RNA biomarkers for use in treating, diagnosing, and monitoring colorectal cancer and colorectal adenoma

Abstract

Methods of treating colorectal cancer or colorectal adenoma, diagnosing colorectal cancer or colorectal adenoma, monitoring treatment in a patient having colorectal cancer or colorectal adenoma, and monitoring risk for developing colorectal cancer or colorectal adenoma in patients can be accomplished through the use of RNA biomarkers, such as let-7c, miR-100, miR-lOb, miR-1246, miR-1290, miR-135b, miR-150p, miR-184, miR-199b, miR-200a, miR-203a, miR- 205, miR-20a, miR-433. miR-455. miR-543, miR-9, miR-18a, miR-19a, miR-4488, miR-107, miR-1228, miR-1229, miR-1323, miR-141, miR-142, miR-1538, miR-17, miR-195, miR-19b, miR-200b, miR-296, miR-34a, miR-3913, miR-5193, and miR-877.

Description

Docket No. 048440-204001 WO / TEC 24-029RNA BIOMARKERS FOR USE IN TREATING, DIAGNOSING, AND MONITORING COLORECTAL CANCER AND COLORECTAL ADENOMACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to US Application No. 63 / 687,123 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 R01 CA227602 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Colorectal cancer is a major public health concern, ranking as the third most common and second deadliest cancer in the United States. Early detection through colorectal cancer screening, universally recommended by professional societies, improves survival rates.However, because participation to screening remains below the ideal 80% target. 76% of colorectal cancer deaths still occur in individuals who are not up to date with screening. Screening can only save the lives of those who participate, and there is still a clinical need for developing a test that is easy to administer and increases compliance. Furthermore, the focus on early cancer detection can reduce the cancer mortality only, but not incidence. To truly reduce colorectal cancer incidence, screening should detect pre-cancerous lesion, but the sensitivity for such lesions has remained limited, especially for blood-based test.

[0004] The low adherence to screening include the time required to perform screening, scheduling challenges, concerns over test invasiveness and pain, discomfort, embarrassment with the endoscopic procedure, distaste for the collection of one’s own feces, lack of insurance, travels, and lack of a phy sician recommendation to screening. However, a blood test may be incorporated into routine health care checks / encounters. and it might complement the screening options already available, and it might make screening a less laborious process, thus improving compliance. The present disclosure is directed to these important needs.BRIEF SUMMARY

[0005] Provided herein are methods of detecting miRNA in a patient with colorectal cancer or colorectal adenoma comprising detecting an elevated expression level, relative to a control, of miRNA in a biological sample obtained from the patient; wherein the miRNA comprises one ormore miRNA set forth in Table A or Table B.

[0006] Provided herein are methods of treating colorectal cancer or colorectal adenoma in a patient in need thereof comprising detecting an elevated expression level, relative to a control, of miRNA in a biological sample obtained from the patient; wherein the miRNA comprises one or more miRNA set forth in Table A or Table B; and administering to the patient an effective amount of an anti-cancer agent.

[0007] Provided herein are methods of diagnosing a patient with colorectal cancer or colorectal adenoma comprising detecting the expression level of miRNA in a biological sample obtained from the patient, and diagnosing the patient as having colorectal cancer or colorectal adenoma when the biological sample has an elevated expression level, relative to a control, of the RNA; wherein the miRNA comprises one or more miRNA set forth in Table A or Table B.

[0008] Provided herein are methods of monitoring treatment in a patient having colorectal cancer or colorectal adenoma or monitoring risk for developing colorectal cancer or colorectal adenoma in a patient comprising: detecting the expression level of miRNA in a biological sample obtained from the patient at a first time point; detecting the expression level of the miRNA in a biological sample obtained from the patient at a second time point, wherein the second time point is later than the first time point; and comparing the expression level of the miRNA at the second time point to the expression level of the miRNA at the first time point, thereby monitoring treatment or monitoring risk; wherein the miRNA comprises one or more miRNA set forth in Table A or Table B. In embodiments, the expression level of the miRNA at the second time point is greater than the expression level of the miRNA at the first time point, indicating that the patient needs a new or modified treatment regimen or that the patient is at risk for developing colorectal cancer or colorectal adenoma.

[0009] These and other embodiments of the disclosure are described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIGS. 1A-1F: Results of the training for the cf / exo-miRNA blood assay. FIGS. 1A-1B: ROC curves with the corresponding AUC values for the final 20-exo-miRNA panel and the 19- cf-miRNA panels alone (FIG. 1A) and combined (FIG. IB) for the detection of colorectal cancer (95% confidence intervals estimated based on 2000 bootstraps are presented as shaded areas). FIG. 1C: Individuals with colorectal cancer have significantly higher cancer-specific risk scores than all other groups. FIGS. 1D-1E: ROC curves with the corresponding AUC values for the final 20-exo-miRNA panel and the 19-cf-miRNA panels alone (FIG. ID) and combined(FIG. IE) for the detection of advanced adenomas (95% confidence intervals estimated based on 2000 bootstraps are presented as shaded areas). FIG. IF: Individuals with advanced adenomas have significantly higher adenoma-specific risk scores than all other groups. ****, pO.OOOl, ns, non-significant

[0011] FIGS. 2A-2B: Reproducibility of the cf / exo-miRNA blood assay. FIG. 2A: The density plot shows controls clustering in the bottom left comer (double-negative area), cases with advanced adenomas cluster in the top left comer, and cases with colorectal cancer cluster in the bottom right. FIG. 2B: In independent testing, the density plot shows reproducibility.

[0012] FIGS. 3A-3F: Results of the testing cohort for the cf / exo-miRNA blood assay. FIGS. 3A-3B: ROC curves with the corresponding AUC values for the final 20-exo-miRNA panel and the 19-cf-miRNA panels alone (FIG. 3A) and combined (FIG. 3B) for the detection of colorectal cancer (95% confidence intervals estimated based on 2000 bootstraps are presented as shaded areas). FIG. 3C: Individuals with colorectal cancer have significantly higher cancerspecific risk scores than all other groups. FIGS. 3D-3E: ROC curves with the corresponding AUC values for the final 20-exo-miRNA panel and the 19-cf-miRNA panels alone (FIG. 3D) and combined (FIG. 3E) for the detection of advanced adenomas (95% confidence intervals estimated based on 2000 bootstraps are presented as shaded areas). FIG. 3F : Individuals with advanced adenomas have significantly higher adenoma-specific risk scores than all other groups. ****, pO.OOOl, ns, non-significant

[0013] FIG. 4 shows the study design.

[0014] FIG. 5 shows the cohort allocation CONSORT flow-chart.

[0015] FIGS. 6A-6F: Discovery of the cf-miRNAs biomarkers. FIGS. 6A-6B: Volcano plot of differentially expressed cf-miRNAs in the plasma samples derived from individuals with (FIG. 6A) colorectal cancer vs. controls (non-disease controls and individuals with low-risk adenomas); and (FIG. 6B) advanced adenomas vs. controls (non-disease controls and individuals with low-risk adenomas); color grading follows significance; panels A and B are entirety derived from the first discovery cohort. FIG. 6C: Volcano plot of differentially expressed cf-miRNAs in the plasma samples derived from cases (with colorectal cancer or advanced adenomas) vs. controls (non-disease controls and individuals with low-risk adenomas); color grading follows significance; panel C is entirely derived from the second and independent discovery cohort. FIG. 6D: Volcano plot of differentially expressed cf-miRNAs in the plasma samples derived from cases (with colorectal cancer or advanced adenomas) vs. non-disease controls; color grading follows significance; panel D is entirely derived from an independent in-silico cohort (GSE25609). FIG. 6E: Volcano plot of differentially expressed miRNAs in the tissue samples derived of cases (colorectal cancer or advanced adenomas) vs. controls tissue samples (low-risk adenomas and normal mucosae); color grading follows significance; panel E is entirely derived from an independent in-silico cohort (GSE41655). FIG. 6F: The ridge-line plot presents the 19 selected cf-miRNAs

[0016] FIGS. 7A-7F: Discovery of the exo-miRNAs biomarkers. FIGS. 7A-B: Volcano plot of differentially expressed exo-miRNAs in the plasma samples derived from individuals with (FIG. 7A) colorectal cancer vs. controls (non-disease controls and individuals with low-risk adenomas); and (FIG. 7B) advanced adenomas vs. controls (non-disease controls and individuals with low-risk adenomas); color grading follows significance; panels A and B are entirely derived from the first discovery cohort. FIG. 7C: Volcano plot of differentially expressed exo-miRNAs in the plasma samples derived from cases (with colorectal cancer or advanced adenomas) vs. controls (non-disease controls and individuals with low-risk adenomas); color grading follows significance; panel C is entirely derived from the second and independent discovery cohort. FIG. 7D: Volcano plot of differentially expressed exo-miRNAs in the plasma samples derived from individuals with colorectal cancer vs. non-disease controls; color grading follows significance; panel D is entirely derived from an independent in-silico cohort (GSE39833). FIG. 7E: Volcano plot of differentially expressed miRNAs in the tissue samples derived of cases (colorectal cancer or advanced adenomas) vs. controls tissue samples (low-risk adenomas and normal mucosae); color grading follows significance; panel E is entirely derived from an independent in-silico cohort (GSE41655). FIG. 7F: The ridge-line plot presents the 20 selected exo-miRNAs

[0017] FIG. 8: Density plots of the two DENEB risk scores (for advanced adenomas and for colorectal cancer) in the two clinical cohorts. The density plots of the two clinical cohorts are displayed side by side to facilitate the comparison of the two cohorts. The density plots display the distribution of the study participants based on the advanced adenoma risk score (y-axis) and the colorectal cancer risk score (x-axis). The comparison between the two cohorts highlight that the results are replicable. The blood-based test clustered the controls (both non-disease controls and individuals with low-risk adenomas) on the bottom-left comer of the diagram (which represents the double-negative test results area). Individuals with advanced adenomas clustered on the top-left comer of the diagram, while those with colorectal cancer on the bottom-right comer. There were very few instances of double-positive results.

[0018] FIGS. 9A-9D: Exploratory analyses for the detection of advanced adenomas, based on the adenoma size. FIG. 9A: In the first clinical cohort (training), the scatter plot demonstrates that larger advanced adenomas had higher risk scores. Furthermore, adenomas that were considered “advanced” for reasons other than size (that is, advanced adenomas <10 mm) still had risk scores higher than the threshold for positivity. FIG. 9B: The violin plots with superimposed box-and-whiskers demonstrated that individuals with advanced adenomas had statistically higher risk score values than individuals with colonoscopy-detected low-risk adenomas. FIG. 9C: In the second clinical cohort (testing), the scatter plot demonstrates that larger advanced adenomas had higher risk scores. Furthermore, adenomas that were considered “advanced” for reasons other than size (that is, advanced adenomas <10 mm) still had risk scores higher than the threshold for positivity. FIG. 9D: The violin plots with superimposed box-and-whiskers demonstrated that individuals with advanced adenomas had statistically higher risk score values than individuals with colonoscopy-detected low-risk adenomas. Abbreviations: ****. p<0.0001; ns, non-significant; LRA, Low-risk adenomas; AA, Advanced adenomas

[0019] FIGS. 10A-10D: Exploratory analyses for the detection of advanced adenomas, based on the adenoma count and the presence of high-grade dysplasia. FIG. 10A: In the first clinical cohort (training), the scatter plot demonstrates that individuals with more adenomas generally had higher risk scores. Furthermore, adenomas that were considered “advanced” for reasons other than count (that is, individuals with <5 adenomas) still had risk scores higher than the threshold for positivity. FIG. 10B: The violin plots with superimposed box-and-whiskers demonstrated that individuals with advanced adenomas had statistically higher risk score values than individuals with colonoscopy-detected low-risk adenomas. FIG. 10C: In the second clinical cohort (testing), the scatter plot demonstrates that individuals with more adenomas generally had higher risk scores. Furthermore, adenomas that were considered “advanced” for reasons other than count (that is. individuals with <5 adenomas) still had risk scores higher than the threshold for positivity. FIG. 10D: The violin plots with superimposed box-and-whiskers demonstrated that individuals with advanced adenomas had statistically higher risk score values than individuals with colonoscopy-detected low-risk adenomas. Abbreviations: ****, p<0.0001; ns, non-significant; LRA, Low-risk adenomas; AA, Advanced adenomasDETAILED 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 etal., 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 “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.

[0022] “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 know n 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.

[0023] “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.

[0024] A “cell” refers to a cell carry ing out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, 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 w ith 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.

[0025] Exemplary RNA described herein include the RNA in Table A or Table B. The RNA in Table A and Table B are from humans and therefore can alternatively be written as hsa-miR,where “hsa” refers to homo sapiens.

[0026] Table A

[0027] Table B

[0028] '‘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 anddouble 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.

[0029] 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.

[0030] 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 of gene 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.

[0031] 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 asintervening 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.

[0032] 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.

[0033] 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 are transcribed 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.

[0034] The terms 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. The control may be any suitable control, as described herein. In embodiments, an “elevated expression level” of the biomarker gene compared to the control (when the expression level of the biomarker is greater than the corresponding control) 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 control. In embodiments, an “elevated expression level” of the biomarker gene is an amount that is statistically significantly greater than the expression level of the gene in a control.

[0035] 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., posttranslational modifications). In embodiments, a biomarker refers to RNA, e.g.. miRNA.

[0036] 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 NanoStnng, 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 1 Oth, 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 Id- 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 intensity level 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.

[0037] “Control'’ is used in accordance with its plain ordinary' meaning and refers to an assay, comparison, or experiment in which the subjects 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 activity7or expression level of RNA. In embodiments, a control is the measurement of the activity or expression level of miRNA. 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-diseased control. 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, a non-diseased patient is a patient that does not have colorectal cancer or colorectal adenoma. 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 a patient that does not have colorectal cancer or colorectal adenoma or a population of patients that do not have colorectal cancer or colorectal adenoma. 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., protein or mRNA) 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, the control is a standard control. In embodiments, a control is a level of expression of the biomarker (e.g., RNA. miRNA) that has been correlated with the diagnosis of colorectal cancer or colorectal adenoma in a subject. In embodiments, a control is a level of expression of the biomarker (e.g., RNA, miRNA) that has been correlated with a healthy7subject (i.e., a subject that does not have colorectal 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 notbe considered as significant.

[0038] The term “healthy patient” refers to a non-diseased patient. In embodiments, a healthy patient is a patient that does not have cancer. In embodiments, a healthy patient is a patient that does not have colorectal cancer or colorectal adenoma. In embodiments, the healthy patient is a control.

