited cohort are detailed in Table 2.

[0150] Table 2: Patient demographics of the study cohort used for the development and validation of lung cancer biomarkers. n = number of patients; SD = standard deviation; % = percentage.

[0151]

[0152]

[0153] miRNA biomarker development and validation

[0154] A total of 525 clinical samples were used for the development and validation of the miRNA classifier, including 261 lung cancer (LC) samples and 264 non-LC samples. Fifty-seven tests were performed at three different sites using three batches of the kit. All samples were run in random order, and the operator was blinded. Run data were collected and normalized, and a logistic regression model (an exemplary method is provided in the specification) was used to calculate the risk score to classify LC and non-LC patients.

[0155] Table 3 details the performance of miRNA biomarkers when used alone or in combination with other miRNA biomarkers, and Tables 4-8 provide (and Figure 1(An exemplary embodiment of such miRNA biomarkers is graphically depicted in FIG. ). As can be seen, the performance of the panel generally peaks when 5 or more miRNA biomarkers are combined, and the addition of additional miRNA biomarkers does not further significantly change the AUC. In addition, exemplary panels of selected 3-miRNA combinations are listed in Table 9.

[0156] Using miRNA biomarkers alone, a performance of 80.5% total area under the curve (AUC), 75.5% sensitivity, and 70.1% specificity was achieved ( Figure 2 ).

[0157] The level of carcinoembryonic antigen (CEA) was further combined with the miRNA classifier obtained in the previous step. Table 10 details the performance of CEA when combined with miRNA biomarkers (single or in combination with other miRNA biomarkers), which is provided in Table 11 (and in Figure 3 (A diagrammatic depiction of an exemplary embodiment of such a miRNA biomarker is provided in FIG. The combination of miRNA and CEA improves the performance of the test, so in addition to measuring the level of miRNA biomarkers in a subject, testing the level of CEA may be valuable.)

[0158] A logistic regression model was used to calculate a new risk score based on miRNA and CEA levels. The new model combining miRNA and CEA achieved an AUC of 86.4%, a sensitivity of 84.3%, and a specificity of 71.6% ( Figure 4 ).

[0159] For the avoidance of doubt, the combinations of miRNAs of the present invention are not limited to the exemplary panels listed in Tables 4-9 and 11. Any suitable combination or number of miRNAs may be selected from the 12 miRNAs to form a panel that gives a desired or sufficiently high AUC.

[0160] Table 3: Mean and maximum AUC values ​​of miRNA biomarkers alone or in combination of up to 12 miRNAs for identifying subjects having or at risk of developing lung cancer.

[0161]

[0162] Table 4: Exemplary panel of miRNA biomarkers for the purpose of identifying subjects having or at risk for lung cancer (with hsa-miR-1280).

[0163]

[0164]

[0165]

[0166] Table 5: Exemplary panel of miRNA biomarkers (with hsa-miR-199b-5p) for the purpose of identifying subjects having or at risk for lung cancer.

[0167]

[0168]

[0169]

[0170] Table 6: Exemplary panel of miRNA biomarkers (hsa-miR-181c-5p) for the purpose of identifying subjects having or at risk for lung cancer.

[0171]

[0172]

[0173]

[0174] Table 7: Exemplary panel of miRNA biomarkers (with hsa-miR-210-3p) for the purpose of identifying subjects having or at risk for lung cancer.

[0175]

[0176]

[0177]

[0178] Table 8: Exemplary panel of miRNA biomarkers (with hsa-miR-16-5p) for the purpose of identifying subjects having or at risk for lung cancer.

[0179]

[0180]

[0181] Table 9: Exemplary panel of selected 3 miRNA combinations for the purpose of identifying subjects having or at risk for lung cancer.

[0182]

[0183]

[0184] Table 10: Mean and maximum AUC values ​​for using miRNA biomarkers in combination with CEA (in a panel of up to 12 miRNAs) to identify subjects having or at risk of developing lung cancer.

