WO2022204378A2

WO2022204378A2 - Identification of rna biomarkers in colorectal cancer

Info

Publication number: WO2022204378A2
Application number: PCT/US2022/021708
Authority: WO
Inventors: Ajay Goel; Yuma Wada
Original assignee: City Of Hope
Priority date: 2021-03-24
Filing date: 2022-03-24
Publication date: 2022-09-29
Also published as: WO2022204378A3

Abstract

Provided herein, inter alia, are methods of detecting an RNA biomarker in a subject, wherein the subject has, or is suspected of having, colorectal cancer, by determining the presence of one or more RNA biomarkers in a biological sample, such as a blood sample, from the subject.

Description

IDENTIFICATION OF RNA BIOMARKERS IN COLORECTAL CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Application No. 63/165,623 filed March 24, 2021, 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 grant nos. CA72851, CA181572, CA184792, CA202797, and CA187956 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII FILE

[0003] The Sequence Listing written in file 048440-805001WO_SL_ST25.txt, created on March 21, 2022, 3,813 bytes, machine format IBM-PC, MS Windows operating system, is hereby incorporated by reference.

BACKGROUND

[0004] In recent years, the diagnosis of invasive submucosal colorectal cancers (T1 CRCs) has increased by up to 15-30% due to the implementation of mass CRC screening and frequent patient examinations. (Refs 1, 2). However, recent advances in endoscopic devices have enabled curative treatment via endoscopic submucosal dissection (ESD) or endoscopic mucosal resection (EMR), for patients with T1 CRC who would have otherwise been treated by radical surgeries. (Ref 3). This has prompted the National Comprehensive Cancer Network to recommend ESD as a preferred treatment modality for patients with suspected T1 CRC. Successful treatment of patients with T1 CRCs starts with accurate diagnosis during endoscopy. However, two prospective studies recently highlighted that 30-40% of these patients are misdiagnosed, and the pre-surgical discrimination of T1 CRC remains clinically challenging. (Refs 4, 5). Although some patients can be successfully treated with ESD or EMR, approximately 70-80% of patients with T1 CRC require radical surgeries to achieve a complete cure, due to the potential risk for lymph node metastasis (LNM) after pathological analysis, which is estimated to occur in as many as 5-15% of patients with high-risk T1 CRC. (Refs 6-8).

[0005] With the implementation of endoscopic treatment for suspected T1 CRCs, it has become necessary to identify the risk of LNM, in order to select patients who truly have high- risk disease and require radical surgery, while sparing others from overtreatment. The currently used pathological criteria to identify LNM in patients with T1 CRC include depth of submucosal invasion (>1000 pm), presence of lymphatic or vascular invasion, high-grade tumor budding, and poorly differentiated histology. (Refs. 9-13). If these factors are absent, endoscopic treatment is considered sufficient to cure patients with T1 CRC who have low-risk for LNM. (Refs. 14, 15). Unfortunately however, in clinical settings, if even one of these pathological risk features is present, the patient is deemed as “high risk for LNM” and is recommended to undergo additional surgery. (Refs 3, 11, 16, 17). Such a dichotomized clinical management approach for patients with T1 CRCs has serious drawbacks, as it often leads to overtreatment, even though the positive predictive value (PPV) for the presence of LNM is quite low. (Ref 18). By using the current clinicopathological criteria, approximately 70-80% of patients with T1 CRC are classified as high-risk, whereas post-surgical pathological results demonstrate that only 5-15% of these patients actually have LNM. (Refs 10, 14, 16, 19-25). This highlights an important clinical challenge: we need more prudent risk assessment for limiting unnecessary radical surgery in 85-95% of patients with T1 CRC. In addition, these data suggest the inadequacy of currently used pathological risk factors and emphasize the need to develop robust molecular biomarkers that can identify the presence of LNM pre-operatively, which would better inform clinical decision-making in patients with T1 CRC, minimize the number of surgeries performed, and reduce the overall burden of costs associated with such invasive surgeries.

[0006] The present disclosure is directed to addressing the need in the art to identify biomarkers, metastatic risks, and appropriate treatment plans for patients having or suspected of having colorectal cancer.

BRIEF SUMMARY

[0007] Provided herein are methods of treating colorectal cancer in a patient in need thereof by administering to the patient an effective amount of an anti-cancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof; wherein a biological sample obtained from the patient comprises an elevated expression level, relative to a control, of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the biological sample is a blood sample.

[0008] Provided herein are methods of identifying an increased risk of developing lymph node metastasis in a patient with colorectal cancer or detecting a lymph node metastasis in a patient with colorectal cancer by detecting an elevated expression level, relative to a control, of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a biological sample obtained from the patient; wherein the elevated expression level of the RNA, indicates an increased risk of developing lymph node metastasis or the presence of a lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the biological sample is a blood sample.

[0009] These and other embodiments of the disclosure are described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIGS. 1A-1B. Training phase of a transcriptomic panel for the identification of LNM in patients with T1 CRC. FIG. 1A: A ROC curve for a 4-miRNA and 5-mRNA panel in serum from training cohort patients (LNP = 5, LNN = 41; AUC: 4-miRNA panel = 0.78, 5-mRNA panel = 0.77, combination panel = 0.86). FIG. IB: Risk score distribution plot in training cohort patients. Modified risk scores were obtained from individual risk scores by using Youden’s index values from the risk model. FIG. 1C: Forest plots with ORs for each panel risk score status in univariate logistic regression analysis in training cohort patients (ORs: 4-miRNA panel = 8.62, 5-mRNA panel = 8.44, combination panel = 14.22). In FIG. 1A, at 15% specificity, the top line is the combination panel, the next lower line is the 4-miRNA panel, and the next lower line is the 5-mRNA panel.

[0011] FIGS. 2A-2D. Validation phase of the transcriptomic panel for the identification of LNM in patients with T1 CRC. FIG. 2A: A ROC curve for the transcriptomic panel in tissue specimens from validation cohort patients (LNP = 12, LNN = 130, AUC = 0.83). FIG. 2B: A ROC curve for the transcriptomic panel in matched serum samples in validation cohort patients (LNP = 12, LNN = 130, AUC = 0.82). FIG. 2C: Risk score distribution plot in serum specimens from validation cohort patients. FIG. 2D: A nomogram illustrating the probability of LNM risk. For clinical purposes, the scores of each covariate are added, and the total score is depicted on the total score point axis.

[0012] FIGS. 3A-3D. Clinical validation of the risk-stratification model in patients with T1 CRC. FIG. 3A: The risk-stratification model, which combines the transcriptomic panel and pathological risk factors, outperformed detection accuracy of the transcriptomic panel or risk factors alone in serum specimens from validation cohort patients (AUC = 0.90). FIGS. 3B-3C: Forest plot with ORs of clinicopathological variables, transcriptomic panel, and risk- stratification model in univariate (FIG. 3B) and multivariate (FIG. 3C) logistic regression analysis in validation cohort patients. FIG. 3D: Currently used pathological factors led to the overtreatment of 92% patients with T1 CRC (left panel). The patients in validation cohort using our transcriptomic classifier divided into high and low risk by Youden’s index. Pie chart shows LNM status of LNP and LNN. The transcriptomic panel would have led to the overtreatment of only 22% patients with T1 CRC (middle panel), and the risk-stratification model would have led to the overtreatment of only 18% patients with T1 CRC (right panel). In FIG. 3 A at 40% specificity, the dashed line is lymphatic invasion, the next lower dashed line is budding grade, the next lower dashed line is vascular invasion, and the next lower dashed line is submucosal invasion. In FIG. 3D from left panel to right panel, high risk is 100%, 29%, and 25%, respectively; low risk is not present, 71%, and 75%, respectively; LNP is 8%, 7%/2%, and 7%/l%, respectively; and LNN is 92%, 22%/69%, and 18%/74% respectively.

[0013] FIG. 4 provides an overview of the study described in the example.

[0014] FIGS. 5A-5B ROC curves for the detection of LNM in T1 CRC training and validation cohorts. FIG. 5A: ROC curve for combined current clinical risk factors (depth of submucosal invasion (>1000 pm), presence of lymphatic or vascular invasion, high-grade tumor budding, and poorly differentiated histology) for LNM without the transcriptomic panel in the training cohort (AUC = 0.73). FIG. 5B: ROC curve for the current clinical risk factors for LNM without the transcriptomic panel in the validation cohort (AUC = 0.76).

[0015] FIGS. 6A-6L show data for the training cohort (LNP=7, LNN=51 ). FIGS 6A-6D show the receiver operating characteristic curve for exosomal miRNAs. FIGS. 6E-6H show receiver operating characteristic curve for cell-free miRNAs. FIG. 61 shows receiver operating characteristic curve for exosomal miRNA panel (AUC, 0.860, 95% Cl, 0.701-1.000). FIG. 6J shows receiver operating characteristic curve for cell-free miRNA panel (AUC, 0.824, 95%CI, 0.664-0.963). FIG. 6K shows receiver operating characteristic curve for exosome and cell-free miRNA panel (AUC, 0.905, 95% Cl, 0.803-1.000). FIG. 6L is a water fall plot for modified risk score distribution

[0016] FIGS. 7A-7D show data for the validation cohort (LNP=12, LNN=132). FIG. 7A shows receiver operating characteristic curve for exosome and cell-free miRNA panel (AUC, 0.844). FIG. 7B is a water fall plot for modified risk score distribution. FIGS 7C-7D show the risk-stratification model (FIG. 7C) and risk-stratification model combines (FIG. 7D).

[0017] FIGS. 8A-8C show performance of transcriptomic panel and risk-stratification model. FIG. 8A: Radar chart plotting for accuracy, sensitivity, specificity, positive predictive value, and negative predictive value. FIG. 8B: Decision curve plotting net benefit against threshold probability. FIG. 8C: Decision curve plotting decrease in operations against threshold probability

[0018] FIGS. 9A-9B show patients undergoing operations based on current guidelines (FIG. 9A) and risk-stratification model described in Example 2 (FIG. 9B).

DETAILED DESCRIPTION

[0019] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et ak, Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et ak, 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.