[0039] 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.

[0040] The singular terms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise.

[0041] A “therapeutic agent” or “anticancer 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 colorectal cancer or colorectal adenoma, 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.

[0042] “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.

[0043] “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.

[0044] 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., colorectal cancer) or outcome in a subject.

[0045] “Image-based screening” refers to methods using imaging technology to detect a cancer or tumor in a patient. Exemplary types of image-based screening include x-rays, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and ultrasound. In embodiments, the image-based screening is CT, MRI, or ultrasound. In embodiments, the ultrasound is endoscopic ultrasonography (EUS). In embodiments, the image-based screening is CT, MRI, or EUS. In embodiments, the image-based screening is MRI or EUS. In embodiments, the image-based screening is CT. In embodiments, the image-based screening is MRI. In embodiments, the image-based screening is EUS.

[0046] The phrase “surgically removing all or a portion of the colon of the patient” includes surgically removing an adenoma (e.g., polyp) in the case of colorectal adenoma. In embodiments for the case of treating a colorectal adenoma, “surgically removing all or a portion of the colon of the patient” refers to removing an adenoma (e.g., neoplastic polyp) during a colonoscopy (e.g.. endoscopic resection).

[0047] 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 anactive agent. The term “treating"’ does not including preventing.

[0048] 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 colorectal cancer, as described above. 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 w ell 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.

[0049] 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.

[0050] 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, dogs, cats, monkeys, and other non-mammalian animals. In embodiments, a patient is human patient. In embodiments, the human patient is at least 50 years old. In embodiments, the human patient is at least 45 years old. In embodiments, the human patient is at least 40 years old. In embodiments, the human patient has Lynch syndrome.

[0051] “Colorectal adenoma” refers to a glandular tumor of the colon and / or rectum that is often a precursor lesion to colon cancer. Colorectal adenomas often present as polyps. Colorectal adenomas include tubular adenoma, tubulovillous adenoma, villous adenoma, and sessile serrated adenoma, with or without dysplasia. A low-risk colorectal adenoma is defined as fewer than 5 adenomas with low-grade dysplasia and all smaller than 10 mm in diameter. A high-risk colorectal adenoma is defined as either 5 or more adenomas or one or more adenoma with highgrade dysplasia or one or more adenoma larger than 10 mm. “

[0052] “Advanced colorectal adenoma” refers to either 5 or more adenomas or one or more adenomas that measure 1.0 cm or more in size, one or more adenomas that contain a substantial villous component (>25%), or one or more adenomas that exhibit high-grade dysplasia.

[0053] “High grade dysplasia” or “HGD” generally refers to colon polyps / abnormal cells in the colon that have an advanced histology and / or appear to be cancerous and / or are associated with a higher risk of developing colorectal cancer. The World Health Organization (WHO) has defined high-grade dysplasia on the basis of both cytological and architectural features, such as high nuclear to cytoplasmic ratio, nuclear pleomorphism, loss of nuclear polarity and intraluminal cribriforming in at least two (or more) glands.

[0054] “Colorectal cancer"’ or “CRC” refers to a cancer that generally begins as grow th (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.

[0055] “Early-onset colorectal cancer” or “EOCRC” refers to colorectal cancer in a patient less than 50 years old at the time of their colorectal cancer diagnosis. In embodiments, the patient ith EOCRC is less than 50 years old and does not have a familial or hereditary disposition for colorectal cancer.

[0056] Methods for treating colorectal cancer, including early-onset colorectal cancer and late- onset colorectal cancer, include: (a) administering to the patient an effective amount of an anticancer agent, (b) administering to the patient an effective amount of radiation therapy, (c) administering to the patient image-based screening, (d) surgically removing all or a portion of the colon of the patient, or (e) a combination of tw o or more thereof. Surgery to remove all or portion of the colon of the patient are known in the art and include, for example, polypectomy, local excision via colonoscope, transanal excision (TAE). transanal endoscopic microsurgery (TEM), low anterior resection (LAR), proctectomy, abdominoperineal resection (APR), pelvic exenteration, and the like. The term “removing all or a portion of the colon” includes: (i) removing all or a portion of the colon, (ii) removing all or a portion of the rectum, and (iii) removing all or a portion of the rectum and all or a portion of the colon.

[0057] In “Stage 1” colorectal cancer, the cancer has grown through the muscularis mucosa into the submucosa (Tl). and it may also have grown into the muscularis propria (T2), but it has not spread to nearby lymph nodes (NO) or to distant sites (M0).

[0058] “Stage 2” colorectal cancer is generally identified by one of the following: (i) the cancer has grown into the outermost layers of the colon or rectum but has not gone through them (T3); it has not reached nearby organs; and it has not spread to nearby lymph nodes (NO) or to distant sites (M0); (ii) the cancer has grown through the w all of the colon or rectum but has not grown into other nearby tissues or organs (T4a). and has not yet spread to nearby lymph nodes (NO) or to distant sites (M0); or (iii) the cancer has grown through the wall of the colon or rectum and is attached to or has grown into other nearby tissues or organs (T4b), but it has not yet spread to nearby lymph nodes (NO) or to distant sites (M0).

[0059] “Stage 3’' colorectal cancer is generally identified by one of the follow ing: (i) the cancer has grown through the mucosa into the submucosa (T 1 ). and it may also have grown into the muscularis propria (T2); it has spread to 1 to 3 nearby lymph nodes (Nl) or into areas of fat near the lymph nodes but not the nodes themselves (Nlc); and it has not spread to distant sites (MO); (ii) the cancer has grown through the mucosa into the submucosa (T 1 ); it has spread to 4 to 6 nearby lymph nodes (N2a); and it has not spread to distant sites (MO); (iii) the cancer has grown into the outermost layers of the colon or rectum (T3) or through the visceral peritoneum (T4a) but has not reached nearby organs; it has spread to 1 to 3 nearby lymph nodes (Nla or Nib) or into areas of fat near the lymph nodes but not the nodes themselves (Nlc); and it has not spread to distant sites (MO); (iv) the cancer has grown into the muscularis propria (T2) or into the outermost layers of the colon or rectum (T3); it has spread to 4 to 6 nearby lymph nodes (N2a); and it has not spread to distant sites (MO); (v) the cancer has grown through the mucosa into the submucosa (Tl), and it might also have grown into the muscularis propria (T2); it has spread to 7 or more nearby lymph nodes (N2b); and it has not spread to distant sites (MO); (vi) the cancer has grown through the wall of the colon or rectum (including the visceral peritoneum) but has not reached nearby organs (T4a); it has spread to 4 to 6 nearby lymph nodes (N2a); and it has not spread to distant sites (MO); (vi) the cancer has grown into the outermost layers of the colon or rectum (T3) or through the visceral peritoneum (T4a) but has not reached nearby organs; it has spread to 7 or more nearby lymph nodes (N2b); and it has not spread to distant sites (MO); or (viii) the cancer has grown through the wall of the colon or rectum and is attached to or has grown into other nearby tissues or organs (T4b); it has spread to at least one nearby lymph node or into areas of fat near the lymph nodes (N 1 or N2); and it has not spread to distant sites (MO).

[0060] “Stage 4’' colorectal cancer is generally identified by one of the following: (i) the cancer may or may not have grown through the wall of the colon or rectum (Any T); it might or might not have spread to nearby lymph nodes (Any N); it has spread to 1 distant organ (such as the liver or lung) or distant set of lymph nodes, but not too distant parts of the peritoneum (the lining of the abdominal cavity) (Mia); (ii) the cancer might or might not have grown through the wall of the colon or rectum (Any T); it might or might not have spread to nearby lymph nodes (Any N); it has spread to more than 1 distant organ (such as the liver or lung) or distant set of lymph nodes, but not too distant parts of the peritoneum (the lining of the abdominal cavity) (Mlb); or (iii) the cancer might or might not have grown through the w all of the colon or rectum (Any T); it might or might not have spread to nearby lymph nodes (Any N); it has spread to distant parts of the peritoneum (the lining of the abdominal cavity), and may or may not havespread to distant organs or lymph nodes (Mlc).

[0061] Methods of Detecting

[0062] Provided herein are methods of detecting RNA in a patient with colorectal cancer comprising detecting an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR- 135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR- 200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR- 433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p. exosomal miR-19a-3p. exosomal miR-4488, cell-free miR-107. cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR- 1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, 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 colorectal cancer is Stage I or Stage II. In embodiments, the colorectal cancer is Stage I. In embodiments, the colorectal cancer is Stage II. In embodiments, the colorectal cancer is Stage III. In embodiments, the colorectal cancer is Stage IV. In embodiments, the method further comprising administering to the patient an effective amount of an anti-cancer agent. In embodiments, the method further comprising diagnosing the patient with colorectal cancer. In embodiments, the method further comprising diagnosing the patient with colorectal cancer and administering to the patient an effective amount of an anti-cancer agent.

[0063] Provided herein are methods of detecting RNA in a patient with colorectal cancer comprising detecting an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient; wherein the RNA comprises one or more RNA set forth in Table B. In embodiments, the colorectal cancer is Stage I or Stage II. In embodiments, the colorectal cancer is Stage I. In embodiments, the colorectal cancer is Stage II. In embodiments, the colorectal cancer is Stage III. In embodiments, the colorectal cancer is Stage IV. In embodiments, the method further comprising administering to the patient an effective amount of an anti-cancer agent. In embodiments, the method further comprising diagnosing the patient with colorectal cancer. In embodiments, the method further comprising diagnosing the patient with colorectal cancer and administering to the patient an effective amount of an anti-cancer agent.

[0064] Provided herein are methods of detecting RNA in a patient with colorectal adenoma comprising detecting an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient: wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR- 135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR- 200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR- 433-3p, exosomal miR-455-3p, exosomal miR-543. exosomal miR-9-5p. exosomal miR-18a-5p. exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR- 1538, cell-free miR-17-5p. cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p. cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof. In embodiment, the colorectal adenoma is high-risk colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older. In embodiments, the method further comprising diagnosing the patient with colorectal adenoma.

[0065] Provided herein are methods of detecting RNA in a patient with colorectal adenoma comprising detecting an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient; wherein the RNA comprises one or more RNA set forth in Table B. In embodiment, the colorectal adenoma is high-risk colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older. In embodiments, the method further comprising diagnosing the patient with colorectal adenoma.

[0066] Methods of Treatment

[0067] Provided herein are methods of treating colorectal cancer in a patient in need thereof comprising: (i) detecting an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p. exosomal miR-184, exosomal miR-199b-5p,exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell-free miR- 107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR- 142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell- free miR-5193. cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR- 4488, or a combination of two or more thereof; and (ii) administering to the patient an effective amount of an anti-cancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, the method comprises administering to the patient the effective amount of the anti-cancer agent. In embodiments, the colorectal cancer is Stage I or Stage II. In embodiments, the colorectal cancer is Stage II. In embodiments, the colorectal cancer is Stage III. In embodiments, the colorectal cancer is Stage IV. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0068] Provided herein are methods of treating colorectal cancer in a patient in need thereof comprising: (i) detecting an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient; wherein the RNA comprises one or more RNA set forth in Table B; and (ii) administering to the patient an effective amount of an anti-cancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, the method comprises administering to the patient the effective amount of the anti-cancer agent. In embodiments, the colorectal cancer is Stage I or Stage II. In embodiments, the colorectal cancer is Stage II. In embodiments, the colorectal cancer is Stage 111. In embodiments, the colorectal cancer is Stage IV. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0069] Provided herein are methods of treating colorectal cancer in a patient in need thereof comprising administering to the patient an effective amount of an anti-cancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof; wherein a biological sample obtained from the patientcomprises an elevated expression level, relative to a control, of a RNA; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p. exosomal miR-10b-5p, exosomal miR- 1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205- 5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p. exosomal miR-19a-3p, exosomal miR-4488, cell- free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p. cell-free miR-1323, cell-free miR- 141 -3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell -free miR-34a-5p, cell- free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, 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 method comprises administering to the patient the effective amount of the anti-cancer agent. In embodiments, the colorectal cancer is Stage I or Stage II. In embodiments, the colorectal cancer is Stage II. In embodiments, the colorectal cancer is Stage III. In embodiments, the colorectal cancer is Stage IV. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0070] Provided herein are methods of treating colorectal cancer in a patient in need thereof comprising administering to the patient an effective amount of an anti-cancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof; wherein a biological sample obtained from the patient comprises an elevated expression level, relative to a control, of a RNA; wherein the RNA comprises one or more RNA set forth in Table B. In embodiments, the method comprises administering to the patient the effective amount of the anti-cancer agent. In embodiments, the colorectal cancer is Stage I or Stage II. In embodiments, the colorectal cancer is Stage II. In embodiments, the colorectal cancer is Stage III. In embodiments, the colorectal cancer is Stage IV. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0071] Provided herein are methods of treating colorectal adenoma in a patient in need thereof comprising: (i) detecting an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p. exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p,exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell-free miR- 107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR- 142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell- free miR-5193. cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR- 4488, or a combination of two or more thereof; and (ii) administering to the patient an effective amount of an anti-cancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, the method comprises surgically removing all or a portion of the colon of the patient, e.g., removing an adenoma during a colonoscopy. In embodiment, the colorectal adenoma is high-risk colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0072] Provided herein are methods of treating colorectal adenoma in a patient in need thereof comprising: (i) detecting an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient; wherein the RNA comprises one or more RNA set forth in Table B; and (ii) administering to the patient an effective amount of an anti-cancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, the method comprises surgically- removing all or a portion of the colon of the patient, e.g., removing an adenoma during a colonoscopy. In embodiments, the colorectal adenoma is high-risk colorectal adenoma. In embodiments, the colorectal adenoma is advanced colorectal adenoma. In embodiments, the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0073] Provided herein are methods of treating colorectal adenoma in a patient in need thereof comprising administering to the patient an effective amount of an anti-cancer agent, administering to the patient an effective amount of radiation therapy, administering to the patientimage-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof; wherein a biological sample obtained from the patient comprises an elevated expression level, relative to a control, of a RNA; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR- 1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205- 5p. exosomal miR-20a-5p. exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543. exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell- free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR- 141-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell- free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, 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 method comprises surgically removing all or a portion of the colon of the patient, e.g., removing an adenoma during a colonoscopy. In embodiment, the colorectal adenoma is high-risk colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0074] Provided herein are methods of treating colorectal adenoma in a patient in need thereof comprising administering to the patient an effective amount of an anti-cancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof; wherein a biological sample obtained from the patient comprises an elevated expression level, relative to a control, of a RNA; surgically removing all or a portion of the colon of the patient. In embodiments, the method comprises surgically removing all or a portion of the colon of the patient, e.g., removing an adenoma during a colonoscopy. In embodiment, the colorectal adenoma is high-risk colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0075] Methods of Diagnosis

