Endogenous reference RNA and uses thereof

Abstract

Endogenous reference RNA, such as miR-15, miR-15b, miR-30e, miR-23a, miR-151a, miR- 221, miR-186, miR-let-7g. miR-185, miR-146a. miR-191, miR-652, miR-93, miR-let-7d, miR- 30e, miR-148b, miR-16. miR-let-7g, miR-142, miR-151b, miR-101, miR-23a, miR-30c. and miR-423, can be used for methods of treating, diagnosing, and monitoring cancer using levels of the endogenous reference RNA for comparison to levels of oncogenic RNA. The endogenous reference RNA can be cell-free RNA and / or exosomal RNA, such as cell-free miR-15b-5p, cell- free miR-30e-5p. cell-free miR-23a-3p, cell-free miR-151a-3p, cell-free miR-221-3p, cell-free miR-186-5p, cell-free miR-let-7g-5p, cell-free miR-185-5p, cell-free miR-146a-5p, cell-free miR-191-5p, cell-free miR-652-3p, cell-free miR-93-5p, cell-free miR-let-7d-5p, exosomal miR- 30e-5p, exosomal miR-15b-5p, exosomal miR-148b-3p, exosomal miR-23a-3p, exosomal miR- 16-5p, exosomal miR-186-5p, exosomal miR-let-7g-5p. exosomal miR-142-5p, exosomal miR- 15 lb, exosomal miR-101-3p, exosomal miR-30c-5p, exosomal miR 146a-5p, exosomal miR- 423-3p, exosomal miR-652-3p, and exosomal miR-221-3p.

Description

Docket: 048440-205001WQ / TEC 24-032ENDOGENOUS REFERENCE RNA AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to US Application No. 63 / 687,128 filed August 26, 2024, the disclosure of which is incorporated by reference herein in its entirety.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under CA214254 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Reference or housekeeping genes used for RNA analysis cannot be consistently detected in circulation due to extensive RNAse mediated degradation. Various reference RNAs (e.g., RNU6) have been used as circulating reference RNA. yet they lack consistency. There is a need in the art to identify suitable endogenous reference RNA for quantifying circulating RNA, such as miRNA. The disclosure is directed to this, as well as other, important ends.BRIEF SUMMARY

[0004] Provided herein are methods of detecting an expression level of RNA in a patient with cancer comprising detecting an expression level of RNA in the biological sample, wherein the RNA comprises miR-15b, miR-23a, miR-30e. or a combination of two or more thereof.

[0005] Provided herein are methods of treating cancer in a patient in need thereof comprising: (i) detecting an expression level of an oncogenic RNA in a biological sample obtained from the patient; (ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises miR-15b, miR-23a, miR-30e. or a combination of two or more thereof; (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; wherein the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA; and (iv) administering to the patient an effective amount of an anti-cancer agent.

[0006] Provided herein are methods of diagnosing a patient with cancer, the method comprising: (i) detecting an expression level of an oncogenic RNA in a biological sample obtained from the patient; (ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises miR-15b, miR-23a, miR-30e. or a combination of two or more thereof; (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA, thereby detecting the normalized expression level of theoncogenic RNA; and (iv) diagnosing the patient as having cancer when the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA.

[0007] These and other embodiments of the disclosure are described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 sets forth the study group statistics for identifying cell-free reference RNA. Cell-free small RNA sequencing was performed on 504 control and 272 cancer samples, and sequenced across 10 batches.

[0009] FIG. 2 sets forth the study design statistics for identifying exosomal reference RNA. Exosomal small RNA sequencing was performed on 506 control and 219 cancer samples, and sequenced across 13 batches.

[0010] FIGS. 3A-3B set forth the study design used to analyze cell-free reference miRNA in healthy patients (i.e., controls) and cancer patients (FIG. 3A) and identifies the top stable reference miRNA resulting from the study (FIG. 3B). The cancers included gastric cancer (GC), pancreatic cancer (PDAC), and colorectal cancer (CRC).

[0011] FIGS. 4A-4B set forth the study design used to analyze cell-free reference miRNA in cancer patients (FIG. 4A) and identifies the top stable cell-free reference miRNA resulting from the study (FIG. 4B). The cancers included gastric cancer (GC), pancreatic cancer (PDAC), and colorectal cancer (CRC). NDC refers to non-diseased control (i.e., healthy patient).

[0012] FIGS. 5A-5B set forth the study design used to analyze exosomal reference miRNA in healthy patients (i.e., controls) and cancer patients (FIG. 5A) and identifies the top stable exosomal reference miRNA resulting from this subgroup analysis (FIG. 5B). The cancers included gastric cancer (GC), pancreatic cancer (PDAC), and colorectal cancer (CRC).

[0013] FIGS. 6A-6B set forth the study design used to analyze exosomal reference miRNA in cancer patients (FIG. 6A) and identifies the top stable exosomal reference miRNA resulting from this subgroup analysis (FIG. 6B). The cancers included gastric cancer (GC), pancreatic cancer (PDAC), and colorectal cancer (CRC). NDC refers to non-diseased control (i.e., healthy patient).

[0014] FIGS. 7A-7B set forth the study design used to analyze cell-free reference miRNA in healthy patients (i.e., controls) and cancer patients (FIG. 7A) and identifies the top reference miRNA resulting from this analysis (FIG. 7B). The cancers included gastric cancer (GC), pancreatic cancer (PDAC), liver cancer (HCC), and colorectal cancer (CRC).

[0015] FIGS. 8A-8B set forth the study design used to analyze cell-free reference miRNA incancer patients (FIG. 8A) and identifies the top cell-free reference miRNA resulting from the batch subgroup analysis (FIG. 8B). The cancers included gastric cancer (GC), pancreatic cancer (PDAC), liver cancer (HCC), and colorectal cancer (CRC). NDC refers to non-diseased control (i.e., healthy patient).

[0016] FIGS. 9A-9B set forth the study design used to analyze exosomal reference miRNA in healthy patients (i.e.. controls) and cancer patients (FIG. 9A) and identifies the top exosomal reference miRNA resulting from this analysis (FIG. 9B). The cancers included gastric cancer (GC), pancreatic cancer (PDAC), esophageal cancer (ESCC), colorectal cancer (CRC), liver cancer (HCC), and cholangiocarcinoma (ICC).

[0017] FIGS. 10A-10B set forth the study design used to analyze exosomal reference miRNA in cancer patients (FIG. 10A) and identifies the top exosomal reference miRNA resulting from the batch subgroup analysis (FIG. 10B). The cancers included gastric cancer (GC), pancreatic cancer (PDAC), esophageal cancer (ESCC). colorectal cancer (CRC), liver cancer (HCC), and cholangiocarcinoma (ICC). NDC refers to non-diseased control (i.e., healthy patient).

[0018] FIG. 11 sets forth an illustrative system 100 including a miRNA detection device 110 communicatively coupled to a computing system 102 for use in a computer-implementation of methods for detecting reference miRNA and / or oncogenic miRNA and methods for diagnosing and treating cancer using reference miRNA and oncogenic miRNA.

[0019] FIG. 12 illustrates a functional block diagram of a machine in the example form of a computer system which comprises instructions for causing the machine to detect reference miRNA and / or oncogenic miRNA and methods for diagnosing and treating cancer using reference miRNA and oncogenic miRNA.DETAILED DESCRIPTION

[0020] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology. 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al, Molecular Cloning. A Laboratory Manual. Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this disclosure. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0021] The term “reference RN A" or “normalizer RNA” or “housekeeping RNA” or “control RNA” or “endogenous RNA” or “endogenous reference RNA” refers to typically endogenous,constitutive RNA that is required for the maintenance of basal cellular function and that are expected to maintain constant expression levels in all cells. For experimental purposes, the expression of one or multiple reference RNA is used as a reference point for the analysis of expression levels of other RNA (e.g., oncogenic RNA). The key criterion for the use of a reference RNA in this manner is that the chosen reference RNA is uniformly expressed with low variance under both control and experimental conditions, i.e., in both healthy patients and diseased patients (e.g.. cancer patients). In embodiments, the reference RNA is miRNA. In embodiments, the reference RNA is any miRNA or combination of miRNA set forth in FIG. 3B, FIG. 4B, FIG. 5B, FIG. 6B, FIG. 7B, FIG. 8B, FIG. 9B, or FIG. 10B.

[0022] The term “oncogenic RNA” refers to RNA involved in biological processes implicated in cancer, such as cell cycle regulation, apoptosis, and / or differentiation. Oncogenic RNA is generally overexpressed in cancers. Oncogenic RNA expression profiles can correlate with cancer prediction, diagnosis, prognosis, and treatment. In embodiments, the oncogenic RNA is oncogenic miRNA.

[0023] The term “tumor-derived exosome” or “exosome” refers to a small (between 20-300 nm in diameter) vesicle comprising a lipid bilayer membrane that encloses an internal space, and which is generated from a cancer cell by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane. The components of tumor-derived exosomes include proteins, DNA, mRNA, microRNA, long noncoding RNA, circular RNA, and the like, which play a role in regulating tumor growth, metastasis, and angiogenesis in the process of cancer development.

[0024] “Exosomal RNA’’ refers to RNA within a tumor-derived exosome or RNA obtained from within a tumor-derived exosome. In embodiments, “exosomal RNA” is exosomal miRNA. Exosomal RNA can be detected and measured by methods known in the art, such as those described herein. In embodiments, exosomal RNA is exosomal miRNA. In embodiments, exosomal RNA is exosomal hsa-miRNA, where “hsa” refers to homo sapiens.

[0025] “Cell-free RNA” or “cf-RNA” refers to RNA that is not within a tumor-derived exosome or RNA that has not been obtained from within a tumor-derived exosome. Cell-free RNA can be detected and measured by methods known in the art. such as those described herein. In embodiments, cell-free RNA is cell-free miRNA. In embodiments, cell-free RNA is cell-free hsa-miRNA, where “hsa” refers to homo sapiens.

[0026] A “cell” refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the artincluding, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian (e.g., human) cells.

[0027] "‘Gastrointestinal cancer’7refers to any cancer that forms in the digestive tract and other abdominal organs. Exemplary types of gastrointestinal cancer include colorectal cancer, gastric cancer, esophageal cancer, liver cancer, pancreatic cancer, pancreatic neuroendocrine tumor, gallbladder cancer, anal cancer, small intestine cancer, gastrointestinal stromal tumor, gastrointestinal neuroendocrine tumor, and cholangiocarcinoma.

[0028] “Colorectal cancer” or “CRC” refers to a cancer that generally begins as growth (e.g., polyp) on the inner lining of the colon or rectum. Over time, the polyps can grow into the wall of the colon or rectum and into blood vessels or lymph nodes. The stage (extent of spread) of a colorectal cancer depends on how deeply it grows into the wall and if it has spread outside the colon or rectum. Colorectal cancer generally occurs when the patient is at least 50 years old, in which case it can also be referred to as late-onset colorectal cancer (LOCRC). The term “colorectal cancer” encompasses colon cancer and rectal cancer. The term “early-onset colorectal cancer” or “EOCRC” refers to colorectal cancer in a patient less than 50 years old.

[0029] “Pancreatic cancer” is a type of cancer that begins with cell growth in the pancreas. In embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). PDAC accounts for more than 90% of cases of pancreatic cancer. Symptoms of pancreatic cancer include diabetes, hyperglycemiajaundice, sudden weight loss, abdominal pain, back pain, persistent loss of appetite, light-colored stools, bloating, nausea, vomiting, or diarrhea.

[0030] “Gastric cancer” is alternatively referred to as stomach cancer, and generally forms in the cells linking the stomach.

[0031] “Esophageal cancer” is a type of cancer that begins with cell growth in the esophagus, generally in the cells that line the inside of the esophagus. In embodiments, the esophageal cancer is esophageal squamous cell carcinoma (ESCC). ESCC is accounts for the majority of all cases of esophageal cancer.

[0032] “Liver cancer” is a type of cancer that begins with cell growth in the liver. In embodiments, the liver cancer is hepatocellular carcinoma (HCC). HCC is the most common type of liver cancer and often occurs in people with chronic liver disease, such as cirrhosis caused by hepatitis B or C.

[0033] “Cholangiocarcinoma’' is a type of cancer that forms in the bile ducts, which are the tubes that go from the liver to the small intestine. In embodiments, the cholangiocarcinoma is intrahepatic cholangiocarcinoma (ICC). ICC occurs in the parts of the bile ducts that are within the liver and can alternatively be referred to as intrahepatic bile duct cancer.

[0034] “Nucleic acid” refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid” does not include nucleosides. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleoside” refers, in the usual and customary sense, to a glycosylamine including a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non limiting examples, of nucleosides include, cytidine, uridine, adenosine, guanosine, thymidine and inosine. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid, e.g. polynucleotides, contemplated herein include any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and any types of DNA. genomic DNA. plasmid DNA. and minicircle DNA, and any fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0035] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and / or modified nucleotides.

[0036] A “microRNA,” “microRNA nucleic acid sequence.” “miR.” “miRNA” as used herein, refers to a nucleic acid that functions in RNA silencing and post-transcriptional regulation ofgene expression. The term includes all forms of a miRNA, such as the pri-, pre-, and mature forms of the miRNA. In embodiments, microRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability’ and translation of mRNAs. miRNAs are transcribed by RNA polymerase II as part of capped and polyadenylated primary transcripts (pri-miRNAs) that can be either protein-coding or non-coding. The primary’ transcript is cleaved by the Drosha ribonuclease III enzyme to produce an approximately 70-nt stem-loop precursor miRNA (pre- miRNA), which is further cleaved by the cytoplasmic Dicer ribonuclease to generate the mature miRNA and antisense miRNA star (miRNA*) products. The mature miRNA is incorporated into a RNA-induced silencing complex (RISC), which recognizes target mRNAs through imperfect base pairing with the miRNA and most commonly results in translational inhibition or destabilization of the target mRNA.

[0037] The term “gene” means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. Further, a “protein gene product” is a protein expressed from a particular gene.

[0038] The word “expression” or “expressed” as used herein in reference to a gene means the transcriptional and / or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding RNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell. The level of expression of non-coding nucleic acid molecules (e.g., miRNA) may be detected by standard PCR or Northern blot methods well know n in the art.

[0039] The terms “expression level,” “amount,” or “level” of a biomarker is a detectable level in a biological sample. “Expression” generally refers to the process by which information (e.g., gene-encoded and / or epigenetic) is converted into the structures present and operating in the cell. Therefore, “expression” may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modifications (e.g., posttranslational modification of a polypeptide). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and / or polypeptide modifications (e.g., post-translational modification of a polypeptide) shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis. “Expressed genes” include those that aretranscribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, miRNA, transfer RNA, ribosomal RNA, IncRNA). Expression levels can be measured by methods known to one skilled in the art and also disclosed herein.

[0040] An “elevated expression level” or “elevated level” or “increased level” of gene expression is an expression level of the gene that is higher than the expression level of the gene in a control or higher than the expression level of an endogenous reference RNA, such as those described herein. In embodiments, an elevated level of oncogenic miRNA is an elevated level after the expression level of oncogenic miRNA is normalized with respect to the expression level of endogenous reference miRNA, such as those described herein. In embodiments, an “elevated expression level” of the biomarker gene compared to a control or the reference RNA (when the expression level of the biomarker is greater than the control or reference RNA) is, for example, an increase in the expression level of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%. 98% or 99% or greater relative to the reference RNA. In embodiments, an “elevated expression level” of the biomarker gene is an amount that is statistically significantly greater than the expression level of the control or reference RNA. In embodiments, an “elevated expression level” of oncogenic miRNA indicates that a patient has cancer when it is known in the art that an elevated level of the oncogenic miRNA is associated with cancer. In embodiments, an “elevated expression level” of oncogenic miRNA indicates that a patient does not have cancer if it is known in the art that an elevated expression level of the oncogenic miRNA is not associated with cancer.

[0041] A “decreased level” or “reduced level” of gene expression is an expression level of the gene that is lower than the expression level of the gene in a control or lower than the expression level of an endogenous reference RNA, such as those described herein. In embodiments, a reduced level of oncogenic miRNA is a reduced level after the expression level of oncogenic miRNA is normalized with respect to the expression level of the endogenous reference miRNA, such as those described herein. In embodiments, a “decreased expression level” of the biomarker gene compared to the control or reference RNA (when the expression level of the biomarker is greater than the control or reference RNA) is, for example, a decrease in the expression level of about 10%. 20%. 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% or greater relative to the control or reference RNA. In embodiments, a “decreased expression level” of the biomarker gene is an amount that is statistically significantly lower than the expression level of the control or reference RNA. In embodiments, a “decreased expression level” of oncogenic miRNA indicates that a patient has cancer when it is known in the art that a decreasedlevel of the oncogenic miRNA is associated with cancer. In embodiments, a “decreased expression level’7of oncogenic miRNA indicates that a patient does not have cancer if it is know n in the art that a decreased expression level of the oncogenic miRNA is not associated with cancer.

[0042] “Comparable” in reference to the level of gene expression refers to an amount or concentration of the gene that is not increased relative to the control or endogenous reference miRNA (such as those described herein) and is not decreased relative to the control or endogenous reference RNA (such as those described herein). In embodiments, a comparable level of oncogenic miRNA is a comparable level after the expression level of oncogenic miRNA is normalized with respect to the expression level of the endogenous reference miRNA, such as those described herein. In embodiments, the expression level of an oncogenic miRNA is + / - 25% (i.e. comparable to) the expression level of the control or reference miRNA. In embodiments, the expression level of an oncogenic miRNA is + / - 20% (i.e. comparable to) the expression level of the control or reference miRNA. In embodiments, the expression level of an oncogenic miRNA is + / - 15% (i.e. comparable to) the expression level of the control or reference miRNA. In embodiments, the expression level of an oncogenic miRNA is + / - 10% (i.e. comparable to) the expression level of the control or reference miRNA. In embodiments, the expression level of an oncogenic miRNA is + / - 5% (i.e. comparable to) the expression level of the control or reference miRNA. In embodiments, a level of the protein that is “comparable” or “not increased” or “not decreased” is an amount or concentration of the oncogenic miRNA that is not statistically significantly different than the amount or concentration of the control or reference miRNA. In embodiments, a “comparable expression level” of oncogenic miRNA indicates that a patient does not have cancer.

[0043] The terms “biomarker gene” and “biomarker” are used interchangeably and in accordance with their plain and ordinary meaning. In embodiments, a biomarker is a gene or a set of genes (i.e., a biomarker gene). Biomarkers include, but are not limited to, polynucleotides (e.g., DNA, and / or RNA), polynucleotide copy number alterations (e.g., DNA copy numbers), polypeptides, or polypeptide and polynucleotide modifications (e.g., post-translational modifications). In embodiments, a biomarker refers to RNA. In embodiments, a biomarker refers to miRNA.

[0044] Biomarker levels may be detected at either the protein or gene expression level. Proteins expressed by biomarkers can be quantified by immunohistochemistry (IHC) or flow' cytometry with an antibody that detects the proteins. Biomarker expression can be quantified by multiple platforms such as real-time polymerase chain reaction (rtPCR), NanoString. RNAseq,or in situ hybridization. There is a range of biomarker expression across as measured by NanoString. In embodiments, quantitative rtPCR, NanoString, RNAseq, and in situ hybridization are platforms to quantitate biomarker gene expression. For NanoString, RNA is extracted from a biological sample and a known quantity of RNA is placed on the NanoString machine for gene expression detection using gene specific probes. The number of counts of biomarkers within a sample is determined and normalized to a set of housekeeping genes. To determine a threshold for increased or decreased biomarker levels, one skilled in the art could assess biomarker levels in a control group of samples and select the 10th, 20th, 25th, 30th, 40th, 50th, 60th, 70th, 75th, 80th or 90th percentile of biomarker gene expression. In embodiments, the increased or decreased expression of biomarkers may be determined by calculating the H- score for the expression of the biomarkers. Thus, the increased or decreased expression of biomarkers may have an H-score. As used herein, an ‘'H-score” or "Histoscore" is a numerical value determined by a semi-quantitative method commonly known for immunohistochemically evaluating protein expression in tumor samples. The H-score may be calculated using the following formula: [1 x (% cells 1+) + 2 x (% cells 2+) + 3 x (% cells 3+)]. According to this formula, the H-score is calculated by determining the percentage of cells having a given staining intensity level (i.e., level 1+, 2+, or 3+ from lowest to highest intensity level), weighting the percentage of cells having the given intensity7level by multiplying the cell percentage by a factor (e.g., 1, 2, or 3) that gives more relative weight to cells with higher-intensity membrane staining, and summing the results to obtain a H-score. Commonly H-scores range from 0 to 300. Further description on the determination of H-scores in tumor cells can be found in Hirsch et al, J Clin Oncol 21: 3798-3807, 2003 and John et al, Oncogene 28:S14-S23, 2009. IHC or other methods known in the art may be used for detecting biomarker expression.