[0185] Number of biomarkers Average AUC variance CEA+1 miRNA 0.52 0.0082 CEA+2 miRNA 0.76 0.0027 CEA+3 miRNA 0.78 0.0024 CEA+4miRNA 0.79 0.0024 CEA+5miRNA 0.80 0.0021 CEA+6miRNA 0.81 0.0021 CEA+7 miRNA 0.81 0.0022 CEA+8miRNA 0.80 0.0022 CEA+9 miRNA 0.80 0.0024 CEA+10miRNA 0.80 0.0025 CEA+11miRNA 0.80 0.0025 CEA+12miRNA 0.80 0.0026

[0186] Table 11: Exemplary panel of CEA and miRNA biomarkers for the purpose of identifying subjects having or at risk for lung cancer.

[0187]

[0188]

[0189]

[0190] discuss

[0191] There is an unmet clinical need for non-invasive methods for early detection of lung cancer. This paper provides circulating biomarkers suitable for identifying subjects with lung cancer or who are at risk of lung cancer. No matter whether individually or in combination, circulating biomarkers that are suitable for distinguishing subjects with lung cancer or who are at risk of lung cancer from subjects without lung cancer have been identified. The use of circulating biomarkers has improved the possibility that this panel may provide an instrument for early detection of illness, because such tests will be more easily used for general testing purposes and are minimally invasive. The biomarkers identified herein also show that it is useful to combine with other cancer biomarkers such as carcinoembryonic antigen (CEA). This study involves the development and validation of biomarkers at 3 different locations to illustrate the variability at different locations, and uses 3 different batches of test kits to illustrate manufacturing differences. When testing in random order, the operator does not know the type of sample, and potential bias is further reduced to ensure the robustness and reliability of the biomarker panel in determining whether a subject suffers from lung cancer or has the risk of lung cancer.

[0192] The examples provided herein relate to exemplary embodiments of the intended uses of the present invention. In some embodiments, biomarkers are intended to be used as an adjunct to the diagnosis of lung cancer in a subject, sometimes in combination with other clinical factors or symptoms. Thus, biomarkers are intended to distinguish subjects who have lung cancer or are at risk of developing lung cancer from subjects who do not have lung cancer.

[0193] Possible embodiments of tests employing such biomarkers can be reagents for research purposes, laboratory-developed tests, or in vitro test kits for early detection or auxiliary diagnosis of lung cancer using the biomarker panels disclosed herein. In other cases, patients classified as being at risk for or suffering from lung cancer can be treated with one or more therapeutic agents or therapies suitable for treating the disease. It is foreseeable that such tests can be used alone or in combination with other methods, including sputum cytology, biopsy or imaging tests (including CT scans, MRI, X-rays, or PET scans), tests for cancer biomarkers (e.g., CEA), or any other method for diagnosing lung cancer recognized by medical practitioners or similar persons in the art.

[0194] References

[0195] 1.Ferlay J,Ervik M,Lam F,Colombet M,Mery L, M, Znaor A, Soerjomataram I, Bray F (2020). Global Cancer Observatory: Cancer Today. Lyon, France: International Agency for Research on Cancer. Available from https: / / gco.iarc.fr / today, accessed 12Dec2022.

[0196] 2.Too HP and Azlinda BA.Modified stem-loop oligonucleotide mediatedreverse transcription and base-spacing constrained quantitative PCR.2011Dec22.(WO2011159256A1).

[0197] 3.NCCN Clinical Practice Guidelines in Oncology for Non-Small CellLung Cancer version 6.2022 National Comprehensive Cancer Network, Inc. 2022. All rights reserved. Accessed December 12, 2022. To view the latest and complete version of the guidelines, visit NCCN.org.

[0198] 4.NCCN Clinical Practice Guidelines in Oncology for Small Cell LungCancer version 2.2023 National Comprehensive Cancer Network, Inc. 2022. All rights reserved. Accessed December 12, 2022. To view the latest and complete version of the guidelines, visit NCCN.org.