[0020] A “cell” refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the 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 with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian (e.g. human) cells. Cells may be useful when they are naturally nonadherent or have been treated not to adhere to surfaces, for example by trypsinization.

[0021] As used herein, 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. In embodiments, “exosomal RNA” is exosomal mRNA. Exosomal RNA can be detected and measured by methods known in the art, such as those described in Example 2 herein.

[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. Thus, the terms miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA are equivalent to the terms cell-free miR-181b, cell-free miR-193b, cell-free miR-195, cell-free miR-411, cell-free AMT mRNA, cell-free FOXA1 mRNA, cell-free PIGR mRNA, cell-free MMP1 mRNA, and cell-free MMP9 mRNA, respectively. Cell-free RNA can be detected and measured by methods known in the art, such as those described in Example 2 herein.

[0024] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid” does not include nucleosides. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleoside” refers, in the usual and customary sense, to a glycosylamine including a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non limiting examples, of nucleosides include, cytidine, uridine, adenosine, guanosine, thymidine and inosine. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid, e.g. polynucleotides, contemplated herein include any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and any types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0025] 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.

[0026] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0027] An "antisense nucleic acid" as referred to herein is a nucleic acid (e.g., DNA or RNA molecule) that is complementary to at least a portion of a specific target nucleic acid and is capable of reducing transcription of the target nucleic acid (e.g. mRNA from DNA), reducing the translation of the target nucleic acid (e.g. mRNA), altering transcript splicing (e.g. single stranded morpholino oligo), or interfering with the endogenous activity of the target nucleic acid. See, e.g., Weintraub, Scientific American, 262:40 (1990). Typically, synthetic antisense nucleic acids (e.g. oligonucleotides) are generally between 15 and 25 bases in length. Thus, antisense nucleic acids are capable of hybridizing to (e.g. selectively hybridizing to) a target nucleic acid. In aspects, the antisense nucleic acid hybridizes to the target nucleic acid in vitro. In aspects, the antisense nucleic acid hybridizes to the target nucleic acid in a cell. In embodiments, the antisense nucleic acid hybridizes to the target nucleic acid in an organism. In aspects, the antisense nucleic acid hybridizes to the target nucleic acid under physiological conditions. Antisense nucleic acids may comprise naturally occurring nucleotides or modified nucleotides such as, e.g., phosphorothioate, methylphosphonate, and anomeric sugar-phosphate, backbone-modified nucleotides.

[0028] In the cell, the antisense nucleic acids hybridize to the corresponding RNA forming a double-stranded molecule. The antisense nucleic acids interfere with the endogenous behavior of the RNA and inhibit its function relative to the absence of the antisense nucleic acid. Furthermore, the double-stranded molecule may be degraded via the RNAi pathway. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus- Sakura, Anal. Biochem., 172:289, (1988)). Further, antisense molecules which bind directly to the DNA may be used. Antisense nucleic acids may be single or double stranded nucleic acids. Non-limiting examples of antisense nucleic acids include siRNAs (including their derivatives or pre-cursors, such as nucleotide analogs), short hairpin RNAs (shRNA), micro RNAs (miRNA), saRNAs (small activating RNAs) and small nucleolar RNAs (snoRNA) or certain of their derivatives or pre-cursors.

[0029] 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.

[0030] The terms “messenger RNA” or “mRNA” refer a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein.

[0031] Nucleic acids can include nonspecific sequences. As used herein, the term "nonspecific sequence" refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to or are only partially complementary to any other nucleic acid sequence. By way of example, a nonspecific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.

[0032] The term “complement,” as used herein, refers to a nucleotide (e.g., RNA or DNA) or a sequence of nucleotides capable of base pairing with a complementary nucleotide or sequence of nucleotides. As described herein and commonly known in the art the complementary (matching) nucleotide of adenosine is thymidine and the complementary (matching) nucleotide of guanosine is cytosine. Thus, a complement may include a sequence of nucleotides that base pair with corresponding complementary nucleotides of a second nucleic acid sequence. The nucleotides of a complement may partially or completely match the nucleotides of the second nucleic acid sequence. Where the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. Where the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence only some of the nucleotides of the complement form base pairs with nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding and a non-coding sequences, wherein the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence. A further example of complementary sequences are sense and antisense sequences, wherein the sense sequence contains complementary nucleotides to the antisense sequence and thus forms the complement of the antisense sequence.

[0033] The term "gene" means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. Further, a "protein gene product" is a protein expressed from a particular gene.

[0034] 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, mRNA) may be detected by standard PCR or Northern blot methods well known in the art. See, Sambrook et ak, 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0035] The terms an “elevated expression level” or “elevated 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. [0036] The terms “does not have an elevated expression level” or an expression level that is “not elevated” is an expression level of the gene that is about the same as (or lower than) the expression level of the gene in a control. The control may be any suitable control, as described herein.

[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 activity or level of RNA. In embodiments, a control is a healthy patient or a healthy population of patients. In embodiments, a control is a patient having colorectal cancer that does not have lymph node metastases or a population of patients having colorectal cancer that does not have lymph node metastases. For example, a test sample can be taken from a patient suspected of having colorectal cancer and compared to samples from a patient having colorectal cancer or a known normal (non-disease) individual. A control can also represent an average value gathered from a population of similar individuals, e.g., cancer patients or healthy individuals with a similar medical background, age, weight, etc.

A control can also be obtained from the same individual, e.g., from an earlier-obtained sample, prior to disease, or prior to treatment. 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, the control is a population of colorectal cancer subjects that do not have metastatic disease. One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.

[0038] The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. Transgenic cells and plants are those that express a heterologous gene or coding sequence, typically as a result of recombinant methods.

[0039] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid including two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein including two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0040] The phrase “specifically (or selectively) binds” to an antibody or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0041] The terms “isolate” or “isolated”, when applied to a nucleic acid, virus, or protein, denotes that the nucleic acid, virus, or protein is essentially free of other cellular components with which it is associated in the natural state. It can be, for example, in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. An RNA that is the predominant species present in a preparation is substantially purified.

[0042] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g-carboxy glutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0043] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0044] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0045] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure. [0046] The following eight groups each contain amino acids that are conservative substitutions for one another: (1) Alanine (A), Glycine (G); (2) Aspartic acid (D), Glutamic acid (E); (3) Asparagine (N), Glutamine (Q); (4) Arginine (R), Lysine (K); (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); (6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (7) Serine (S), Threonine (T); and (8) Cysteine (C), Methionine (M) (see, e.g.. Creighton, Proteins (1984)).

[0047] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions ( i.e ., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0048] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (e.g., www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then the to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0049] An amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N- terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0050] The terms "numbered with reference to" or "corresponding to," when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.

[0051] As used herein, 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.

[0052] A “detectable agent” or “detectable moiety” is a compound or composition detectable by appropriate means such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means. The RNA described herein and the expression level of the RNA described herein may be accomplished through the use of a detectable moiety in an assay or kit. A detectable moiety is a monovalent detectable agent or a detectable agent bound (e.g. covalently and directly or via a linking group) with another compound, e.g., a nucleic acid. Exemplary detectable agents/moieties for use in the present disclosure include an antibody ligand, a peptide, a nucleic acid, radioisotopes, 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 (e.g., DYNABEADS® by ThermoFisher, encompassing functionalized magnetic beads such as DYNABEADS® M-270 amine by ThermoFisher), 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 (e.g. carbon-11, nitrogen-13, oxygen-15, fluorine-18, rubidium-82), fluorodeoxyglucose (e.g. fluorine-18 labeled), any gamma ray emitting radionuclides, positron- emitting radionuclide, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biocolloids, microbubbles (e.g. including microbubble shells including albumin, galactose, lipid, and/or polymers; microbubble gas core including air, heavy gas(es), perfluorcarbon, nitrogen, octafluoropropane, perflexane lipid microsphere, perflutren, etc.), iodinated contrast agents (e.g. iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate), 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.

[0053] 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 prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of colorectal cancer or lymph node metastasis, or reducing the likelihood of the onset (or reoccurrence) of colorectal cancer or lymph node metastasis, or their symptoms or the intended therapeutic effect, e.g., treatment or amelioration of colorectal cancer, or their symptoms including any objective or subjective parameter of treatment such as abatement; remission; diminishing of symptoms or making the cancer more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a patient’s physical or mental well-being.

[0054] “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 and autopsy samples, and frozen sections taken for histological purposes. Such samples 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 RNA obtained from blood. In embodiments, a biological sample is an exosome obtained from a blood sample, wherein the exosome comprises RNA. In embodiments, a biological sample is an exosome obtained from a serum sample, wherein the exosome comprises RNA. In embodiments, a biological sample is an exosome obtained from a plasma sample, wherein the exosome comprises RNA.

[0055] “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.

[0056] The singular terms "a," "an," and "the" include the plural reference unless the context clearly indicates otherwise.

[0057] 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 with or without lymph node metastases) or outcome in a subject.

[0058] 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, prevention of a disease’s transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable. In other words, "treatment" as used herein includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; 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.

[0059] The terms "treating" and "treatment" may include prophylactic treatment. Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The administering step may consist of a single administration or may include a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the risk or condition, the age of the patient, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In embodiments, chronic administration is required. For example, the therapeutic agents are administered to the subject in an amount and for a duration sufficient to treat the patient.

[0060] The term “prevent” is used in accordance with its plain and ordinary meaning and refers to a decrease in the occurrence of disease symptoms in a patient. The prevention may be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would likely occur absent treatment.

[0061] 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). A “prophylactically effective amount” of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of a disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of a disease, pathology, or condition, or their symptoms. The full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques.

[0062] For any compound 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.

[0063] As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.

[0064] Dosages may be varied depending upon the requirements of the patient and the compound being employed. The dose administered to a patient, in the context of the present disclosure, 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.

[0065] As used herein, 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.

[0066] The terms “patient” or “subject” are used in accordance with its plain and ordinary meaning and refer to a living organism suffering from or prone to a disease that can be treated by administration of a pharmaceutical composition, such as anti-cancer agents and chemotherapeutic agents. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, cats, monkeys, and other non-mammalian animals. In embodiments, a patient is human. In embodiments, the patient is a human with colorectal cancer. In embodiments, the subject is a human with invasive submucosal colorectal cancer.