[0045] '‘Control’’ is used in accordance with its plain ordinary7meaning and refers to an assay, comparison, or experiment in which the subj ects or reagents of the experiment are treated as in a parallel experiment except for omission of a procedure, reagent, or variable of the experiment. In embodiments, the control is used as a standard of comparison in evaluating experimental effects. In embodiments, a control is the measurement of the activity or expression level of RNA. In embodiments, a control is the measurement of the activity7or expression level of oncogenic miRNA. In embodiments, a control is the measurement of the activity7or expression level of reference RNA (e.g., endogenous RNA). In embodiments, a control is a healthy patient or a healthy population of patients. In embodiments, a control is an average value from a population of similar patients, e.g., healthy patients with a similar medical background, age, weight, etc. In embodiments, a healthy patient can be referred to as a non-diseased patient or non-diseasedcontrol. In embodiments, the control is a population of non-diseased patients. In embodiments, a non-diseased patient is a patient that does not have cancer. In embodiments, the control is a patient that does not have cancer or a population of patients that do not have cancer. In embodiments, the control is an average value from population of healthy patients. A control can also be obtained from the same patient, e.g., from an earlier-obtained sample, prior to disease, prior to treatment, or a normalized miRNA expression level relative to the expression level of a reference miRNA. One of skill will recognize that controls can be designed for assessment of any number of parameters. In embodiments, a control is a negative control. In embodiments, such as some embodiments relating to detecting the level of expression of a gene / protein or a subset of genes / proteins, a control comprises the average amount of expression (e.g., miRNA) in a population of subjects (e.g., with cancer) or in a healthy or general population. In embodiments, the control comprises an average amount (e.g. amount of expression) in a population in which the number of subjects (n) is 5 or more, 20 or more, 50 or more, 100 or more, 1,000 or more, and the like. In embodiments, a control is a level of expression of the biomarker (e g., RNA, miRNA) that has been correlated with the diagnosis of cancer in a subject. In embodiments, a control is a level of expression of the biomarker (e.g., RNA, miRNA) that has been correlated with ahealthy subject (i.e., a subject that does not have cancer). One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.

[0046] The term “healthy patient’' refers to a non-diseased patient. In embodiments, a healthy patient is a patient that does not have cancer.

[0047] The term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, “about” means within a standard deviation using measurements generally acceptable in the art. In embodiments, “about” means a range extending to + / - 10% of the specified value. In embodiments, “about” includes the specified value.

[0048] The singular terms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise.

[0049] A “therapeutic agent” or “anti-cancer agent” as used herein refer to an agent (e.g., compound, pharmaceutical composition) that when administered to a subject will have the intended therapeutic effect, e.g., treatment or amelioration of cancer, or their symptoms including any objective or subjective parameter of treatment such as abatement; remission;diminishing of symptoms or making the cancer more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a patient’s physical or mental well-being.

[0050] “Biological sample” or “sample” refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy samples, and frozen sections taken for histological purposes. A biological sample include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages. T cells, etc. In embodiments, a biological sample is blood. In embodiments, a biological sample is a serum sample (e.g., the fluid and solute component of blood without the clotting factors). In embodiments, a biological sample is a plasma sample (e.g, the liquid portion of blood). In embodiments, a biological sample is cell-free miRNA obtained from blood. In embodiments, a biological sample is an exosome obtained from a blood sample, wherein the exosome comprises miRNA. In embodiments, a biological sample is an exosome obtained from a serum sample, wherein the exosome comprises miRNA. In embodiments, a biological sample is an exosome obtained from a plasma sample, wherein the exosome comprises miRNA.

[0051] “Liquid biological sample” refers to liquid materials obtained or derived from a subject or patient. Liquid biological samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, urine, synovial fluid, and the like. In embodiments, a liquid biological sample is a blood sample.

[0052] The term “diagnosis” is used in accordance with its plain and ordinary meaning and refers to an identification or likelihood of the presence of a disease (e.g., cancer) or outcome in a subject.

[0053] The terms “treating” or “treatment” are used in accordance with their plain and ordinary meaning and broadly includes any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission, whether partial or total and whether detectable or undetectable. Treatment may inhibit the disease’s spread; relieve the disease’s symptoms, fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The term “treating” does not including preventing.

[0054] An “effective amount” is an amount sufficient to accomplish a stated purpose (e.g. achieve the effect for which it is administered, treat a disease). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing of the severity' or frequency of the symptom(s), or elimination of the symptom(s). The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. In embodiments, “therapeutically effective amount” refers to the amount of the therapeutic agent sufficient to treat or ameliorate cancer. For any therapeutic agent described herein, the therapeutically effective amount can be initially determined from cell culture assays. Target concentrations will be those concentrations of active compound(s) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art. As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan. Dosages may be varied depending upon the requirements of the patient and the therapeutic agent being employed. The dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached.Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state. A “therapeutically effective amount” can also be found on the label or Prescribing Information for commercially available therapeutic agents.

[0055] The term “administering'’ means oral administration, administration as a suppository,topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. In embodiments, the administering does not include administration of any active agent other than the recited active agent.

[0056] The terms "‘patient’' or “subject” are used in accordance with its plain and ordinary meaning and refer to a living organism suffering from or prone to a disease that can be treated by administration of a pharmaceutical composition, such as anti-cancer agents and chemotherapeutic agents. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, cats, monkeys, and other non-mammalian animals. In embodiments, a patient is human patient.

[0057] The term “NDC” or “non-diseased control” or “healthy patient” refer to a non-diseased or healthy patient. In embodiments, a healthy patient is a patient that does not have cancer.

[0058] Reference RNA

[0059] In embodiments of the methods described herein, the reference RNA comprise one or more miRNA set forth in FIG. 3B. FIG. 4B. FIG. 5B, FIG. 6B, FIG. 7B, FIG. 8B, FIG. 9B, or FIG. 10B.

[0060] In embodiments, the reference RNA comprise one or more miRNA set forth in FIG. 3B. In embodiments, the reference RNA comprise one or more of the first 9 miRNA set forth in FIG. 3B. In embodiments, the reference RNA comprise one or more of the first 6 miRNA set forth in FIG. 3B. In embodiments, the reference RNA comprise the first 3 miRNA set forth in FIG. 3B and one or more of the next 6 miRNA set forth in FIG. 3B. The term “first” in the phrase “first 9 miRNA” means the first 9 rows in the table. The term “next” in the phrase “next 6 miRNA” refers to rows 4-10 in the table. In other words, the words “first” and “next” in this context refers to the rows wherein the first row is the top row in the table.

[0061] In embodiments, the reference RNA comprise one or more miRNA set forth in FIG. 4B. In embodiments, the reference RNA comprise one or more miRNA set forth in FIG. 4B. In embodiments, the reference RNA comprise one or more of the first 10 miRNA set forth in FIG. 4B. In embodiments, the reference RNA comprise one or more of the first 6 miRNA set forth inFIG. 4B. In embodiments, the reference RNA comprise the first 3 miRNA set forth in FIG. 4B and one or more of the next 7 miRNA set forth in FIG. 4B.

[0062] In embodiments, the reference RNA comprise one or more miRNA set forth in FIG. 5B. In embodiments, the reference RNA comprise one or more of the first 10 miRNA set forth in FIG. 5B. In embodiments, the reference RNA comprise one or more of the first 6 miRNA set forth in FIG. 5B. In embodiments, the reference RNA comprise the first 3 miRNA set forth in FIG. 5B and one or more of the next 7 miRNA set forth in FIG. 5B.

[0063] In embodiments, the reference RNA comprise one or more miRNA set forth in FIG. 6B. In embodiments, the reference RNA comprise one or more of the first 10 miRNA set forth in FIG. 6B. In embodiments, the reference RNA comprise one or more of the first 6 miRNA set forth in FIG. 6B. In embodiments, the reference RNA comprise the first 3 miRNA set forth in FIG. 6B and one or more of the next 7 miRNA set forth in FIG. 6B.

[0064] In embodiments, the reference RNA comprise one or more miRNA set forth in FIG. 7B. In embodiments, the reference RNA comprise one or more of the first 10 miRNA set forth in FIG. 7B. In embodiments, the reference RNA comprise one or more of the first 6 miRNA set forth in FIG. 7B. In embodiments, the reference RNA comprise the first 2 miRNA set forth in FIG. 7B and one or more of the next 8 miRNA set forth in FIG. 7B.

[0065] In embodiments, the reference RNA comprise one or more miRNA set forth in FIG. 8B. In embodiments, the reference RNA comprise one or more of the first 10 miRNA set forth in FIG. 8B. In embodiments, the reference RNA comprise one or more of the first 6 miRNA set forth in FIG. 8B. In embodiments, the reference RNA comprise the first 3 miRNA set forth in FIG. 8B and one or more of the next 7 miRNA set forth in FIG. 6B.

[0066] In embodiments, the reference RNA comprise one or more miRNA set forth in FIG. 9B. In embodiments, the reference RNA comprise one or more of the first 10 miRNA set forth in FIG. 8B. In embodiments, the reference RNA comprise one or more of the first 6 miRNA set forth in FIG. 8B.

[0067] In embodiments, the reference RNA comprise one or more miRNA set forth in FIG. 10B. In embodiments, the reference RNA comprise one or more of the first 10 miRNA set forth in FIG. 10B. In embodiments, the reference RNA comprise one or more of the first 6 miRNA set forth in FIG. 10B. In embodiments, the reference RNA comprise the first 3 miRNA set forth in FIG. 10B and one or more of the next 7 miRNA set forth in FIG. 10B.

[0068] In embodiments of the methods described herein, the reference RNA comprise miR-15, miR-15b, miR-30e, miR-23a, miR-151a, miR-221, miR-186, miR-let-7g, miR-185, miR-146a,miR-191. miR-652, miR-93, miR-let-7d, miR-30e, miR-148b, miR-16, miR-let-7g, miR-142, miR-15 lb, miR-101. miR-23a, miR-30c, miR-423, or a combination of two or more thereof.

[0069] In embodiments of the methods described herein, the reference RNA comprise cell-free miR-15b-5p, cell-free miR-30e-5p, cell-free miR-23a-3p, cell-free miR-151a-3p, cell-free miR- 221-3p, cell-free miR-186-5p, cell-free miR-let-7g-5p, cell-free miR-185-5p. cell-free miR- 146a-5p, cell-free miR-191-5p, cell-free miR-652-3p. cell-free miR-93-5p. cell-free miR-let-7d- 5p, exosomal miR-30e-5p, exosomal miR-15b-5p, exosomal miR-148b-3p, exosomal miR- 23a- 3p, exosomal miR-16-5p, exosomal miR-186-5p, exosomal miR-let-7g-5p, exosomal miR-142- 5p, exosomal miR-15 lb, exosomal miR-101-3p, exosomal miR-30c-5p, exosomal miR 146a-5p. exosomal miR-423-3p, exosomal miR-652-3p, exosomal miR-221-3p, or a combination of two or more thereof.

[0070] In embodiments of the methods described herein, the reference RNA comprise miR- 15b, miR-23a, and miR-30e.

[0071] In embodiments of the methods described herein, the reference RNA comprise miR- 15b-5p, miR-23a-3p, and miR-30e-5p.

[0072] In embodiments of the methods described herein, the reference RNA comprises exosomal miR-15b-5p, cell free miR-I5b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p, cell-free miR-30e-5p, or a combination of two or more thereof.

[0073] In embodiments of the methods described herein, the reference RNA comprises exosomal miR-15b-5p, exosomal miR-23a-3p, exosomal miR-30e-5p, or a combination of two or more thereof.

[0074] In embodiments of the methods described herein, the reference RNA comprises cell free miR-15b-5p, cell-free miR-23a-3p, cell-free miR-30e-5p, or a combination of two or more thereof.

[0075] In embodiments of the methods described herein, the reference RNA comprises two miR selected from the group consisting of exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p, and cell-free miR-30e-5p. In embodiments, the reference RNA comprises three miR selected from the group consisting of exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p. and cell-free miR-30e-5p. In embodiments, the reference RNA comprises four miR selected from the group consisting of exosomal miR-I5b-5p, cell free miR- 15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p, and cell-free miR- 30e-5p. In embodiments, the reference RNA comprises five miR selected from the groupconsisting of exosomal miR-15b-5p. cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR- 23a-3p. exosomal miR-30e-5p, and cell-free miR-30e-5p.

[0076] In embodiments of the methods described herein, the reference RNA comprises at least two miR selected from the group consisting of exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p. cell-free miR-23a-3p, exosomal miR-30e-5p, and cell-free miR-30e-5p. In embodiments, the reference RNA comprises at least three miR selected from the group consisting of exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR- 23a-3p, exosomal miR-30e-5p, and cell-free miR-30e-5p. In embodiments, the reference RNA comprises at least four miR selected from the group consisting of exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p. and cell- free miR-30e-5p. In embodiments, the reference RNA comprises at least five miR selected from the group consisting of exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p, and cell-free miR-30e-5p.

[0077] In embodiments of the methods described herein, the reference RNA comprises exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p, and cell-free miR-30e-5p.

[0078] In embodiments of the methods described herein, the reference RNA comprises exosomal miR-15b-5p, exosomal miR-23a-3p, and exosomal miR-30e-5p.

[0079] In embodiments of the methods described herein, the reference RNA comprises cell free miR-15b-5p, cell-free miR-23a-3p, and cell-free miR-30e-5p.

[0080] In embodiments of the methods described herein, the reference RNA consist of exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p. and cell-free miR-30e-5p.

[0081] In embodiments of the methods described herein, the reference RNA consist of exosomal miR-15b-5p, exosomal miR-23a-3p, and exosomal miR-30e-5p.

[0082] In embodiments of the methods described herein, the reference RNA consist of cell free miR-15b-5p, cell-free miR-23a-3p, and cell-free miR-30e-5p.

[0083] Oncogenic RNA

[0084] In embodiments of the methods described herein, oncogenic RNA is any oncogenic RNA known in the art. In embodiments, oncogenic RNA is oncogenic miRNA.

[0085] In embodiments of the methods described herein, the oncogenic RNA is miR-21, miR- 101-1, miR-101-2. miR-143. miR-145, miR-16-1. miR-26Al, miR-26a2. miR-29C, miR-155, miR-30A, miR-100, miR-106b, miR-lOb, miR-126, miR-15a, miR-16-2, miR-17, miR-181al,miR-181a2, miR-199al, miR-199a2, miR-19a, miR-205, miR-221, miR-29bl, miR-29b2, miR- 30e, miR-31. miR-34a, miR-99a, miR-let-7b, miR-let-7c, miR-133al, miR-133a2, miR-141. miR-183, miR-195, miR-200a, miR-200b, miR-200c, miR-203a, miR-20a, miR-222, miR-29a, miR-30cl, miR-30c2, miR-451a, miR-7-1, miR-7-2, miR-7-3, miR-9-1, miR-9-2, miR-92al, miR-92a2, miR-9-3, miR-let-7al, miR-let-7a2, miR-let-7a3, miR-1-1, miR-1-2, miR-125a, miR- 125bl, miR-125b2, miR-139, miR-140, miR-142. miR-150, miR-15b, miR-181bl. miR-181b2, miR-182. miR-18a, miR-19bl, miR-19b2. miR-214, miR-223, miR-25. miR-27a, miR-375, miR-let-7d, miR-let-fl, miR-let-712, miR-122, miR-128-1, miR-128-2, miR-146A, miR-192, miR-194-1, miR-194-2, miR-210, miR-224, miR-23a, miR-26b, miR-27b, miR-301a, miR-32, miR-335, miR-342, miR-497, miR-96, miR-let-7e, miR-let-7i, miR-103al, miR-103a2, miR- 107, miR-lOa, miR-124-1. miR-124-2, miR-124-3, miR-130b, miR-133b, miR-144, miR-148a, miR-152, miR-193b, miR-199b, miR-204, miR-218-1, miR-218-2, miR-23b, miR-24-1, miR- 24-2, miR-28, miR-30b, miR-30d, miR-324, miR-378a, miR-93, miR-let-7g, miR-129-1, miR- 129-2, miR-130a, miR-135b, miR-137, miR-146b, miR-181c, miR-193A, miR-22, miR-34b, miR-34c. miR-423, miR-455, miR-106a, miR-132, miR-135al. miR-135a2, miR-138-1, miR- 138-2, miR-148b, miR-149, miR-206, miR-215, miR-320a, miR-331, miR-345, miR-373, miR- 449a, miR-483, miR-486-1, miR-486-2, miR-494, miR-625, miR-98, miR-99b, or a combination of two or more thereof. See Suszynska et al, CMC: Cancer miRNA Census - a list of cancer- related miRNA genes,” Nucleic Acids Research, 52: 1628-1644 (2024). the disclosure of which is incorporated by reference herein in its entirety. In embodiments, the oncogenic RNA is is exosomal miRNA, cell-free miRNA, or a combination thereof. ’‘Or a combination thereof’ includes a combination of exosomal miRNA and cell-free miRNA, where the miRNA can be the same and / or different. For example, the miRNA can comprise exosomal miR-99b and cell-free miR-99b (i.e., the miRNA comprises exosomal miRNA and cell-free miRNA, and the miRNA are the same). As another example, the miRNA can comprise exosomal miR-99b and cell-free miR-98 (i.e., the miRNA comprises exosomal miRNA and cell-free miRNA, and the miRNA are different). As another example, the miRNA can comprise only exosomal miRNA or only cell- free miRNA.

[0086] In embodiments of the methods described herein, the oncogenic RNA comprises miR- 32, miR-625, miR-486, miR-550a, miR-30d, or a combination of two or more thereof. In embodiments, the oncogenic RNA is is exosomal miRNA, cell-free miRNA, or a combination thereof. In embodiments, the oncogenic RNA comprises exosomal miR-32-5p. cell-free miR- 625-3p, exosomal miR-486-3p, exosomal miR-550a-3-5p, exosomal miR-625-3p, cell-free miR- 30d-5p, or a combination of two or more thereof. In embodiments, the cancer is colorectalcancer. In embodiments, the cancer is early onset colorectal cancer. In embodiments, the cancer is high grade dysplasia.

[0087] In embodiments of the methods described herein, the oncogenic RNA comprises miR- 4659b, miR-296, miR-4685, miR-550a, miR-4446, miR-432, miR-151a, miR-199a, miR-146a, miR-24, miR-223, miR-556, miR-2355, miR-181. miR-3120, miR-7-1, miR-99b, miR-425, or a combination of two or more thereof. In embodiments, the oncogenic RNA is is exosomal miRNA, cell-free miRNA, or a combination thereof. In embodiments, the oncogenic RNA comprises cell-free miR-4659b-3p, cell-free miR-296-5p, cell-free miR-4685-3p, cell-free miR- 550a-5p, cell-free miR-4446-3p, cell-free miR-432-5p, cell-free miR-151a-3p, cell-free miR- 199a-3p, cell-free miR-146a-5p. cell-free miR-24-3p. cell-free miR-223-3p, cell-free miR- 4659b-3p, cell-free miR-296-5p, exosomal miR-556-3p, exosomal miR-2355-5p, exosomal miR-181a-3p, exosomal miR-3120-3p, exosomal miR-7-l-3p, exosomal miR-99b-3p, exosomal miR-425-3p, or a combination of two or more thereof. In embodiments, the cancer is colorectal cancer. In embodiments, the cancer is early onset colorectal cancer. In embodiments, the cancer is high grade dysplasia.