[0076] Provided herein are methods of diagnosing a patient with colorectal cancer comprising:(i) detecting the expression level of RNA in a biological sample obtained from the patient; and(ii) diagnosing the patient as having colorectal cancer or colorectal adenoma when the biological sample has an elevated expression level, relative to a control, of the RNA; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR- 1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p. exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205- 5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell- free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR- 14 l-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell- free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, 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 method further comprises administering to the patient the effective amount of an anti-cancer agent. In embodiments, the colorectal cancer is Stage I or Stage II. In embodiments, the colorectal cancer is Stage II. In embodiments, the colorectal cancer is Stage III. In embodiments, the colorectal cancer is Stage IV. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0077] Provided herein are methods of diagnosing a patient with colorectal cancer comprising:(i) detecting the expression level of RNA in a biological sample obtained from the patient; and(ii) diagnosing the patient as having colorectal cancer or colorectal adenoma when the biological sample has an elevated expression level, relative to a control, of the RNA; wherein the RNA comprises one or more RNA set forth in Table B. In embodiments, the method further comprises administering to the patient the effective amount of an anti-cancer agent. In embodiments, the colorectal cancer is Stage I or Stage II. In embodiments, the colorectal cancer is Stage II. In embodiments, the colorectal cancer is Stage III. In embodiments, the colorectal cancer is Stage IV. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0078] Provided herein are methods of diagnosing a patient with colorectal adenoma comprising: (i) detecting the expression level of RNA in a biological sample obtained from the patient; and (ii) diagnosing the patient as having colorectal cancer or colorectal adenoma whenthe biological sample has an elevated expression level, relative to a control, of the RNA; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488. cell-free miR-107. cell-free miR-1228-5p. cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17- 5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, 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 method further comprises administering to the patient the effective amount of an anti-cancer agent. In embodiments, the colorectal adenoma is high-risk colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0079] Provided herein are methods of diagnosing a patient with colorectal adenoma comprising: (i) detecting the expression level of RNA in a biological sample obtained from the patient: and (ii) diagnosing the patient as having colorectal cancer or colorectal adenoma when the biological sample has an elevated expression level, relative to a control, of the RNA; wherein the RNA comprises one or more RNA set forth in Table B. In embodiments, the method comprises surgically removing all or a portion of the colon of the patient, e.g., removing an adenoma during a colonoscopy. In embodiments, the colorectal adenoma is high-risk colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0080] Methods of Monitoring

[0081] Provided herein are methods of monitoring treatment in a patient having colorectal cancer comprising (i) detecting the expression level of RNA in a biological sample obtained from the patient at a first time point; (ii) detecting the expression level of the RNA in a biological sample obtained from the patient at a second time point, wherein the second timepoint is later than the first time point; and (iii) comparing the expression level of the RNA at the second time point to the expression level of the RNA at the first time point, thereby monitoring treatment; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR- 150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal rniR- 203a-3p, exosomal miR-205-5p. exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR- 455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p. exosomal miR-19a-3p. exosomal miR-4488, cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17- 5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof. In embodiments, an elevated expression level of RNA at the second point in time when compared to the expression level of RNA at the first point in time indicates that the patient has developed colorectal cancer or that the colorectal cancer is no longer in remission, and optionally that the patient needs a new or modified treatment for colorectal cancer. In embodiments, the new or modified treatment is the replacing the existing anti-cancer agent(s) with a new anti-cancer agent(s), adding a new anti-cancer agent(s) to the existing anti-cancer agent(s), increasing the dosage of the existing anti-cancer agent(s), or adding anew anti-cancer agent(s) to the existing anti-cancer agent(s) and increasing the dosage of the existing anti-cancer agent(s). In embodiments, the new treatment can further include or alternatively include administering to the patient an effective amount of radiation therapy, surgically removing all or a portion of the colon of the patient, or a combination thereof. In embodiments, the colorectal cancer is Stage I or Stage II. In embodiments, the colorectal cancer is Stage II. In embodiments, the colorectal cancer is Stage III. In embodiments, the colorectal cancer is Stage IV. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0082] Provided herein are methods of monitoring treatment in a patient having colorectal cancer comprising (i) detecting the expression level of RNA in a biological sample obtained from the patient at a first time point; (ii) detecting the expression level of the RNA in a biological sample obtained from the patient at a second time point, wherein the second time point is later than the first time point; and (iii) comparing the expression level of the RNA at the second time point to the expression level of the RNA at the first time point, thereby monitoring treatment; wherein the RNA comprises one or more RNA set forth in Table B. In embodiments,an elevated expression level of RNA at the second point in time when compared to the expression level of RNA at the first point in time indicates that the patient has developed colorectal cancer or that the colorectal cancer is no longer in remission, and optionally that the patient needs a new or modified treatment for colorectal cancer. In embodiments, the new or modified treatment is the replacing the existing anti-cancer agent(s) with a new anti-cancer agent(s), adding a new anti-cancer agent(s) to the existing anti-cancer agent(s), increasing the dosage of the existing anti-cancer agent(s), or adding a new anti-cancer agent(s) to the existing anti-cancer agent(s) and increasing the dosage of the existing anti-cancer agent(s). In embodiments, the new treatment can further include or alternatively include administering to the patient an effective amount of radiation therapy, surgically removing all or a portion of the colon of the patient, or a combination thereof. In embodiments, the colorectal cancer is Stage I or Stage II. In embodiments, the colorectal cancer is Stage II. In embodiments, the colorectal cancer is Stage III. In embodiments, the colorectal cancer is Stage IV. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0083] Provided herein are methods of monitoring risk for developing colorectal cancer in a patient comprising (i) detecting the expression level of RNA in a biological sample obtained from the patient at a first time point; (ii) detecting the expression level of the RNA in a biological sample obtained from the patient at a second time point, wherein the second time point is later than the first time point; and (iii) comparing the expression level of the RNA at the second time point to the expression level of the RNA at the first time point, thereby monitoring treatment or monitoring risk; wherein the RNA comprises exosomal let-7c-5p exosomal miR- 100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b- 5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p. exosomal miR-433-3p, exosomal miR-455-3p. exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR- 1538, cell-free miR-17-5p. cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p. cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof. In embodiments, an elevated expression level of RNA at the second point in time when compared to the expression level of RNA at the first point in time indicates that the patient is at risk of developing colorectal cancer. In embodiments, the method furthercomprises administering to the patient an effective amount of an anti-cancer agent. In embodiments, the colorectal cancer is Stage I or Stage II. In embodiments, the colorectal cancer is Stage II. In embodiments, the colorectal cancer is Stage III. In embodiments, the colorectal cancer is Stage IV. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0084] Provided herein are methods of monitoring risk for developing colorectal cancer in a patient comprising (i) detecting the expression level of RNA in a biological sample obtained from the patient at a first time point; (ii) detecting the expression level of the RNA in a biological sample obtained from the patient at a second time point, wherein the second time point is later than the first time point; and (iii) comparing the expression level of the RNA at the second time point to the expression level of the RNA at the first time point, thereby monitoring treatment or monitoring risk; wherein the RNA comprises one or more RNA set forth in Table B. In embodiments, an elevated expression level of RNA at the second point in time when compared to the expression level of RNA at the first point in time indicates that the patient is at risk of developing colorectal cancer. In embodiments, the method further comprises administering to the patient an effective amount of an anti-cancer agent. In embodiments, the colorectal cancer is Stage I or Stage II. In embodiments, the colorectal cancer is Stage II. In embodiments, the colorectal cancer is Stage III. In embodiments, the colorectal cancer is Stage IV. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0085] Provided herein are methods of monitoring treatment in a patient having colorectal adenoma comprising (i) detecting the expression level of RNA in a biological sample obtained from the patient at a first time point; (ii) detecting the expression level of the RNA in a biological sample obtained from the patient at a second time point, wherein the second time point is later than the first time point; and (iii) comparing the expression level of the RNA at the second time point to the expression level of the RNA at the first time point, thereby monitoring treatment; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p. exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR- 150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR- 203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR- 455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488. cell-free miR-107. cell-free miR-1228-5p. cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof. In embodiments, an elevated expression level of RNA at the second point in time when compared to the expression level of RNA at the first point in time indicates that the patient has developed colorectal adenoma. In embodiments, the colorectal adenoma is high-risk colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0086] Provided herein are methods of monitoring treatment in a patient having colorectal adenoma comprising (i) detecting the expression level of RNA in a biological sample obtained from the patient at a first time point; (ii) detecting the expression level of the RNA in a biological sample obtained from the patient at a second time point, wherein the second time point is later than the first time point; and (iii) comparing the expression level of the RNA at the second time point to the expression level of the RNA at the first time point, thereby monitoring treatment; wherein the RNA comprises one or more RNA set forth in Table B. In embodiments, the treatment is surgically removing all or a portion of the colon of the patient, e.g., removing an adenoma during a colonoscopy. In embodiments, an elevated expression level of RNA at the second point in time when compared to the expression level of RNA at the first point in time indicates that the patient has developed colorectal adenoma. In embodiments, the colorectal adenoma is high-risk colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0087] Provided herein are methods of monitoring risk for developing colorectal adenoma in a patient comprising (i) detecting the expression level of RNA in a biological sample obtained from the patient at a first time point; (ii) detecting the expression level of the RNA in a biological sample obtained from the patient at a second time point, wherein the second time point is later than the first time point; and (iii) comparing the expression level of the RNA at the second time point to the expression level of the RNA at the first time point, thereby monitoring treatment or monitoring risk; wherein the RNA comprises exosomal let-7c-5p exosomal miR- 100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p. exosomal miR-20a-5p. exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR- 1538, cell-free miR-17-5p. cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p. cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof. In embodiments, an elevated expression level of RNA at the second point in time when compared to the expression level of RNA at the first point in time indicates that the patient is at risk of developing colorectal cancer. In embodiments, an elevated expression level of RNA at the second point in time when compared to the expression level of RNA at the first point in time indicates that the patient is at risk of developing colorectal adenoma. In embodiments, the method further comprises administering to the patient an effective amount of an anti-cancer agent. In embodiments, the colorectal adenoma is high-risk colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0088] Provided herein are methods of monitoring risk for developing colorectal adenoma in a patient comprising (i) detecting the expression level of RNA in a biological sample obtained from the patient at a first time point; (ii) detecting the expression level of the RNA in a biological sample obtained from the patient at a second time point, wherein the second time point is later than the first time point; and (iii) comparing the expression level of the RNA at the second time point to the expression level of the RNA at the first time point, thereby monitoring treatment or monitoring risk; wherein the RNA comprises one or more RNA set forth in Table B. In embodiments, an elevated expression level of RNA at the second point in time when compared to the expression level of RNA at the first point in time indicates that the patient is at risk of developing colorectal cancer. In embodiments, an elevated expression level of RNA at the second point in time when compared to the expression level of RNA at the first point in time indicates that the patient is at risk of developing colorectal adenoma. In embodiments, the treatment is surgically removing all or a portion of the colon of the patient, e.g., removing an adenoma during a colonoscopy. In embodiments, the colorectal adenoma is high-risk colorectal adenoma. In embodiment, the colorectal adenoma is advanced colorectal adenoma. Inembodiment, the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia. In embodiments, the patient is 40 years old or older. In embodiments, the patient is 45 years old or older. In embodiments, the patient is 50 years old or older.

[0089] Methods of Treatment

[0090] Provided herein is a method of treating colorectal cancer in a patient in need thereof comprising: (i) selecting a patient having a diagnosis of colorectal cancer based on a risk score or an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient, wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-107. cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323. cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR- 1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, 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, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, step (ii) comprises administering to the patient an effective amount of an anti-cancer agent. In embodiments, the method further comprises detecting the expression level of reference RNA, as described herein.

[0091] Provided herein is a method of treating colorectal cancer in a patient in need thereof comprising: (i) selecting a patient having a diagnosis of colorectal cancer based on a risk score or an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient, wherein the RNA comprises one or more RNA set forth in Table B; and (ii) treating the patient from step (i) by administering to the patient an effective amount of an anticancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, step (ii) comprises administering to the patient an effective amount of an anti-cancer agent. In embodiments, themethod further comprises detecting the expression level of reference RNA, as described herein.

[0092] Provided herein is a method of treating colorectal cancer in a patient in need thereof comprising: (i) receiving or obtaining a risk score or an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient, wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205- 5p, exosomal miR-20a-5p. exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543. exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell- free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR- 141-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell -free miR-34a-5p, cell- free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof; and (ii) diagnosing a patient with colorectal cancer based on the risk score or the elevated expression level of RNA, monitoring a patient who is at risk of developing colorectal cancer based on the risk score or the elevated expression level of RNA. monitoring efficacy of treatment for colorectal cancer in a patient based on the risk score or the elevated expression level of RNA; and (iii) treating the patient from step (ii) by administering to the patient an effective amount of an anti-cancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, the colorectal cancer is early colorectal cancer. In embodiments, step (iii) comprises administering to the patient an effective amount of an anti-cancer agent. In embodiments, the method further comprises detecting the expression level of reference RNA, as described herein.

[0093] Provided herein is a method of treating colorectal cancer in a patient in need thereof comprising: (i) receiving or obtaining a risk score or an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient, wherein the RNA comprises one or more RNA set forth in Table B; and (ii) diagnosing a patient with colorectal cancer based on the risk score or the elevated expression level of RNA, monitoring a patient who is at risk of developing colorectal cancer based on the risk score or the elevated expression level of RNA, monitoring efficacy of treatment for colorectal cancer in a patient based on the risk score or the elevated expression level of RNA; and (iii) treating the patient from step (ii) by administering tothe patient an effective amount of an anti-cancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, the colorectal cancer is early colorectal cancer. In embodiments, step (iii) comprises administering to the patient an effective amount of an anti-cancer agent. In embodiments, the method further comprises detecting the expression level of reference RNA, as described herein.