[0067] The terms "marker," "RNA marker," and “biomarker” are used interchangeably throughout the disclosure, and are used in accordance with their plain and ordinary meaning. A marker refers generally to RNA (e.g., miRNA or mRNA), the level or concentration of which is associated with a particular biological state, particularly a state associated with colorectal cancer. Panels, assays, kits and methods described herein may comprise antibodies, binding fragments thereof or other types of target-binding agents, which are specific for the RNA markers described herein (e.g., miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA and/or MMP9 mRNA). [0068] The terms "metastasis," "metastatic," and "metastatic cancer" can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. Cancer occurs at an originating site, e.g., colon, which site is referred to as a primary tumor, e.g., primary colon cancer. Some cancer cells in the primary tumor or originating site acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and/or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites and tissues in the body. A second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if colorectal cancer metastasizes to the lymph nodes, the secondary tumor at the site of the lymph nodes consist of colorectal cells and not abnormal lymph node cells. The secondary tumor in the lymph nodes is referred to as lymph node metastasis. Thus, the phrase metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors. The phrases non-metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors.

[0069] Methods of Treatment

[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, surgically removing all or a portion of the colon of the patient, or a combination thereof; wherein a biological sample obtained from the patient comprises an elevated expression level, relative to a control, of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR- 411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods of treating colorectal cancer in a patient in need thereof comprises administering to the patient an effective amount of an anti-cancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof; wherein a blood sample obtained from the patient comprises an elevated expression level, relative to a control, of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the method comprises administering to the patient an effective amount of an anti-cancer agent.

In embodiments, the method comprises surgically removing all or a portion of the colon of the patient. In embodiments, the method comprises administering to the patient an effective amount of an anti-cancer agent and surgically removing all or a portion of the colon of the patient. In embodiments, the blood sample is a serum sample or a plasma sample. In embodiments, the blood sample is a serum sample. In embodiments, the blood sample is a plasma sample. In embodiments, the colorectal cancer is colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is colorectal cancer without lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer without lymph node metastasis. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of one miRNA selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of two miRNA selected from the group consisting of miR-181b, miR- 193b, miR-195, and miR-411. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of three miRNA selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of miR-181b, miR-193b, miR-195, and miR-411. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of one RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of two RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of three RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of four RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of five RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of six RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of seven RNA selected from the group consisting of miR- 181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of eight RNA selected from the group consisting of miR- 181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of one mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of two mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of three mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of four mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the RNA is exosomal RNA. In embodiments, the RNA is cell-free RNA. In embodiments, the RNA is exosomal RNA and cell-free RNA. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR- 411. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR- 195, and exosomal miR-411. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR- 411, and one cell-free RNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, and two cell-free RNA selected from the group consisting of cell-free miR- 181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR- 195, and exosomal miR-411, and three cell-free RNA selected from the group consisting of cell- free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the blood sample comprises an elevated expression level, relative to a control, of exosomal miR- 181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell- free miR-193b, cell-free miR-195, and cell-free miR-411.

[0071] 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 an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a biological sample obtained from the patient; and (ii) administering to the patient an effective amount of an anti-cancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof. In embodiments, the methods of treating colorectal cancer in a patient in need thereof comprise: (i) detecting an elevated expression level, relative to a control, of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient; and (ii) administering to the patient an effective amount of an anti-cancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof. In embodiments, the method comprises administering to the patient an effective amount of an anti-cancer agent. In embodiments, the method comprises surgically removing all or a portion of the colon of the patient. In embodiments, the method comprises administering to the patient an effective amount of an anti-cancer agent and surgically removing all or a portion of the colon of the patient. In embodiments, the blood sample is a serum sample or a plasma sample. In embodiments, the blood sample is a serum sample. In embodiments, the blood sample is a plasma sample. In embodiments, the colorectal cancer is colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is colorectal cancer without lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer without lymph node metastasis. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one miRNA selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two miRNA selected from the group consisting of miR-181b, miR- 193b, miR-195, and miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three miRNA selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of miR-181b, miR-193b, miR-195, and miR- 411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR- 411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of four RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of five RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of six RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of seven RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of eight RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of four mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the RNA is exosomal RNA. In embodiments, the RNA is cell-free RNA. In embodiments, the RNA is exosomal RNA and cell-free RNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, and one cell-free RNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR- 411, and two cell-free RNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, and three cell-free RNA selected from the group consisting of cell -free miR- 181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of exosomal miR- 181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell- free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the anti-cancer agent is a chemotherapeutic agent. 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 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 method further comprises surgically removing all or a portion of the colon of the patient. In embodiments, the method does not comprise detecting the expression level of miR-32, LYZ mRNA, C2CD4A mRNA, and RCC1 mRNA.

[0072] Methods of Detecting LNM or Identifying Increased Risk of LNM

[0073] Provided herein are methods of detecting a lymph node metastasis or identifying an increased risk of developing lymph node metastasis in a patient with colorectal cancer comprising detecting an elevated expression level, relative to a control, of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a biological sample obtained from the patient; wherein the elevated expression level of the RNA indicates a lymph node metastasis or an increased risk of developing lymph node metastasis. In embodiments, the disclosure provides methods of detecting a lymph node metastasis or identifying an increased risk of developing lymph node metastasis in a patient with colorectal cancer comprising detecting an elevated expression level, relative to a control, of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient; wherein the elevated expression level of the RNA indicates a lymph node metastasis or an increased risk of developing lymph node metastasis. In embodiments, the methods comprise detecting a lymph node metastasis in the patient with colorectal cancer. In embodiments, the methods comprise identifying an increased risk of developing lymph node metastasis in a patient with colorectal cancer. In embodiments, the blood sample is a serum sample or a plasma sample. In embodiments, the blood sample is a serum sample. In embodiments, the blood sample is a plasma sample. In embodiments, the colorectal cancer is colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is colorectal cancer without lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer without lymph node metastasis. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one miRNA selected from the group consisting of miR- 181b, miR-193b, miR-195, and miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two miRNA selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three miRNA selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of miR-181b, miR-193b, miR-195, and miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one RNA selected from the group consisting of miR- 181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two RNA selected from the group consisting of miR- 181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three RNA selected from the group consisting of miR- 181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of four RNA selected from the group consisting of miR- 181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of five RNA selected from the group consisting of miR- 181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of six RNA selected from the group consisting of miR- 181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of seven RNA selected from the group consisting of miR- 181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of eight RNA selected from the group consisting of miR- 181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of four mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the RNA is exosomal RNA. In embodiments, the RNA is cell-free RNA. In embodiments, the RNA is exosomal RNA and cell-free RNA. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR- 195, and exosomal miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR- 411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR- 195, and exosomal miR-411, and one cell-free RNA selected from the group consisting of cell- free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411, and two cell-free RNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three exosomal miRNA selected from the group consisting of exosomal miR- 181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, and three cell-free RNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the method further comprises administering to the patient an effective amount of an anticancer agent. In embodiments, the method further comprises administering to the patient an effective amount of a chemotherapeutic agent. 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 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. In embodiments, the method further comprises surgically removing all or a portion of the colon of the patient. In embodiments, the method does not comprise detecting the expression level of miR-32, LYZ mRNA, C2CD4A mRNA, and RCC1 mRNA.

[0074] Provided herein are methods of detecting a lymph node metastasis or identifying an increased risk of developing lymph node metastasis in a patient with colorectal cancer comprising detecting an elevated expression level, relative to a control, of an exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR- 195, and exosomal miR-411, in a blood sample obtained from the patient; wherein the elevated expression level of the exosomal miRNA indicates a lymph node metastasis or an increased risk of developing lymph node metastasis. In embodiments, the methods comprise detecting a lymph node metastasis in the patient with colorectal cancer. In embodiments, the methods comprise identifying an increased risk of developing lymph node metastasis in a patient with colorectal cancer. In embodiments, the blood sample is a serum sample or a plasma sample. In embodiments, the blood sample is a serum sample. In embodiments, the blood sample is a plasma sample. In embodiments, the colorectal cancer is colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer with lymph node metastasis. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411. In embodiments, the method further comprises administering to the patient an effective amount of an anticancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof. In embodiments, the anticancer agent is a chemotherapeutic agent. In embodiments, the method does not comprise detecting the expression level of miR-32, LYZ mRNA, C2CD4A mRNA, and RCC1 mRNA.

[0075] Provided herein are methods of detecting a lymph node metastasis or identifying an increased risk of developing lymph node metastasis in a patient with colorectal cancer comprising detecting an elevated expression level, relative to a control, of an exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR- 195, and exosomal miR-411, and a cell-free miRNA selected from the group consisting of cell- free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411, in a blood sample obtained from the patient; wherein the elevated expression level of the exosomal miRNA and the cell-free miRNA indicates a lymph node metastasis or an increased risk of developing lymph node metastasis. In embodiments, the methods comprise detecting a lymph node metastasis in the patient with colorectal cancer. In embodiments, the methods comprise identifying an increased risk of developing lymph node metastasis in a patient with colorectal cancer. In embodiments, the blood sample is a serum sample or a plasma sample. In embodiments, the blood sample is a serum sample. In embodiments, the blood sample is a plasma sample. In embodiments, the colorectal cancer is colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer with lymph node metastasis. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, and one cell-free RNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two exosomal miRNA selected from the group consisting of exosomal miR- 181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, and two cell-free RNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, and three cell-free RNA selected from the group consisting of cell-free miR-181b, cell-free miR- 193b, cell-free miR-195, and cell-free miR-411. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the method further comprises administering to the patient an effective amount of an anti cancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof. In embodiments, the anticancer agent is a chemotherapeutic agent. In embodiments, the method does not comprise detecting the expression level of miR-32, LYZ mRNA, C2CD4A mRNA, and RCC1 mRNA.