[0088] In embodiments of the methods described herein, the oncogenic RNA comprises miR- 100, miR-184, miR-203a, miR-1290, miR-20a, miR-205, miR-135b, miR-150, miR-433, miR- 543, miR-199b, miR-18a, miR-19a, miR-4488, miR-200b, miR-1229, miR-296, miR-34a, miR- 15, miR-5193, miR-1228, miR-141 , miR-1 7, miR-195, miR-142, miR-3913, miR-1538, miR- 1323, miR-877, miR-19b, or a combination of two or more thereof. In embodiments, the oncogenic RNA is is exosomal miRNA, cell-free miRNA, or a combination thereof. In embodiments, the oncogenic RNA comprises exosomal miR-100-5p, exosomal miR-184, exosomal miR-203a-3p, exosomal miR-1290, exosomal miR-20a-5p, exosomal miR-205-5p, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-433-3p, exosomal miR-543, exosomal miR-199b-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-200b-3p, cell-free miR-1229-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell- free miR-15-5p, cell-free miR-5193, cell-free miR-1228-5p, cell-free miR-141-3p, cell-free miR-107, cell-free miR-195-5p, cell-free miR-142-3p, cell-free miR-3913-5p, cell-free miR- 1538, cell-free miR-1323, cell-free miR-877-3p, cell-free miR-19b-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof. In embodiments, the cancer is colorectal cancer. In embodiments, the cancer is colorectal adenoma. In embodiments, the cancer is advanced colorectal adenoma.

[0089] In embodiments of the methods described herein, the oncogenic RNA comprises miR- 30c, miR-142, miR-340, miR-335, -145, miR-200a, miR-200b, miR-429, or acombination of two or more thereof. In embodiments, the oncogenic RNA is is exosomal miRNA, cell-free miRNA. or a combination thereof. In embodiments, the oncogenic RNA comprises cell-free miR-30c-5p, cell-free miR-142-3p, cell-free miR-340-5p, cell-free miR-335- 5p, exosomal miR-1260b, exosomal miR-145-5p, exosomal miR-200a-3p, exosomal miR-200b- 3p, exosomal miR-429, exosomal miR-145-3p, or a combination of two or more thereof. In embodiments, the cancer is pancreatic cancer. In embodiments, the cancer is pancreatic ductal adenocarcinoma.

[0090] In embodiments of the methods described herein, the oncogenic RNA comprises miR- 23b, miR-216b, miR-217, let-7e, miR-26a, miR-223, miR-340, miR-1260a, miR-141, miR-143, miR-148a, miR-200c, miR-216a, miR-34a, let-7f. miR-369, miR-125a. miR-495, miR-375, miR-199a, or a combination of two or more thereof. In embodiments, the oncogenic RNA is is exosomal miRNA, cell-free miRNA, or a combination thereof. In embodiments, the oncogenic RNA comprises cell-free miR-23b-3p. exosomal miR-216b-5p, exosomal miR-217-5p, cell-free let-7e-5p, cell-free miR-26a-5p, cell-free miR-223-3p, cell-free miR-340-3p, exosomal miR- 1260a, exosomal miR-141-3p, exosomal miR-143-3p, exosomal miR-148a-3p, exosomal miR- 200c-3p, exosomal miR-216a-5p, exosomal miR-34a-5p, cell -free let-7f-5p, cell-free miR-369- 3p, cell-free miR-125a-5p, cell-free miR-495-3p, exosomal miR-375-3p, exosomal miR-199a- 5p, or a combination of two or more thereof. In embodiments, the cancer is pancreatic cancer. In embodiments, the cancer is pancreatic ductal adenocarcinoma. See WO 2023 / 239920, the disclosure of which is incorporated by reference herein in its entirety.

[0091] In embodiments of the methods described herein, the oncogenic RNA comprises miR- 21, miR-215. miR-335, miR-27a, miR-95. miR-181b. miR-431, miR-1246. miR-192, miR-196a, miR-183, miR-135b, or a combination of two or more thereof. In embodiments, the oncogenic RNA is is exosomal miRNA, cell-free miRNA, or a combination thereof. In embodiments, the oncogenic RNA comprises cell-free miR-21-3p, cell-free miR-21-5p, cell-free miR-215-5p, cell- free miR-335-3p, cell-free miR-27a-3p, cell-free miR-95-3p, cell-free miR-181b-5p, cell-free miR-431-5p, exosomal miR-21-3p, exosomal miR-21-5p, exosomal miR-1246, exosomal miR- 192-3p, exosomal miR-215-5p, exosomal miR-27a-3p, exosomal miR-95-3p, exosomal miR- 196a-5p, exosomal miR-183-5p, exosomal miR-135b-5p, or a combination of two or more thereof. In embodiments, the cancer is gastric cancer.

[0092] In embodiments of the methods described herein, the oncogenic RNA comprises miR- 513a, miR-628, miR-193a, miR-210, miR-4304, miR-194, miR-4453, or a combination of two or more thereof. In embodiments, the oncogenic RNA comprises miR-513a-5p, miR-628-3p, miR-193a-5p, miR-210, miR-4304, miR-194-3p, miR-4453, or a combination of two or morethereof. In embodiments, the oncogenic RNA is is exosomal miRNA, cell-free miRNA, or a combination thereof. In embodiments, the cancer is colorectal cancer. See WO 2024 / 006581, the disclosure of which is incorporated by reference herein in its entirety.

[0093] In embodiments of the methods described herein, the oncogenic RNA comprises miR- 181b, miR-193b. miR-195, miR-411, AMT mRNA. FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA or a combination of two or more thereof. In embodiments, the oncogenic RNA is is exosomal miRNA, cell-free miRNA, or a combination thereof. In embodiments, the oncogenic RNA comprises exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR- 195, cell-free miR-411, or a combination of two or more thereof. In embodiments, the cancer is colorectal cancer. See WO 2022 / 204378, the disclosure of which is incorporated by reference herein in its entirety.

[0094] Normalized Expression Levels

[0095] In embodiments of the methods described herein, the methods comprise normalizing the expression level of the oncogenic miRNA to the expression level of the reference miRNA, thereby obtaining (or determining) a normalized expression level of the oncogenic miRNA. In embodiments, an elevated expression level of oncogenic miRNA relative to the expression level of a control indicates that the patient has cancer. In embodiments, the expression level of the reference miRNA is used as a control to the normalized expression level of the oncogenic miRNA. In embodiments, an elevated expression level of oncogenic miRNA relative to the expression level of reference miRNA indicates that the patient has cancer. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 1. 1 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 1.2 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 1.3 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 1.4 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 1 .5 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 1.6 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is atleast 1.7 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 1.8 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 1.9 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 2 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 2.5 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 3 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 3.5 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 4 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 5 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 6 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 7 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 8 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 9 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of oncogenic miRNA that is at least 10 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level of oncogenic miRNA refers to a normalized expression level of oncogenic miRNA that is statistically significantly greater than the expression level of the reference miRNA.

[0096] In embodiments of the methods described herein, the methods comprise normalizing the expression level of the oncogenic miRNA to the expression level of the reference miRNA. thereby obtaining (or determining) a normalized expression level of the oncogenic miRNA. In embodiments, the expression level of the reference miRNA is used as a control to thenormalized expression level of the oncogenic miRNA. In embodiments, a reduced expression level of oncogenic miRNA relative to the expression level of reference miRNA indicates that the patient has cancer. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 1.1 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 1.2 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 1.3 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 1.4 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 1.5 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 1.6 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 1.7 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 1.8 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 1.9 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 2 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 2.5 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 3 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 3.5 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 4 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 5 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 6 times less than the expression level of the reference miRNA. Inembodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 7 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 8 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 9 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level refers to a normalized expression level of oncogenic miRNA that is at least 10 times less than the expression level of the reference miRNA. In embodiments, a reduced expression level of oncogenic miRNA refers to a normalized expression level of oncogenic miRNA that is statistically significantly less than the expression level of the reference miRNA.

[0097] Methods

[0098] Provided herein is a method of detecting an expression level of reference RNA in a patient with cancer comprising detecting an expression level of reference RNA in the biological sample, wherein the reference RNA comprises one or more miRNA set forth in FIG. 3B, FIG. 4B, FIG. 5B, FIG. 6B, FIG. 7B, FIG. 8B, FIG. 9B, or FIG. 10B. In embodiments, the reference RNA is miRNA. In embodiments, the reference RNA is not oncogenic. In embodiments, the reference miRNA is any reference miRNA as described herein or any combination of two or more reference miRNA as described herein, including embodiments thereof. In embodiments, the method further comprises detecting an expression level of oncogenic RNA. In embodiments, the expression level of oncogenic RNA is increased or decreased relative to the expression level of reference RNA.

[0099] Provided herein is a method of detecting an expression level of reference RNA in a patient with cancer comprising detecting an expression level of reference RNA in the biological sample, wherein the reference RNA comprises miR-15, miR-15b, miR-30e, miR-23a, miR-151a, miR-221. miR-186, miR-let-7g, miR-185. miR-146a, miR-191. miR-652, miR-93. miR-let-7d. miR-30e, miR-148b, miR-16, miR-let-7g, miR-142, miR-151b, miR-101, miR-23a, miR-30c, miR-423, or a combination of two or more thereof. In embodiments, the reference RNA is miRNA. In embodiments, the reference RNA is not oncogenic. In embodiments, the reference RNA comprises cell-free miR-15b-5p. cell-free miR-30e-5p, cell-free miR-23a-3p. cell-free miR-151 a-3p, cell-free miR-221 -3p, cell-free miR-186-5p, cell-free miR-let-7g-5p, cell-free miR-185-5p, cell-free miR-146a-5p, cell-free miR-191-5p, cell-free miR-652-3p, cell-free miR- 93-5p, cell-free miR-let-7d-5p, exosomal miR-30e-5p, exosomal miR-15b-5p, exosomal miR- 148b-3p, exosomal miR-23a-3p. exosomal miR-16-5p, exosomal miR-186-5p, exosomal miR-let-7g-5p, exosomal miR-142-5p, exosomal miR-151b, exosomal miR-101-3p, exosomal miR- 30c-5p. exosomal miR 146a-5p, exosomal miR-423-3p. exosomal miR-652-3p, and exosomal miR-221-3p, or a combination of two or more thereof. In embodiments, the method further comprises detecting an expression level of oncogenic RNA. In embodiments, the expression level of oncogenic RNA is increased or decreased relative to the expression level of reference RNA.

[0100] Provided herein is a method of detecting an expression level of RNA in a patient with cancer comprising detecting an expression level of RNA in the biological sample, wherein the RNA comprises miR-15b-5p, miR-23a-3p, miR-30e-5p, or a combination of two or more thereof. In embodiments, the RNA is miRNA. In embodiments, the RNA is not oncogenic. In embodiments, the RNA is reference miRNA. In embodiments, the reference RNA comprises exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p, cell-free miR-30e-5p, or a combination of two or more thereof. In embodiments, the method further comprises detecting an expression level of oncogenic RNA. In embodiments, the expression level of oncogenic RNA is increased or decreased relative to the expression level of reference RNA.

[0101] Provided herein is a method of detecting an expression level of RNA in a patient with cancer, the method comprising: (i) detecting an expression level of reference RNA in the biological sample, wherein the reference RNA comprises miR-15b, miR-23a, miR-30e, or a combination of two or more thereof, and (ii) detecting an expression level of oncogenic RNA in the biological sample. In embodiments, the expression level of oncogenic RNA is increased or decreased relative to the expression level of the reference RNA. In embodiments, the reference RNA is miRNA. In embodiments, the reference RNA comprises exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p, cell-free miR- 30e-5p, or a combination of two or more thereof.

[0102] Provided herein is a method of detecting an expression level of an oncogenic RNA in a patient with cancer comprising: (i) detecting an expression level of the oncogenic RNA in a biological sample obtained from the patient with cancer; (ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises one or more miRNA set forth in FIG. 3B. FIG. 4B. FIG. 5B. FIG. 6B, FIG. 7B, FIG. 8B, FIG. 9B, or FIG.10B; and (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; thereby detecting the normalized expression level of the oncogenic RNA. In embodiments, the reference miRNA is any reference miRNA as described herein or any combination of two or more reference miRNA as described herein, including embodimentsthereof. In embodiments, the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA. In embodiments, the oncogenic miRNA is any oncogenic miRNA known in the art and described herein. The skilled artisan would understand the type of cancer the patient has based on the types of oncogenic miRNA detected in the biological sample.

[0103] Provided herein is a method of detecting an expression level of an oncogenic RNA in a patient with cancer comprising: (i) detecting an expression level of the oncogenic RNA in a biological sample obtained from the patient with cancer; (ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises miR-15, miR- 15b, miR-30e, miR-23a, miR-151a, miR-221, miR-186. miR-let-7g, miR-185, miR-146a, miR- 191, miR-652, miR-93, miR-let-7d, miR-30e, miR-148b, miR-16, miR-let-7g, miR-142, miR- 151b, miR-101, miR-23a, miR-30c, miR-423, or a combination of two or more thereof; and (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; thereby detecting the normalized expression level of the oncogenic RNA. In embodiments, the reference RNA comprises cell-free miR-15b-5p, cell-free miR-30e-5p, cell- free miR-23a-3p, cell-free miR-151a-3p, cell-free miR-221-3p, cell-free miR-186-5p, cell-free miR-let-7g-5p, cell-free miR-185-5p, cell-free miR-146a-5p, cell-free miR-191-5p, cell-free miR-652-3p, cell-free miR-93-5p, cell-free miR-let-7d-5p, exosomal miR-30e-5p, exosomal miR-15b-5p. exosomal miR-148b-3p, exosomal miR-23a-3p. exosomal miR-16-5p, exosomal miR-186-5p, exosomal miR-let-7g-5p, exosomal miR-142-5p, exosomal miR-151b, exosomal miR-101-3p, exosomal miR-30c-5p, exosomal miR 146a-5p, exosomal miR-423-3p, exosomal miR-652-3p, exosomal miR-221-3p. or a combination of two or more thereof. In embodiments, the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA. In embodiments, the oncogenic miRNA is any oncogenic miRNA known in the art and described herein. The skilled artisan would understand the type of cancer the patient has based on the types of oncogenic miRNA detected in the biological sample.

[0104] Provided herein is a method of detecting an expression level of an oncogenic RNA in a patient with cancer comprising: (i) detecting an expression level of the oncogenic RNA in a biological sample obtained from the patient with cancer; (ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises miR-15b-5p, miR-23a-3p, miR-30e-5p, or a combination of two or more thereof; and (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; thereby detecting the normalized expression level of the oncogenic RNA. In embodiments, the reference RNA comprises exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-freemiR-23a-3p, exosomal miR-30e-5p, cell-free miR-30e-5p, or a combination of two or more thereof. In embodiments, the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA. In embodiments, the oncogenic miRNA is any oncogenic miRNA known in the art and described herein. The skilled artisan would understand the type of cancer the patient has based on the types of oncogenic miRNA detected in the biological sample.

[0105] Provided herein is a method of treating cancer in a patient in need thereof comprising: (i) detecting an expression level of an oncogenic RNA in a biological sample obtained from the patient; (ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises one or more miRNA set forth in FIG. 3B, FIG. 4B, FIG. 5B. FIG. 6B, FIG. 7B, FIG. 8B, FIG. 9B, or FIG. 10B; (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; wherein the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA; and (iv) administering to the patient an effective amount of an anti-cancer agent. In embodiments, the reference miRNA is any reference miRNA as described herein or any combination of two or more reference miRNA as described herein, including embodiments thereof. In embodiments, the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA. In embodiments, the oncogenic miRNA is any oncogenic miRNA known in the art and described herein. The skilled artisan would understand the ty pe of cancer the patient has based on the types of oncogenic miRNA detected in the biological sample.

[0106] Provided herein is a method of treating cancer in a patient in need thereof comprising: (i) detecting an expression level of an oncogenic RNA in a biological sample obtained from the patient; (ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises miR-15, miR-15b, miR-30e, miR-23a, miR-151a, miR-221, miR- 186, miR-let-7g, miR-185. miR-146a, miR-191. miR-652, miR-93, miR-let-7d, miR-30e, miR- 148b, miR-16, miR-let-7g, miR-142, miR-151b, miR-101, miR-23a, miR-30c, miR-423, or a combination of two or more thereof; (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; wherein the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA; and (iv) administering to the patient an effective amount of an anti-cancer agent. In embodiments, the reference RNA comprises cell-free miR-15b-5p, cell-free miR-30e-5p, cell- free miR-23a-3p, cell-free miR-151a-3p, cell-free miR-221-3p, cell-free miR-186-5p, cell-free miR-let-7g-5p, cell-free miR-185-5p, cell-free miR-146a-5p, cell-free miR-191-5p, cell-freemiR-652-3p, cell-free miR-93-5p, cell-free miR-let-7d-5p, exosomal miR-30e-5p, exosomal miR-15b-5p, exosomal miR-148b-3p, exosomal miR-23a-3p, exosomal miR-16-5p, exosomal miR-186-5p, exosomal miR-let-7g-5p, exosomal miR-142-5p, exosomal miR-151b, exosomal miR-101-3p, exosomal miR-30c-5p, exosomal miR 146a-5p, exosomal miR-423-3p, exosomal miR-652-3p, exosomal miR-221-3p, or a combination of two or more thereof. In embodiments, the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA. In embodiments, the oncogenic miRNA is any oncogenic miRNA known in the art and described herein. The skilled artisan w ould understand the type of cancer the patient has based on the types of oncogenic miRNA detected in the biological sample.

[0107] Provided herein is a method of treating cancer in a patient in need thereof comprising: (i) detecting an expression level of an oncogenic RNA in a biological sample obtained from the patient; (ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises miR-15b-5p, miR-23a-3p, miR-30e-5p, or a combination of tw o or more thereof; (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; wherein the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA; and (iv) administering to the patient an effective amount of an anti-cancer agent. In embodiments, the reference RNA comprises exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR- 23a-3p. exosomal miR-30e-5p, cell-free miR-30e-5p. or a combination of two or more thereof. In embodiments, the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA. In embodiments, the oncogenic miRNA is any oncogenic miRNA known in the art and described herein. The skilled artisan would understand the type of cancer the patient has based on the types of oncogenic miRNA detected in the biological sample.

[0108] Provided herein is a method of diagnosing a patient with cancer, the method comprising: (i) detecting an expression level of an oncogenic RNA in a biological sample obtained from the patient; (ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises one or more miRNA set forth in FIG. 3B, FIG. 4B, FIG. 5B, FIG. 6B, FIG. 7B, FIG. 8B, FIG. 9B, or FIG. 10B; (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA, thereby detecting the normalized expression level of the oncogenic RNA; and (iv) diagnosing the patient as having cancer when the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA. In embodiments, the reference miRNA is any reference miRNA as described herein or any combination of tw o or more reference miRNAas described herein, including embodiments thereof. In embodiments, the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA. In embodiments, the oncogenic miRNA is any oncogenic miRNA known in the art and described herein. The skilled artisan w ould understand the type of cancer the patient has based on the types of oncogenic miRNA detected in the biological sample.

[0109] Provided herein is a method of diagnosing a patient with cancer, the method comprising: (i) detecting an expression level of an oncogenic RNA in a biological sample obtained from the patient; (ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises miR-15, miR-15b, miR-30e, miR-23a, miR-151a, miR-221, miR-186, miR-let-7g, miR-185. miR-146a, miR-191. miR-652, miR-93, miR-let-7d, miR-30e, miR-148b, miR-16, miR-let-7g, miR-142, miR-151b, miR-101, miR-23a, miR-30c, miR-423, or a combination of two or more thereof; (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA, thereby detecting the normalized expression level of the oncogenic RNA; and (iv) diagnosing the patient as having cancer when the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA. In embodiments, the reference RNA comprises cell-free miR-15b-5p, cell-free miR-30e-5p, cell-free miR-23a-3p, cell-free miR-151a-3p, cell-free miR- 221-3p, cell-free miR-186-5p, cell-free miR-let-7g-5p, cell-free miR-185-5p. cell-free miR- I46a-5p, cell-free miR-191-5p, cell-free miR-652-3p. cell-free miR-93-5p. cell-free miR-let-7d- 5p, exosomal miR-30e-5p, exosomal miR-15b-5p, exosomal miR-148b-3p, exosomal miR-23a- 3p, exosomal miR-16-5p, exosomal miR-186-5p, exosomal miR-let-7g-5p, exosomal miR-142- 5p, exosomal miR-15 lb, exosomal miR-101-3p, exosomal miR-30c-5p, exosomal miR 146a-5p. exosomal miR-423-3p, exosomal miR-652-3p, exosomal miR-221 -3p, or a combination of two or more thereof. In embodiments, the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA. In embodiments, the oncogenic miRNA is any oncogenic miRNA known in the art and described herein. The skilled artisan would understand the type of cancer the patient has based on the types of oncogenic miRNA detected in the biological sample.