[0094] Provided herein is a method of treating colorectal cancer in a patient in need thereof comprising: (i) receiving or obtaining a risk score or an elevated expression level of RNA, wherein the risk score or elevated expression level of RNA is produced by a non-transi ton 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 w hich comprises detecting an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient, wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290. exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205- 5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell- free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR- 14 l-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell- free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of tw o or more thereof; and (ii) diagnosing a patient with colorectal cancer based on the risk score or the elevated expression level of RNA, monitoring a patient who is at risk of developing colorectal cancer based on the risk score or the elevated expression level of RNA, monitoring efficacy of treatment for colorectal cancer in a patient based on the risk score or the elevated expression level of RNA; and (iii) treating the patient from step (ii) by administering to the patient an effective amount of an anti-cancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, step (iii) comprises administering to the patient an effective amount of an anti-cancer agent. In embodiments, the method further comprises detecting the expression level of RNA, as described herein.

[0095] Provided herein is a method of treating colorectal cancer in a patient in need thereof comprising: (i) receiving or obtaining a risk score or an elevated expression level of RNA, wherein the risk score or elevated expression level of 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 elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient, wherein the RNA comprises one or more RNA set forth in Table B; and (ii) diagnosing a patient with colorectal cancer based on the risk score or the elevated expression level of RNA, monitoring a patient who is at risk of developing colorectal cancer based on the risk score or the elevated expression level of RNA, monitoring efficacy of treatment for colorectal cancer in a patient based on the risk score or the elevated expression level of RNA; and (iii) treating the patient from step (ii) by administering to the patient an effective amount of an anti-cancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, step (iii) comprises administering to the patient an effective amount of an anticancer agent. In embodiments, the method further comprises detecting the expression level of RNA, as described herein.

[0096] Provided herein is a method of treating colorectal adenoma in a patient in need thereof comprising: (i) selecting a patient having a diagnosis of colorectal adenoma based on a risk score or an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient, wherein the RNA comprises exosomal let-7c-5p exosomal miR-100- 5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p. exosomal miR-433-3p, exosomal miR-455-3p. exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR- 1538, cell-free miR-17-5p. cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p. cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, 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-adenoma agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing allor a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, step (ii) comprises surgically removing all or a portion of the colon of the patient, e.g., removing an adenoma during a colonoscopy. In embodiments, the method further comprises detecting the expression level of reference RNA, as described herein.

[0097] Provided herein is a method of treating colorectal adenoma in a patient in need thereof comprising: (i) selecting a patient having a diagnosis of colorectal adenoma based on a risk score or an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient, wherein the RNA comprises one or more RNA set forth in Table B; and (ii) treating the patient from step (i) by administering to the patient an effective amount of an anti-adenoma agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, step (ii) comprises surgically removing all or a portion of the colon of the patient, e.g., removing an adenoma during a colonoscopy. In embodiments, the method further comprises detecting the expression level of reference RNA, as described herein.

[0098] Provided herein is a method of treating colorectal adenoma in a patient in need thereof comprising: (i) receiving or obtaining a risk score or an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient, wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205- 5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p. exosomal miR-18a-5p. exosomal miR-19a-3p, exosomal miR-4488, cell- free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR- 141-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell- free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p. cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof; and (ii) diagnosing a patient with colorectal adenoma based on the risk score or the elevated expression level of RNA, monitoring a patient who is at risk of developing colorectal adenoma based on the risk score or the elevated expression level of RNA, monitoring efficacy of treatment for colorectal adenoma in a patient based on the risk score or the elevated expression level of RNA; and (lii) treating the patient from step (ii) by administering to the patient an effective amount of an anti-adenomaagent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, the colorectal adenoma is early colorectal adenoma. In embodiments, step (iii) comprises surgically removing all or a portion of the colon of the patient, e.g., removing an adenoma during a colonoscopy. In embodiments, the method further comprises detecting the expression level of reference RNA. as described herein.

[0099] Provided herein is a method of treating colorectal adenoma in a patient in need thereof comprising: (i) receiving or obtaining a risk score or an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient, wherein the RNA comprises one or more RNA set forth in Table B; and (ii) diagnosing a patient with colorectal adenoma based on the risk score or the elevated expression level of RNA, monitoring a patient who is at risk of developing colorectal adenoma based on the risk score or the elevated expression level of RNA, monitoring efficacy of treatment for colorectal adenoma in a patient based on the risk score or the elevated expression level of RNA; and (iii) treating the patient from step (ii) by administering to the patient an effective amount of an anti-adenoma agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, the colorectal adenoma is early colorectal adenoma. In embodiments, step (iii) comprises surgically removing all or a portion of the colon of the patient, e.g., removing an adenoma during a colonoscopy. In embodiments, the method further comprises detecting the expression level of reference RNA. as described herein.

[0100] Provided herein is a method of treating colorectal adenoma in a patient in need thereof comprising: (i) receiving or obtaining a risk score or an elevated expression level of RNA. wherein the risk score or elevated expression level of 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 elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient, wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205- 5p, exosomal miR-20a-5p. exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543. exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR- 14 l-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell- free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof; and (ii) diagnosing a patient with colorectal adenoma based on the risk score or the elevated expression level of RNA, monitoring a patient who is at risk of developing colorectal adenoma based on the risk score or the elevated expression level of RNA, monitoring efficacy of treatment for colorectal adenoma in a patient based on the risk score or the elevated expression level of RNA; and (iii) treating the patient from step (ii) by administering to the patient an effective amount of an anti-adenoma agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, step (iii) comprises surgically removing all or a portion of the colon of the patient, e.g., removing an adenoma during a colonoscopy. In embodiments, the method further comprises detecting the expression level of RNA, as described herein.

[0101] Provided herein is a method of treating colorectal adenoma in a patient in need thereof comprising: (i) receiving or obtaining a risk score or an elevated expression level of RNA, wherein the risk score or elevated expression level of RNA is produced by a non-transi ton 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 elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient, wherein the RNA comprises one or more RNA set forth in Table B; and (ii) diagnosing a patient with colorectal adenoma based on the risk score or the elevated expression level of RNA, monitoring a patient who is at risk of developing colorectal adenoma based on the risk score or the elevated expression level of RNA, monitoring efficacy of treatment for colorectal adenoma in a patient based on the risk score or the elevated expression level of RNA; and (iii) treating the patient from step (ii) by administering to the patient an effective amount of an anti-adenoma agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof. In embodiments, step (iii) comprises surgically removing all or a portion of the colon of the patient, e.g., removing an adenoma during a colonoscopy. In embodiments, the method further comprises detecting the expression level of RNA, as described herein.

[0102] Provided herein are methods of processing data generated from the RNA levels in the biological sample obtained from a patient for establishing a risk score (composite risk score), e.g., a score indicative colorectal cancer or colorectal adenoma. In embodiments, the method comprises the steps of (i) normalizing and / or scaling numeric values of the RNA level data, (ii) refining the discriminatory power of individual 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. In embodiments, the composite risk score obtained from step (iii) is compared to a control and the comparison allows the sample to be designated as positive or negative for colorectal cancer or colorectal adenoma or a scale of likelihood of colorectal cancer or colorectal adenoma. 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 risk score can be based on a comparison to a control, such as a healthy patient, a population of healthy patients, a patient with colorectal cancer, or a population of patients with colorectal cancer.

[0103] In embodiments, the 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.

[0104] 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 RNA and / or risk scores. 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 colorectal cancer or colorectal adenoma, diagnose a patient with colorectal cancer or colorectal adenoma, monitor a patient who is at risk of developing colorectal cancer or colorectal adenoma, or monitoring efficacy of treatment for colorectal cancer or colorectal adenoma in a patient. Receiving and obtaining can be used interchangeably herein and can refer to physically receiving / 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.

[0105] RNA Biomarkers

[0106] In embodiments of the methods described herein, the RNA biomarkers are miRNA. Inembodiments, the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR- 10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150- 5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a- 3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455- 3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488. cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323. cell-free miR-141-3p, cell-free miR-142-3p. cell-free miR-1538, cell-free miR-17- 5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof

[0107] In embodiments of the methods described herein, the RNA comprise at least one miRNA from Table A. In embodiments, the RNA comprise at least two miRNA from Table A. In embodiments, the RNA comprise at least three miRNA from Table A. In embodiments, the RNA comprise at least four miRNA from Table A. In embodiments, the RNA comprise at least five miRNA from Table A. In embodiments, the RNA comprise at least six miRNA from Table A. In embodiments, the RNA comprise at least seven miRNA from Table A. In embodiments, the RNA comprise at least eight miRNA from Table A. In embodiments, the RNA comprise at least nine miRNA from Table A. In embodiments, the RNA comprise at least ten miRNA from Table A. In embodiments, the RNA comprise at least eleven miRNA from Table A. In embodiments, the RNA comprise at least twelve miRNA from Table A. In embodiments, the RNA comprise at least thirteen miRNA from Table A. In embodiments, the RNA comprise at least fourteen miRNA from Table A. In embodiments, the RNA comprise at least fifteen miRNA from Table A. In embodiments, the RNA comprise at least sixteen miRNA from Table A. In embodiments, the RNA comprise at least seventeen miRNA from Table A. In embodiments, the RNA comprise at least eighteen miRNA from Table A. In embodiments, the RNA comprise at least nineteen miRNA from Table A. In embodiments, the RNA comprise at least twenty miRNA from Table A. In embodiments, the RNA comprise at least twenty-one miRNA from Table A. In embodiments, the RNA comprise at least twenty-two miRNA from Table A. In embodiments, the RNA comprise at least twenty-three miRNA from Table A. In embodiments, the RNA comprise at least twenty-four miRNA from Table A. In embodiments, the RNA comprise at least twenty -five miRNA from Table A. In embodiments, the RNA comprise at least twenty-six miRNA from Table A. In embodiments, the RNA comprise at least twenty-seven miRNA from Table A. In embodiments, the RNA comprise at least twenty-eight miRNA from Table A. In embodiments, the RNA comprise at least twenty-nine miRNA fromTable A. In embodiments, the RNA comprise at least thirty miRNA from Table A. In embodiments, the RNA comprise at least thirty -one miRNA from Table A. In embodiments, the RNA comprise at least thirty-two miRNA from Table A. In embodiments, the RNA comprise at least thirty -three miRNA from Table A. In embodiments, the RNA comprise at least thirty-four miRNA from Table A. In embodiments, the RNA comprise at least thirty-five miRNA from Table A. In embodiments, the RNA comprise at least thirty-six miRNA from Table A. In embodiments, the RNA comprise at least thirty -seven miRNA from Table A. In embodiments, the RNA comprise at least thirty-eight miRNA from Table A.

[0108] In embodiments of the methods described herein, the RNA comprise one miRNA from Table A. In embodiments, the RNA comprise two miRNA from Table A. In embodiments, the RNA comprise three miRNA from Table A. In embodiments, the RNA comprise four miRNA from Table A. In embodiments, the RNA comprise five miRNA from Table A. In embodiments, the RNA comprise six miRNA from Table A. In embodiments, the RNA comprise seven miRNA from Table A. In embodiments, the RNA comprise eight miRNA from Table A. In embodiments, the RNA comprise nine miRNA from Table A. In embodiments, the RNA comprise ten miRNA from Table A. In embodiments, the RNA comprise eleven miRNA from Table A. In embodiments, the RNA comprise twelve miRNA from Table A. In embodiments, the RNA comprise thirteen miRNA from Table A. In embodiments, the RNA comprise fourteen miRNA from Table A. In embodiments, the RNA comprise fifteen miRNA from Table A. In embodiments, the RNA comprise sixteen miRNA from Table A. In embodiments, the RNA comprise seventeen miRNA from Table A. In embodiments, the RNA comprise eighteen miRNA from Table A. In embodiments, the RNA comprise nineteen miRNA from Table A. In embodiments, the RNA comprise twenty miRNA from Table A. In embodiments, the RNA comprise twenty -one miRNA from Table A. In embodiments, the RNA comprise twenty -two miRNA from Table A. In embodiments, the RNA comprise twenty-three miRNA from Table A. In embodiments, the RNA comprise twenty-four miRNA from Table A. In embodiments, the RNA comprise twenty-five miRNA from Table A. In embodiments, the RNA comprise twenty- six miRNA from Table A. In embodiments, the RNA comprise twenty-seven miRNA from Table A. In embodiments, the RNA comprise twenty-eight miRNA from Table A. In embodiments, the RNA comprise twenty-nine miRNA from Table A. In embodiments, the RNA comprise thirty' miRNA from Table A. In embodiments, the RNA comprise thirty-one miRNA from Table A. In embodiments, the RNA comprise thirty-tw o miRNA from Table A. In embodiments, the RNA comprise thirty-three miRNA from Table A. In embodiments, the RNA comprise thirty-four miRNA from Table A. In embodiments, the RNA comprise thirty-fivemiRNA from Table A. In embodiments, the RNA comprise thirty-six miRNA from Table A. In embodiments, the RNA comprise thirty-seven miRNA from Table A. In embodiments, the RNA comprise thirty-eight miRNA from Table A.

[0109] In embodiments, the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p. exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR- 1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p. cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, and cell-free miR-4488.

[0110] In embodiments, the RNA consists of exosomal let-7c-5p exosomal miR-100-5p. exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR- 1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p. cell-free miR-18a-5p, cell-free miR-19a-3p. and cell-free miR-4488.[OHl] In embodiments, the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p. and exosomal miR-4488. In embodiments, the RNA consists of exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205- 5p, exosomal miR-20a-5p. exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543.exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, and exosomal miR-4488.

[0112] In embodiments, the RNA comprises cell-free miR-107, cell-free miR-1228-5p, cell- free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p. cell-free miR- 200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR- 5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, and cell-free miR- 4488.

[0113] In embodiments, the RNA consists of cell-free miR-107, cell-free miR-1228-5p, cell- free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p. cell-free miR- 200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR- 5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, and cell-free miR- 4488.

[0114] In embodiments of the methods described herein, the RNA comprises one or more RNA from Set forth in Table B. In embodiments, the the RNA comprises the RNA from Set forth in Table B. In embodiments, the RNA consists of the RNA from Set forth in Table B.