[0076] Methods of Diagnosing LNM Risk

[0077] Provided herein are methods of diagnosing a patient having colorectal cancer as high risk for lymph node metastasis or low risk for lymph node metastasis, the method comprising:

(i) detecting the expression level of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a biological sample obtained from the patient; and (ii) diagnosing the patient as having: (a) a high risk for lymph node metastasis when the biological sample has an elevated expression level, relative to a control, of the RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, or (b) a low risk for lymph node metastasis when the biological sample does not have an elevated expression level, relative to a control, of the RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise diagnosing a patient having colorectal cancer as high risk for lymph node metastasis or low risk for lymph node metastasis, the method comprising: (i) detecting the expression level of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient; and (ii) diagnosing the patient as having: (a) a high risk for lymph node metastasis when the blood sample has an elevated expression level, relative to a control, of the RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, or (b) a low risk for lymph node metastasis when the blood sample does not have an elevated expression level, relative to a control, of the RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the blood sample is a serum sample or a plasma sample. In embodiments, the blood sample is a serum sample. In embodiments, the blood sample is a plasma sample. In embodiments, the colorectal cancer is colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is colorectal cancer without lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer without lymph node metastasis. In embodiments, the methods comprise detecting the expression level of one miRNA selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the one miRNA is elevated relative to the control, and as not having an increased risk for lymph node metastasis when the expression level of the one miRNA is not elevated relative to the control. In embodiments, the methods comprise detecting the expression level of two miRNA selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the two miRNA is elevated relative to the control and as not having an increased risk for lymph node metastasis when the expression level of the two miRNA is not elevated relative to the control. In embodiments, the methods comprise detecting the expression level of three miRNA selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the three miRNA is elevated relative to the control and as not having an increased risk for lymph node metastasis when the expression level of the three miRNA is not elevated relative to the control. In embodiments, the methods comprise detecting the expression level of miR-181b, miR-193b, miR-195, and miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of miR-181b, miR-193b, miR-195, and miR-411 are elevated relative to the control and as not having an increased risk for lymph node metastasis when the expression level of miR-181b, miR-193b, miR-195, and miR-411 are not elevated relative to the control. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the mRNA is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the mRNA is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the mRNA is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of four mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the mRNA is elevated. In embodiments, the methods comprise detecting the expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in the biological sample obtained from the patient, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA are elevated relative to the control and as not having an increased risk for lymph node metastasis when the expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA are not elevated relative to the control. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the RNA is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two RNA selected from the group consisting of miR- 181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the RNA is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the RNA is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of four RNA selected from the group consisting of miR-181b, miR-

193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the RNA is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of five RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the RNA is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of six RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the RNA is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of seven RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the RNA is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of eight RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the RNA is elevated. In embodiments, the methods comprise detecting the expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in the biological sample obtained from the patient, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA are elevated relative to the control and as not having an increased risk for lymph node metastasis when the expression level of miR-18 lb, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA are not elevated relative to the control. In embodiments, the RNA is exosomal RNA. In embodiments, the RNA is cell-free RNA. In embodiments, the RNA is exosomal RNA and cell- free RNA. In embodiments, the methods comprise detecting the expression level of one exosomal miRNA selected from the group consisting of exosomal miR-18 lb, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the one exosomal miRNA is elevated relative to the control, and as not having an increased risk for lymph node metastasis when the expression level of the one exosomal miRNA is not elevated relative to the control. In embodiments, the methods comprise detecting the expression level of two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR- 195, and exosomal miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the two exosomal miRNA is elevated relative to the control and as not having an increased risk for lymph node metastasis when the expression level of the two exosomal miRNA is not elevated relative to the control. In embodiments, the methods comprise detecting the expression level of three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR- 195, and exosomal miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the three exosomal miRNA is elevated relative to the control and as not having an increased risk for lymph node metastasis when the expression level of the three exosomal miRNA is not elevated relative to the control. In embodiments, the methods comprise detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 are elevated relative to the control and as not having an increased risk for lymph node metastasis when the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 are not elevated relative to the control. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR- 195, and exosomal miR-411, and one cell-free RNA selected from the group consisting of cell- free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, and two cell-free RNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR- 195, and exosomal miR-411, and three cell-free RNA selected from the group consisting of cell- free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level is elevated. In embodiments, the method further comprises administering to the patient an effective amount of an anticancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof. In embodiments, the anticancer agent is a chemotherapeutic agent. In embodiments, the method does not comprise detecting the expression level of miR-32, LYZ mRNA, C2CD4A mRNA, and RCC1 mRNA.

[0078] Provided herein are methods of diagnosing a patient having colorectal cancer as high risk for lymph node metastasis, the method comprising: (i) detecting the expression level of an exosomal RNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 in a blood sample obtained from the patient; and (ii) diagnosing the patient as having a high risk for lymph node metastasis when the blood sample has an elevated expression level, relative to a control, of the exosomal RNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411. In embodiments, the blood sample is a serum sample or a plasma sample. In embodiments, the blood sample is a serum sample. In embodiments, the blood sample is a plasma sample. In embodiments, the colorectal cancer is colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is colorectal cancer without lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer without lymph node metastasis. In embodiments, the methods comprise detecting the expression level of one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the one exosomal miRNA is elevated relative to the control. In embodiments, the methods comprise detecting the expression level of two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR- 411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the two exosomal miRNA is elevated relative to the control. In embodiments, the methods comprise detecting the expression level of three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR- 195, and exosomal miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of the three exosomal miRNA is elevated relative to the control. In embodiments, the methods comprise detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 are elevated relative to the control. In embodiments, the method further comprises administering to the patient an effective amount of an anti cancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof. In embodiments, the anticancer agent is a chemotherapeutic agent. In embodiments, the method does not comprise detecting the expression level of miR-32, LYZ mRNA, C2CD4A mRNA, and RCC1 mRNA.

[0079] Provided herein are methods of diagnosing a patient having colorectal cancer as high risk for lymph node metastasis, the method comprising: (i) detecting the expression level of an exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411, and a cell-free RNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 in a blood sample obtained from the patient; and (ii) diagnosing the patient as having a high risk for lymph node metastasis when the blood sample has an elevated expression level, relative to a control, of the exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, and the cell-free RNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the blood sample is a serum sample or a plasma sample. In embodiments, the blood sample is a serum sample. In embodiments, the blood sample is a plasma sample. In embodiments, the colorectal cancer is colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is colorectal cancer without lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer without lymph node metastasis. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR- 411, and one cell-free RNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, and two cell-free RNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, and three cell-free RNA selected from the group consisting of cell -free miR- 181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level is elevated. In embodiments, the methods comprise detecting an elevated expression level, relative to a control, of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR- 411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411, wherein the patient is diagnosed as having an increased risk for lymph node metastasis when the expression level is elevated. In embodiments, the method further comprises administering to the patient an effective amount of an anticancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof. In embodiments, the anticancer agent is a chemotherapeutic agent. In embodiments, the method does not comprise detecting the expression level of miR-32, LYZ mRNA, C2CD4A mRNA, and RCC1 mRNA.

[0080] Methods of Monitoring for LNM Risk

[0081] Provided herein is a method of monitoring a patient having colorectal cancer for an increased risk of lymph node metastasis, the method comprising: (i) detecting the expression level of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR- 411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a biological sample obtained from the patient at a first time point; and (i) detecting the expression level of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR- 411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a biological sample obtained from the patient at a second time point later than the first time point; and (ii) diagnosing the patient as having an increased risk for lymph node metastasis when the biological sample at the second time point has an elevated expression level of the RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level of the RNA at the first time point. In embodiments, the blood sample is a serum sample or a plasma sample. In embodiments, the blood sample is a serum sample. In embodiments, the blood sample is a plasma sample. In embodiments, the colorectal cancer is colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is colorectal cancer without lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer without lymph node metastasis. In embodiments, the RNA is exosomal RNA. In embodiments, the RNA is cell-free RNA. In embodiments, the RNA is exosomal RNA and cell- free RNA. In embodiments, comprising detecting the expression level of one miRNA selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411 at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the one miRNA selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411 compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the one miRNA compared to the expression level at the first time point. In embodiments, comprising detecting the expression level of two miRNA selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411 at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the two miRNA selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411 compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the two miRNA compared to the expression level at the first time point. In embodiments, comprising detecting the expression level of three miRNA selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411 at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the three miRNA selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411 compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the three miRNA compared to the expression level at the first time point. In embodiments, comprising detecting the expression level of miR-181b, miR-193b, miR- 195, and miR-411 at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the miR-181b, miR-193b, miR-195, and miR-411 compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of miR-181b, miR-193b, miR-195, and miR-411 compared to the expression level at the first time point. In embodiments, the methods comprise detecting the expression level of one RNA selected from the group consisting of miR-181b, miR-193b, miR- 195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the one RNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the one RNA compared to the expression level at the first time point. In embodiments, the methods comprise detecting the expression level of two RNA selected from the group consisting of miR-181b, miR-193b, miR- 195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the two RNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the two RNA compared to the expression level at the first time point. In embodiments, the methods comprise detecting the expression level of three RNA selected from the group consisting of miR-181b, miR-193b, miR- 195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the three RNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the three RNA compared to the expression level at the first time point. In embodiments, the methods comprise detecting the expression level of four RNA selected from the group consisting of miR-181b, miR-193b, miR- 195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the four RNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the four RNA compared to the expression level at the first time point. In embodiments, the methods comprise detecting the expression level of five RNA selected from the group consisting of miR-181b, miR-193b, miR- 195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the five RNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the five RNA compared to the expression level at the first time point. In embodiments, the methods comprise detecting the expression level of six RNA selected from the group consisting of miR-181b, miR-193b, miR- 195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the six RNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the six RNA compared to the expression level at the first time point. In embodiments, the methods comprise detecting the expression level of seven RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the seven RNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the seven RNA compared to the expression level at the first time point. In embodiments, the methods comprise detecting the expression level of eight RNA selected from the group consisting of miR-181b, miR-193b, miR- 195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the eight RNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the eight RNA compared to the expression level at the first time point. In embodiments, the methods comprise detecting the expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of miR-181b, miR- 193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the RNA compared to the expression level at the first time point. In embodiments, the methods comprise detecting the expression level of an mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the mRNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the mRNA compared to the expression level at the first time point. In embodiments, the methods comprise detecting the expression level of one mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the one mRNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the one mRNA compared to the expression level at the first time point. In embodiments, the methods comprise detecting the expression level of two mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the two mRNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the two mRNA compared to the expression level at the first time point. In embodiments, the methods comprise detecting the expression level of three mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the three mRNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the three mRNA compared to the expression level at the first time point. In embodiments, the methods comprise detecting the expression level of four mRNA selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the four mRNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the four mRNA compared to the expression level at the first time point. In embodiments, the methods comprise detecting the expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the mRNA compared to the expression level at the first time point. In embodiments, the method further comprises administering to the patient an effective amount of an anti cancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof. In embodiments, the anticancer agent is a chemotherapeutic agent. In embodiments, the method does not comprise detecting the expression level of miR-32, LYZ mRNA, C2CD4A mRNA, and RCC1 mRNA. In embodiments, the patient is diagnosed as having an increased risk of lymph node metastasis when the expression level of the RNA in the biological sample obtained from the patient at the first time point is not elevated, and the expression level of the RNA in the biological sample obtained from the patient at the second time point is elevated. In embodiments, the patient is diagnosed as not having an increased risk of lymph node metastasis when the expression level of the RNA in the biological sample obtained from the patient at the first time point is not elevated, and the expression level of the RNA in the biological sample obtained from the patient at the second time point is not elevated.