[0110] Provided herein is a method of diagnosing a patient with cancer, the method comprising: (i) detecting an expression level of an oncogenic RNA in a biological sample obtained from the patient; (ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises miR-15b-5p, miR-23a-3p, miR-30e-5p, or a combination of two or more thereof; (iii) normalizing the expression level of the oncogenicRNA to the expression level of the reference RNA, thereby detecting the normalized expressionlevel of the oncogenic RNA; and (iv) diagnosing the patient as having cancer when the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA. In embodiments, the reference RNA comprises exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR- 30e-5p, cell-free miR-30e-5p, or a combination of two or more thereof. In embodiments, the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA. In embodiments, the oncogenic miRNA is any oncogenic miRNA known in the art and described herein. The skilled artisan would understand the type of cancer the patient has based on the types of oncogenic miRNA detected in the biological sample.[OHl] Provided herein is a computer-implemented method of administering an effective amount of an anti-cancer agent to a patient with cancer, the method comprising: (i) obtaining a sample data set comprising an expression level of oncogenic RNA from a biological sample obtained from the patient with cancer; (ii) obtaining a reference data set comprising an expression level of a reference RNA from the biological sample, wherein the reference RNA comprises one or more miRNA set forth in FIG. 3B, FIG. 4B, FIG. 5B, FIG. 6B, FIG. 7B, FIG. 8B, FIG. 9B, or FIG. 10B; (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; (iv) determining an elevated expression level of the normalized expression level of the oncogenic RNA; and (v) administering to the patient an effective amount of the anti-cancer agent based on the determined normalized expression level. In embodiments, the reference miRNA is any reference miRNA as described herein or any combination of two or more reference miRNA as described herein, including embodiments thereof. In embodiments, the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA. In embodiments, the oncogenic miRNA is any oncogenic miRNA known in the art and described herein. The skilled artisan would understand the ty pe of cancer the patient has based on the types of oncogenic miRNA detected in the biological sample.

[0112] Provided herein is a computer-implemented method of administering an effective amount of an anti-cancer agent to a patient with cancer, the method comprising: (i) obtaining a sample data set comprising an expression level of oncogenic RNA from a biological sample obtained from the patient with cancer; (ii) obtaining a reference data set comprising an expression level of a reference RNA from the biological sample, wherein the reference RNA comprises miR-15, miR-15b, miR-30e, miR-23a, miR-151a, miR-221 , miR-186, miR-let-7g, miR-185, miR-146a, miR-191, miR-652, miR-93, miR-let-7d, miR-30e, miR-148b, miR-16, miR-let-7g, miR-142, miR-15 lb, miR-101, miR-23a, miR-30c, miR-423. or a combination oftwo or more thereof; (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; (iv) determining an elevated expression level of the normalized expression level of the oncogenic RNA; and (v) administering to the patient an effective amount of the anti-cancer agent based on the determined normalized expression level. In embodiments, the reference RNA comprisescell-free miR-15b-5p, cell-free miR-30e-5p, cell- free miR-23a-3p. cell-free miR-151a-3p, cell-free miR-221-3p, cell-free miR-186-5p, cell-free miR-let-7g-5p. cell-free miR-185-5p, cell-free miR-146a-5p, cell-free miR-191-5p, cell-free miR-652-3p, cell-free miR-93-5p, cell-free miR-let-7d-5p, exosomal miR-30e-5p, exosomal miR-15b-5p, exosomal miR-148b-3p, exosomal miR-23a-3p, exosomal miR-16-5p, exosomal miR-186-5p, exosomal miR-let-7g-5p, exosomal miR-142-5p, exosomal miR-151b, exosomal miR-101-3p. exosomal miR-30c-5p. exosomal miR 146a-5p, exosomal miR-423-3p. exosomal miR-652-3p, exosomal miR-221-3p, or a combination of two or more thereof. In embodiments, the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA. In embodiments, the oncogenic miRNA is any oncogenic miRNA known in the art and described herein. The skilled artisan would understand the type of cancer the patient has based on the types of oncogenic miRNA detected in the biological sample.

[0113] Provided herein is a computer-implemented method of administering an effective amount of an anti-cancer agent to a patient with cancer, the method comprising: (i) obtaining a sample data set comprising an expression level of oncogenic RNA from a biological sample obtained from the patient with cancer; (ii) obtaining a reference data set comprising an expression level of a reference RNA from the biological sample, wherein the reference RNA comprises miR-15b-5p, miR-23a-3p, miR-30e-5p, or a combination of two or more thereof; (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; (iv) determining an elevated expression level of the normalized expression level of the oncogenic RNA; and (v) administering to the patient an effective amount of the anti-cancer agent based on the determined normalized expression level. In embodiments, the reference RNA comprises exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR- 23a-3p, exosomal miR-30e-5p, cell-free miR-30e-5p, or a combination of two or more thereof. In embodiments, the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA. In embodiments, the oncogenic miRNA is any oncogenic miRNA known in the art and described herein. The skilled artisan would understand the type of cancer the patient has based on the types of oncogenic miRNA detected in the biological sample.

[0114] Provided herein is a computer-implemented system for administering an effectiveamount of an anti-cancer agent to a patient with cancer, wherein the computer-implemented system comprises: (i) a computer-readable medium holding a control data set of normalized expression levels of oncogenic RNA from a population of patients that do not have cancer; (ii) a processing device; (iii) a computer-readable medium containing programming instructions that are configured to instruct the processing device to: (a) receive a sample data set comprising an expression level of oncogenic RNA from a biological sample obtained from a patient with cancer; (b) receive a reference data set comprising an expression level of a reference RNA from the biological sample, wherein the reference RNA comprises one or more miRNA set forth in FIG. 3B, FIG. 4B, FIG. 5B, FIG. 6B, FIG. 7B, FIG. 8B, FIG. 9B, or FIG. 10B; (c) normalize the expression level of the oncogenic RNA to the expression level of the reference RNA; (d) receive the control data set of normalized expression levels of oncogenic RNA from the population of patients that do not have cancer; and (e) determine an elevated expression level of the normalized expression level of the oncogenic RNA in the sample data set when compared to the normalized expression level of oncogenic RNA in the control data set. In embodiments, the patient is administered an effective amount of the anti-cancer agent based on the elevated expression level of the normalized expression level of the oncogenic RNA in the sample data set received from the computer-readable medium. In embodiments, the reference miRNA is any reference miRNA as described herein or any combination of two or more reference miRNA as described herein, including embodiments thereof. In embodiments, the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA. In embodiments, the computer-readable medium generates a report providing results and instructions for administering to the patient an effective amount of the anti-cancer agent. In embodiments, the computer-readable medium generates a report providing results and instructions (and / or recommendations) providing the type of anti-cancer agent to administer to the patient. In embodiments, the oncogenic miRNA is any oncogenic miRNA known in the art and described herein. The skilled artisan would understand the type of cancer the patient has based on the types of oncogenic miRNA detected in the biological sample.

[0115] Provided herein is a computer-implemented system for administering an effective amount of an anti-cancer agent to a patient with cancer, wherein the computer-implemented system comprises: (i) a computer-readable medium holding a control data set of normalized expression levels of oncogenic RNA from a population of patients that do not have cancer; (ii) a processing device; (iii) a computer-readable medium containing programming instructions that are configured to instruct the processing device to: (a) receive a sample data set comprising an expression level of oncogenic RNA from a biological sample obtained from a patient withcancer; (b) receive a reference data set comprising an expression level of a reference RNA from the biological sample, wherein the reference RNA comprises miR-15, miR-15b, miR-30e, miR- 23a, miR-15 la, miR-221, miR-186, miR-let-7g, miR-185, miR-146a, miR-191, miR-652, miR- 93, miR-let-7d, miR-30e, miR-148b, miR-16, miR-let-7g, miR-142, miR-151b, miR-101, miR- 23a, miR-30c, miR-423, or a combination of two or more thereof; (c) normalize the expression level of the oncogenic RNA to the expression level of the reference RNA; (d) receive the control data set of normalized expression levels of oncogenic RNA from the population of patients that do not have cancer; and (e) determine an elevated expression level of the normalized expression level of the oncogenic RNA in the sample data set when compared to the normalized expression level of oncogenic RNA in the control data set. In embodiments, the patient is administered an effective amount of the anti-cancer agent based on the elevated expression level of the normalized expression level of the oncogenic RNA in the sample data set received from the computer-readable medium. In embodiments, the reference RNA comprises cell-free miR-15b- 5p, cell-free miR-30e-5p, cell-free miR-23a-3p, cell-free miR-15 la-3p, cell-free miR-221-3p, cell-free miR-186-5p, cell-free miR-let-7g-5p, cell-free miR-185-5p, cell-free miR-146a-5p, cell-free miR-191-5p, cell-free miR-652-3p, cell-free miR-93-5p, cell-free miR-let-7d-5p, exosomal miR-30e-5p, exosomal miR-15b-5p, exosomal miR-148b-3p, exosomal miR-23a-3p, exosomal miR-16-5p, exosomal miR-186-5p, exosomal miR-let-7g-5p, exosomal miR-142-5p, exosomal miR-15 lb. exosomal miR-101-3p, exosomal miR-30c-5p, exosomal miR 146a-5p, exosomal miR-423-3p, exosomal miR-652-3p, exosomal miR-221 -3p, or a combination of two or more thereof. In embodiments, the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA. In embodiments, the computer- readable medium generates a report providing results and instructions for administering to the patient an effective amount of the anti-cancer agent. In embodiments, the computer-readable medium generates a report providing results and instructions (and / or recommendations) providing the type of anti-cancer agent to administer to the patient. In embodiments, the oncogenic miRNA is any oncogenic miRNA known in the art and described herein. The skilled artisan would understand the type of cancer the patient has based on the types of oncogenic miRNA detected in the biological sample.

[0116] Provided herein is a computer-implemented system for administering an effective amount of an anti-cancer agent to a patient with cancer, wherein the computer-implemented system comprises: (i) a computer-readable medium holding a control data set of normalized expression levels of oncogenic RNA from a population of patients that do not have cancer; (ii) a processing device; (iii) a computer-readable medium containing programming instructions thatare configured to instruct the processing device to: (a) receive a sample data set comprising an expression level of oncogenic RNA from a biological sample obtained from a patient with cancer; (b) receive a reference data set comprising an expression level of a reference RNA from the biological sample, wherein the reference RNA comprises miR-15b-5p, miR-23a-3p, miR- 30e-5p, or a combination of two or more thereof; (c) normalize the expression level of the oncogenic RNA to the expression level of the reference RNA; (d) receive the control data set of normalized expression levels of oncogenic RNA from the population of patients that do not have cancer; and (e) determine an elevated expression level of the normalized expression level of the oncogenic RNA in the sample data set when compared to the normalized expression level of oncogenic RNA in the control data set. In embodiments, the patient is administered an effective amount of the anti-cancer agent based on the elevated expression level of the normalized expression level of the oncogenic RNA in the sample data set received from the computer- readable medium. In embodiments, the reference RNA comprises exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p, cell-free miR-30e-5p, or a combination of two or more thereof. In embodiments, the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA. In embodiments, the computer-readable medium generates a report providing results and instructions for administering to the patient an effective amount of the anti-cancer agent. In embodiments, the computer-readable medium generates a report providing results and instructions (and / or recommendations) providing the type of anti-cancer agent to administer to the patient. In embodiments, the reference miRNA is any reference miRNA as described herein, including embodiments thereof. In embodiments, the oncogenic miRNA is any oncogenic miRNA known in the art and described herein. The skilled artisan would understand the type of cancer the patient has based on the types of oncogenic miRNA detected in the biological sample.

[0117] Provided herein is a method of treating cancer in a patient in need thereof comprising: (i) selecting a patient having a diagnosis of cancer based on an elevated expression level of oncogenic RNA in a biological sample obtained from the patient, wherein the expression level of the oncogenic RNA is normalized to the expression level of reference RNA, wherein the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA, and wherein the reference RNA comprises one or more miRNA set forth in FIG. 3B. FIG. 4B, FIG. 5B, FIG. 6B, FIG. 7B, FIG. 8B, FIG 9B, or FIG. 10B; and (ii) treating the patient from step (i) by administering to the patient an effective amount of an anti-cancer agent. In embodiments, the reference miRNA is any reference miRNA as described herein or any combination of two or more reference miRNA as described herein,including embodiments thereof.

[0118] Provided herein is a method of treating cancer in a patient in need thereof comprising: (i) selecting a patient having a diagnosis of cancer based on an elevated expression level of oncogenic RNA in a biological sample obtained from the patient, wherein the expression level of the oncogenic RNA is normalized to the expression level of reference RNA, wherein the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA, and wherein the reference RNA comprises miR-15, miR- 15b, miR-30e, miR-23a, miR-151a, miR-221, miR-186, miR-let-7g, miR-185, miR-146a, miR- 191, miR-652, miR-93, miR-let-7d, miR-30e, miR-148b, miR-16, miR4et-7g, miR-142, miR- 15 lb, miR-101, miR-23a, miR-30c, miR-423. or a combination of two or more thereof; and (ii) treating the patient from step (i) by administering to the patient an effective amount of an anticancer agent. In embodiments, the reference RNA comprises cell-free miR-15b-5p, cell-free miR-30e-5p, cell-free miR-23a-3p, cell-free miR-15 la-3p, cell-free miR-221-3p, cell-free miR- 186-5p, cell-free miR-let-7g-5p, cell-free miR-185-5p, cell-free miR-146a-5p, cell-free miR- 19 l-5p, cell-free miR-652-3p, cell-free miR-93-5p, cell-free miR-let-7d-5p, exosomal miR-30e- 5p, exosomal miR-15b-5p, exosomal miR-148b-3p, exosomal miR-23a-3p, exosomal miR-16- 5p, exosomal miR-186-5p, exosomal miR-let-7g-5p, exosomal miR-142-5p, exosomal miR- 151b, exosomal miR-101-3p, exosomal miR-30c-5p, exosomal miR 146a-5p, exosomal miR- 423-3p, exosomal miR-652-3p, exosomal miR-221 -3p.

[0119] Provided herein is a method of treating cancer in a patient in need thereof comprising: (i) selecting a patient having a diagnosis of cancer based on an elevated expression level of oncogenic RNA in a biological sample obtained from the patient, wherein the expression level of the oncogenic RNA is normalized to the expression level of reference RNA, wherein the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA, and wherein the reference RNA comprises miR- 15b, miR-23a. miR-30e, or a combination of two or more thereof; and (ii) treating the patient from step (i) by administering to the patient an effective amount of an anti-cancer agent. In embodiments, the reference RNA comprises exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p, cell-free miR-30e-5p, or a combination of two or more thereof.

[0120] Provided herein is a method of treating cancer in a patient in need thereof comprising: (i) receiving or obtaining an expression level of oncogenic RNA in a biological sample obtained from the patient, wherein the expression level of the oncogenic RNA is normalized to the expression level of reference RNA, wherein the normalized expression level of the oncogenicRNA is elevated or reduced relative to the expression level of the reference RNA, and wherein the reference RNA comprises one or more miRNA set forth in FIG. 3B, FIG. 4B, FIG. 5B. FIG. 6B, FIG. 7B, FIG. 8B, FIG. 9B, or FIG. 10B; and (ii) diagnosing a patient with cancer based on the elevated and / or reduced expression level of oncogenic RNA, monitoring a patient who is at risk of developing cancer based on the elevated or reduced expression level of oncogenic RNA, or monitoring efficacy of treatment for cancer in a patient based on the elevated or reduced expression level of oncogenic RNA; and (iii) treating the patient from step (ii) by administering to the patient an effective amount of an anti-cancer agent. In embodiments, the reference miRNA is any reference miRNA as described herein or any combination of two or more reference miRNA as described herein, including embodiments thereof

[0121] Provided herein is a method of treating cancer in a patient in need thereof comprising: (i) receiving or obtaining an expression level of oncogenic RNA in a biological sample obtained from the patient, wherein the expression level of the oncogenic RNA is normalized to the expression level of reference RNA, wherein the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA, and wherein the reference RNA comprises miR-15, miR-15b, miR-30e, miR-23a, miR-151a, miR-221, miR- 186, miR-let-7g, miR-185, miR-146a, miR-191, miR-652, miR-93, miR-let-7d, miR-30e, miR- 148b, miR-16, miR-let-7g. miR-142, miR-151b, miR-101, miR-23a, miR-30c, miR-423, or a combination of two or more thereof; and (ii) diagnosing a patient with cancer based on the elevated and / or reduced expression level of oncogenic RNA, monitoring a patient who is at risk of developing cancer based on the elevated or reduced expression level of oncogenic RNA, or monitoring efficacy of treatment for cancer in a patient based on the elevated or reduced expression level of oncogenic RNA; and (iii) treating the patient from step (ii) by administering to the patient an effective amount of an anti-cancer agent. In embodiments, the reference RNA comprises cell-free miR-15b-5p, cell-free miR-30e-5p, cell-free miR-23a-3p, cell-free miR- 151a-3p, cell-free miR-221-3p, cell-free miR-186-5p, cell-free miR-let-7g-5p, cell-free miR- 185-5p, cell-free miR-146a-5p, cell-free miR-191-5p, cell-free miR-652-3p, cell-free miR-93- 5p, cell-free miR-let-7d-5p, exosomal miR-30e-5p, exosomal miR-15b-5p, exosomal miR-148b- 3p, exosomal miR-23a-3p, exosomal miR-16-5p, exosomal miR-186-5p, exosomal miR-let-7g- 5p, exosomal miR-142-5p, exosomal miR-151b, exosomal miR-101-3p, exosomal miR-30c-5p, exosomal miR 146a-5p, exosomal miR-423-3p, exosomal miR-652-3p. exosomal miR-221 -3p, or a combination of two or more thereof.

[0122] Provided herein is a method of treating cancer in a patient in need thereof comprising:(i) receiving or obtaining an expression level of oncogenic RNA in a biological sample obtainedfrom the patient, wherein the expression level of the oncogenic RNA is normalized to the expression level of reference RNA, wherein the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA, and wherein the reference RNA comprises miR-15b, miR-23a, miR-30e, or a combination of two or more thereof; and (ii) diagnosing a patient wi th cancer based on the elevated and / or reduced expression level of oncogenic RNA, monitoring a patient who is at risk of developing cancer based on the elevated or reduced expression level of oncogenic RNA, or monitoring efficacy of treatment for cancer in a patient based on the elevated or reduced expression level of oncogenic RNA; and (iii) treating the patient from step (ii) by administering to the patient an effective amount of an anti-cancer agent. In embodiments, the reference RNA comprises exosomal miR- 15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p. cell-free miR-23a-3p, exosomal miR-30e- 5p, cell-free miR-30e-5p, or a combination of two or more thereof.

[0123] Provided herein is a method of treating cancer in a patient in need thereof comprising: (i) receiving or obtaining an expression level of oncogenic RNA, wherein the expression level of oncogenic RNA is produced by a non-transitory computer-readable storage medium having instructions stored thereon which, when executed by a processor, causes the processor to perform an operation comprising applying an algorithm to the results of a method which comprises detecting an expression level of oncogenic RNA in a biological sample obtained from the patient, wherein the expression level of the oncogenic RNA is normalized to the expression level of reference RNA, wherein the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA, and wherein the reference RNA comprises one or more miRNA set forth in FIG. 3B, FIG. 4B, FIG. 5B, FIG. 6B, FIG. 7B, FIG. 8B, FIG. 9B, or FIG. 10B; and (ii) diagnosing a patient with cancer based on the elevated and / or reduced expression level of oncogenic RNA, monitoring a patient who is at risk of developing cancer based on the elevated or reduced expression level of oncogenic RNA, or monitoring efficacy of treatment for cancer in a patient based on the elevated or reduced expression level of oncogenic RNA; and (iii) treating the patient from step (ii) by administering to the patient an effective amount of an anti-cancer agent. In embodiments, the reference miRNA is any reference miRNA as described herein or any combination of two or more reference miRNA as described herein, including embodiments thereof.