[0115] In embodiments of the methods described herein, the RNA comprise at least one miRNA from Table B. In embodiments, the RNA comprise at least two miRNA from Table B. In embodiments, the RNA comprise at least three miRNA from Table B. In embodiments, the RNA comprise at least four miRNA from Table B. In embodiments, the RNA comprise at least five miRNA from Table B. In embodiments, the RNA comprise at least six miRNA from Table B. In embodiments, the RNA comprise at least seven miRNA from Table B. In embodiments, the RNA comprise at least eight miRNA from Table B. In embodiments, the RNA comprise at least nine miRNA from Table B. In embodiments, the RNA comprise at least ten miRNA from Table B. In embodiments, the RNA comprise at least eleven miRNA from Table B. In embodiments, the RNA comprise at least twelve miRNA from Table B. In embodiments, the RNA comprise at least thirteen miRNA from Table B. In embodiments, the RNA comprise at least fourteen miRNA from Table B. In embodiments, the RNA comprise at least fifteen miRNA from Table B. In embodiments, the RNA comprise at least sixteen miRNA from Table B. In embodiments, the RNA comprise at least seventeen miRNA from Table B. In embodiments, the RNA comprise at least eighteen miRNA from Table B. In embodiments, the RNA comprise at least nineteen miRNA from Table B. In embodiments, the RNA comprise at least twenty miRNA from Table B. In embodiments, the RNA comprise at least twenty-one miRNA fromTable B. In embodiments, the RNA comprise at least twenty -two miRNA from Table B. In embodiments, the RNA comprise at least twenty-three miRNA from Table B. In embodiments, the RNA comprise at least twenty -four miRNA from Table B. In embodiments, the RNA comprise at least twenty -five miRNA from Table B. In embodiments, the RNA comprise at least twenty -six miRNA from Table B. In embodiments, the RNA comprise at least twenty -seven miRNA from Table B. In embodiments, the RNA comprise at least twenty-eight miRNA from Table B. In embodiments, the RNA comprise at least twenty-nine miRNA from Table B. In embodiments, the RNA comprise at least thirty miRNA from Table B. In embodiments, the RNA comprise at least thirty -one miRNA from Table B. In embodiments, the RNA comprise at least thirty-two miRNA from Table B. In embodiments, the RNA comprise at least thirty-three miRNA from Table B. In embodiments, the RNA comprise at least thirty-four miRNA from Table B. In embodiments, the RNA comprise at least thirty-five miRNA from Table B.

[0116] In embodiments of the methods described herein, the RNA comprise one miRNA from Table B. In embodiments, the RNA comprise two miRNA from Table B. In embodiments, the RNA comprise three miRNA from Table B. In embodiments, the RNA comprise four miRNA from Table B. In embodiments, the RNA comprise five miRNA from Table B. In embodiments, the RNA comprise six miRNA from Table B. In embodiments, the RNA comprise seven miRNA from Table B. In embodiments, the RNA comprise eight miRNA from Table B. In embodiments, the RNA comprise nine miRNA from Table B. In embodiments, the RNA comprise ten miRNA from Table B. In embodiments, the RNA comprise eleven miRNA from Table B. In embodiments, the RNA comprise twelve miRNA from Table B. In embodiments, the RNA comprise thirteen miRNA from Table B. In embodiments, the RNA comprise fourteen miRNA from Table B. In embodiments, the RNA comprise fifteen miRNA from Table B. In embodiments, the RNA comprise sixteen miRNA from Table B. In embodiments, the RNA comprise seventeen miRNA from Table B. In embodiments, the RNA comprise eighteen miRNA from Table B. In embodiments, the RNA comprise nineteen miRNA from Table B. In embodiments, the RNA comprise twenty miRNA from Table B. In embodiments, the RNA comprise twenty -one miRNA from Table B. In embodiments, the RNA comprise twenty -two miRNA from Table B. In embodiments, the RNA comprise twenty-three miRNA from Table B. In embodiments, the RNA comprise twenty-four miRNA from Table B. In embodiments, the RNA comprise twenty-five miRNA from Table B. In embodiments, the RNA comprise twenty- six miRNA from Table B. In embodiments, the RNA comprise twenty -seven miRNA from Table B. In embodiments, the RNA comprise tw enty -eight miRNA from Table B. In embodiments, the RNA comprise twenty -nine miRNA from Table B. In embodiments, the RNAcomprise thirty miRNA from Table B. In embodiments, the RNA comprise thirty-one miRNA from Table B. In embodiments, the RNA comprise thirty-two miRNA from Table B. In embodiments, the RNA comprise thirty-three miRNA from Table B. In embodiments, the RNA comprise thirty-four miRNA from Table B. In embodiments, the RNA comprise thirty -five miRNA from Table B.

[0117] miR Controls

[0118] The term “reference RNA” or “normalizer RNA” or “housekeeping RNA” or “control RNA” refers to ty pically 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 (e.g., in both healthy patients and cancer patients). In embodiments, the reference RNA is miRNA. In embodiments, the reference RNA is hsa-miRNA.

[0119] “Reference RNA” described herein include miR-15b-5p, miR-23a-3p, and exosomal miR-30e-5p. 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, and cell-free miR-30e-5p.

[0120] In embodiments, the methods described herein (including embodiments thereof) further comprise detecting the expression level of a reference RNA, 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, or a combination of two or more thereof in the biological sample obtained from the patient. In embodiments, the methods described herein further comprising detecting the expression level 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 the biological sample obtained from the patient. In embodiments, the methods described herein (including embodiments thereof) further comprise normalizing the expression levels of oncogenic RNA to the expression level of the reference RNA. In embodiments, the expression level of the reference RNA are used as a control to the expression level of the RNA. In embodiments, RNA is miRNA.

[0121] In embodiments, the methods described herein (including embodiments thereof)further comprise detecting the expression level of a reference miRNA in the biological sample, wherein the reference miRNA is as described herein. In embodiments, the methods described herein (including embodiments thereof) further comprise normalizing the expression level of the miRNA to the expression level of the reference miRNA, thereby obtaining a normalized expression level of the miRNA. In embodiments, the expression level of the reference miRNA is used as a control to the normalized expression level of the miRNA.

[0122] In embodiments, an elevated expression level refers to a normalized expression level of 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 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 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 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 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 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 miRNA that is at least 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 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 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 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 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 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 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 miRNA that is at least 4 times greater than the expression level of the reference miRNA. Inembodiments, an elevated expression level refers to a normalized expression level of 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 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 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 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 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 miRNA that is at least 10 times greater than the expression level of the reference miRNA. In embodiments, an elevated expression level refers to a normalized expression level of miRNA that is statistically significantly greater than the expression level of the reference miRNA.

[0123] Methods

[0124] In embodiments of all 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.

[0125] Anti-Cancer Agents

[0126] 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. In embodiments, the anti-cancer agent is a chemotherapeutic agent. In embodiments, the anti-cancer agent is adagrasib, bevacizumab, irinotecan, capecitabine, ramucirumab, oxaliplatin, cetuximab, fluorouracil, fruquintinib, ipilimumab, pembrolizumab, leucovorin, trifluridine, tipiracil, nivolumab, panitumumab, regorafenib, tucatinib, ziv-aflibercept, encorafenib, trastuzumab,pertuzumab, lapatinib, larotrectinib, entrectinib, selpercatinib, sotorasib, regorafenib, or a combination of two or more thereof.

[0127] 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 alkydating 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, ixabepilone, patupilone, or sagopilone. the chemotherapeutic agent comprises 5-fluorouracil, leucovorin, oxaliplatin, irinotecan, capecitabine, or a combination of two or more thereof.

[0128] 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. In embodiments, 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.

[0129] “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.

[0130] “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, selumetinib / AZD6244, GSK1120212 / trametinib, 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., hexamethly melamine, 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, fl oxouridine, cytarabine), purine 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, PD 184352, 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 growth 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 toniIn,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, epidermalgrowth factor receptor (EGFR)-targeted therapy or therapeutic (e.g. gefitinib. erlotinib, cetuximab, lapatinib. panitumumab, vandetanib, afatinib, 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.

[0131] Kits

[0132] 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.

[0133] “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).

[0134] 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 lengthdepending 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., a Southern 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.

[0135] 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.

[0136] 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.

[0137] In embodiments, methods include detecting a level of a biomarker with 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 ohgonucleotide / 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 enzy me or functional fragment thereof, or a radioactive agent. In embodiments, an antibody is detectably 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.

[0138] 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 thereof) 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, polyacry lamides, 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.

[0139] 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 sitesare 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.

[0140] 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.

[0141] 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.

[0142] 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).

[0143] 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 andthe 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, paramagnetic metal 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.

[0144] 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 type of assay to be performed with the oligonucleotide and is readily determined by the skilled artisan.

[0145] 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.

[0146] 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 thesecomponents 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 colorectal cancer in a patent, including reagents for detecting miRNA markers in a biological (e.g., blood) sample from a patient.

[0147] 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.

[0148] miRNA Expression

[0149] In embodiments of the methods described herein, the elevated level of gene expression is an elevated 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).

[0150] In embodiments of the methods described herein, the individual elevated expression level of the miRNA described herein are used. In embodiments, the individual elevatedexpression level of the exosomal miRNA described herein are used. In embodiments, the individual elevated expression level of the cell-free miRNA described are used. In embodiments, the individual elevated expression level of the cell-free miRNA and exosomal miRNA described herein are used.

[0151] In embodiments, the elevated expression levels of the miRNA are weighted and combined to form a risk score. In embodiments, the elevated expression levels of the exosomal RNA described herein are weighted and combined to form a risk score. In embodiments, the elevated expression levels of the cell-free miRNA described herein are weighted and combined to form a risk score. In embodiments, the elevated expression levels of the cell-free and exosomal miRNA described herein are weighted and combined to form a risk score. In embodiments, the expression levels of the miRNA are normalized to the expression level of reference miRNA, and the normalized expression levels of the miRNA (cell-free miRNA and / or exosomal miRNA) are weighted.

[0152] 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 2< tmethod 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 quantifies the absolute expression of each miRNAs in each sample analyzed and then calculates the different expression of each miRNA in sample versus the controls. These expression values of the 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 RNA.

[0153] 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 colorectal cancer), comprising the steps of (i) normalizing and / or scaling numeric values of the RNA expression data (e.g.. the exosomal miRNA expression data and / or cell-free miRNA expression data), (ii) refining the discriminatory power of individual 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 a control and the comparison allows thesample to be designated as positive or negative for colorectal cancer and / or colorectal adenoma. 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 profde. In embodiments, the expression of level of each miRNA is calculated using 2'ACtmethod, the normalized expression values are log10transformed, and then used in the equations herein.

[0154] Embodiments 1 to 77

[0155] Embodiment 1. A method of detecting RNA in a patient with colorectal cancer or colorectal adenoma, the method comprising detecting an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290. exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205- 5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell- free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR- 14 l-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p. cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell- free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof.

[0156] Embodiment 2. The method of embodiment 1 for detecting RNA in a patient with colorectal cancer.

[0157] Embodiment 3. The method of embodiment 2, wherein the colorectal cancer is Stage I.

[0158] Embodiment 4. The method of embodiment 2, wherein the colorectal cancer is StageII.

[0159] Embodiment 5. The method of embodiment 2, wherein the colorectal cancer is StageIII.

[0160] Embodiment 6. The method of embodiment 2. wherein the colorectal cancer is StageIV.

[0161] Embodiment 7. The method of embodiment 1 for detecting RNA in a patient withcolorectal adenoma.

[0162] Embodiment 8. The method of embodiment 7, wherein the colorectal adenoma is high- risk colorectal adenoma.

[0163] Embodiment 9. The method of embodiment 7, wherein the colorectal adenoma is advanced colorectal adenoma.

[0164] Embodiment 10. The method of embodiment 7, wherein the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia.

[0165] Embodiment 11. The method of any one of embodiments 1 to 10, further comprising administering to the patient an effective amount of an anti-cancer agent.

[0166] Embodiment 12. A method of treating colorectal cancer or colorectal adenoma in a patient in need thereof, the method comprising: (i) detecting an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR- 1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205- 5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell- free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR- 14 l-3p, cell-free miR-142-3p. cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p. cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell- free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof; and (ii) administering to the patient an effective amount of an anti-cancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof.

[0167] Embodiment 13. A method of treating colorectal cancer or colorectal adenoma in a patient in need thereof, the method comprising administering to the patient an effective amount of an anti-cancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof; wherein a biological sample obtained from the patient comprises an elevated expression level, relative to a control, of aRNA; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR- 10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150- 5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a- 3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455- 3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488. cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323. cell-free miR-141-3p, cell-free miR-142-3p. cell-free miR-1538, cell-free miR-17- 5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof.

[0168] Embodiment 14. The method of embodiment 12 or 13 for treating colorectal cancer.

[0169] Embodiment 15. The method of embodiment 14, wherein the colorectal cancer is StageI.

[0170] Embodiment 16. The method of embodiment 14, wherein the colorectal cancer is StageII.

[0171] Embodiment 17. The method of embodiment 14, wherein the colorectal cancer is StageIII.

[0172] Embodiment 18. The method of embodiment 14, wherein the colorectal cancer is StageIV.

[0173] Embodiment 19. The method of embodiment 12 or 13 for treating colorectal adenoma.

[0174] Embodiment 20. The method of embodiment 19, wherein the colorectal adenoma is high-risk colorectal adenoma.

[0175] Embodiment 21. The method of embodiment 19, wherein the colorectal adenoma is advanced colorectal adenoma.

[0176] Embodiment 22. The method of embodiment 19, wherein the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia.

[0177] Embodiment 23. The method of any one of embodiments 12 to 22, comprising administering to the patient the effective amount of the anti -cancer agent.

[0178] Embodiment 24. A method of diagnosing a patient with colorectal cancer or colorectal adenoma, the method comprising: (i) detecting the expression level of RNA in a biological sample obtained from the patient; and (ii) diagnosing the patient as having colorectal cancer orcolorectal adenoma when the biological sample has an elevated expression level, relative to a control, of the RNA; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p. exosomal miR-4488, cell-free miR-107. cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR- 1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p. cell-free miR-18a-5p, cell-free miR-19a-3p. cell-free miR-4488, or a combination of two or more thereof.

[0179] Embodiment 25. The method of embodiment 24 for diagnosing a patient with colorectal cancer.

[0180] Embodiment 26. The method of embodiment 25, wherein the colorectal cancer is StageI.

[0181] Embodiment 27. The method of embodiment 25, wherein the colorectal cancer is StageII.

[0182] Embodiment 28. The method of embodiment 25, wherein the colorectal cancer is StageIII.

[0183] Embodiment 29. The method of embodiment 25, wherein the colorectal cancer is StageIV.

[0184] Embodiment 30. The method of embodiment 24 for diagnosing a patient with colorectal adenoma.

[0185] Embodiment 31. The method of embodiment 30, wherein the colorectal adenoma is high-risk colorectal adenoma.