[0082] Provided herein is a method of monitoring a patient having colorectal cancer for an increased risk of lymph node metastasis comprising: (i) detecting the expression level of an exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411 in a blood sample obtained from the patient at a first time point; and (i) detecting the expression level of an exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 in a blood sample obtained from the patient at a second time point later than the first time point; and (ii) diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 compared to the expression level of the exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411 at the first time point. In embodiments, the blood sample is a serum sample or a plasma sample. In embodiments, the blood sample is a serum sample. In embodiments, the blood sample is a plasma sample. In embodiments, the colorectal cancer is colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is colorectal cancer without lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer without lymph node metastasis. In embodiments, the method of monitoring a patient having colorectal cancer for an increased risk of lymph node metastasis comprises: (i) detecting the expression level of one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR- 195, and exosomal miR-411 in a blood sample obtained from the patient at a first time point; and (i) detecting the expression level of one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR- 411 in a blood sample obtained from the patient at a second time point later than the first time point; and (ii) diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 compared to the expression level of the one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411 at the first time point. In embodiments, the one exosomal miRNA obtained at the first time point is the same as the one exosomal miRNA obtained at the second time point (e.g., the expression level of exosomal miR-181b is measured at the first time point and the expression level of exosomal miR-181b is measured as the second time point). In embodiments, the method of monitoring a patient having colorectal cancer for an increased risk of lymph node metastasis comprises: (i) detecting the expression level of two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411 in a blood sample obtained from the patient at a first time point; and (i) detecting the expression level of two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 in a blood sample obtained from the patient at a second time point later than the first time point; and (ii) diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 compared to the expression level of the two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 at the first time point. In embodiments, the two exosomal miRNA obtained at the first time point are the same as the two exosomal miRNA obtained at the second time point. In embodiments, the method of monitoring a patient having colorectal cancer for an increased risk of lymph node metastasis comprises: (i) detecting the expression level of three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 in a blood sample obtained from the patient at a first time point; and (i) detecting the expression level of three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411 in a blood sample obtained from the patient at a second time point later than the first time point; and (ii) diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 compared to the expression level of the three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR- 411 at the first time point. In embodiments, the three exosomal miRNA obtained at the first time point are the same as the three exosomal miRNA obtained at the second time point. In embodiments, the method of monitoring a patient having colorectal cancer for an increased risk of lymph node metastasis comprises: (i) detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 in a blood sample obtained from the patient at a first time point; and (i) detecting the expression level of exosomal miR- 181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 in a blood sample obtained from the patient at a second time point later than the first time point; and (ii) diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 compared to the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 at the first time point. In embodiments, the method further comprises administering to the patient an effective amount of an anticancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof. In embodiments, the anticancer agent is a chemotherapeutic agent. In embodiments, the method does not comprise detecting the expression level of miR-32, LYZ mRNA, C2CD4A mRNA, and RCC1 mRNA.

[0083] Provided herein is a method of monitoring a patient having colorectal cancer for an increased risk of lymph node metastasis comprising: (i) detecting the expression level of an exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411 and a cell-free miRNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 in a blood sample obtained from the patient at a first time point; and (i) detecting the expression level of an exosomal miRNA selected from the group consisting of exosomal miR- 181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 and a cell-free miRNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 in a blood sample obtained from the patient at a second time point later than the first time point; and (ii) diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 and the cell-free miRNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 compared to the expression level of the exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR- 411 and a cell-free miRNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 at the first time point. In embodiments, the blood sample is a serum sample or a plasma sample. In embodiments, the blood sample is a serum sample. In embodiments, the blood sample is a plasma sample. In embodiments, the colorectal cancer is colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is colorectal cancer without lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer without lymph node metastasis. In embodiments, the method of monitoring a patient having colorectal cancer for an increased risk of lymph node metastasis comprises: (i) detecting the expression level of one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR- 195, and exosomal miR-411 and one cell-free miRNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 in a blood sample obtained from the patient at a first time point; and (i) detecting the expression level of one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 and one cell-free miRNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 in a blood sample obtained from the patient at a second time point later than the first time point; and (ii) diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411 and the one cell-free miRNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 compared to the expression level of the one exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR- 411 and the one cell-free miRNA selected from the group consisting of cell-free miR-181b, cell- free miR-193b, cell-free miR-195, and cell-free miR-411 at the first time point. In embodiments, the one exosomal miRNA obtained at the first time point is the same as the one exosomal miRNA obtained at the second time point (e.g., the expression level of exosomal miR-181b is measured at the first time point and the expression level of exosomal miR-181b is measured as the second time point). In embodiments, the method of monitoring a patient having colorectal cancer for an increased risk of lymph node metastasis comprises: (i) detecting the expression level of two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 and two cell-free miRNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 in a blood sample obtained from the patient at a first time point; and (i) detecting the expression level of two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 and two cell-free miRNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 in a blood sample obtained from the patient at a second time point later than the first time point; and (ii) diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 and two cell-free miRNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 compared to the expression level of the two exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR- 195, and exosomal miR-411 and two cell-free miRNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 at the first time point. In embodiments, the two exosomal miRNA obtained at the first time point are the same as the two exosomal miRNA obtained at the second time point. In embodiments, the method of monitoring a patient having colorectal cancer for an increased risk of lymph node metastasis comprises: (i) detecting the expression level of three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 and three cell-free miRNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 in a blood sample obtained from the patient at a first time point; and (i) detecting the expression level of three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411 and three cell-free miRNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 in a blood sample obtained from the patient at a second time point later than the first time point; and (ii) diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411 and three cell-free miRNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 compared to the expression level of the three exosomal miRNA selected from the group consisting of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 and three cell-free miRNA selected from the group consisting of cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 at the first time point. In embodiments, the three exosomal miRNA obtained at the first time point are the same as the three exosomal miRNA obtained at the second time point and the three cell-free miRNA at the first time point are the same as the three cell-free miRNA at the second time point. In embodiments, the method of monitoring a patient having colorectal cancer for an increased risk of lymph node metastasis comprises: (i) detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR- 193b, cell-free miR-195, and cell-free miR-411 in a blood sample obtained from the patient at a first time point; and (i) detecting the expression level of exosomal miR-181b, exosomal miR-

193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 in a blood sample obtained from the patient at a second time point later than the first time point; and (ii) diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 compared to the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR- 195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell- free miR-411 at the first time point. In embodiments, the method further comprises administering to the patient an effective amount of an anti cancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof. In embodiments, the anticancer agent is a chemotherapeutic agent. In embodiments, the method does not comprise detecting the expression level of miR-32, LYZ mRNA, C2CD4A mRNA, and RCC1 mRNA.

[0084] In embodiments of the methods that comprise monitoring a patient having colorectal cancer for an increased risk of lymph node metastasis, the RNA that is monitored at the first time point is the same RNA that is monitored at the second time point. For example, if the expression levels of miR-181b and miR-195 are detected at the first time point, then the expression levels of miR-181b and miR-195 are detected at the second time point so that a comparison of the expression levels of the same RNA can be made.

[0085] In embodiments of the methods that comprise an exosomal RNA and a cell-free RNA, the RNA is the same in each case. For example, when the exosomal RNA is miR-181b, then the cell-free RNA is miR-181b. When the methods comprise two exosomal RNA and two cell-free RNA, then the RNA are the same in each case. For example, if the two exosomal RNA are exosomal miR-181b and exosomal miR-193b, then the cell-free RNA are cell-free miR-181b and cell-free miR-193b. When the methods comprise three exosomal RNA and three cell-free RNA, then the RNA are the same in each case. For example, if the three exosomal RNA are exosomal miR-181b, exosomal miR-193b, and exosomal miR-411, then the cell-free RNA are cell-free miR-181b, cell-free miR-193b, and cell-free miR-411.

[0086] In embodiments of the methods that comprise an exosomal RNA and a cell-free RNA, the RNA are different in each case. For example, when the exosomal RNA is miR-181b, then the cell-free RNA is miR-411. When the methods comprise two exosomal RNA and two cell- free RNA, then the RNA are different in each case. For example, if the two exosomal RNA are exosomal miR-181b and exosomal miR-193b, then the cell-free RNA are cell-free miR-195 and cell-free miR-411. [0087] Methods of Detection

[0088] Provided herein are methods of detecting an RNA biomarker in a subject, wherein the subject has, or is suspected of having, colorectal cancer, by determining the presence of one or more biomarkers in a biological sample from the subject, wherein the one or more biomarkers are miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, MMP9 mRNA, or a combination of two or more thereof. In embodiments, the subject has colorectal cancer. In embodiments, the subject is suspected of having colorectal cancer. In embodiments, the colorectal cancer is colorectal cancer with lymph node metastasis.