[0124] Provided herein is a method of treating cancer in a patient in need thereof comprising: (i) receiving or obtaining an expression level of oncogenic RNA, wherein the expression level of oncogenic RNA is produced by a non-transitory' computer-readable storage medium having instructions stored thereon which, when executed by a processor, causes the processor toperform an operation comprising applying an algorithm to the results of a method which comprises detecting an expression level of oncogenic RNA in a biological sample obtained from the patient, wherein the expression level of the oncogenic RNA is normalized to the expression level of reference RNA, wherein the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA, and wherein the reference RNA comprises miR-15, miR-15b, miR-30e, miR-23a, miR-151a, miR-221, miR-186. miR-let-7g, miR-185, miR-146a, miR-191, miR-652, miR-93, miR-let-7d, miR-30e. miR-148b, miR-16, miR-let-7g, miR-142, miR-151b, miR-101, miR-23a, miR-30c, miR-423, or a combination of two or more thereof; and (ii) diagnosing a patient with cancer based on the elevated and / or reduced expression level of oncogenic RNA, monitoring a patient who is at risk of developing cancer based on the elevated or reduced expression level of oncogenic RNA, or monitoring efficacy of treatment for cancer in a patient based on the elevated or reduced expression level of oncogenic RNA; and (iii) treating the patient from step (ii) by administering to the patient an effective amount of an anti-cancer agent. In embodiments, the reference miRNA is any reference miRNA as described herein or any combination of two or more reference miRNA as described herein, including embodiments thereof. In embodiments, the reference RNA comprises cell-free miR-15b-5p, cell-free miR-30e-5p, cell-free miR-23a-3p, cell-free miR-151a-3p, cell-free miR-221-3p, cell-free miR-186-5p, cell-free miR-let-7g-5p, cell-free miR-185-5p, cell-free miR-146a-5p, cell-free miR-191-5p, cell-free miR-652-3p, cell- free miR-93-5p, cell-free miR-let-7d-5p, exosomal miR-30e-5p, exosomal miR-15b-5p, exosomal miR-148b-3p, exosomal miR-23a-3p, exosomal miR-16-5p, exosomal miR-186-5p, exosomal miR-let-7g-5p, exosomal miR-142-5p, exosomal miR-151b, exosomal miR-101-3p, exosomal miR-30c-5p, exosomal miR 146a-5p, exosomal miR-423-3p, exosomal miR-652-3p, exosomal miR-221 -3 p, or a combination of two or more thereof.

[0125] Provided herein is a method of treating cancer in a patient in need thereof comprising: (i) receiving or obtaining an expression level of oncogenic RNA, wherein the expression level of oncogenic RNA is produced by a non-transitory computer-readable storage medium having instructions stored thereon which, when executed by a processor, causes the processor to perform an operation comprising applying an algorithm to the results of a method which comprises detecting an expression level of oncogenic RNA in a biological sample obtained from the patient, wherein the expression level of the oncogenic RNA is normalized to the expression level of reference RNA, wherein the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA, and wherein the reference RNA comprises miR-15b, miR-23a, miR-30e, or a combination of two or morethereof; and (ii) diagnosing a patient with cancer based on the elevated and / or reduced expression level of oncogenic RNA. monitoring a patient who is at risk of developing cancer based on the elevated or reduced expression level of oncogenic RNA, or monitoring efficacy of treatment for cancer in a patient based on the elevated or reduced expression level of oncogenic RNA; and (iii) treating the patient from step (ii) by administering to the patient an effective amount of an anti-cancer agent. In embodiments, the reference RNA comprises exosomal miR- 15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p. cell-free miR-23a-3p, exosomal miR-30e- 5p, cell-free miR-30e-5p, or a combination of two or more thereof.

[0126] Provided herein are methods of processing data generated from the RNA levels in the biological sample obtained from a patient for establishing a cancer risk score (composite risk score), e.g., a score indicative cancer. In embodiments, the method comprises the steps of (i) normalizing numeric values of the oncogenic RNA level data, (ii) refining the discriminatory power of individual oncogenic RNA by statistically weighting some of the numeric values associated therewith, and (iii) summating the numeric values obtained from step (ii) to provide a composite risk score. The composite risk score obtained from step (iii) is compared to reference RNA and the comparison allows the sample to be designated as positive or negative for cancer or a scale of likelihood of cancer. In embodiments, the composite risk score is normalized. In embodiments, the composite risk score is scaled. In embodiments, the composite risk score is weighted. Weighted refers to the relevant value being adjusted to more appropriately reflect its contribution to the risk score. The cancer risk score can be based on a comparison to a control, such as a healthy patient, a population of healthy patients, a patient with cancer, or a population of patients with cancer.

[0127] In embodiments, the cancer risk score is produced by a non- transitory computer- readable storage medium having instructions stored thereon which, when executed by a processor, causes the processor to perform an operation comprising applying an algorithm to the protein levels.

[0128] In embodiments of the methods described herein, a medical provider instructs a patient to obtain laboratory tests. The laboratory analyzes a biological sample provided by the patient to produce test results, e.g., levels of oncogenic RNA and / or cancer risk scores, e.g., that are normalized based on reference RNA as described herein. The medical provider receives the test results from the laboratory or obtains the test results from a patient so that the medical provider can use the test results to treat a patient with cancer, diagnose a patient with cancer, monitor a patient who is at risk of developing cancer, or monitoring efficacy of treatment for cancer in a patient. Receiving and obtaining can be used interchangeably herein and can refer to physicallyreceiving / obtaining paper documents or receiving / obtaining files via an electronic device (e.g., computer, phone). Medical provider refers to any person or entity that provides medical services to a patient. In embodiments, the medical provider is a medical doctor, a nurse, a nurse practitioner, a physician’s assistant, a hospital, a doctor’s office, and the like.

[0129] Methods

[0130] In embodiments of the methods described herein, the biological sample is any biological sample. In embodiments, the biological sample is a liquid biological sample. In embodiments, the biological sample is a blood sample or a tissue sample. In embodiments, the biological sample is a tissue sample. In embodiments, the tissue sample is a tumor tissue sample. In embodiments, the biological sample is a stool sample. In embodiments, the biological sample is a liquid biological sample. In embodiments, the biological sample is a blood sample. In embodiments, the blood sample is a serum sample or a plasma sample. In embodiments, the biological sample is a serum sample. In embodiments, the biological sample is a plasma sample. In embodiments, the biological sample is a stool sample.

[0131] In embodiments of the methods described herein, the cancer is any cancer. The skilled artisan would understand the type of cancer being detected, treated, or diagnosed in the methods described herein based on the oncogenic miRNA detected in the biological sample obtained from the patient. In embodiments, the cancer is a gastrointestinal cancer. In embodiments, the gastrointestinal cancer is colorectal cancer, stomach cancer, esophageal cancer, liver cancer, pancreatic cancer, a pancreatic neuroendocrine tumor, gallbladder cancer, anal cancer, small intestine cancer, a gastrointestinal stromal tumor, a gastrointestinal neuroendocrine tumor, or cholangiocarcinoma. In embodiments, the cancer is colorectal cancer. In embodiments, the colorectal cancer is colon cancer. In embodiments, the colorectal cancer is rectal cancer. In embodiments, the cancer is stomach cancer. In embodiments, the cancer is esophageal cancer. In embodiments, the cancer is liver cancer. In embodiments, the cancer is pancreatic cancer. In embodiments, the cancer is a pancreatic neuroendocrine tumor. In embodiments, the cancer is gallbladder cancer. In embodiments, the cancer is anal cancer. In embodiments, the cancer is small intestine cancer. In embodiments, the cancer is a gastrointestinal stromal tumor. In embodiments, the cancer is a gastrointestinal neuroendocrine tumor. In embodiments, the cancer is a cholangiocarcinoma. In embodiments, the cancer is colorectal cancer, stomach cancer, esophageal cancer, liver cancer, pancreatic cancer, a pancreatic neuroendocrine tumor, gallbladder cancer, anal cancer, small intestine cancer, a gastrointestinal stromal tumor, a gastrointestinal neuroendocrine tumor, cholangiocarcinoma, kidney cancer, lymphoma, breast cancer, prostate cancer, lung cancer, bladder cancer, non-Hodgkin’s lymphoma, melanoma,thyroid cancer, endometrial cancer, ovarian cancer, cervical cancer, pancreatic cancer, leukemia, brain cancer, myeloma, head and neck cancer, or testicular cancer.

[0132] Anti-Cancer Agents

[0133] In embodiments, the methods described herein comprise administering to a patient an effective amount of an anti-cancer agent. The anticancer treatment can be any drug known in the art as useful for treating cancer, such as chemotherapy, immunotherapy, or a combination thereof. In embodiments, the anti-cancer agent is a chemotherapeutic agent. The specific type of anti-cancer agent used in methods of treatment will be based on the ty pe of cancer being treated, as is known in the art.

[0134] In embodiments, the chemotherapeutic agent is an alkylating agent, an antimetabolite compound, an anthracy cline compound, an antitumor antibiotic, a platinum compound, a topoisomerase inhibitor, a vinca alkaloid, a taxane compound, an epothilone compound, or a combination of two or more thereof. In embodiments, the alkylating agent is carboplatin, chlorambucil, cyclophosphamide, melphalan, mechlorethamine, procarbazine, or thiotepa. In embodiments, the antimetabolite compound is azacitidine, capecitabine, cytarabine, gemcitabine, doxifluridine, hydroxyurea, methotrexate, pemetrexed, 6-thioguanine, 5- fluorouracil, or 6-mercaptopurine. In embodiments, the anthracy cline compound is daunorubicin, doxorubicin, idarubicin, epirubicin, or mitoxantrone. In embodiments, the antitumor antibiotic is actinomycin, bleomycin, mitomycin, or valrubicin. In embodiments, the platinum compound is cisplatin or oxaliplatin. In embodiments, the topoisomerase inhibitor is irinotecan, topotecan, amsacrine, etoposide, teniposide, or eribulin. In embodiments, the vinca alkaloid is vincristine, vinblastine, vinorelbine, or vindesine. In embodiments, the taxane compound is paclitaxel or docetaxel. In embodiments, the epothilone compound is epothilone, ixabepi lone, patupilone, or sagopilone. the chemotherapeutic agent comprises 5-fluorouracil, leucovorin, oxaliplatin, irinotecan, capecitabine. or a combination of two or more thereof.

[0135] In embodiments, the chemotherapeutic agent comprises 5-fluorouracil, leucovorin, oxaliplatin, irinotecan, capecitabine, or a combination of two or more thereof. In embodiments, the chemotherapeutic agent comprises everolimus, erlotinib, olaparib, mitomycin, sunitinib, gemcitabine, 5-fluorouracil, irinotecan, oxaliplatin, paclitaxel, capecitabine, cisplatin, docetaxel, or a combination of two or more thereof. In embodiments, the chemotherapeutic agent comprises 5-fluorouracil, oxaliplatin, irinotecan, capecitabine, or a combination of two or more thereof. In embodiments, the chemotherapeutic agent comprises gemcitabine, 5-fluorouracil, irinotecan, oxaliplatin, paclitaxel, capecitabine, cisplatin, docetaxel, or a combination of two or more thereof. In embodiments, the chemotherapeutic agent comprises gemcitabine. Inembodiments, the chemotherapeutic agent comprises 5 -fluorouracil. In embodiments, the chemotherapeutic agent comprises irinotecan. In embodiments, the chemotherapeutic agent comprises oxaliplatin. In embodiments, the chemotherapeutic agent comprises paclitaxel. In embodiments, the chemotherapeutic agent comprises capecitabine. In embodiments, the chemotherapeutic agent comprises cisplatin. In embodiments, the chemotherapeutic agent comprises docetaxel. In embodiments, the chemotherapeutic agent comprises further leucovorin.

[0136] “Chemotherapeutic” or “chemotherapeutic agent” is used in accordance with its plain ordinary meaning and refers to a chemical composition or compound having antineoplastic properties or the ability to inhibit the grow th or proliferation of cells.

[0137] “Anti-cancer agent” is used in accordance with its plain ordinary meaning and refers to a composition (e.g. compound, drug, antagonist, inhibitor, modulator) having antineoplastic properties or the ability to inhibit the grow th or proliferation of cells. In some embodiments, an anti-cancer agent is a chemotherapeutic. In embodiments, an anti-cancer agent is an agent identified herein having utility in methods of treating cancer. In embodiments, an anti-cancer agent is an agent approved by the FDA or similar regulatory agency of a country other than the USA, for treating cancer. Examples of anti-cancer agents include, but are not limited to, MEK (e.g. MEK1, MEK2, or MEK1 and MEK2) inhibitors (e.g. XL518, CI-1040, PD035901, selumetimb, trametimb, GDC-0973. ARRY-162, ARRY-300. AZD8330. PD0325901. U0126. PD98059, TAK-733, PD318088, AS703026, BAY 869766), alkylating agents (e g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan). ethylenimine and methylmelamines (e.g., hexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, semustine, streptozocin), triazenes (decarbazine)), anti-metabolites (e.g., 5- azathioprine, leucovorin, capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid analog (e.g.. methotrexate), or pyrimidine analogs (e.g., fluorouracil, floxouridine, cytarabine), punne analogs (e.g., mercaptopurine, thioguanine, pentostatin), etc.), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin. etc.), platinum-based compounds (e.g. cisplatin, oxaloplatin, carboplatin), anthracenedione (e.g., mitoxantrone), substituted urea (e.g., hydroxyurea), methyl hydrazine derivative (e.g., procarbazine), adrenocortical suppressant (e.g., mitotane, aminoglutethimide), epipodophyllotoxins (e.g.,etoposide), antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes (e.g., L- asparaginase), inhibitors of mitogen-activated protein kinase signaling (e.g. U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY 43-9006, wortmannin, or LY294002), mTOR inhibitors, antibodies (e.g., rituxan), 5-aza-2'-deoxy cytidine, doxorubicin, vincristine, etoposide, gemcitabine, imatinib, geldanamycin, 15-N-allylamino-15- demethoxygeldanamycin (15-AAG), bortezomib, trastuzumab, anastrozole; angiogenesis inhibitors; antiandrogen, antiestrogen; antisense oligonucleotides; apoptosis gene modulators; apoptosis regulators; arginine deaminase; BCR / ABL antagonists; beta lactam derivatives; bFGF inhibitor; bicalutamide; camptothecin derivatives; casein kinase inhibitors (ICOS); clomifene analogues; cytarabine dacliximab; dexamethasone; estrogen agonists: estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; finasteride; fludarabine; fluorodaunorunicin hydrochloride; gadolinium texaphyrin; gallium nitrate; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; immunostimulant peptides; insulin-like grow th factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; matrilysin inhibitors; matrix metalloproteinase inhibitors; MIF inhibitor; mifepristone; mismatched double stranded RNA; monoclonal antibody; mycobacterial cell wall extract; nitric oxide modulators; oxaliplatin; panomifene; pentrozole; phosphatase inhibitors; plasminogen activator inhibitor; platinum complex; platinum compounds; prednisone; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; ras famesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; ribozymes; signal transduction inhibitors; signal transduction modulators; single chain antigen-binding protein; stem cell inhibitor; stem-cell division inhibitors; stromelysin inhibitors; synthetic glycosaminoglycans; tamoxifen methiodide; telomerase inhibitors; thyroid stimulating hormone; translation inhibitors; tyrosine kinase inhibitors; urokinase receptor antagonists; steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, gonadotropin-releasing hormone agonists (GnRH) such as goserelin or leuprolide, adrenocorticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen), androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogen (e.g., flutamide), immunostimulants (e.g., Bacillus Calmette-Guerin, levamisole, interleukin-2, alpha-interferon, etc.), monoclonal antibodies (e.g., anti-CD20, anti-HER2. anti-CD52. anti-HLA-DR. and anti-VEGF monoclonal antibodies), immunotoxins (e g., anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-pseudomonas exotoxin conjugate, etc.), radioimmunotherapy (e.g., anti-CD20 monoclonal antibody conjugated toi nIn,90Y, or131I, etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin, epirubicin, topotecan, itraconazole, vindesine, cerivastatin, vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan, clofazimine, 5- nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib, gefitinib, EGFR inhibitors, epidermal growth factor receptor (EGFR)-targeted therapy or therapeutic (e.g. gefitinib. erlotinib, cetuximab, lapatinib. pamtumumab. vandetanib, afatimb. canertinib, neratinib. CP-724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, dacomitinib, desmethyl erlotinib, AZD8931, AEE788, pelitinib, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035. BMS-599626), sorafenib, imatinib, sunitinib, dasatinib. or the like.

[0138] Kits

[0139] Provided here are kits comprising components, such as reagents and reaction mixtures, to conduct the assays to detect the miRNA as described herein. As part of the kit, materials and instruction are provided, e.g., for storage and use of kit components. In embodiments, the kits comprise one or more of the following: a RNA probe that can hybridize to a RNA biomarker, pairs of primers that under appropriate reaction conditions can prime amplification of at least a portion of a RNA marker or a RNA encoding a polypeptide marker (e.g., by PCR). instructions on how to use the kit, and a label or insert indicating regulatory approval for diagnostic or therapeutic use. In embodiments, the kit further includes RNA microarrays comprising RNA of the disclosure or molecules which specifically bind to the RNA described herein. In embodiments, standard techniques of microarray technology are utilized to assess expression of the RNA. Polynucleotide arrays, particularly arrays that bind RNA described herein, also can be used for diagnostic applications.

[0140] Provided herein is a kit comprising reagents capable of detecting an expression level of reference RNA in a biological sample: wherein the RNA comprises one or more miRNA set forth in FIG. 3B. FIG. 4B. FIG. 5B, FIG. 6B, FIG. 7B, FIG. 8B, FIG. 9B, or FIG. 10B.

[0141] Provided herein is a kit comprising reagents capable of detecting an expression level of reference RNA in a biological sample: wherein the RNA comprises miR-15, miR-15b, miR-30e, miR-23a. miR-151a. miR-221. miR-186, miR-let-7g, miR-185. miR-146a, miR-191. miR-652, miR-93, miR-let-7d, miR-30e, miR-148b, miR-16, miR-let-7g, miR-142, miR-151b, miR-101, miR-23a, miR-30c, miR-423, or a combination of two or more thereof. In embodiments, the reference RNA comprises cell-free miR-15b-5p, cell-free miR-30e-5p, cell-free miR-23a-3p, cell-free miR-15 la-3p. cell-free miR-221-3p, cell-free miR-186-5p, cell-free miR-let-7g-5p, cell-free miR-I85-5p, cell-free miR-146a-5p, cell-free miR-I9I-5p, cell-free miR-652-3p, cell-free miR-93-5p, cell-free miR-let-7d-5p, exosomal miR-30e-5p, exosomal miR-15b-5p, exosomal miR-148b-3p, exosomal miR-23a-3p. exosomal miR-16-5p, exosomal miR-186-5p, exosomal miR-let-7g-5p, exosomal miR-142-5p, exosomal miR-151b, exosomal miR-101-3p, exosomal miR-30c-5p, exosomal miR 146a-5p, exosomal miR-423-3p, exosomal miR-652-3p, and exosomal miR-221-3p, or a combination of two or more thereof.

[0142] Provided herein is a kit comprising reagents capable of detecting an expression level of reference RNA in a biological sample; wherein the RNA comprises miR-15b, miR-23a, miR- 30e, or a combination of two or more thereof. In embodiments, the reference RNA comprises exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p. cell-free miR-30e-5p, or a combination of two or more thereof.

[0143] Provided herein is a kit comprising reagents capable of detecting: (i) an expression level of reference RNA in a biological sample; wherein the RNA comprises miR-15b, miR-23a, miR-30e. or a combination of two or more thereof, and (ii) an expression level of oncogenic RNA in a biological sample. In embodiments, the reference RNA comprises exosomal miR- 15b- 5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p, cell-free miR-30e-5p, or a combination of two or more thereof. In embodiments, the oncogenic RNA is any oncogenic RNA described herein.

[0144] '‘Assaying” or ‘'detecting” means using an analytic procedure to qualitatively assess or quantitatively measure the presence or amount or the functional activity of a target entity' (e.g., miRNA). For example, detecting the level of RNA (such as miRNA) means using an analytic procedure (such as an in vitro procedure) to qualitatively assess or quantitatively measure the presence or amount of the miRNA. In embodiments, raw expression values are normalized by performing quantile normalization relative to the reference distribution and subsequent log 10- transformation. In embodiments, when miRNA expression is detected using the nCounter® Analysis System marketed by Nanostring Technologies, the reference distribution is generated by pooling reported (i.e.. raw) counts for the test sample and one or more control samples (preferably at least 2 samples, more preferably at least any of 4, 8 or 16 samples) after excluding values for technical (both positive and negative control) probes and without performing intermediate normalization relying on negative (background-adjusted) or positive (synthetic sequences spiked with known titrations).