[0186] Embodiment 32. The method of embodiment 30, wherein the colorectal adenoma is advanced colorectal adenoma.

[0187] Embodiment 33. The method of embodiment 30, wherein the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia.

[0188] Embodiment 34. A method of monitoring treatment in a patient having colorectalcancer or colorectal adenoma or monitoring risk for developing colorectal cancer or colorectal adenoma in a patient, the method comprising: (i) detecting the expression level of RNA in a biological sample obtained from the patient at a first time point; (ii) detecting the expression level of the RNA in a biological sample obtained from the patient at a second time point, wherein the second time point is later than the first time point; and (iii) comparing the expression level of the RNA at the second time point to the expression level of the RNA at the first time point, thereby monitoring treatment or monitoring risk; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205- 5p, exosomal miR-20a-5p. exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543. exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell- free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR- 141-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell -free miR-34a-5p, cell- free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof.

[0189] Embodiment 35. The method of embodiment 34, wherein an elevated expression level of RNA at the second point in time when compared to the expression level of RNA at the first point in time indicates that the patient has an increased risk of developing colorectal cancer or colorectal adenoma.

[0190] Embodiment 36. The method of embodiment 34 or 35 for monitoring treatment in a patient having colorectal cancer or monitoring risk for developing colorectal cancer in a patient.

[0191] Embodiment 37. The method of embodiment 34 or 35 for monitoring treatment in a patient having colorectal cancer.

[0192] Embodiment 38. The method of embodiment 34 or 35 for monitoring risk for developing colorectal cancer in the patient.

[0193] Embodiment 39. The method of any one of embodiments 34 to 38, wherein the colorectal cancer is Stage I.

[0194] Embodiment 40. The method of any one of embodiments 34 to 38, wherein the colorectal cancer is Stage II.

[0195] Embodiment 41. The method of any one of embodiments 34 to 38, wherein thecolorectal cancer is Stage III.

[0196] Embodiment 42. The method of any one of embodiments 34 to 38, wherein the colorectal cancer is Stage IV.

[0197] Embodiment 43. The method of embodiment 34 or 35 for monitoring treatment in a patient having colorectal adenoma or monitoring risk for developing colorectal adenoma in a patient.

[0198] Embodiment 44. The method of embodiment 34 or 35 for monitoring treatment in a patient having colorectal adenoma.

[0199] Embodiment 45. The method of embodiment 34 or 35 for monitoring risk for developing colorectal adenoma in the patient.

[0200] Embodiment 46. The method of embodiment 34, 35, 43, 44, or 45, wherein the colorectal adenoma is high-risk colorectal adenoma.

[0201] Embodiment 47. The method of embodiment 34, 35, 43, 44, or 45, wherein the colorectal adenoma is advanced colorectal adenoma.

[0202] Embodiment 48. The method of embodiment 34, 35, 43, 44, or 45, wherein the colorectal adenoma is advanced colorectal adenoma with high grade dysplasia.

[0203] Embodiment 49. The method of any one of embodiments 1 to 48, wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR- 1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184. exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205- 5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell- free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p. cell-free miR-1323, cell-free miR- 141 -3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell- free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, and cell-free miR-4488.

[0204] Embodiment 50. The method of any one of embodiments 1 to 48, wherein the RNA consists of exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR- 1246, exosomal miR-1290, exosomal miR-I35b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p. exosomal miR-19a-3p, exosomal miR-4488, cell- free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR- 141-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell -free miR-34a-5p, cell- free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p. and cell-free miR-4488.

[0205] Embodiment 51. The method of any one of embodiments 1 to 48. wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR- 1246. exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205- 5p, exosomal miR-20a-5p. exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, and exosomal miR-4488.

[0206] Embodiment 52. The method of any one of embodiments 1 to 48. wherein the RNA consists of exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR- 1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205- 5p, exosomal miR-20a-5p. exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543. exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, and exosomal miR-4488.

[0207] Embodiment 53. The method of any one of embodiments 1 to 48. wherein the RNA comprises cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR- 34a-5p. cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p. cell-free miR-19a-3p, and cell-free miR-4488.

[0208] Embodiment 54. The method of any one of embodiments 1 to 48. wherein the RNA consists of cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR- 1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell- free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p. cell-free miR-5193, cell-free miR-877-3p, cell-free miR- 18a-5p, cell-free miR-19a-3p, and cell-free miR-4488.

[0209] Embodiment 55. The method of any one of embodiments 1 to 54. further comprisingdetecting the 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.

[0210] Embodiment 56. The method of any one of embodiments 1 to 54. further comprising detecting the expression level of a reference RNA in the biological sample, wherein the reference RNA comprises miR-15b-5p, miR-23a-3p, and miR-30e-5p.

[0211] Embodiment 57. The method of any one of embodiments 1 to 54, further comprising detecting the expression level of a reference RNA in the biological sample, 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.

[0212] Embodiment 58. The method of any one of embodiments 1 to 54, further comprising detecting the expression level of a reference RNA in the biological sample, 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.

[0213] Embodiment 59. The method of any one of embodiments 55 to 58, wherein the reference RNA further comprises exosomal miR-16-5p. cell-free miR-16-5p. exosomal miR- 103a-3p, cell-free miR-103a-3p, exosomal U6, cell-free U6, or a combination of two or more thereof.

[0214] Embodiment 60. The method of any one of embodiments 55 to 58, wherein the reference RNA further comprises exosomal miR-16-5p, cell-free miR-16-5p, exosomal miR- 103a-3p, cell-free miR-103a-3p, exosomal U6, and cell-free U6.

[0215] Embodiment 61. The method of any one of embodiments 1 to 60, wherein the biological sample is a liquid biological sample.

[0216] Embodiment 62. The method of any one of embodiments 1 to 60, wherein the biological sample is a blood sample.

[0217] Embodiment 63. The method of any one of embodiments 1 to 60, wherein the biological sample is a plasma sample.

[0218] Embodiment 64. The method of any one of embodiments 1 to 60. wherein the biological sample is a serum sample.

[0219] Embodiment 65. The method of any one of embodiments 1 to 60. wherein thebiological sample is a tissue sample.

[0220] Embodiment 66. The method of any one of embodiments 1 to 65, wherein the patient is 45 years old or older.

[0221] Embodiment 67. The method of any one of embodiments 1 to 66, wherein the patient is of Caucasian descent.

[0222] Embodiment 68. The method of any one of embodiments 1 to 65, wherein the patient is of Asian descent.

[0223] Embodiment 69. The method of any one of embodiments 1 to 68, wherein the control is a healthy patient.

[0224] Embodiment 70. The method of any one of embodiments 1 to 68. wherein the control is a healthy patient that does not have cancer

[0225] Embodiment 71. The method of any one of embodiments 1 to 68. wherein the control is a healthy patient 45 years old or older that does not have cancer.

[0226] Embodiment 72. The method of any one of embodiments 1 to 71, wherein the patient is a human patient.

[0227] Embodiment 73. The method of any one of embodiments 1 to 72, wherein the anticancer agent is adagrasib, bevacizumab, irinotecan, capecitabine, ramucirumab, oxaliplatin, cetuximab, fluorouracil, fruquintinib, ipilimumab. pembrolizumab, leucovorin, trifl uridine, tipiracil, nivolumab, panitumumab, regorafenib. tucatinib, ziv-aflibercept, encorafenib, trastuzumab, pertuzumab, lapatinib, larotrectinib, entrectinib, selpercatinib, sotorasib, regorafenib, or a combination of two or more thereof.

[0228] Embodiment 74. The method of any one of embodiments 1 to 72, wherein the anticancer agent is a chemotherapeutic agent.

[0229] Embodiment 75. The method of embodiment 74, 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 76. The method of embodiment 75, 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 anthracy cline 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 vindesine; the taxane compound is paclitaxel or docetaxel; and the epothilone compound is epothilone, ixabepilone. patupilone, or sagopilone.

[0231] Embodiment 77. A kit comprising reagents capable of detecting an expression level of RNA from a biological sample; wherein the RNA comprises exosomal let-7c-5p exosomal miR- 100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b- 5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR- 1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p. cell-free miR-18a-5p, cell-free miR-19a-3p. cell-free miR-4488, or a combination of two or more thereof.EXAMPLE

[0232] The inventors developed and validated a cell-free and exosomal microRNA (cf- miRNA and exo-miRNA) blood-based test in a population eligible for colorectal cancer screening. The co-pnmary outcomes were sensitivity for colorectal cancer, sensitivity for colorectal advanced adenomas, and specificity for advanced neoplasia (cancer and advanced adenomas), compared to colonoscopy.

[0233] Of 1602 study-participants, 837 were utilized for a genome-wide discovery of circulating biomarkers for advanced adenomas and colorectal cancer and 765 to train and test a blood-based test powered by XGBoost. In the testing cohort, the blood-based test showed an area under the receiver operating characteristic curve (AUC) of 95.2% for distinguishing colorectal cancer and advanced adenomas from controls. A total of 90.9% of individuals with colorectal cancer and 81.0% of individuals with advanced adenomas were detected by the bloodtest. Sensitivity’ for stage I, II, or III colorectal cancer was 92.3% (95% confidence intervals, 79.7 to 97.4) and sensitivity for advanced adenomas was 81.0% (95% confidence intervals, 69.2 to 89. 1). A total of 84.6% of participants without any advanced colorectal neoplasia (cancer oradvanced adenoma) had a negative blood-based test, therefore specificity for advanced neoplasia was 84.6% (95% confidence intervals. 77.0 to 90.0). Specificity’ for negative colonoscopy was 83.1% (95% confidence intervals, 71.5 to 90.5)

[0234] In an average-risk population, this blood-based test had 92.3% sensitivity for colorectal cancer, 81.0% sensitivity’ for advanced adenomas, and 84.6% specificity for advanced colorectal neoplasia.

[0235] Materials and Methods

[0236] Study Design. The DENEB study (DEtection of colorectal NEoplasias in Blood) was a prospective, observational, STARD-compliant, multicenter study designed according to the EDRN (Early Detection Research Network) recommendations to complete phases I, II, and III. This study is aimed at developing and testing a cf-miRNA and exo-miRNA blood test for detecting early-stage colorectal cancer and advanced adenomas in a screening-relevant population (FIG. 4). Eligible individuals were enrolled from two European and Japanese sites. The study was approved by each participating center's institutional review board. All participants or their legal representatives provided written informed consent.

[0237] Study Population. Eligible persons were 45 years of age or older at the time of consent, at average risk of colorectal cancer, and undergoing routine screening with colonoscopy. Exclusion criteria included a history of cancer, a known diagnosis of inflammatory bowel disease, and a hereditary’ colorectal cancer syndrome.

[0238] Clinical Procedures. Participants provided written informed consent and a blood sample before undergoing colonoscopy. Colonoscopy preparation followed established guidelines, with the endoscopist assessing the quality of bowel preparation for each participant. The colonoscopy had to be complete to the appendiceal orifice or ileocecal valve, except in cases where a cancerous stenosis prevented completion. The size and location of any colonoscopy -identified lesions were recorded, and resected lesions underwent histopathological review. If multiple lesions were present, the most advanced lesion was considered the primary one for final data analysis. According to endoscopic and histopathological review, the study participants were classified according to the European Society of Gastrointestinal Endoscopy (ESGE) recommendations as non-disease control (negative colonoscopy), low-risk adenomas (individuals with colonoscopy-detected four or fewer adenomas with low-grade dysplasia and smaller than 10 mm in diameter), high-risk adenomas (defined as individuals with colonoscopy- detected five or more adenomas, or one or more adenoma with high-grade dysplasia, or one ormore adenoma > 10 mm), or or colorectal cancer.

[0239] Outcome Measures. The outcome measures were sensitivity for colorectal cancer and advanced adenomas, and specificity for advanced neoplasia in average-risk participants, aged 45 years or older, compared to colonoscopy as a reference. Secondary outcome measures included sensitivity for stage I-III colorectal cancer. The exploratory analyses included the sensitivity for the detection of different subty pes of colorectal advanced adenomas (according to adenoma size, degree of dysplasia, or adenoma count).

[0240] Laboratory Procedures. Plasma samples (0.5 rnL) were collected in the recruiting centers, shipped with dry ice to the central biorepository, and stored at -80° C. The test that was herein developed and tested is a blood-based test interrogates the transcriptional expression of 19 cf-miRNAs and 20 exo-miRNAs using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results are integrated into a binary “abnormal signal detected” (positive test) or “normal signal detected” (negative test). Furthermore, when an “abnormal signal” was detected, the test provided the most likely result as “advanced adenoma” or “colorectal cancer”.

[0241] Statistical Analyses. The performance of the blood-based test was evaluated in terms of ROC curve analysis, sensitivity for colorectal cancer, sensitivity for advanced adenomas, and specificity for advanced neoplasia, compared to screening colonoscopy, with the threshold for low vs. high-risk scores defined by minimizing the distance from the point of perfect discrimination (0,1). Confidence intervals around the sensitivity and specificity values were estimated by the Wilson method, while confidence intervals for the ROC curves were estimates with 2000 stratified bootstrap replicates. Statistical significance defined at p<0.05 for all analyses and was tested by the student t-test for two groups and by anova for multiple groups. All analyses were computed in R.

[0242] Results

[0243] Participants. A total of 3083 participants were enrolled, and the present study included 1613 study participants (FIG. 5). 848 study participants were allocated to the biomarker discovery’ cohorts and 765 to the training (clinical cohort #1) and testing (clinical cohort #2) of the blood-based test. There was no overlap of study participants across study cohorts and all biospecimens were prospectively collected. The mean age of the participants in the two final clinical cohorts was 63.5 (range: 21-100, training) and 63.5 (range: 29-87, validation; Table 4). Because of the geographical origin of the participating centers, there was an equal representation of individuals of Asian and European / Caucasian heritage in the study population.

[0244] Biomarker Panel Development. Cell-free transcriptomic differential gene expression analysis identified 193 cell-free candidate biomarkers for advanced adenoma detection and 150 cell-free candidates for colorectal cancer detection (FIGS. 6A-6B). Of these, 33 were in common between both and were given consideration as potentially diagnostic cell-free biomarkers. We utilized three additional independent cohorts (FIGS. 6C-6E) to reduce the number of candidate biomarkers to a panel of 19 cell-free transcripts (FIG. 6F). Exosomal transcriptomic differential gene expression analysis identified 226 exosomal candidate biomarkers for advanced adenoma detection and 104 exosomal candidates for colorectal cancer detection (FIGS. 7A-7B). Of these, 46 were shared and were given consideration as potentially diagnostic. Utilizing three additional independent cohorts (FIGS. 7C-7E), the number of candidate biomarkers was reduced to 20 exosomal biomarkers of diagnostic potential (FIG. 7F).