In embodiments, the colorectal cancer is colorectal cancer without lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer without lymph node metastasis. 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 blood sample is a serum sample. In embodiments, the blood sample is a plasma sample.

[0089] Provided herein are methods of detecting a lymph node metastasis in a patient with colorectal cancer or a patient suspected of having colorectal cancer, the method comprising determining the presence of one or more biomarkers in a biological sample from the subject, wherein the one or more biomarkers are miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, MMP9 mRNA, or a combination of two or more thereof; wherein the presence of the one or more biomarkers indicates a lymph node metastasis. In embodiments, the method comprises detecting a lymph node metastasis in a patient with colorectal cancer. In embodiments, the methods comprise detecting a lymph node metastasis in a patient suspected of having colorectal cancer. In embodiments, the colorectal cancer is colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is colorectal cancer without lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer with lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer without lymph node metastasis. 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 blood sample is a serum sample. In embodiments, the blood sample is a plasma sample. In embodiments, the miRNA is exosomal miRNA, cell-free miRNA, or a combination thereof. In embodiments, the mRNA is exosomal mRNA, cell-free mRNA, or a combination thereof. [0090] In embodiments of the methods described herein, determining the presence of one or more biomarkers comprises detecting the presence of one or more of the biomarkers. In embodiments when one or more of the biomarkers are detected, the method further comprises surgically removing all or a portion of the colon of the subject. The skilled artisan will artisan will recognize that the method comprises surgically removing at least the cancerous portion of the colon of the subject. The cancerous portion can include cancerous cells, tissues, or tumors. In embodiments, the surgical procedure to remove all or a portion of the colon is a colectomy. In embodiments, the surgical procedure to remove all or a portion of the colon is hemicolectomy.

In embodiments, the surgical procedure to remove all or a portion of the colon is radical resection. In embodiments, the surgical procedure to remove all or a portion of the colon is radical surgery.

[0091] In embodiments of the methods described herein, when one or more of the biomarkers are detected, the method further comprises administering to the subject an effective amount of an anticancer treatment. In embodiments when one or more of the biomarkers are detected, the method further comprises surgically removing a portion of the colon of the subject and administering to the subject an effective amount of an anticancer treatment. The anticancer treatment can be any known in the art, such as anti-cancer agent, radiation, chemotherapy, immunotherapy, or a combination thereof. In embodiments, the anticancer treatment comprises administering an effective amount of a chemotherapeutic agent.

[0092] In embodiments, detecting the presence of one or more of the biomarkers comprises surgically removing a portion of the colon and/or administering to the subject an effective amount of an anti cancer treatment, and thereafter monitoring the subject for the presence of colorectal cancer and/or for the progression of the colorectal cancer. In embodiments, monitoring the subject is conducted once per month, once every two months, once every three months, once every four months, once every six months, once every year, once every five years, and the like. If monitoring reveals the presence of one or more of the biomarkers, the methods further comprise surgically removing a portion of the colon of the subject and/or continuing administering to the subject an effective amount of an anticancer treatment (e.g., anti-cancer agent, radiation, chemotherapy, immunotherapy, etc.). If the method comprising continuing administration of the anticancer treatment, the method may comprise administering to the subject an increased dose of an anticancer agent and/or administering a new treatment regimen (new/different anticancer treatment) to the subject.

[0093] In embodiments of the methods described herein, determining the presence of one or more biomarkers comprises detecting the absence of the biomarkers (i.e., not detecting any biomarkers in the biological sample). In embodiments when no biomarkers are detected, the method comprises monitoring the subject for the presence of colorectal cancer and/or for the progression of the colorectal cancer. In embodiments, monitoring the subject is conducted once per month, once every two months, once every three months, once every four months, once every six months, or once every year. If monitoring reveals the presence of one or more of the biomarkers, the methods further comprise surgically removing a portion of the colon of the subject and/or administering to the subject an effective amount of an anticancer treatment. The anticancer treatment can be any known in the art, such as anti-cancer agent, radiation, chemotherapy, immunotherapy, or a combination thereof.

[0094] In embodiments, the methods comprise determining the presence of one biomarker selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise determining the presence of two biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise determining the presence of three biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise determining the presence of four biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise determining the presence of five biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise determining the presence of six biomarkers selected from the group consisting of miR- 181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise determining the presence of seven biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR- 411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise determining the presence of eight biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise determining the presence miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise determining the presence of elevated levels of the biomarker, relative to a control. In embodiments, the miRNA is exosomal miRNA. In embodiments, the miRNA is cell- free miRNA. In embodiments, the miRNA is exosomal miRNA and cell-free miRNA.

[0095] In embodiments, the methods comprise determining the presence of one or more biomarkers in a biological sample from the subject, wherein the one or more biomarkers are miR-181b, miR-193b, miR-195, miR-411, or a combination of two or more thereof. In embodiments, the methods comprise determining the presence of one biomarker selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411. In embodiments, the methods comprise determining the presence of two biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411. In embodiments, the methods comprise determining the presence of three biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411. In embodiments, the methods comprise determining the presence of miR-181b, miR-193b, miR-195, and miR-411. In embodiments, the methods comprise determining the presence of miR-181b. In embodiments, the methods comprise determining the presence of miR-193b. In embodiments, the methods comprise determining the presence of miR-195. In embodiments, the methods comprise determining the presence of miR-411. In embodiments, the methods comprise determining the presence of elevated levels of the biomarker, relative to a control. In embodiments, the miRNA is exosomal miRNA. In embodiments, the miRNA is cell-free miRNA. In embodiments, the miRNA is exosomal miRNA and cell-free miRNA.

[0096] In embodiments, the methods comprise determining the presence of one or more biomarkers in a biological sample from the subject, wherein the one or more biomarkers are AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, MMP9 mRNA, or a combination of two or more thereof. In embodiments, the methods comprise determining the presence of one biomarker selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise determining the presence of two biomarkers selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise determining the presence of three biomarkers selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise determining the presence of four biomarkers selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise determining the presence of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the methods comprise determining the presence of AMT mRNA. In embodiments, the methods comprise determining the presence of FOXA1 mRNA. In embodiments, the methods comprise determining the presence of PIGR mRNA. In embodiments, the methods comprise determining the presence of MMP1 mRNA. In embodiments, the methods comprise determining the presence of MMP9 mRNA. In embodiments, the methods comprise determining the presence of elevated levels of the biomarker, relative to a control.

[0097] Provided herein are methods of treating colorectal cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an anticancer treatment; wherein a biological sample from the subject has one or more biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, MMP9 mRNA, and a combination of two or more thereof. The anticancer treatment can be any known in the art, such as anti-cancer agent, radiation, chemotherapy, immunotherapy, or a combination thereof. In embodiments, the methods further comprise monitoring the subject for the presence or progression of colorectal cancer. In embodiments, the subject is at risk for developing lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer (T1 CRC). In embodiments, the biological sample is a blood sample.

[0098] Provided herein are methods of treating colorectal cancer in a subject having a lymph node metastasis in need thereof, the method comprising administering to the subject an effective amount of an anti cancer treatment; wherein a biological sample from the subject has one or more biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR- 411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, MMP9 mRNA, and a combination of two or more thereof. The anticancer treatment can be any known in the art, such as anti-cancer agent, radiation, chemotherapy, immunotherapy, or a combination thereof. In embodiments, the methods further comprise monitoring the subject for the presence or progression of colorectal cancer. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer (T1 CRC). In embodiments, the biological sample is a blood sample.

[0099] Provided herein are methods of treating colorectal cancer in a subject in need thereof, the method comprising determining the presence of one or more biomarkers in a biological sample from the subject, wherein the one or more biomarkers are miR-181b, miR-193b, miR- 195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, MMP9 mRNA, or a combination of two or more thereof; detecting the presence of the one or more biomarkers; and administering to the subject an effective amount of an anticancer treatment. The anticancer treatment can be any known in the art, such as anti-cancer agent, radiation, chemotherapy, immunotherapy, or a combination thereof. In embodiments, the methods further comprise monitoring the subject for the presence or progression of colorectal cancer. In embodiments, the subject is at risk for developing lymph node metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer (T1 CRC). In embodiments, the biological sample is a blood sample.

[0100] Provided herein are methods of treating colorectal cancer in a patient having a lymph node metastasis, the method comprising determining the presence of one or more biomarkers in a biological sample from the subject, wherein the one or more biomarkers are miR-181b, miR- 193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, MMP9 mRNA, or a combination of two or more thereof; detecting the presence of the one or more biomarkers; and administering to the subject an effective amount of an anticancer treatment.

The anti cancer treatment can be any known in the art, such as anti-cancer agent, radiation, chemotherapy, immunotherapy, or a combination thereof. In embodiments, the methods further comprise monitoring the subject for the presence or progression of colorectal cancer. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer (T1 CRC). In embodiments, the biological sample is a blood sample.

[0101] In embodiments, the subject has one biomarker selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the subject has two biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the subject has three biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the subject has four biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the subject has five biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the subject has six biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the subject has seven biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the subject has eight biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the subject has all nine biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the biomarkers have an elevated expression level relative to a control.

[0102] In embodiments, the subject has one biomarker selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411. In embodiments, the subject has two biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411. In embodiments, the subject has three biomarkers selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411. In embodiments, the subject has the four biomarkers of miR- 181b, miR-193b, miR-195, and miR-411. In embodiments, the subject has the biomarker of miR-181b. In embodiments, the subject has the biomarker of miR-193b. In embodiments, the subject has the biomarker of miR-195. In embodiments, the subject has the biomarker of miR- 411. In embodiments, the miRNA have an elevated expression level relative to a control. In embodiments, the miRNA are exosomal miRNA. In embodiments, the miRNA are cell-free miRNA. In embodiments, the miRNA are exosomal miRNA and cell-free miRNA.