[0145] The terms ‘'probe” or “primer” refer to one or more nucleic acid fragments whose specific hybridization to a sample can be detected. A probe or primer can be of any length depending on the particular technique it will be used for. For example, PCR primers are generally between 10 and 40 nucleotides in length, while nucleic acid probes for, e.g., aSouthern blot, can be more than a hundred nucleotides in length. The probe or primers can be unlabeled or labeled as described below so that its binding to a target sequence can be detected (e.g., with a FRET donor or acceptor label). The probe or primer can be designed based on one or more particular (preselected) portions of a chromosome, e.g., one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase chain reaction (PCR) amplification products. One of skill can adjust these factors to provide optimum hybridization and signal production for a given hybridization and detection procedures, and to provide the required resolution among different genes or genomic locations.

[0146] Probes and primers can also be immobilized on a solid surface (e.g., nitrocellulose, glass, quartz, fused silica slides), as in an array. Techniques for producing high density arrays can also be used for this purpose. One of skill will recognize that the precise sequence of particular probes and primers can be modified from the target sequence to a certain degree to produce probes that are “substantially identical’' or “substantially complementary to” a target sequence, but retain the ability to specifically bind to (i.e., hybridize specifically to) the same targets from which they were derived.

[0147] The term “capable of hybridizing to” refers to a polynucleotide sequence that forms Watson-Crick bonds with a complementary sequence. One of skill will understand that the percent complementarity need not be 100% for hybridization to occur, depending on the length of the polynucleotides, length of the complementary region(e.g. 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more bases in length), and stringency of the conditions. For example, a polynucleotide (e.g., primer or probe) can be capable of binding to a polynucleotide having 60%. 65%. 70%. 75%. 80%. 85%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99% or 100% complementarity over the stretch of the complementary region.

[0148] In embodiments, methods include detecting a level of a biomarker w ith a specific binding agent (e.g., an agent that binds to a nucleic acid molecule). Exemplary binding agents include an antibody or a fragment thereof, a detectable protein or a fragment thereof, a nucleic acid molecule such as an oligonucleotide / polynucleotide comprising a sequence that is complementary' to patient genomic DNA, miRNA or a cDNA produced from patient mRNA, or any combination thereof. In embodiments, an antibody is labeled with detectable moiety, e.g., a fluorescent compound, an enzyme or functional fragment thereof, or a radioactive agent. In embodiments, an antibody is delectably labeled by coupling it to a chemiluminescent compound. In embodiments, the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of chemical reaction. Nonlimiting examples of useful chemiluminescent labeling compounds are luminol, isoluminol,theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

[0149] In embodiments, the subject matter provides a composition comprising a binding agent, wherein the binding agent is attached to a solid support, (e.g., a strip, a polymer, a bead, a nanoparticle, a plate such as a multiwell plate, or an array such as a microarray). In embodiments relating to the use of a nucleic acid probe attached to a solid support (such as a microarray), a nucleic acid in a test sample may be amplified (e.g., using PCR) before or after the nucleic acid to be measured is hybridized with the probe. In embodiments, reverse transcription polymerase chain reaction (RT-PCR) is used to detect miRNA levels. In embodiments, a probe on a solid support is used, and miRNA (or a portion thereol) in a biological sample is converted to cDNA or partial cDNA and then the cDNA or partial cDNA is hybridized to a probe (e.g., on a microarray), hybridized to a probe and then amplified, or amplified and then hybridized to a probe. In embodiments, a strip may be a nucleic acid-probe coated porous or non-porous solid support strip comprising linking a nucleic acid probe to a carrier to prepare a conjugate and immobilizing the conjugate on a porous solid support. In embodiments, the support or carrier comprises glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. In embodiments, the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present subject matter. In embodiments, the support material may have any structural configuration so long as the coupled molecule is capable of binding to a binding agent (e.g., an antibody). In embodiments, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. In embodiments, the surface may be flat such as a plate (or a well within a multiwell plate), sheet, test strip, polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

[0150] In embodiments, a solid support comprises a polymer, to which an agent is chemically bound, immobilized, dispersed, or associated. In embodiments, a polymer support may be, e.g., a network of polymers, and may be prepared in bead form (e.g., by suspension polymerization). In embodiments, the location of active sites introduced into a polymer support depends on the type of polymer support. In embodiments, in a swollen-gel-bead polymer support the active sites are distributed uniformly throughout the beads, whereas in a macroporous-bead polymer support they are predominantly on the internal surfaces of the macropores. In embodiments, the solid support, e.g., a device, may contain a biomarker binding agent alone or together with a binding agent for at least one, two, three or more other biomarkers.

[0151] In embodiments, the cells in a biological sample are lysed to release a protein or nucleic acid. Numerous methods for lysing cells and assessing protein and nucleic acid levels are known in the art. In embodiments, cells are physically lysed, such as by mechanical disruption, liquid homogenization, high frequency sound waves, freeze / thaw cycles, with a detergent, or manual grinding. Non-limiting examples of detergents include Tween 20, Triton X- 100, and sodium dodecyl sulfate (SDS). Non-limiting examples of assays for determining the level of a protein include HPLC. LC / MS, ELISA, immunoelectrophoresis. Western blot, immunohistochemistry, and radioimmunoassays. Non-limiting examples of assays for determining the level of an miRNA include Northern blotting, RT-PCR, RNA sequencing, and qRT-PCR.

[0152] In embodiments, once a suitable biological sample has been obtained, it is analyzed to quantitate the expression level of each of the biomarker genes. In embodiments, determining the expression level of a gene comprises detecting and quantifying RNA transcribed from that gene or a protein translated from such RNA. In embodiments, the RNA includes miRNA transcribed from the gene, and / or specific spliced variants thereof and / or fragments of such miRNA and spliced variants.

[0153] In embodiments, raw expression values are normalized to a reference miRNA. In embodiments, raw expression values are normalized by performing quantile normalization relative to the reference distribution and subsequent log 10-transformation. In embodiments, when the gene expression is detected using the nCounter® Analysis System marketed by NanoString® Technologies, the reference distribution is generated by pooling reported (i.e., raw) counts for the test sample and one or more control samples (preferably at least 2 samples, more preferably at least any of 4, 8 or 16 samples) after excluding values for technical (both positive and negative control) probes and without performing intermediate normalization relying on negative (background-adjusted) or positive (synthetic sequences spiked with known titrations).

[0154] A “detectable agent” or “detectable moiety” is a compound or composition detectable by appropriate means such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means. The RNA described herein and the expression level of the RNA described herein may be accomplished through the use of a detectable moiety in an assay or kit. A detectable moiety is a monovalent detectable agent or a detectable agent bound (e.g., covalently and directly or via a linking group) with another compound, e g., a nucleic acid. Exemplary' detectable agents / moieties for use in the present disclosure include an antibody ligand, a peptide, a nucleic acid, radioisotopes, paramagneticmetal ions, fluorophore (e.g. fluorescent dyes), electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, a biotin-avidin complex, a biotin-streptavidin complex, digoxigenin, magnetic beads, paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic iron oxide nanoparticles, ultrasmall superparamagnetic iron oxide nanoparticle aggregates, superparamagnetic iron oxide nanoparticles, superparamagnetic iron oxide nanoparticle aggregates, monocrystalline iron oxide nanoparticles, monocrystalline iron oxide, nanoparticle contrast agents, liposomes or other delivery vehicles containing Gadolinium chelate molecules, gadolinium, radionuclides, fluorodeoxy glucose, any gamma ray emitting radionuclides, positron-emitting radionuclide, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biocolloids, microbubbles, iodinated contrast agents, barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two- photon fluorophores, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide.

[0155] In embodiments, oligonucleotides in kits are capable of specifically hybridizing to a target region of a polynucleotide, such as for example, an RNA transcript or cDNA generated therefrom. As used herein, specific hybridization means the oligonucleotide forms an antiparallel double-stranded structure with the target region under certain hybridizing conditions, while failing to form such a structure with non-target regions when incubated with the polynucleotide under the same hybridizing conditions. The composition and length of each oligonucleotide in the kit will depend on the nature of the transcript containing the target region as well as the ty pe of assay to be performed with the oligonucleotide and is readily determined by the skilled artisan.

[0156] In embodiments, the kit comprises reagents capable of detecting an expression level of RNA from a biological sample; wherein the RNA is as described herein.

[0157] In embodiments, the disclosure provides a kit for detecting the RNA (e.g., miRNA) described herein. In embodiments, the kit is an assay system including any one of assay reagents, assay controls, protocols, exemplary' assay results, or combinations of these components designed to provide the user with means to evaluate the expression level of the RNA (e.g.. miRNA) described herein. In embodiments, the disclosure provides a kit for diagnosing cancer in a patent, including reagents for detecting miRNA markers in a biological (e.g., blood) sample from a patient.

[0158] In embodiments, the kits comprise one or more of the following: a RNA probe that can hybridize to a RNA biomarker, pairs of primers that under appropriate reaction conditions canprime amplification of at least a portion of a RNA marker or a RNA encoding a polypeptide marker (e.g., by PCR). instructions on how to use the kit, and a label or insert indicating regulatory approval for diagnostic or therapeutic use. In embodiments, the kit further includes RNA microarrays comprising RNA of the disclosure or molecules which specifically bind to the RNA described herein. In embodiments, standard techniques of microarray technology are utilized to assess expression of the RNA. Polynucleotide arrays, particularly arrays that bind RNA described herein, also can be used for diagnostic applications.

[0159] miRNA Expression

[0160] In embodiments of the methods described herein, the level of gene expression is a level of RNA (e.g., miRNA) expression. Levels of gene expression can be determined by methods known in the art, such as those described herein. In embodiments, the RNA is miRNA. In embodiments, RNA expression is detected by direct digital counting of nucleic acids, RNA sequencing (RNA-seq), quantitative reverse transcriptase polymerase chain reaction (RT-qPCR), quantitative polymerase chain reaction (qPCR), multiplex qPCR, microarray analysis, or a combination thereof. In embodiments, RNA expression is detected by RNA sequencing. RNA sequencing is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample. In embodiments, the gene expression level is an average of the gene expression level of the biomarker genes. In embodiments, the average of the gene expression level of the biomarker genes is an average of the normalized gene expression level of the biomarker genes. In embodiments, the gene expression level of the biomarker genes is a median of the gene expression level of the biomarker genes. In embodiments, the median of the gene expression level of the biomarker genes is a median of a normalized gene expression level of the biomarker genes. In embodiments, the gene expression level of the biomarker genes is the gene expression level of the biomarker genes normalized to a reference gene (e.g., reference miRNA as described herein).

[0161] In embodiments of the methods described herein, the individual expression level of the miRNA described herein are used. In embodiments, the individual expression level of the exosomal miRNA described herein are used. In embodiments, the individual expression level of the cell-free miRNA described are used. In embodiments, the individual expression level of the cell-free miRNA and exosomal miRNA described herein are used.

[0162] In embodiments, the expression levels of the oncogenic miRNA are weighted and combined to form a risk score. In embodiments, the expression levels of the oncogenic exosomal RNA described herein are weighted and combined to form a risk score. In embodiments, the expression levels of the oncogenic cell-free miRNA described herein are weighted andcombined to form a risk score. In embodiments, the elevated expression levels of the oncogenic cell-free and exosomal miRNA described herein are weighted and combined to form a risk score. In embodiments, the expression levels of the oncogenic miRNA are normalized to the expression level of reference miRNA (as described herein), and the normalized expression levels of the oncogenic miRNA (cell-free miRNA and / or exosomal miRNA) are weighted.

[0163] Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as the control (e.g., healthy individuals). The 2tmethod is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The Ct (threshold cycle) method quantification was used for the evaluation of the expression level of each miRNA. The threshold cycle (Ct) is defined as the PCR cycle at which the fluorescent signal of the reporter dye crosses an arbitrarily placed threshold. This method allows to quantify the absolute expression of each miRNAs in each sample analyzed and then to calculate the different expression of each miRNA in sample versus the controls. These expression values of the oncogenic RNA can be used individually to produce a risk score, can be added together to produce a risk score, or logistic regression analysis can be applied to produce a risk score based on weighted values of the expression levels of the oncogenic RNA.

[0164] In embodiments, the disclosure provides methods of processing miRNA expression data generated from the expression levels of the miRNA in the biological sample obtained from a patient as described herein, for establishing the presence of a signature indicative of cancer), comprising the steps of (i) normalizing numeric values of the oncogenic RNA expression data (e.g., the exosomal miRNA expression data and / or cell-free miRNA expression data), (ii) refining the discriminatory power of individual oncogenic miRNA by statistically weighting some of the numeric values associated therewith, and (iii) summating the numeric values obtained from step (ii) to provide a composite expression score. In embodiments, the composite expression score obtained from step (iii) is compared to reference RNA and the comparison allows the sample to be designated as positive or negative for cancer. In embodiments, the composite expression score is normalized. In embodiments, the composite expression score is scaled. In embodiments, the composite expression score is weighted. Weighted refers to the relevant value being adjusted to more appropriately reflect its contribution to the profile. In embodiments, the expression of level of each miRNA is calculated using 2'ACtmethod, the normalized expression values are log10transformed.

[0165] Computer-Implemented System

[0166] FIG. 11 sets forth an illustrative system 100 including a miRNA detection device 110 communicatively coupled to a computing system 102. miRNA detection device 110 is coupled to computing system 102 either directly (e.g., through one or more communication cables) or through network 130, which may be the internet or any other combination of wide-area, localarea, wired, and / or wireless networks. In embodiments, computing system 102 is included in or integrated with the miRNA detection device 110. In embodiments, miRNA detection device 110 processes a biological sample and produces data regarding the expression level of predetermined miRNA. The data can be sent to computing system 102 (e.g., through network 130) or stored on a storage device and at a later stage transferred to computing system 102 (e.g., through network 130). In embodiments, computing system 102 may or may not include a display 108 and one or more input devices (not illustrated) for receiving commands from a user or operator (e.g., a technician). In embodiments, computing system 102 and / or miRNA detection device 1 10 is accessed by users or other devices remotely through network 130. Thus, in embodiments, the methods described herein run remotely on computing system 102.

[0167] In embodiments, computing system 102 includes one computing device or a combination of a number of computing devices of any type, such as personal computers, laptops, network servers (e.g., local servers or servers included on a public / private / hybrid cloud), mobile devices, etc., where some or all of the devices are interconnected. In embodiments, computing system 102 includes one or more processors (not illustrated), each of which has one or more cores. In embodiments, computing system 102 includes one or more general-purpose processors (e.g., CPUs), special-purpose processors such as graphics processors (GPUs), digital signal processors, or any combination of these and other types of processors. In embodiments, some or all processors in computing system are implemented using customized or customizable circuitry, such as application specific integrated circuits (ASICs) or field programmable gate arrays (FPGAs). In embodiments, computing system 102 retrieves and executes non-transitory, computer-readable instructions stored in one or more memories or storage devices (not illustrated) integrated into or otherwise communicatively coupled to computing system 102. The memory / storage devices include any combination of non-transitory computer readable storage media including semiconductor memory chips of various types (DRAM, SRAM, SDRAM, flash memory, programmable read-only memory7) and so on. In embodiments, magnetic and / or optical disks are used. In embodiments, the memory / storage devices include removable storage media that is readable and / or writeable: examples of such media include compact disc (CD), read-only digital versatile disc (e.g., DVD-ROM, dual-layer DVD-ROM), read-only and recordable Blu- ray® disks, ultra-density' optical disks, flash memory' cards (e.g., SD cards, mini-SD cards,micro-SD cards, etc.), and so on. In embodiments, data and other information (e.g. sequencing data) is stored in one or more remote locations, e.g.. cloud storage, and synchronized with other the components of system 100.

[0168] FIG. 12 illustrates a functional block diagram of a machine in the example form of a computer system which comprises instructions for causing the machine to perform any of the methods described herein. In examples, the machine is connected (e.g.. networked) to other machines as described above. In embodiments, the machine operates in the capacity of a server or a client machine in a client-server network environment, or as a peer machine in a peer-to- peer (or distributed) network environment. In embodiments, the machine is any special-purpose machine capable of executing a set of instructions (sequential or otherwise) that specify actions to be taken by that machine for performing the functions describe herein. Further, while only a single machine is illustrated, the term “machine” includes any collection of machines that individually or jointly execute a set (or multiple sets) of instructions to perform any one or more of the methodologies discussed herein. In embodiments, the computing system 102 of FIG. 11 is implemented by the example machine shown in FIG. 12 (or a combination of two or more of such machines).

[0169] Example computer system 400 may include processing device 403, memory 407. data storage device 409 and communication interface 415. which communicate with each other via data and control bus 401 . In embodiments, computer system 400 includes display device 413 and / or user interface 411. In embodiments, the user interface 411 includes a graphical user interface.

[0170] Processing device 403 includes, without limitation, a microprocessor, a central processing unit, an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), a digital signal processor (DSP) and / or a network processor. Processing device 403 is configured to execute processing logic 405 for performing the methods described herein. In general, processing device 403 includes any suitable special-purpose processing device specially programmed with processing logic 405 to perform the methods described herein.

[0171] Memory 407 includes, for example, without limitation, at least one of a read-only memory (ROM), a random access memory (RAM), a flash memory, a dynamic RAM (DRAM) and a static RAM (SRAM), storing computer-readable instructions 417 executable by processing device 403. In general, memory 407 includes any suitable non-transitory computer readable storage medium storing computer-readable instructions 417 executable by processing device 403 for performing the methods described herein. Although one memory device 407 is illustrated in FIG. 12, in some examples, computer system 400 includes two or more memory devices (e.g..dynamic memory and static memory).

[0172] In embodiments, computer system 400 includes communication interface device 415, for direct communication with other computers (including wired and / or wireless communication), and / or for communication with a network. In embodiments, computer system 400 includes display device 413 (e.g., a liquid crystal display (LCD), a touch sensitive display, etc.). In embodiments, computer system 400 includes user interface 411 (e.g., an alphanumeric input device, a cursor control device, etc.).

[0173] In embodiments, computer system 400 includes data storage device 409 storing instructions (e.g., software) for performing any one or more of the methods described herein. Data storage device 409 includes any suitable non-transitory computer-readable storage medium, including solid-state memories, optical media and magnetic media.

[0174] Embodiments 1 to 60

[0175] Embodiment 1. A method of detecting an expression level of RNA in a patient with cancer, the method comprising detecting an expression level of RNA in the biological sample, wherein the RNA comprises miR-15b-5p, miR-23a-3p, miR-30e-5p, or a combination of two or more thereof.

[0176] Embodiment 2. A method of detecting an expression level of an oncogenic RNA in a patient with cancer, the method comprising: (i) detecting an expression level of the oncogenic RNA in a biological sample obtained from the patient with cancer; (ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises miR- 15b-5p, miR-23a-3p, miR-30e-5p, or a combination of two or more thereof; and (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; thereby detecting the normalized expression level of the oncogenic RNA.

[0177] Embodiment 3. A method of diagnosing a patient with cancer, the method comprising: (i) detecting an expression level of an oncogenic RNA in a biological sample obtained from the patient: (ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises miR-15b-5p, miR-23a-3p, miR-30e-5p, or a combination of two or more thereof; (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA, thereby detecting the normalized expression level of the oncogenic RNA; and (iv) diagnosing the patient as having cancer when the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA.

[0178] Embodiment 4. The method of any one of Embodiments 1 to 3, further comprising administering to the patient an effective amount of an anti-cancer agent.

[0179] Embodiment 5. A method of treating cancer in a patient in need thereof, the method comprising: (i) detecting an expression level of an oncogenic RNA in a biological sample obtained from the patient; (ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises miR-15b-5p, miR-23a-3p, miR-30e-5p, or a combination of two or more thereof; (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; wherein the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA; and (iv) administering to the patient an effective amount of an anti-cancer agent.

[0180] Embodiment 6. A computer-implemented method of administering an effective amount of an anti-cancer agent to a patient with cancer, the method comprising: (i) obtaining a sample data set comprising an expression level of oncogenic RNA from a biological sample obtained from the patient with cancer; (ii) obtaining a reference data set comprising an expression level of a reference RNA from the biological sample, wherein the reference RNA comprises miR-15b- 5p, miR-23a-3p, miR-30e-5p, or a combination of two or more thereof; (iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; (iv) determining an elevated expression level of the normalized expression level of the oncogenic RNA; and (v) administering to the patient an effective amount of the anti-cancer agent based on the determined normalized expression level.