[0199] 5.NCCN Clinical Practice Guidelines in Oncology for Lung CancerScreening version 1.2023 National Comprehensive Cancer Network, Inc. 2022. All rights reserved. Accessed December 12, 2022. To view the latest and complete version of the guidelines, visit NCCN.org.

Claims

1. A method for determining whether a subject has lung cancer or is at risk of developing lung cancer, wherein the method comprises detecting the expression level of at least one miRNA in a biological sample obtained from the subject, wherein the at least one miRNA is selected from the group consisting of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p, and hsa-miR-877-5p.

2. The method of claim 1, wherein the method comprises detecting at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve miRNAs.

3. The method of claim 1 or claim 2, wherein the method comprises detecting the expression levels of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p.

4. The method of any one of the preceding claims, wherein a differential expression level of the one or more miRNAs compared to a control indicates that the subject has or is at risk of developing lung cancer.

5. The method of any one of the preceding claims, comprising: a. contacting a biological sample obtained from the subject with an isolated probe set suitable for detecting one or more miRNAs selected from the group consisting of hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p. b. comparing the expression level of the one or more miRNAs in the biological sample with the level of the same miRNA in a control; c. determining whether the subject has lung cancer or is at risk of developing lung cancer by differential expression of the one or more miRNAs compared to a control.

6. The method of any of the preceding claims, wherein the method further comprises detecting the expression level of at least one additional biomarker from a biological sample obtained from the subject.

7. The method of claim 6, wherein the additional biomarker is one or more biomarkers selected from carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 125 (CA-125), and cytokeratin 19 fragment antigen (CYFRA 21-1).

8. The method of any one of the preceding claims, wherein the biological sample is a non-cellular biological fluid.

9. The method of claim 8, wherein the acellular biological fluid is plasma and / or serum.

10. The method of any of the preceding claims, wherein the control comprises one or more biological samples obtained from a healthy subject, a non-diseased subject, a cancer-free subject, a lung cancer-free subject, and / or a subject that does not have or is not at risk for developing lung cancer.

11. The method of any of the preceding claims, further comprising imaging testing, sputum cytology, biopsy, or a combination thereof.

12. A method of treating a subject suffering from lung cancer, comprising: a. using the method of any one of claims 1 to 11 to determine whether a subject has lung cancer or is at risk of developing lung cancer; as well as b. treating the subject identified as having or being at risk for developing lung cancer with one or more therapies selected from administration of an anti-cancer compound, surgery, and / or radiation therapy.

13. The method of any of the preceding claims, wherein the at least one miRNA is detected by one or more methods selected from sequencing, nucleic acid hybridization, microarray, and nucleic acid amplification.

14. A kit for determining whether a subject has lung cancer or is at risk of developing lung cancer, the kit comprising an isolated probe set and / or reagent set capable of detecting one or more miRNAs, wherein the one or more miRNAs are selected from hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p.

15. The kit of claim 14, wherein the separate probe set and / or reagent set is capable of detecting hsa-miR-1280, hsa-miR-487b-3p, hsa-miR-199b-5p, hsa-miR-205-5p, hsa-miR-16-5p, hsa-miR-320a-3p, hsa-miR-23b-3p, hsa-miR-181c-5p, hsa-miR-92a-3p, hsa-miR-210-3p, hsa-miR-342-3p and hsa-miR-877-5p.

16. The kit of claim 14 or 15, wherein the probe is selected from the group consisting of an aptamer, an antibody, an affibody, a peptide, and a nucleic acid.

17. The kit of any one of claims 14 to 16, wherein the at least one miRNA is detected by one or more methods selected from sequencing, nucleic acid hybridization, microarray, and nucleic acid amplification.

18. The kit of any one of claims 14 to 17, wherein the kit further comprises probes and / or reagents for detecting at least one additional biomarker.

19. The kit of claim 18, wherein the biomarker is one or more selected from carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 125 (CA-125), and cytokeratin 19 fragment antigen (CYFRA 21-1).

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