[0103] In embodiments, the subject has one biomarker selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the subject has two biomarkers selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the subject has three biomarkers selected from the group consisting of AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the subject has four biomarkers selected from the group consisting of AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the subject has five biomarkers of AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the subject has the biomarker of AMT mRNA. In embodiments, the subject has the biomarker of FOXAl mRNA. In embodiments, the subject has the biomarker of PIGR mRNA. In embodiments, the subject has the biomarker of MMP1 mRNA. In embodiments, the subject has the biomarker of MMP9 mRNA. In embodiments, the biomarker has an elevated expression level relative to a control.

[0104] In embodiments of the methods described herein, the methods comprise administering to the subject an effective amount of an anti-cancer agent (e.g., as defined herein). In embodiments, the method comprises administering to the subject an effective amount of chemotherapy (e.g., a chemotherapeutic agent). In embodiments, the method comprises administering to the subject an effective amount of a chemotherapeutic agent and radiation therapy. In embodiments, the method comprises administering to the subject an effective amount of an immunotherapy. In embodiments, the method comprises administering to the subject an effective amount of an immunotherapy and radiation therapy. In embodiments, the method comprises administering to the subject an effective amount of a chemotherapeutic agent and an immunotherapy. In embodiments, the method comprises administering to the subject an effective amount of a chemotherapeutic agent, an immunotherapy, and radiation therapy.

[0105] In embodiments, the chemotherapeutic agent is an alkylating agent, an antimetabolite compound, an anthracy cline compound, an antitumor antibiotic, a platinum compound, a topoisomerase inhibitor, a vinca alkaloid, a taxane compound, an epothilone compound, or a combination of two or more thereof. In embodiments, the alkylating agent is carboplatin, chlorambucil, cyclophosphamide, melphalan, mechlorethamine, procarbazine, or thiotepa. In embodiments, the antimetabolite compound is azacitidine, capecitabine, cytarabine, gemcitabine, doxifluridine, hydroxyurea, methotrexate, pemetrexed, 6-thioguanine, 5- fluorouracil, or 6-mercaptopurine. In embodiments, the anthracycline 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, amscarine, 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 epothiolone compound is epithilone, ixabepilone, patupilone, or sagopilone.

[0106] In embodiments, the method comprises administering to the subject an effective amount of 5-fluorouracil. In embodiments, the method comprises administering to the subject an effective amount of 5-fluorouracil, leucovorin, oxaliplatin, irinotecan, capecitabine, or a combination of two or more thereof. In embodiments, the method comprises administering to the subject an effective amount of 5-fluorouracil, and an effective amount of leucovorin, oxaliplatin, irinotecan, capecitabine, or a combination of two or more thereof. In embodiments, the method comprises administering to the subject an effective amount of 5-fluorouracil, leucovorin, oxaliplatin, irinotecan, capecitabine, or a combination of two or more thereof, and also administering to the patient an effective amount of bevacizumab, ziv-aflibercept, ramucirumab, cetuximab, or panitumumab. In embodiments, the method comprises administering to the subject an effective amount of 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.

[0107] In embodiments, the method comprises administering to the subject an effective amount of 5-fluorouracil, leucovorin, oxaliplatin, irinotecan, capecitabine, bevacizumab, ziv- aflibercept, ramucirumab, cetuximab, panitumumab, encorafenib, entrectinib, ipilumamab, trifluridine, tipiracil, nivolumab, pembrolizumab, regorafenib, trastuzumab, pertuzumab, atezolizumab, savolitinib, tucatinib, pralsetinib, cibisatamab, cabozantinib-S-malate, cevumeran, patritumab deruxtecan, taminadenant, spartalizumab, ciforadenant, binimetinib, methotrexate, masitinib, napabucasin, or a combination of two or more thereof.

[0108] In embodiments of the methods described herein, the methods comprise administering to the subject an effective amount of atezolizumab, bevacizumab, capecitabine, cetuximab, encorafenib, binimetinib, entrectinib, fluorouracil, ipilumamab, irinotecan, levoleucovorin, leucovorin, methotrexate, trifluridine/tipiracil, nivolumab, oxaliplatin, panitumumab, pembrolizumab, ramucirumab, regorafenib, ziv-aflibercept, methotrexate, masitinib, napabucasin, or a combination of two or more thereof. In embodiments of the methods described herein, the methods comprise administering to the subject an effective amount of bevacizumab, capecitabine, cetuximab, encorafenib, entrectinib, fluorouracil, ipilumamab, irinotecan, leucovorin, trifluridine, tipiracil, nivolumab, oxaliplatin, panitumumab, pembrolizumab, ramucirumab, regorafenib, ziv-aflibercept, methotrexate, or a combination of two or more thereof. In embodiments, the method comprises administering to the subject an effective amount of bevacizumab, capecitabine, cetuximab, encorafenib, entrectinib, fluorouracil, ipilumamab, irinotecan, leucovorin, trifluridine/tipiracil, nivolumab, oxaliplatin, panitumumab, pembrolizumab, ramucirumab, regorafenib, ziv-aflibercept, vitamin D3, trastuzumab, pertuzumab, atezolizumab, savolitinib, tucatinib, pralsetinib, cibisatamab, cabozantinib-S- malate, patritumab deruxtecan, spartalizumab, ciforadenant, or a combination of two or more thereof.

[0109] In embodiments, the method comprises administering to the subject an effective amount of bevacizumab, capecitabine, cetuximab, encorafenib, entrectinib, fluorouracil, ipilumamab, irinotecan, leucovorin, trifluridine, tipiracil, nivolumab, oxaliplatin, panitumumab, pembrolizumab, ramucirumab, regorafenib, ziv-aflibercept, vitamin D3, trastuzumab, pertuzumab, atezolizumab, savolitinib, tucatinib, pralsetinib, cibisatamab, cabozantinib-S- malate, cevumeran (R07198457), PSB205, patritumab deruxtecan, taminadenant , spartalizumab, ciforadenant, COM902, PY314, binimetinib, methotrexate, masitinib, napabucasin, or a combination of two or more thereof.

[0110] In embodiments, the method comprises administering to the subject an effective amount of bevacizumab, capecitabine, cetuximab, encorafenib, entrectinib, fluorouracil, ipilumamab, irinotecan, leucovorin, trifluridine, tipiracil, nivolumab, oxaliplatin, panitumumab, pembrolizumab, ramucirumab, regorafenib, ziv-aflibercept, vitamin D3, trastuzumab, pertuzumab, atezolizumab, savolitinib, tucatinib, pralsetinib, cibisatamab, cabozantinib-S- malate, cevumeran, patritumab deruxtecan, spartalizumab, ciforadenant, binimetinib, methotrexate, masitinib, napabucasin, or a combination of two or more thereof. In embodiments, the irinotecan is irinotecan hydrochloride.

[0111] In embodiments, the methods comprise administering to the subject an effective amount of a combination of trifluridine and tipiracil, a combination of capecitabine and oxaliplatin, a combination of leucovorin calcium, fluorouracil, and irinotecan; a combination of leucovorin calcium, fluorouracil, irinotecan, and bevacizumab; a combination of leucovorin calcium, fluorouracil, irinotecan, and cetuximab; a combination of leucovorin calcium, fluorouracil, and oxaliplatin; a combination of salcaprozate sodium, cisplatin, and vinblastine; a combination of leucovorin calcium and fluorouracil; a combination of capecitabine and irinotecan; a combination of capecitabine and oxaliplatin; a combination of cetuximab and panitumumab; or a combination of encorafenib and binimetinib. In embodiments, the irinotecan is irinotecan hydrochloride.

[0112] In embodiments, the methods further comprise measuring the tumor size, measuring submucosal invasion, identifying the presence of lymphatic invasion, identifying the presence of vascular invasion, detecting the grade of tumor budding, identifying tumor histology, or a combination of two or more thereof. In embodiments, the methods further comprise measuring the tumor size. In embodiments, the methods further comprise measuring submucosal invasion. In embodiments, the methods further comprise identifying the presence of lymphatic invasion.

In embodiments, the methods further comprise identifying the presence of vascular invasion. In embodiments, the methods further comprise detecting the grade of tumor budding. In embodiments, the methods further comprise identifying tumor histology.

[0113] In embodiments of the methods described herein, the subject has a tumor size >20 mm, a depth of submucosal invasion >1000 pm, presence of lymphatic invasion, presence of vascular invasion, high-grade tumor budding (>2), poorly differentiated histology, or a combination of two or more thereof. In embodiments, the subject has a tumor size >20 mm. In embodiments, the subject has a depth of submucosal invasion >1000 pm. In embodiments, the subject has a presence of lymphatic invasion. In embodiments, the subject has a presence of vascular invasion. In embodiments, the subject has high-grade tumor budding (>2). In embodiments, the subject has poorly differentiated histology.

[0114] “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 growth or proliferation of cells.

[0115] “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 growth 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, floxouridine, 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 (VP 16), etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.), platinum-based compounds (e.g. cisplatin, oxaloplatin, carboplatin), anthracenedione (e.g., mitoxantrone), substituted urea (e.g., hydroxyurea), methyl hydrazine derivative (e.g., procarbazine), adrenocortical suppressant (e.g., mitotane, aminoglutethimide), epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), inhibitors of mitogen- activated protein kinase signaling (e.g. U0126, PD98059, PD184352, PD0325901, ARRY- 142886, SB239063, SP600125, BAY 43-9006, wortmannin, or LY294002), mTOR inhibitors, antibodies (e.g., rituxan), 5-aza-2'-deoxycytidine, doxorubicin, vincristine, etoposide, gemcitabine, imatinib (Gleevec.RTM.), geldanamycin, 17-N-Allylamino-17- Demethoxygeldanamycin (17-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 (BCG), 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 to ⁱⁿIn, ⁹⁰Y, or ¹³¹I, etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin, epirubicin, topotecan, itraconazole, vindesine, cerivastatin, vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan, clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib, gefitinib, EGFR inhibitors, epidermal growth factor receptor (EGFR)-targeted therapy or therapeutic (e.g. gefitinib (Iressa™), erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™), panitumumab (Vectibix™), vandetanib (Caprelsa™), afatinib/BIBW2992, CI-1033/canertinib, neratinib/HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, dacomitinib/PF299804, OSI-420/desmethyl erlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib, dasatinib, or the like.