[0181] Embodiment 7. A computer-implemented system for administering an effective amount of an anti-cancer agent to a patient with cancer, the computer-implemented system comprising: (i) a computer-readable medium holding a control data set of normalized expression levels of oncogenic RNA from a population of patients that do not have cancer; (ii) a processing device: (iii) a computer-readable medium containing programming instructions that are configured to instruct the processing device to: (a) receive a sample data set comprising an expression level of oncogenic RNA from a biological sample obtained from a patient with cancer; (b) receiving a reference data set comprising an expression level of a reference RNA from the biological sample, wherein the reference RNA comprises miR-15b-5p, miR-23a-3p, miR-30e-5p, or a combination of two or more thereof; (a) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; (b) receiving the control data set of normalized expression levels of oncogenic RNA from the population of patients that do not have cancer; and (c) determining an elevated expression level of the normalized expression level of the oncogenic RNA in the sample data set when compared to the normalized expression level of oncogenic RNA in the control data set; and (i) administering to the patient an effective amount of the anti-cancer agent based on the elevated expression level of the normalizedexpression level of the oncogenic RNA in the sample data set received from the computer- readable medium.

[0182] Embodiment 8. The method of any one of Embodiments 2 to 7, wherein the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA.

[0183] Embodiment 9. The method of of any one of Embodiments 2 to 7, wherein the normalized expression level of the oncogenic RNA is reduced relative to the expression level of the reference RNA.

[0184] Embodiment 10. The method of any one of Embodiments 1 to 9, wherein the reference RNA comprises exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p, cell-free miR-30e-5p, or a combination of two or more thereof.

[0185] Embodiment 11. The method of any one of Embodiments 1 to 9, wherein the reference RNA comprises exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p, and cell-free miR-30e-5p.

[0186] Embodiment 12. The method of any one of Embodiments 1 to 9, wherein the reference RNA consists of exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p, and cell-free miR-30e-5p.

[0187] Embodiment 13. The method of any one of Embodiments 1 to 9, wherein the reference RNA comprises exosomal miR-15b-5p, exosomal miR-23a-3p, and exosomal miR-30e-5p.

[0188] Embodiment 14. The method of any one of Embodiments 1 to 9, wherein the reference RNA consists of exosomal miR-15b-5p, exosomal miR-23a-3p. and exosomal miR-30e-5p.

[0189] Embodiment 15. The method of any one of Embodiments 1 to 9, wherein the reference RNA comprises cell free miR-15b-5p, cell-free miR-23a-3p, and cell-free miR-30e-5p.

[0190] Embodiment 16. The method of any one of Embodiments 1 to 9, wherein the reference RNA consists of cell free miR-15b-5p, cell-free miR-23a-3p, and cell-free miR-30e-5p.

[0191] Embodiment 17. The method of any one of Embodiments 1 to 16, wherein the oncogenic RNA is oncogenic miRNA.

[0192] Embodiment 18. The method of any one of Embodiments 1 to 17, wherein the oncogenic RNA comprises miR-32, miR-625, miR-486, miR-550a, miR-30d-5p, or a combination of two or more thereof; or wherein the oncogenic RNA comprises exosomal miR- 32-5p, cell-free miR-625-3p, exosomal miR-486-3p, exosomal miR-550a-3-5p, exosomal miR- 625-3p, cell-free miR-30d-5p, or a combination of two or more thereof.

[0193] Embodiment 19. The method of any one of Embodiments 1 to 17, wherein the oncogenic RNA comprises miR-4659b, miR-296, miR-4685, miR-550a, miR-4446, miR-432, miR-151a, miR-199a, miR-146a, miR-24, miR-223, miR-556, miR-2355, miR-181a, miR-3120, miR-7-1, miR-99b, miR-425, or a combination of two or more thereof; or wherein the oncogenic RNA comprises cell-free miR-4659b-3p, cell-free miR-296-5p, cell-free miR-4685-3p, cell-free miR-550a-5p, cell-free miR-4446-3p, cell-free miR-432-5p, cell-free miR-151a-3p, cell-free miR-199a-3p, cell-free miR-146a-5p, cell-free miR-24-3p, cell-free miR-223-3p. cell-free miR- 4659b-3p, cell-free miR-296-5p, exosomal miR-556-3p, exosomal miR-2355-5p, exosomal miR-181a-3p, exosomal miR-3120-3p, exosomal miR-7-l-3p, exosomal miR-99b-3p, exosomal miR-425-3p, or a combination of two or more thereof.

[0194] Embodiment 20. The method of any one of Embodiments 1 to 17, wherein the oncogenic RNA comprises miR-100, miR-184, miR-203a, miR-1290, miR-20a, miR-205, miR- 135b, miR-150, miR-433, miR-543, miR-199b, miR-18a, miR-19a, miR-4488, miR-200b, miR- 1229, miR-296, miR-34a, miR-15, miR-5193. miR-1228, miR-141, miR-107, miR-195, miR- 142, miR-3913, miR-1538, miR-1323, miR-877, miR-19b, or a combination of two or more thereof; or wherein the oncogenic RNA comprises exosomal miR-100-5p, exosomal miR-184, exosomal miR-203a-3p, exosomal miR-1290, exosomal miR-20a-5p, exosomal miR-205-5p, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-433-3p, exosomal miR-543, exosomal miR-199b-5p, exosomal miR-18a-5p. exosomal miR-19a-3p. exosomal miR-4488, cell-free miR-200b-3p, cell-free miR-1229-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell- free miR-15-5p, cell-free miR-5193, cell-free miR-1228-5p, cell-free miR-141-3p, cell-free miR-107, cell-free miR-195-5p, cell-free miR-142-3p, cell-free miR-3913-5p, cell-free miR- 1538, cell-free miR-1323, cell-free miR-877-3p, cell-free miR-19b-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof.

[0195] Embodiment 21. The method of any one of Embodiments 1 to 17, whrein the oncogenic RNA comprises miR-30c, miR-142, miR-340, miR-335. miR-1260b, miR-145, miR- 200a, miR-200b, miR-429, or a combination of two or more thereof; or wherein the oncogenic RNA comprises cell-free miR-30c-5p, cell-free miR-142-3p, cell-free miR-340-5p, cell-free miR-335-5p, exosomal miR-1260b, exosomal miR-145-5p, exosomal miR-200a-3p, exosomal miR-200b-3p, exosomal miR-429, exosomal miR-145-3p, or a combination of two or more thereof.

[0196] Embodiment 22. The method of any one of Embodiments 1 to 17, wherein the oncogenic RNA comprises miR-23b, miR-216b, miR-217, let-7e, miR-26a, miR-223, miR-340, miR-1260a, miR-141, miR-143, let-7f, miR-369.miR-125a, miR-495, miR-375, miR-199a, or a combination of two or more thereof; or wherein the oncogenic RNA comprises cell-free miR-23b-3p, exosomal miR-216b-5p, exosomal miR- 217-5p, cell-free let-7e-5p, cell-free miR-26a-5p, cell-free miR-223-3p, cell-free miR-340-3p, exosomal miR-1260a, exosomal miR-141-3p, exosomal miR-143-3p, exosomal miR-148a-3p, exosomal miR-200c-3p, exosomal miR-216a-5p, exosomal miR-34a-5p, cell-free let-7f-5p, cell- free miR-369-3p, cell-free miR-125a-5p, cell-free miR-495-3p, exosomal miR-375-3p, exosomal miR-199a-5p, or a combination of two or more thereof.

[0197] Embodiment 23. The method of any one of Embodiments 1 to 17, wherein the oncogenic RNA comprises miR-21, miR-215, miR-335, miR-27a, miR-95, miR-181b, miR-431. miR-1246, miR-192. miR-196a, miR-183. miR-135b. or a combination of two or more thereof; or wherein the oncogenic RNA comprises cell-free miR-21-3p, cell-free miR-21-5p, cell-free miR-215-5p, cell-free miR-335-3p, cell-free miR-27a-3p, cell-free miR-95-3p, cell-free miR- 181b-5p, cell-free miR-431-5p, exosomal miR-21-3p, exosomal miR-21-5p, exosomal miR- 1246, exosomal miR-192-3p, exosomal miR-215-5p, exosomal miR-27a-3p, exosomal miR-95- 3p, exosomal miR-196a-5p, exosomal miR-183-5p, exosomal miR-135b-5p, or a combination of two or more thereof.

[0198] Embodiment 24. The method of any one of Embodiments 1 to 17, wherein the oncogenic RNA comprises miR-513a. miR-628, miR-193a. miR-210, miR-4304. miR-194, miR- 4453, or a combination of two or more thereof; or wherein the oncogenic RNA comprises miR- 513a-5p, miR-628-3p, miR-193a-5p, miR-210, miR-4304, miR-194-3p, miR-4453, or a combination of two or more thereof.

[0199] Embodiment 25. The method of any one of Embodiments 1 to 17, wherein the oncogenic RNA comprises miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA or a combination of two or more thereof; or wherein the oncogenic RNA comprises exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411. cell-free miR-181b, cell-free miR-193b. cell-free miR- 195, cell-free miR-411, or a combination of two or more thereof.

[0200] Embodiment 26. The method of any one of Embodiments 1 to 17, wherein the oncogenic RNA comprises miR-21, miR-101-1. miR-101-2, miR-143, miR-145, miR-16-1, miR-26Al, miR-26a2, miR-29C, miR-155, miR-30A, miR-100, miR-106b, miR-lOb, miR-126, miR-15a, miR-16-2, miR-17, miR-181al, miR-181a2, miR-199al, miR-199a2, miR-19a, miR- 205, miR-221, miR-29bl, miR-29b2, miR-30e, miR-31, miR-34a, miR-99a, miR-let-7b, miR- let-7c, miR-133al, miR-133a2, miR-141, miR-183, miR-195. miR-200a, miR-200b, miR-200c, miR-203a, miR-20a, miR-222,miR-7-3, miR-9-1, miR-9-2, miR-92al, miR-92a2, miR-9-3, miR-let-7al, miR-let-7a2, miR-let- 7a3, miR-1-1, miR-1-2, miR-125a, miR-125bl, miR-125b2, miR-139, miR-140, miR-142. miR- 150, miR-15b, miR-181bl, miR-181b2, miR-182, miR-18a, miR-19bl, miR-19b2, miR-214, miR-223, miR-25, miR-27a, miR-375, miR-let-7d, miR-let-fl, miR-let-7f2, miR-122, miR-128- 1, miR- 128-2, miR-146A, miR-192, miR- 194-1, miR- 194-2, miR-210, miR-224, miR-23a, miR- 26b, miR-27b, miR-301a, miR-32, miR-335, miR-342, miR-497, miR-96, miR-let-7e, miR-let- 71, miR-103al. miR-103a2, miR-107, miR-lOa. miR-124-1, miR-124-2, miR-124-3. miR-130b, miR-133b, miR-144, miR-148a, miR-152, miR-193b, miR-199b, miR-204, miR-218-1, miR- 218-2, miR-23b, miR-24-1, miR-24-2, miR-28, miR-30b, miR-30d, miR-324, miR-378a, miR- 93, miR-let-7g, miR-129-1, miR-129-2, miR-130a, miR-135b, miR-137, miR-146b, miR-181c, miR-193A. miR-22, miR-34b. miR-34c. miR-423, miR-455, miR-106a, miR-132, miR-135al. miR-135a2, miR-138-1, miR-138-2, miR-148b, miR-149, miR-206, miR-215, miR-320a, miR- 331, miR-345, miR-373, miR-449a, miR-483, miR-486-1, miR-486-2, miR-494, miR-625, miR- 98, miR-99b, or a combination of two or more thereof.

[0201] Embodiment 27. The method of Embodiment 26, wherein the oncogenic RNA is exosomal miRNA, cell-free miRNA, or a combination thereof.

[0202] Embodiment 28. The method of any one of Embodiments 1 to 27, wherein the cancer is a gastrointestinal cancer.

[0203] Embodiment 29. The method of Embodiment 28, wherein the gastrointestinal cancer is colorectal cancer, stomach cancer, esophageal cancer, liver cancer, pancreatic cancer, a pancreatic neuroendocrine tumor, gallbladder cancer, anal cancer, small intestine cancer, a gastrointestinal stromal tumor, a gastrointestinal neuroendocrine tumor, or cholangiocarcinoma.

[0204] Embodiment 30. The method of any one of Embodiments 1 to 27, wherein the cancer is colorectal cancer.

[0205] Embodiment 31. The method of Embodiment 30, wherein the colorectal cancer is colon cancer.

[0206] Embodiment 32. The method of Embodiment 30, wherein the colorectal cancer is rectal cancer.

[0207] Embodiment 33. The method of any one of Embodiments 1 to 27, wherein the cancer is stomach cancer.

[0208] Embodiment 34. The method of any one of Embodiments 1 to 27, wherein the cancer is esophageal cancer.

[0209] Embodiment 35. The method of any one of Embodiments 1 to 27, wherein the canceris liver cancer.

[0210] Embodiment 36. The method of any one of Embodiments 1 to 27, wherein the cancer is pancreatic cancer.

[0211] Embodiment 37. The method of any one of Embodiments 1 to 27, wherein the cancer is a pancreatic neuroendocrine tumor.

[0212] Embodiment 38. The method of any one of Embodiments 1 to 27, wherein the cancer is gallbladder cancer.

[0213] Embodiment 39. The method of any one of Embodiments 1 to 27, wherein the cancer is anal cancer.

[0214] Embodiment 40. The method of any one of Embodiments 1 to 27, wherein the cancer is small intestine cancer.

[0215] Embodiment 41. The method of any one of Embodiments 1 to 27, wherein the cancer is a gastrointestinal stromal tumor.

[0216] Embodiment 42. The method of any one of Embodiments 1 to 27, wherein the cancer is a gastrointestinal neuroendocrine tumor.

[0217] Embodiment 43. The method of any one of Embodiments 1 to 27, wherein the cancer is a cholangiocarcinoma.

[0218] Embodiment 44. The method of any one of Embodiments 1 to 27, wherein the cancer is colorectal cancer, stomach cancer, esophageal cancer, liver cancer, pancreatic cancer, a pancreatic neuroendocrine tumor, gallbladder cancer, anal cancer, small intestine cancer, a gastrointestinal stromal tumor, a gastrointestinal neuroendocrine tumor, cholangiocarcinoma, kidney cancer, lymphoma, breast cancer, prostate cancer, lung cancer, bladder cancer, nonHodgkin's lymphoma, melanoma, thyroid cancer, endometrial cancer, ovarian cancer, cervical cancer, pancreatic cancer, leukemia, brain cancer, myeloma, head and neck cancer, or testicular cancer.

[0219] Embodiment 45. The method of Embodiment 18, 19, 20, 24, or 25, wherein the cancer is colorectal cancer.

[0220] Embodiment 46. The method of Embodiment 21 or 22, wherein the cancer is pancreatic cancer.

[0221] Embodiment 47. The method of Embodiment 23, wherein the cancer is gastric cancer.

[0222] Embodiment 48. The method of any one of Embodiments 1 to 47, wherein the biological sample is a liquid biological sample.

[0223] Embodiment 49. The method of any one of Embodiments 1 to 47, wherein thebiological sample is a blood sample.

[0224] Embodiment 50. The method of any one of Embodiments 1 to 47, wherein the biological sample is a plasma sample.

[0225] Embodiment 51. The method of any one of Embodiments 1 to 47, wherein the biological sample is a serum sample.

[0226] Embodiment 52. The method of any one of Embodiments 1 to 47, wherein the biological sample is a tissue sample.

[0227] Embodiment 53. The method of any one of Embodiments 1 to 50, wherein the patient is a human patient.

[0228] Embodiment 54. The method of any one of Embodiments 4 to 51 , wherein the anticancer agent is a chemotherapeutic agent.

[0229] Embodiment 55. The method of Embodiment 54, wherein the chemotherapeutic agent is an alkylating agent, an antimetabolite compound, an anthracycline compound, an antitumor antibiotic, a platinum compound, a topoisomerase inhibitor, a vinca alkaloid, a taxane compound, an epothilone compound, or a combination of two or more thereof.

[0230] Embodiment 56. The method of Embodiment 55, wherein the alkylating agent is carboplatin, chlorambucil, cyclophosphamide, melphalan, mechlorethamine, procarbazine, or thiotepa; the antimetabolite compound is azacitidine, capecitabine, cytarabine, gemcitabine, doxifluridine, hydroxyurea, methotrexate, pemetrexed, 6-thioguanine, 5-fluorouracil, or 6- mercaptopurine; the anthracycline compound is daunorubicin, doxorubicin, idarubicin, epirubicin, or mitoxantrone; the antitumor antibiotic is actinomycin, bleomycin, mitomycin, or valrubicin; the platinum compound is cisplatin or oxaliplatin; the topoisomerase inhibitor is irinotecan, topotecan, amsacrine, etoposide, teniposide, or eribulin; the vinca alkaloid is vincristine, vinblastine, vinorelbine, or vind esine; the taxane compound is paclitaxel or docetaxel; and the epothilone compound is epothilone, ixabepilone. patupilone, or sagopilone.

[0231] Embodiment 57. The method of any one of Embodiments 4 to 53, wherein the anticancer agent is an immune checkpoint inhibitor, a chemotherapeutic agent, or combination thereof.

[0232] Embodiment 58. A kit comprising reagents capable of detecting an expression level of RNA in a biological sample; wherein the RNA comprises exosomal miR-15b-5p, cell free miR- 15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p, cell-free miR-30e- 5p, or a combination of two or more thereof.

[0233] Embodiment 59. The kit of Embodiment 58, wherein the RNA comprise exosomalmiR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR- 30e-5p. and cell-free miR-30e-5p.

[0234] Embodiment 60. The kit of Embodiment 58, wherein the RNA consist of exosomal miR-15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR- 30e-5p. and cell-free miR-30e-5p.EXAMPLES

[0235] The inventors sough to identify suitable endogenous reference genes (e g., reference RNA) for circulating miRNA quantification in cancer detection using small RNA sequencing data. Expression stabilities of the reference genes were evaluated using NormFinder as described, for example, by Andersen et al, Cancer Research, 64:5245-5250 (2004), the disclosure of which is incorporated by reference herein. The strategy was to identify reference genes with low overall variation and with low variation between sample subgroups of the sample set, e.g., control vs. cancer and across different types of cancer.

[0236] Example 1

[0237] The study design is shown in FIG. 1 for cell-free miRNA and FIG. 2 for exosomal miRNA. Control refers to a healthy human patient (i.e., a patient that does not have cancer). “GC” is gastric cancer. “PDAC” is pancreatic ductal adenocarcinoma. “CRC” is colorectal cancer.

[0238] The analysis for cell-free RNA where the comparison is between control and cancer is shown in FIG. 3A and the results are shown in FIG. 3B. The top ten candidates for reference cell-free miRNA included miR-15b, miR-30e, miR-23a, miR-151a, miR-221, miR-186, miR-let- 7g, miR-185. miR-146a, and miR-191. More particularly, the top ten candidates were cell-free miR-15b-5p. cell-free miR-30e-5p, cell-free miR-23a-3p. cell-free miR-151a-3p. cell-free miR- 221-3p, cell-free miR-186-5p, cell-free miR-let-7g-5p, cell-free miR-185-5p, cell-free miR- 146a-5p, and cell-free miR-191-5p.

[0239] The analysis for cell-free RNA where the comparison is between different types of cancers is shown in FIG. 4A and the results are shown in FIG. 4B. The top ten candidates for reference cell-free miRNA included miR-15, miR-30e, miR-23a, miR-151a, miR-652, miR- 146a, miR-93, miR-186, and miR-let-7d. More particularly, the top ten candidates for reference cell-free miRNA included cell-free miR-15b-5p, cell-free miR-30e-5p, cell-free miR-23a-3p, cell-free miR-151a-3p. cell-free miR-652-3p, cell-free miR-146a-5p. cell-free miR-93-5p. cell- free miR-186-5p, and cell-free miR-let-7d-5p.

[0240] The analysis for exosomal RNA where the comparison is between control and cancer isshown in FIG. 5A and the results are shown in FIG. 5B. The top ten candidates for reference exosomal miRNA included miR-30e, miR-15b, miR-148b, miR-23a, miR-16, miR-186, miR-let- 7g, miR-142, miR-151b, and miR-101. More particularly, the top ten candidates were exosomal miR-30e-5p, exosomal miR-15b-5p, exosomal miR-148b-3p, exosomal miR-23a-3p, exosomal miR-16-5p, exosomal miR-186-5p, exosomal miR4et-7g-5p, exosomal miR-142-5p, exosomal miR-151b, and exosomal miR-101-3p.