[0245] Development and testing of a blood-based transcriptomic assay for colorectal cancer and advanced adenoma detection.

[0246] The diagnostic test, named DENEB, was trained using an XGBoost-based model, based on the RT-qPCR results from the clinical cohort #1 (training), to predict the presence of advanced adenomas (“advanced adenoma risk score”) and the presence of colorectal cancer (“colorectal cancer risk score”). The model was then locked before transitioning it to the clinical cohort #2 (testing).

[0247] In the clinical cohort #1, the cf-miRNA-only and the exo-miRNA-only cancer-specific blood-based tests achieved AUC values of 92.4% (CI95%: 89.9 - 94.9%) and 94.8% (CI95%: 92.9 - 96.8%) for the detection of colorectal cancer, with their combination reaching an AUC value of 97.7% (CI95%: 96.6 - 98.8%; FIGS. 1A-1B). Individuals with colorectal cancer, both early- and late-stage, demonstrated significantly higher cancer-specific risk scores than all other groups (FIG. 1C). For the advanced adenoma risk score, the cf-miRNA-only and the exo- miRNA-only tests achieved AUC values of 94.8% (CI95%: 92.7 - 96.9%) and 90.4% (CI95%: 87.5 - 93.3%), and their combination reached an AUC value of 97. 1% (CI95%: 95.8 - 98.4%; FIGS. 1D-1E). Individuals with colonoscopy-detected advanced adenomas demonstrated significantly higher risk scores than all other groups (FIG. IF). The results of the cancerspecific and adenoma-specific risk scores were dichotomized as “abnormal signal detected” and “normal signal detected” by utilizing the point of minimum distance from the (1,0) coordinate (thresholds= -0.49019373 for the colorectal cancer risk score and 0.57667491 for the advanced adenoma risk score). The overall test categorized results as “normal” (test results negative for both colorectal cancer and advanced adenomas), or “abnormal” (test results positive for eithercolorectal cancer or advanced adenomas or both).

[0248] In the transition from the clinic cohort # 1 (training) to the clinic cohort #2 (independent testing), the diagnostic model and the positivity thresholds were fully locked. Individuals with negative colonoscopy findings and individuals with low-risk adenomas both clustered in the double negative area of the density plots in both cohorts (FIG. 2). Study participants who had advanced adenomas or colorectal cancer, instead, clustered separately (FIG. 8)

[0249] In the second clinic cohort (testing), the blood-based test confirmed the results obtained in training. The cf-miRNA-only and exo-miRNA-only cancer-specific models achieved AUC values of 82.7% (CI95%: 76.5 - 88.9%) and 91.5% (CI95%: 87.2 - 95.8%), respectively (FIG. 3A). Their combination achieved an AUC value of 95.2% (CI95%: 92.4 - 98.0%), with higher cancer-specific risk scores for those with colorectal cancer than all other colonoscopy findings (FIGS. 3B-3C). For the adenoma-specific model, the cf-miRNA-only and the exo- miRNA-only models achieved AUC values of 90.2% (CI95%: 85.8 - 94.6%) and 89.0% (CI95%: 84.0 - 94.0%), respectively (FIG. 3D). Their combination demonstrated an AUC value of 95.2% (CI95%: 92.2 - 98.2%), with higher adenoma-specific risk scores for those with adenomas, compared with all other study participants (FIGS. 3E-3F).

[0250] Primary outcomes analyses

[0251] In the clinical cohort 1, a total of 123 of 134 participants (91.8%) with colonoscopy- detected colorectal cancer had a positive blood-based test, and 11 (8.2%) had a negative test, which indicates an overall sensitivity of 91.8% (CI95%: 85.9 - 95.4) for colorectal cancer. In the clinical cohort 2. a total of 50 of 55 participants (90.1%) with colonoscopy-detected colorectal cancer had a positive test, and 5 (9.9%) had a negative test, which confirmed an overall sensitivity of 90. 1% (CI95%: 80.4 - 96. 1) for colorectal cancer. The cf / exo-miRNA blood-test also identified 72 of 80 screening-relevant (stage I, II. or III) colorectal cancers in the clinical cohort 1 (sensitivity: 90.0%; CI95%: 81.5 - 94.9) and 36 of 39 in the clinical cohort 2 (sensitivity: 92.3%; CI95%: 79.7 - 97.4). The test identified 26 of 28 stage IV cancer in the cohort 1 (sensitivity: 92.8%; CI95%: 77.4 - 98.0) and 9 of 9 in the cohort 2 (Table 1).

[0252] For the advanced adenomas, in the clinical cohort 1, atotal of 1 10 of 134 participants (82.1%) with colonoscopy-detected advanced adenomas had a positive blood-based test, and 24 (17.9%) had a negative test, which indicates a sensitivity of 82. 1% (CI95%: 74.7 - 87.7) for advanced adenomas. In the clinical cohort 2, a total of 47 of 58 participants (81.0%) withcolonoscopy -detected advanced adenomas had a positive test, and 11 (19.0%) demonstrated negative test results, which indicates a sensitivity of 81.0% (CI95%: 69.2 - 89. 1) for advanced adenomas (Table 1).

[0253] For specificity analyses, in the clinical cohort 1. a total of 241 of 267 participants (90.3%) without colorectal advanced neoplasias (cancer or advanced adenomas) had negative test results, which indicates that the cf / exo-miRNA blood-based test had an overall specificity of 90.3% (CI95%: 86. 1 - 93.3). Moreover, when sub-analyzing these results for the absence of any colorectal lesion. 116 of 133 participants (87.2%) with negative colonoscopy findings had a negative blood test result, which indicates a specificity of 87.2% (CI95%:80.5 - 91.9) for the absence of colorectal lesions at colonoscopy. In the clinical cohort 2, a total of 99 of 117 participants (84.6%) without colorectal advanced neoplasias had a negative cf / exo-miRNA test, which indicates that an overall specificity of 84.6% (CI95%:77.0 - 90.0). At sub-analysis for individuals with negative colonoscopy findings, 49 of 59 participants (83.1%) with negative colonoscopy findings had a negative cf / exo-miRNA blood test, which indicates a specificity of 83.1% (CI95%:71.5 - 91.6, Table 1).

[0254] Exploratory' analyses

[0255] The sensitivity of this blood test for stage 0 / 1 colorectal cancer was 92.5% (CI95%:80. 1 - 97.4) in the clinical cohort 1 and 95.5% (CI95%:78.2 - 99.2) in the clinical cohort 2. For stage II colon cancer, it was 97. 1% (CI95%:85. 1 - 99.5) in cohort 1 and 90.9% (CI95%:62.3 - 98.4) in cohort 2. For stage III colorectal cancer, it was 76.5% (CI95%:52.7 - 90.4) in cohort 1 and 66.7% (CI95%:30 - 90.3) in cohort 2. The sensitivity of the cf / exo-miRNA blood-based test for distal cancers was 88.9% (CI95%:78.8 - 94.5) in the clinical cohort 1 and 96.0% (CI95%: 80.5 - 99.3) in the clinical cohort 2 (Table 3).

[0256] For the advanced adenomas sub-analyses, the cf / exo-miRNA blood test was validated in the clinical cohort 2 with a sensitivity of 100% for advanced adenomas larger than 30 mm in diameter. 85.7% (CI95%: 48.7 - 97.4) for advanced adenomas of size 20-29 mm, 84.0% (CI95%:65.4 - 93.6) for advanced adenomas of size 10-19 mm. For individuals whose adenomas were considered “advanced” for reasons other than size (either high-grade dysplasia or five or more adenomas), the cf / exo-miRNA blood test still detected 42 of 53 participants in the clinical cohort 1 and 17 of 23 participants in the clinical cohort 2. which indicates that the test maintained a sensitivity of 79.3% (CI95%:66.5 - 88.0) and 73.9% (CI95%:83.5 - 87.5), respectively. In fact, a total of 44 of 55 participants with advanced adenomas with high-grade dysplasia had a positive cf / exo-miRNA blood test in the clinical cohort 1, which indicates asensitivity of 80.0% (CI95%:67.6 - 88.5). In the clinical cohort 2, a total of 18 of 27 participants with colonoscopy-detected advanced adenomas with high-grade dysplasia had a positive cf / exo- miRNA blood based test, which validates the sensitivity’ for the presence of high-grade dysplasia at 66.7% (CI95%:47.8 - 81.4) (FIGS. 9-10). Finally, in the clinical cohort 2, the cf / exo-miRNA blood-based test demonstrated a sensitivity of 88.5% (CI95%: 71.0 - 96.0) for Asian patients with advanced adenomas and 75% (CI95%:57.9 - 86.8) for Caucasian patients (Table 4).

[0257] Discussion

[0258] This study reports on a new generation of exosome-based blood test using transcriptomic analysis to non-invasively detect colorectal cancer and advanced adenomas. The test achieved 84.6% specificity, 92.3% sensitivity for stage I-III colorectal cancers, and 81.0% sensitivity for advanced adenomas. Powered by machine learning, the test was developed and independently tested in a large, international cohort of 1613 participants. This non-invasive approach will complement existing screening strategies and appeal to individuals who resist traditional or more invasive screening methods.

[0259] Despite screening recommendations, colorectal cancer remains a leading cause of cancer-related deaths, with most fatalities occurring in unscreened individuals. Colonoscopy remains the most effective tool to reduce one’s risk of colorectal cancer, but the low compliance drives the need for alternative, less invasive tests. Blood-based tests offer a potential solution, as they are preferred over stool or endoscopic methods. However, existing blood-based tests (including methylation-, cfDNA-, fragmentomic-. and multiomic-based approaches) reported high sensitivity' values for colorectal cancer but not advanced adenomas, a crucial endpoint to reduce both colorectal cancer mortality and incidence. The most recent cfDNA-based blood test (Shield) recently reported a sensitivity of 87.5% (CI95% = 75.3 - 94.1) for screening-relevant colorectal cancer stages but 13.2% (CI95% = 11.3 - 15.3) for advanced adenomas. Similarly, the recently-announced Freenome test PREEMPT-CRC trial results reported a sensitivity of 75.0% (CI95% = 62.8 - 84.2%) for stage I-III colorectal cancer but 12.5% (CI95% = 11.3 - 13.8%) for advanced adenomas. This disparity informed our hypothesis that advanced adenomas and colorectal cancer, sitting at opposite ends of the adenoma-carcinoma sequence, necessitate distinct biomarkers for accurate detection of both. Therefore, our cf / exo-miRNA blood-based test incorporates both adenoma-specific and cancer-specific biomarkers. Although blood RNA- based tests have not been studied as extensively as cfDNA-based technologies, studies have already reported that transcriptomic assays can detect pre-cancerous lesions non-invasively. Furthermore, our group has, in the past several years, demonstrated that a combination ofcirculating cell-free and exosomal trans criptomic analyses can improve a test’s performance by leveraging the abundance of cell-free analytes with the tissue-specificity of exosomes.

[0260] This blood test demonstrated at least several advantages. It maintained a stable sensitivity for all colorectal cancer stages, including early-stage lesions, and locations. The sensitivity values for advanced adenomas were high, and we noticed a trend for higher values for individuals with larger adenomas, suggesting a degree of size-specificity, but could still detect advanced adenomas smaller than 20 mm.

[0261] In conclusion, this study introduces a novel exosome-based transcriptomic assay capable of detecting both early-stage colorectal cancer and advanced adenomas from a single blood draw. Our study was founded on the hypothesis that distinct biomarkers are required for the detection of colorectal cancer and advanced adenomas, given their distinct biological characteristics. By addressing the limitations of existing screening methods, this approach offers a complementary strategy that may contribute to colorectal cancer screening campaigns.

[0262] Supplemental Methods

[0263] The DENEB study was a multi-centric, international, ERDN phase I to III, PRoBE- compliant, retro-prospective cohort biomarkers study that encompassed a systematic miRNA- based biomarker discovery (ERDN phase I), a clinical assay development and training phase (ERDN phase II), and final performance evaluation (ERDN phase III). The study was conducted in accordance with the Declaration of Helsinki, it was registered and completed on clinicaltrials.gov (NCT06342401). and it was STARD-compliant. The prospective collection of the patient datasets in each cohort was approved by the institutional review board (IRB) at each institution and at the leading center (IRB No. 23228).

[0264] The study design is depicted in FIG. 4. This multi-center, prospective, international, cohort biomarker study comprised two main phases: first, a blood-based discovery phase which was used to discover biomarkers of potential diagnostic interest for the detection of early-stage colorectal cancer as well as advanced adenomas. Second, an RT-qPCR phase during which the diagnostic test was trained and externally tested. There was no overlap of patients in any of the study phases.

[0265] The discovery phase of this study was designed to identify the biomarkers that were most likely to be tumor-derived and it utilized two independent clinical cohorts and three additional publicly available cohorts. The clinic-based discovery cohort included 570 individuals from two independent cohorts (N=268 and N=302, respectively). The discovery' efforts werethen assessed and further refined by comparison with three external and independent in-silico cohorts (GSE 39833. GSE 25609, and GSE 41655). This third step in the discovery’ approaches comprised 267 individuals.

[0266] Candidate biomarkers that demonstrated a statistically significant association wi th the presence of AA and CRC at the conclusion of the discovery phase were formally included in the construction of the RT-qPCR blood-based test. In transitioning our findings from small RNA sequencing to RT-qPCR, we first trained a machine learning model on the RT-qPCR results from a first clinic-based cohort (N=535). After the machine learning-based diagnostic assay was established and locked, based on a combination of cf- and exo-miRNAs, the assay was transitioned to the validation cohort (N=230). All the 785 participants in both the training and testing cohorts were enrolled at the Hospital Clinic de Barcelona (Barcelona, Spain) and the Mie University Graduate School of Medicine (Mie, Japan) between 2018 and 2023. Participant randomization to the training and testing cohorts (7:3 ratio) utilized a block-based strategy to ensure a representative balance of sex, age, disease-stage, and ethnicity.