[0241] The analysis for exosomal RNA where the comparison is between different types of cancers is shown in FIG. 6A and the results are shown in FIG. 6B. The top ten candidates for reference exosomal miRNA included miR-23a, miR-30e, miR-15b. miR-30c, miR- 15 lb. miR 146a. miR-423, miR 101, miR-652, and miR-221. More particularly, the top ten candidates for reference exosomal miRNA included exosomal miR-23a-3p, exosomal miR-30e-5p, exosomal miR-15b-5p, exosomal miR-30c-5p, exosomal miR-151b, exosomal miR 146a-5p, exosomal miR-423-3p, exosomal miR 101-3p, exosomal miR-652-3p, and exosomal miR-221-3p.

[0242] Results

[0243] The results show that cell free-miR-23a-3p, cell-free miR-15b-5p, cell-free miR-30e- 5p, exosomal miR-23a-3p, exosomal miR-15b-5p, exosomal miR-30e-5p, and exosomal miR- 148b-3p were the most stable candidates in the sequencing dataset across healthy controls and cancer patients. The most stable overlapping reference RNA were miR-23a-3p, miR-15b-5p, and miR-30e-5p, which particularly included cell-free miR-23a-3p, cell-free miR-15b-5p, cell-free miR-30e-5p, exosomal miR-23a-3p, exosomal miR-15b-5p, and exosomal miR-30e-5p.

[0244] Example 2

[0245] The studies in Example 1 w ere reevaluated with additional new cases, in which the cell-free datasets included 158 patients having liver cancer (hepatocellular carcinoma or HCC) and 20 more patients having colorectal cancer (CRC). The exosomal datasets included 35 patients having esophageal cancer (esophageal squamous cell carcinoma or ESCC), 39 patients having cholangiocarcinoma (intrahepatic cholangiocarcinoma or ICC), and 183 patients having liver cancer (hepatocellular carcinoma or HCC).

[0246] The second analysis for cell-free RNA where the comparison is between control and cancer is shown in FIG. 7A and the results are shown in FIG. 7B. The top ten candidates for reference cell-free miRNA included cell-free miR-30e, cell-free miR- 15b, cell-free miR-1 et-7g, cell-free miR-let-7f, cell-free miR-26b, cell-free miR-142, cell-fee miR-126, cell-free miR-186, cell-free miR-103a, and cell-free miR-let-7d. More particularly, the top ten candidates were cell- free miR-30e-5p, cell-free miR-15b-5p, cell-free miR-let-7g-5p, cell-free miR-1 et-7f-5p, cell-free miR-26b-5p, cell-free miR-142-5p, cell-fee miR-126-3p, cell-free miR-186-5p, cell-free miR-103a-3p, and cell-free miR-let-7d-5p.

[0247] The second analysis for cell-free RNA where the comparison is between different types of cancers is shown in FIG. 8A and the results are shown in FIG. 8B. The top ten candidates for reference cell-free miRNA included cell-free miR-30e, cell-free miR-15b. cell- free miR-23a, cell-free miR-151a. cell-free miR-146a, cell-free miR-652. cell-fee miR-let-7g, cell-free miR-93, cell-free miR-186, and cell-free miR-let-7d. More particularly, the top ten candidates were cell-free miR-30e-5p, cell-free miR-15b-5p, cell-free miR-23a-3p, cell-free miR-151a-3p, cell-free miR-146a-5p, cell-free miR-652-3p, cell-fee miR-let-7g-5p, cell-free miR-93-5p, cell-free miR-186-5p, and cell-free miR-let-7d-5p.

[0248] The second analysis for exosomal RNA where the comparison is between control and cancer is shown in FIG. 9A and the results are shown in FIG. 9B. The top ten candidates for reference exosomal miRNA included exosomal miR-30e, exosomal miR-186, exosomal miR- let-7i, exosomal miR-142, exosomal miR-23a, exosomal miR-151b, exosomal miR-let-7g, exosomal miR-15b, exosomal miR-30c, and exosomal miR-16. More particularly, the top ten candidates were exosomal miR-30e-5p, exosomal miR-186-5p, exosomal miR-let-7i-5p, exosomal miR-142-5p, exosomal miR-23a-3p, exosomal miR-151b, exosomal miR-let-7g-5p, exosomal miR-15b-5p, exosomal miR-30c-5p, and exosomal miR-16-5p.

[0249] The second analysis for exosomal RNA where the comparison is between different types of cancers is shown in FIG. 10A and the results are shown in FIG. 10B. The top ten candidates for reference exosomal miRNA included exosomal miR-30e, exosomal miR-23a. exosomal miR- 15b, exosomal miR-423, exosomal miR-30c, exosomal miR 146a, exosomal miR-151b, exosomal miR 101, exosomal miR-185, and exosomal miR-186. More particularly, the top ten candidates for reference exosomal miRNA included exosomal miR-30e-5p, exosomal miR-23a-3p, exosomal miR-15b-5p. exosomal miR-423-3p, exosomal miR-30c-5p, exosomal miR 146a-5p, exosomal miR-151b, exosomal miR 101-3p, exosomal miR-185-5p. and exosomal miR-186-5p.

[0250] Results

[0251] The results show that cell-free miR-30e-5p, exosomal miR-30e-5p, cell-free miR-15b- 5p, exosomal miR-15b-5p, cell-free miR-23a-3p, and exosomal miR-23a-3p robustly expressed across different disease and different ty pes of cancer and batch subgroups in the sequencing datasets. Thus, miR-30e-5p, miR-15b-5p, and miR-23a-3p are reference miRNA for detection of diseases, such as cancer, in biological samples, such as blood.

[0252] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. The headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMSWhat is claimed is:

1. A method of detecting an expression level of reference RNA in a patient wi th cancer, the method comprising detecting an expression level of reference RNA in the biological sample, wherein the reference RNA comprises miR-15b, miR-23a, miR-30e, or a combination of two or more thereof.

2. A method of detecting an expression level of an oncogenic RNA in a patient with cancer, the method comprising:(i) detecting an expression level of the oncogenic RNA in a biological sample obtained from the patient with cancer;(ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises miR-15b, miR-23a, miR-30e, or a combination of two or more thereof; and(iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; thereby detecting the normalized expression level of the oncogenic RNA.

3. A method of diagnosing a patient with cancer, the method comprising:(i) detecting an expression level of an oncogenic RNA in a biological sample obtained from the patient;(ii) detecting an expression level of a reference RNA in the biological sample, wherein the reference RNA comprises miR-15b. miR-23a, miR-30e, or a combination of two or more thereof;(iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA, thereby detecting the normalized expression level of the oncogenic RNA; and(iv) diagnosing the patient as having cancer when the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA.

4. A method of treating cancer in a patient in need thereof, the method comprising:(i) detecting an expression level of an oncogenic RNA in a biological sample obtained from the patient;(ii) detecting an expression level of a reference RNA in the biological sample,wherein the reference RNA comprises miR-15b, miR-23a, miR-30e, or a combination of two or more thereof;(iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA; wherein the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA; and(iv) administenng to the patient an effective amount of an anti-cancer agent.

5. A method of treating cancer in a patient in need thereof the method comprising:(i) selecting a patient having a diagnosis of cancer based on an expression level of oncogenic RNA in a biological sample obtained from the patient, wherein the expression level of the oncogenic RNA is normalized to the expression level of reference RNA. wherein the normalized expression level of the oncogenic RNA is elevated or reduced relative to the expression level of the reference RNA, and wherein the reference RNA comprises miR-15b, miR-23a, miR-30e, or a combination of tw o or more thereof; and(ii) treating the patient from step (i) by administering to the patient an effective amount of an anti-cancer agent.

6. A computer-implemented method of administering an effective amount of an anti-cancer agent to a patient with cancer, the method comprising:(i) obtaining a sample data set comprising an expression level of oncogenic RNA from a biological sample obtained from the patient with cancer;(ii) obtaining a reference data set comprising an expression level of a reference RNA from the biological sample, wherein the reference RNA comprises miR-15b, miR-23a, miR-30e, or a combination of two or more thereof;(iii) normalizing the expression level of the oncogenic RNA to the expression level of the reference RNA;(iv) determining an elevated expression level of the normalized expression level of the oncogenic RNA; and(v) administering to the patient an effective amount of the anti-cancer agent based on the determined normalized expression level.

7. A computer-implemented system for administering an effective amount of an anti-cancer agent to a patient with cancer, the computer-implemented system comprising:(i) a computer-readable medium holding a control data set of normalized expression levels of oncogenic RNA from a population of patients that do not have cancer;(ii) a processing device:(iii) a computer-readable medium containing programming instructions that are configured to instruct the processing device to:(a) receive a sample data set comprising an expression level of oncogenic RNA from a biological sample obtained from a patient with cancer;(b) receive a reference data set comprising an expression level of a reference RNA from the biological sample, wherein the reference RNA comprises miR-15b, miR-23a, miR-30e. or a combination of two or more thereof;(c) normalize the expression level of the oncogenic RNA to the expression level of the reference RNA;(d) receive the control data set of normalized expression levels of oncogenic RNA from the population of patients that do not have cancer; and(e) determine an elevated expression level of the normalized expression level of the oncogenic RNA in the sample data set when compared to the normalized expression level of oncogenic RNA in the control data set.

8. The method of claim 2, wherein the normalized expression level of the oncogenic RNA is elevated relative to the expression level of the reference RNA.

9. The method of claim 2, wherein the normalized expression level of the oncogenic RNA is reduced relative to the expression level of the reference RNA.

10. The method of claim 1, wherein wherein the reference RNA comprises miR-15b- 5p, miR-23a-3p, miR-30e-5p, or a combination of two or more thereof11. The method of claim 9, wherein the reference RNA comprises exosomal miR- 15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e- 5p. cell-free miR-30e-5p, or a combination of two or more thereof.

12. The method of claim 9. wherein the reference RNA comprises exosomal miR- 15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e- 5p, and cell-free miR-30e-5p.

13. The method of claim 9, wherein the reference RNA consists of exosomal miR- 15b-5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e- 5p, and cell-free miR-30e-5p.

14. The method of claim 9, wherein the reference RNA comprises exosomal miR-15b-5p, exosomal miR-23a-3p, and exosomal miR-30e-5p.

15. The method of claim 9. wherein the reference RNA consists of exosomal miR- 15b-5p, exosomal miR-23a-3p, and exosomal miR-30e-5p.

16. The method of claim 9. wherein the reference RNA comprises cell free miR-15b- 5p, cell-free miR-23a-3p, and cell-free miR-30e-5p.

17. The method of claim 9. wherein the reference RNA consists of cell free miR-15b- 5p, cell-free miR-23a-3p, and cell-free miR-30e-5p.

18. The method of claim 1, wherein the oncogenic RNA is oncogenic miRNA.

19. The method of claim 17, wherein the oncogenic RNA comprises:(a) miR-32, miR-625, miR-486, miR-550a, miR-30d-5p, or a combination of two or more thereof(b) exosomal miR-32-5p, cell-free miR-625-3p, exosomal miR-486-3p, exosomal miR-550a-3-5p, exosomal miR-625-3p, cell-free miR-30d-5p, or a combination of two or more thereof.(c) miR-4659b, miR-296, miR-4685, miR-550a, miR-4446, miR-432, miR-151a, miR-199a, miR-146a, miR-24, miR-223, miR-556, miR-2355, miR-181a, miR-3120, miR-7-1, miR-99b, miR-425, or a combination of two or more thereof(d) cell-free miR-4659b-3p, cell-free miR-296-5p. cell-free miR-4685-3p, cell-free miR-550a-5p, cell-free miR-4446-3p, cell-free miR-432-5p, cell-free miR-151a-3p, cell-free miR-199a-3p, cell-free miR-146a-5p, cell-free miR-24-3p, cell-free miR-223-3p, cell-free miR- 4659b-3p, cell-free miR-296-5p. exosomal miR-556-3p, exosomal miR-2355-5p, exosomal miR-181a-3p, exosomal miR-3120-3p, exosomal miR-7-l-3p, exosomal miR-99b-3p, exosomal miR-425-3p, or a combination of two or more thereof.(e) miR-100, miR-184, miR-203a, miR-1290, miR-20a, miR-205, miR-135b, miR- 150, miR-433, miR-543, miR-199b, miR-18a, miR-19a, miR-4488, miR-200b, miR-1229, miR- 296, miR-34a, miR-15, miR-5193, miR-1228. miR-141, miR-107, miR-195, miR-142. miR- 3913, miR-1538, miR-1323, miR-877, miR-19b, or a combination of two or more thereof(f) exosomal miR-100-5p, exosomal miR-184, exosomal miR-203a-3p, exosomal miR-1290, exosomal miR-20a-5p, exosomal miR-205-5p, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-433-3p. exosomal miR-543, exosomal miR-199b-5p, exosomal miR-18a-5p, exosomal miR-19a-3p. exosomal miR-4488. cell-free miR-200b-3p, cell-free miR-1229-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-15-5p, cell-free miR-5193, cell-free miR-1228-5p, cell-free miR-141-3p, cell-free miR-107, cell-free miR-195-5p, cell-free miR-142-3p, cell-free miR-3913-5p, cell-free miR-1538, cell-free miR-1323, cell-free miR-877- 3p, cell-free miR-19b-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof.(g) miR-30c, miR-142. miR-340, miR-335, miR-1260b. miR-145, miR-200a. miR- 200b, miR-429, or a combination of two or more thereof;(h) cell-free miR-30c-5p, cell-free miR-142-3p, cell-free miR-340-5p, cell-free miR- 335-5p, exosomal miR-1260b, exosomal miR-145-5p, exosomal miR-200a-3p, exosomal miR- 200b-3p, exosomal miR-429, exosomal miR-145-3p, or a combination of two or more thereof.(i) miR-23b. miR-216b, miR-217. let-7e, miR-26a, miR-223. miR-340, miR-1260a, miR-141, miR-143, miR-148a, miR-200c, miR-216a, miR-34a, let-7f, miR-369, miR-125a, miR-495, miR-375, miR-199a, or a combination of two or more thereof;(j) cell-free miR-23b-3p, exosomal miR-216b-5p, exosomal miR-217-5p, cell-free let-7e-5p, cell-free miR-26a-5p, cell-free miR-223-3p, cell-free miR-340-3p, exosomal miR- 1260a, exosomal miR-141 -3p, exosomal miR-143-3p, exosomal miR-148a-3p, exosomal miR- 200c-3p, exosomal miR-216a-5p, exosomal miR-34a-5p, cell -free let-7f-5p, cell-free miR-369- 3p, cell-free miR-125a-5p, cell-free miR-495-3p, exosomal miR-375-3p, exosomal miR-199a- 5p, or a combination of two or more thereof.(k) miR-21, miR-215, miR-335, miR-27a. miR-95, miR-181b, miR-431, miR-1246, miR-192, miR-196a, miR-183, miR-135b, or a combination of two or more thereof;(l) cell-free miR-21-3p, cell-free miR-21-5p, cell-free miR-215-5p, cell-free miR- 335-3p, cell-free miR-27a-3p, cell-free miR-95-3p, cell-free miR-181b-5p, cell-free miR-431- 5p, exosomal miR-21-3p, exosomal miR-21-5p. exosomal miR-1246, exosomal miR-192-3p, exosomal miR-215-5p, exosomal miR-27a-3p, exosomal miR-95-3p, exosomal miR-196a-5p, exosomal miR-183-5p, exosomal miR-135b-5p, or a combination of two or more thereof.(m) miR-513a, miR-628, miR-193a, miR-210, miR-4304, miR-194, miR-4453, or a combination of two or more thereof;(n) miR-513a-5p, miR-628-3p, miR-193a-5p, miR-210, miR-4304, miR-194-3p, miR-4453, or a combination of two or more thereof.(o) miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA or a combination of two or more thereof;(p) exosomal miR-181b. exosomal miR-193b. exosomal miR-195, exosomal miR- 411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, cell-free miR-411, or acombination of two or more thereof.(q) miR-21, miR-101-1, miR-101-2, miR-143, miR-145, miR-16-1. miR-26Al, miR-26a2, miR-29C, miR-155, miR-30A, miR-100, miR-106b, miR-lOb, miR-126, miR-15a, miR- 16-2, miR-17, miR-181al, miR-181a2, miR-199al, miR-199a2, miR-19a, miR-205, miR-221, miR-29bl, miR-29b2, miR-30e, miR-31, miR-34a, miR-99a, miR-let-7b, miR-let-7c, miR- 133al, miR-133a2, miR-141, miR-183, miR-195, miR-200a, miR-200b, miR-200c, miR-203a, miR-20a. miR-222, miR-29a, miR-30cl, miR-30c2. miR-451a. miR-7-1. miR-7-2. miR-7-3. miR-9-1, miR-9-2, miR-92al, miR-92a2, miR-9-3, miR-let-7al, miR-let-7a2, miR-let-7a3, miR- 1-1, miR-1-2, miR-125a, miR-125bl, miR-125b2, miR-139, miR-140, miR-142, miR-150, miR- 15b, miR-181bl, miR-181b2, miR-182, miR-18a, miR-19bl, miR-19b2, miR-214, miR-223, miR-25, miR-27a, miR-375. miR-let-7d, miR-let-fl, miR-let-712. miR-122, miR-128-1, miR- 128-2, miR-146A, miR-192, miR-194-1, miR-194-2, miR-210, miR-224, miR-23a, miR-26b, miR-27b, miR-301a, miR-32, miR-335, miR-342, miR-497, miR-96, miR-let-7e, miR-let-7i, miR-103al, miR-103a2, miR-107, miR-lOa, miR-124-1, miR-124-2, miR-124-3, miR-130b, miR-133b, miR-144. miR-148a, miR-152. miR-193b. miR-199b, miR-204. miR-218-1, miR- 218-2, miR-23b, miR-24-1, miR-24-2, miR-28, miR-30b, miR-30d, miR-324, miR-378a, miR- 93, miR-let-7g, miR-129-1, miR-129-2, miR-130a, miR-135b, miR-137, miR-146b, miR-181c, miR-193A, miR-22, miR-34b, miR-34c, miR-423, miR-455, miR-106a, miR-132, miR-135al, miR-135a2, miR-138-1, miR-138-2, miR-148b, miR-149, miR-206, miR-215, miR-320a, miR- 331, miR-345, miR-373. miR-449a, miR-483. miR-486-1, miR-486-2, miR-494. miR-625. miR- 98, miR-99b, or a combination of two or more thereof.

20. The method of claim 1, wherein the cancer is a gastrointestinal cancer, wherein the gastrointestinal cancer is colorectal cancer, stomach cancer, esophageal cancer, liver cancer, pancreatic cancer, a pancreatic neuroendocrine tumor, gallbladder cancer, anal cancer, small intestine cancer, a gastrointestinal stromal tumor, a gastrointestinal neuroendocrine tumor, or cholangiocarcinoma.

21. The method of claim 1. wherein the cancer is a gastrointestinal cancer, colorectal cancer, stomach cancer, esophageal cancer, liver cancer, pancreatic cancer, a pancreatic neuroendocrine tumor, gallbladder cancer, anal cancer, small intestine cancer, a gastrointestinal stromal tumor, a gastrointestinal neuroendocrine tumor, cholangiocarcinoma. kidney cancer, lymphoma, breast cancer, prostate cancer, lung cancer, bladder cancer, non-Hodgkin’s lymphoma, melanoma, thyroid cancer, endometrial cancer, ovarian cancer, cervical cancer, pancreatic cancer, leukemia, brain cancer, myeloma, head and neck cancer, or testicular cancer.

22. The method of claim 1, wherein the biological sample is a liquid biological sample.

23. A kit comprising reagents capable of detecting an expression level of reference RNA in a biological sample; wherein the RNA comprises miR-15b, miR-23a, miR-30e, or a combination of two or more thereof.

24. The kit of claim 23, further comprising reagents capable of detecting an expression level of oncogenic RNA in the biological sample.

25. The kit of claim 23, wherein the reference RNA comprises exosomal miR-15b- 5p, cell free miR-15b-5p, exosomal miR-23a-3p, cell-free miR-23a-3p, exosomal miR-30e-5p, cell-free miR-30e-5p, or a combination of two or more thereof.

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