[0267] Definitions, inclusion and exclusion criteria, and study endpoints. Eligible participants were 18 to 100 years of age at the time of consent. Cases were defined as having a histological diagnosis of colorectal cancer (TNM classification, 8thedition) or advanced adenomas, according to the ESGE 2020 recommendations. Therefore, colonoscopy results were classified as non-disease control (negative colonoscopy), low-risk adenomas (individuals with colonoscopy -detected four or fewer adenomas with low-grade dysplasia and smaller than 10 mm in diameter), high-risk adenomas (defined as individuals with colonoscopy-detected five or more adenomas, or one or more adenoma with high-grade dysplasia, or one or more adenoma > 10 mm), or colorectal cancer. Key exclusion criteria were a history of cancer, a known diagnosis of inflammatory bowel disease, a hereditary predisposition to colorectal cancer (identified through genetic testing), and have received adequate quality colonoscopy, locally assessed by the endoscopist, in terms of bowel preparation and cecal intubation. All patients underwent standard diagnostic, staging, and therapeutic procedures per local guidelines, received stage-specific curative-intent resection, with or without systemic therapy (as appropriate), or upfront systemic therapy (as appropriate). Importantly, all biospecimens were collected before the administration of any treatment.

[0268] Laboratory procedures for RNA sequencing (discovery phase). For the biomarker discovery' phase, total exosomal-RNA content was isolated from 200 pL of plasma by first isolating the exosomes, and then extracting the RNA content of such exosomes usingexoRNeasy Midi Kit (Qiagen). Total cell-free-RNA content was isolated from 200 pL of plasma directly using exoRNeasy Midi Kit (Qiagen). Construction of next-generation sequencing libraries for miRNAs from plasma was performed using the Small RNA-Seq Kit V3 (Revvity, Waltham, MA, USA). After size selection, libraries’ quality and quantity was assessed using the TapeStation (Agilent, Santa Clara, CA, USA) and then equimolar-pooled prior to sequencing on an Illumina HighS eq 2500 with single-end 35-base read lengths at an average of 10 million reads per sample. Based on the discovery cohort sample size (N=8 7), this study phase was adequately powered (>80%) to detect at least 50 differentially expressed miRNAs, under the assumptions of a 5% false discovery' rate, 30x coverage (Zo), and a 5% significance level (a) ('RNASeqSampleSize' , Version 2.12.0).

[0269] Illumina small RNA-seq 3' adapters were trimmed using cutadapt software, and all retained sequences were confirmed to contain high-quality scores and peaks concentrated at 22 nt, representing miRNAs. Preprocessed reads were aligned to a human reference genome (human genome build 38) and annotated using GENCODE miRNA annotation. For the biomarker discovery phase, the initial candidate miRNAs were selected based on differential gene expression (|Log2(Fold-Change)| >0.5 and Benjamini-Hochberg-adjusted p values < 0.05). This step identified 193 adenoma-specific and 150 cancer specific cell-free biomarkers, and 33 were shared (FIG. 4) Small RNA Sequencing, in a similar manner, was also conducted in a second and independent cohort, and the same statistical approaches and biomarker selection strategy could replicate similar results for all but one of these 33 biomarkers. We then tested whether any of these remaining 32 biomarkers were downregulated in GSE39833 (N=99) or GSE 41655 (N=107), which removed another 5 candidates. Of these 27 remaining candidates, we selected only those that demonstrated a statistically significant over-expression in at least two independent cohorts, which resulted in a final panel of 19 cf-miRNAs. The exosome biomarker selection process followed the same strategy. The initial candidate exo-miRNAs were selected based on differential gene expression (|Log2(Fold-Change)| >0.5 and Benjamini- Hochberg-adjusted p values < 0.05), which identified 226 adenoma-specific and 104 cancer specific cell-free biomarkers, and 46 were shared (FIG. 4). Exosome-directed small RNA Sequencing, in a similar manner, was conducted in the same independent cohort, and the same statistical approaches and biomarker selection strategy could replicate similar results for all biomarkers. We then tested whether any of these remaining 32 biomarkers were downregulated in GSE25609 (N=61) or GSE41655 (N=107), which removed 5 candidates. Of these 40 remaining candidates, we selected only those that demonstrated a statistically significant overexpression in at least two independent cohorts, which resulted in a final panel of 20 exo-miRNAs.

[0270] Laboratory7procedures for the diagnostic assay based on RT-qPCR (training and validation). After completion of the biomarker discovery phase, we proceeded to develop a plasma-based assay based on RT-qPCR. From the two clinical cohorts (training and testing), total circulating cell-free RNA was isolated from 200 pL of plasma using miRNeasy serum / plasma kit (Qiagen). Exosome-bound total RNA was isolated in a two-step process that involved both centrifugation, precipitation, and resuspension of exosomes from 200 pL of plasma, followed by exosome lysis and RNA extraction with (Total Exosome Precipitation Reagent by Invitrogen, Waltham, MA, USA, followed by miRNeasy serum / plasma kit). Complementary DNA was synthesized from total RNA using the miRCURY LNA RT Kit (Qiagen) and then miRNA expression was assessed by quantitative reverse transcription PCR (RT-qPCR) on a QuantStudio 7 Flex Real-Time PCR system (Thermo Fisher Scientific, Irwindale, CA, USA) using high-quality miRNA probes and high-sensitivity SYBR Green Master Mix (Thermo Fisher Scientific). The relative abundance of target transcripts was determined and normalized to the expression levels of hsa-miR-15b-5p, hsa-miR-23a-3p, and hsa-miR-30e-5p as internal controls using the by 2'ACtmethod. ACt refers to the difference of Ct values between the transcript of interest and the average of the three normalizers.

[0271] Model architecture and hyperparameters. The final diagnostic assay, named DENEB. employed XGBoost, a popular ML model that utilizes sequential iterative boosting as a strategy to convert weak learners (decision trees) into a more robust model. XGBoost is particularly suited for complex and high-dimensional data because of the faster computation, its many optimization options, and its built-in methods to handle missing data. In this study, two independent and stacked XGBoost-based models (one for CRC and one for AA) were allowed to undergo a maximum of 500 training rounds with gradient boosting trees implemented by the ‘xgboost’ package in R (Version 0.1.3). The expression levels of the cf-miRNAs and exo- miRNAs were the independent variables based on which the model predictions were made. We utilized the cross-entropy loss evaluation metric to train two binary discriminatory assays (one for CRC vs. controls, and the other for AA vs. controls). In order to minimize overtraining, each tree was allowed to reach a maximum of 3 branches with high low-pruning strategy (y=15) and a slow the learning rate (s=l%), with each decision tree (i.e., each training round) allowed to utilize only 75% of samples available. At the end of the training phase, the two models were fully locked, the results were dichotomized in “positive7’ and “negative” based on the point with the minimum distance for the (1,0) point.

[0272] If both the CRC and AA assays predicted the absence of colorectal neoplasias (i.e. , double negative), the final model prediction was ‘‘normal signal detected / ’ If the CRC assay gave a result above the CRC threshold, but the AA was negative, the final model prediction was “abnormal signal detected,” with a subscript indicating that the patient most likely had a CRC. If the CRC assay gave a result above the AA threshold, but the CRC was negative, the final model prediction was “abnormal signal detected”, with a subscript indicating that the patient most likely had a AA. Finally, if both the CRC and the AA assay gave a result above the corresponding thresholds, the final model prediction was “abnormal signal detected”, and the final decision of CRC vs. AA was based on which signal was higher (higher numerical probability).

[0273] Table 1: Demographic Characteristics of the Participants

[0274] Table 2: Sensitivity and Specificity of the Cell-free and Exosomal miRNA (cf / exo- miRNA) Blood-Based Test for the Most Advanced Findings on Colonoscopy

[0275] (A) Excluded were 28 stage IV and 15 pathologically confirmed, incompletely staged colorectal cancer cases from cohort 1, and 9 stage IV and 7 pathologically confirmed, incompletely staged colorectal cancer cases from cohort 2

[0276] Table 3

[0277] Table 4: Sub-Analyses

[0278] It is understood that the examples 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 scope of this application and claims. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application are expressly incorporated by reference herein in their entirety and for all purposes.

Claims

CLAIMSWhat is claimed is:

1. A method of detecting RNA in a patient with colorectal cancer or colorectal adenoma, the method comprising detecting an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR- 1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b- 5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a- 5p, exosomal miR-433-3p. exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p. exosomal miR-19a-3p, exosomal miR-4488, cell-free miR- 107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR- 142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell- free miR-5193. cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR- 4488, or a combination of two or more thereof.

2. The method of claim 1 for detecting RNA in the patient with colorectal cancer, wherein the colorectal cancer is Stage I colorectal cancer or Stage II colorectal cancer.

3. The method of claim 1 for detecting RNA in the patient with colorectal adenoma, wherein the colorectal adenoma is advanced colorectal adenoma.

4. The method of claim 1. wherein the biological sample is blood.

5. The method of claim 1. wherein the control is a healthy patient.

6. A method of diagnosing a patient with colorectal cancer or colorectal adenoma, the method comprising:(i) detecting the expression level of RNA in a biological sample obtained from the patient; and(ii) diagnosing the patient as having colorectal cancer or colorectal adenoma when the biological sample has an elevated expression level, relative to a control, of the RNA; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184. exosomal miR-199b- 5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p,exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p. exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-107, cell-free miR-1228-5p, cell-free miR- 1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell- free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR-5193. cell-free miR-877-3p, cell-free miR-18a-5p. cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof.

7. A method of treating colorectal cancer or colorectal adenoma in a patient in need thereof, the method comprising:(i) detecting an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p. exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433- 3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-107, cell- free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141- 3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR- 195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell- free miR-34a-5p, cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877- 3p, cell-free miR-18a-5p. cell-free miR-19a-3p, cell-free miR-4488. or a combination of two or more thereof; and(ii) administering to the patient an effective amount of an anti-cancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof.

8. A method of treating colorectal cancer or colorectal adenoma in a patient in need thereof, the method comprising administering to the patient an effective amount of an anticancer agent, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of thepatient, or a combination of two or more thereof; wherein a biological sample obtained from the patient comprises an elevated expression level, relative to a control, of a RNA; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR- 1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205- 5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p. exosomal miR-18a-5p. exosomal miR-19a-3p. exosomal miR-4488, cell- free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR- 141-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell- free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p. cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof9. A method of treating colorectal cancer in a patient in need thereof, the method comprising:(i) selecting a patient having a diagnosis of colorectal cancer based on a risk score or an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient, wherein the RNA comprises exosomal let-7 c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p. exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-107, cell-free miR- 1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell- free miR-142-3p. cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p. cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, 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, administering to the patient an effective amount of radiation therapy, administering to the patient image-based screening, surgically removing all or a portion of the colon of the patient, or a combination of two or more thereof.

10. A method of monitoring treatment in a patient having colorectal cancer or colorectal adenoma or monitoring risk for developing colorectal cancer or colorectal adenoma in a patient, the method comprising:(i) detecting the expression level of RNA in a biological sample obtained from the patient at a first time point;(ii) detecting the expression level of the RNA in a biological sample obtained from the patient at a second time point, wherein the second time point is later than the first time point; and(iii) comparing the expression level of the RNA at the second time point to the expression level of the RNA at the first time point, thereby monitoring treatment or monitoring risk; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR- 10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150- 5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a- 3p, exosomal miR-205-5p. exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455- 3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17- 5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof; wherein an elevated expression level of RNA at the second point in time when compared to the expression level of RNA at the first point in time indicates that the patient has an increased risk of developing colorectal cancer or colorectal adenoma.

11. The method of claim 1. wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543. exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR- 142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell- free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, and cell-freemiR-4488.

12. The method of claim 1. wherein the RNA consists of exosomal let-7 c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p. exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p. exosomal miR-19a-3p, exosomal miR-4488, cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR- 142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell- free miR-5193. cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, and cell-free miR-4488.

13. The method of claim 1, wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, and exosomal miR-4488.

14. The method of claim 1, wherein the RNA consists of exosomal let-7 c-5p exosomal miR-100-5p, exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p. exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p. exosomal miR-20a-5p. exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p, and exosomal miR-4488.

15. The method of claim 1, wherein the RNA comprises cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR- 142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p. cell-free miR-200b-3p, cell-free miR-296-5p, cell -free miR-34a-5p, cell-free miR-3913-5p, cell- free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, and cell-free miR-4488.

16. The method of claim 1. wherein the RNA consists of cell-free miR-107, cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323, cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR-1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell- free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, and cell-free miR-4488.

17. A method of detecting RNA in a patient with colorectal cancer or colorectal adenoma, the method comprising detecting an elevated expression level, relative to a control, of RNA in a biological sample obtained from the patient; wherein the RNA comprises let-7c. miR- 100, miR-lOb, miR-1246, miR-1290, miR-135b, miR-150p, miR-184, miR-199b, miR-200a, miR-203a, miR-205, miR-20a, miR-433, miR-455, miR-543, miR-9, miR-18a, miR-19a, miR- 4488, miR-107, miR-1228, miR-1229, miR-1323, miR-141, miR-142, miR-1538, miR-17, miR- 195, miR-19b, miR-200b, miR-296, miR-34a. miR-3913, miR-5193, and miR-877, or a combination of two or more thereof.

18. The method of claim 1, further comprising detecting the 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.

19. The method of claim 1 , further comprising detecting the expression level of a reference RNA in the biological sample, 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.

20. A kit comprising reagents capable of detecting an expression level of RNA from a biological sample; wherein the RNA comprises exosomal let-7c-5p exosomal miR-100-5p. exosomal miR-10b-5p, exosomal miR-1246, exosomal miR-1290, exosomal miR-135b-5p, exosomal miR-150-5p, exosomal miR-184, exosomal miR-199b-5p, exosomal miR-200a-3p, exosomal miR-203a-3p, exosomal miR-205-5p, exosomal miR-20a-5p, exosomal miR-433-3p, exosomal miR-455-3p, exosomal miR-543, exosomal miR-9-5p, exosomal miR-18a-5p, exosomal miR-19a-3p. exosomal miR-4488, cell-free miR-107. cell-free miR-1228-5p, cell-free miR-1229-3p, cell-free miR-1323. cell-free miR-141-3p, cell-free miR-142-3p, cell-free miR- 1538, cell-free miR-17-5p, cell-free miR-195-5p, cell-free miR-19b-3p, cell-free miR-200b-3p, cell-free miR-296-5p, cell-free miR-34a-5p, cell-free miR-3913-5p, cell-free miR-5193, cell-free miR-877-3p, cell-free miR-18a-5p, cell-free miR-19a-3p, cell-free miR-4488, or a combination of two or more thereof.

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