[0116] Additionally, the compounds described herein can be co-administered with conventional immunotherapeutic agents including, but not limited to, immunostimulants (e.g., Bacillus Calmette-Guerin (BCG), 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.), and radioimmunotherapy (e.g., anti-CD20 monoclonal antibody conjugated to ^mIn, ⁹⁰Y, or ¹³¹I, etc.).

[0117] Kits

[0118] Provided here are kits comprising components, such as reagents and reaction mixtures, to conduct the assays to detect the miRNA and mRNA as described herein. As part of the kit, materials and instruction are provided, e.g., for storage and use of kit components.

[0119] “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, mRNA). For example, detecting the level of RNA (such as miRNA or mRNA) means using an analytic procedure (such as an in vitro procedure) to qualitatively assess or quantitatively measure the presence or amount of the RNA. In embodiments, raw expression values are normalized by performing quantile normalization relative to the reference distribution and subsequent log 10-transformation. In embodiments, when RNA 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).

[0120] In embodiments, the kit comprises reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is selected from the group consisting of: (i) miR- 181b, miR-193b, miR-195, and miR-411; (ii) miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA; and (iii) AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the kit comprises reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is selected from the group consisting of miR-181b, miR-193b, miR- 195, and miR-411. In embodiments, the kit comprises reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is miR-181b, miR-193b, miR- 195, and miR-411. In embodiments, the kit comprises reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the kit comprises reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the kit comprises reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is selected from the group consisting of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA. In embodiments, the kit comprises reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

[0121] In embodiments, the kit comprises reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is (i) exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411; or (ii) exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR- 195, and cell-free miR-411. In embodiments, the kit comprises reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411. In embodiments, the kit comprises reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is selected from the group consisting of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411. In embodiments, the kit comprises reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411. In embodiments, the kit comprises reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is selected from the group consisting of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, exosomal miR-411, exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR- 195, and cell-free miR-411.

[0122] In embodiments, the disclosure provides a kit for detecting the RNA (e.g., miRNA, mRNA) described herein. In embodiments, the kit is an assay system including any one of assay reagents, assay controls, protocols, exemplary assay results, or combinations of these components designed to provide the user with means to evaluate the expression level of the RNA (e.g., miRNA, mRNA) described herein.

[0123] In embodiments, the disclosure provides a kit for diagnosing colorectal cancer in a patent, including reagents for detecting RNA markers in a biological (e.g., blood) sample from a patient.

[0124] 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.

[0125] 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, such as for identifying subjects that have a condition characterized by expression of polypeptide biomarkers, e.g., interstitial lung disease.

[0126] In addition, the means for detecting of the assay system of the present disclosure can be immobilized on a substrate. Such a substrate can include any suitable substrate for immobilization of a detection reagent such as would be used in any of the previously described methods of detection. Briefly, a substrate suitable for immobilization of a means for detecting includes any solid support, such as any solid organic, biopolymer or inorganic support that can form a bond with the means for detecting without significantly affecting the activity and/or ability of the detection means to detect the desired target molecule. Exemplary organic solid supports include polymers such as polystyrene, nylon, phenol-formaldehyde resins, and acrylic copolymers (e.g., polyacrylamide). The kit can also include suitable reagents for the detection of the reagent and/or for the labeling of positive or negative controls, wash solutions, dilution buffers and the like. The assay system can also include a set of written instructions for using the system and interpreting the results. [0127] Embodiments 1 to 62

[0128] Embodiment 1. A method of treating colorectal cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of an anti-cancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof; wherein a blood sample obtained from the patient comprises an elevated expression level, relative to a control, of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

[0129] Embodiment 2. A method of treating colorectal cancer in a patient in need thereof, the method comprising: (i) detecting an elevated expression level, relative to a control, of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient; and (ii) administering to the patient an effective amount of an anti-cancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof.

[0130] Embodiment 3. The method of Embodiment 1 or 2, comprising detecting an elevated expression level, relative to the control, of miR-181b, miR-193b, miR-195, and miR-411.

[0131] Embodiment 4. The method of Embodiment 1 or 2, comprising detecting an elevated expression level, relative to the control, of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

[0132] Embodiment 5. The method of Embodiment 1 or 2, comprising detecting an elevated expression level, relative to the control, of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

[0133] Embodiment 6. The method of any one of Embodiments 1 to 5, wherein the RNA is exosomal RNA.

[0134] Embodiment 7. The method of Embodiment 1 or 2, comprising detecting an elevated expression level, relative to the control, of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411.

[0135] Embodiment 8. A method of identifying an increased risk of developing lymph node metastasis in a patient with colorectal cancer or detecting a lymph node metastasis in a patient with colorectal cancer, the method comprising detecting an elevated expression level, relative to a control, of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient; wherein the elevated expression level of the RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, indicates an increased risk of developing lymph node metastasis or the presence of a lymph node metastasis.

[0136] Embodiment 9. The method of Embodiment 8, wherein the method comprises identifying an increased risk of developing lymph node metastasis in a patient with colorectal cancer.

[0137] Embodiment 10. The method of Embodiment 8, wherein the method comprises detecting a lymph node metastasis in a patient with colorectal cancer.

[0138] Embodiment 11. The method of any one of Embodiments 8 to 10, comprising detecting an elevated expression level, relative to the control, of miR-181b, miR-193b, miR-195, and miR-411.

[0139] Embodiment 12. The method of any one of Embodiments 8 to 10, comprising detecting an elevated expression level, relative to the control, of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

[0140] Embodiment 13. The method of any one of Embodiments 8 to 10, comprising detecting an elevated expression level, relative to the control, of miR-181b, miR-193b, miR-195, miR- 411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

[0141] Embodiment 14. The method of any one of Embodiments 8 to 13, wherein the RNA is exosomal RNA.

[0142] Embodiment 15. The method of any one of Embodiments 8 to 10, comprising detecting an elevated expression level, relative to the control, of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal and miR-411.

[0143] Embodiment 16. The method of any one of Embodiments 8 to 10, comprising detecting an elevated expression level, relative to the control, of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, exosomal and miR-411, cell-free miR-181b, cell-free miR-193b, cell- free miR-195, and cell-free and miR-411.

[0144] Embodiment 17. A method of diagnosing a patient having colorectal cancer as high risk for lymph node metastasis or low risk for lymph node metastasis, the method comprising:

(i) detecting the expression level of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient; and (ii) diagnosing the patient as having: (a) a high risk for lymph node metastasis when the blood sample has an elevated expression level, relative to a control, of the RNA selected from the group consisting of miR- 181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, or (b) a low risk for lymph node metastasis when the blood sample does not have an elevated expression level, relative to a control, of the RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

[0145] Embodiment 18. The method of Embodiment 17, comprising: (i) detecting the expression level of miR-181b, miR-193b, miR-195, and miR-411 in the blood sample, and (ii) diagnosing the patient as having: (a) a high risk for lymph node metastasis when the blood sample has an elevated expression level of miR-181b, miR-193b, miR-195, and miR-411, relative to the control, or (b) a low risk for lymph node metastasis when the blood sample does not have an elevated expression level of miR-181b, miR-193b, miR-195, miR-411, relative to the control.

[0146] Embodiment 19. The method of Embodiment 17, comprising; (i) detecting the expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in the blood sample, and (ii) diagnosing the patient as having: (a) a high risk for lymph node metastasis when the blood sample has an elevated expression level of AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, relative to the control, or (b) a low risk for lymph node metastasis when the blood sample does not have an elevated expression level of AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, relative to the control.

[0147] Embodiment 20. The method of Embodiment 17, comprising: (i) detecting the expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in the blood sample, and (ii) diagnosing the patient as having: (a) a high risk for lymph node metastasis when the blood sample has an elevated expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, relative to the control, or (b) a low risk for lymph node metastasis when the blood sample does not have an elevated expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, relative to the control.

[0148] Embodiment 21. The method of any one of Embodiments 17 to 20, wherein the RNA is exosomal RNA.

[0149] Embodiment 22. The method of Embodiment 17, comprising: (i) detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 in the blood sample, and (ii) diagnosing the patient as having: (a) a high risk for lymph node metastasis when the blood sample has an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, relative to the control, or (b) a low risk for lymph node metastasis when the blood sample does not have an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, relative to the control.

[0150] Embodiment 23. The method of Embodiment 17, comprising: (i) detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411, in the blood sample, and (ii) diagnosing the patient as having: (a) a high risk for lymph node metastasis when the blood sample has an elevated expression level of e exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR- 193b, cell-free miR-195, and cell-free miR-411, relative to the control, or (b) a low risk for lymph node metastasis when the blood sample does not have an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411, relative to the control.

[0151] Embodiment 24. A method of diagnosing a patient having colorectal cancer as high risk for lymph node metastasis, the method comprising: (i) detecting the expression level of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient; and (ii) diagnosing the patient as having a high risk for lymph node metastasis when the blood sample has an elevated expression level, relative to a control, of the RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

[0152] Embodiment 25. The method of Embodiment 24, comprising: (i) detecting the expression level of miR-181b, miR-193b, miR-195, and miR-411 in the blood sample, and (ii) diagnosing the patient as having a high risk for lymph node metastasis when the blood sample has an elevated expression level of miR-181b, miR-193b, miR-195, and miR-411, relative to the control.

[0153] Embodiment 26. The method of Embodiment 24, comprising: (i) detecting the expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in the blood sample, and (ii) diagnosing the patient as having a high risk for lymph node metastasis when the blood sample has an elevated expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, relative to the control.

[0154] Embodiment 27. The method of Embodiment 24, comprising: (i) detecting the expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in the blood sample, and (ii) diagnosing the patient as having a high risk for lymph node metastasis when the blood sample has an elevated expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, relative to the control.

[0155] Embodiment 28. The method of any one of Embodiments 24 to 27, wherein the RNA is exosomal RNA.

[0156] Embodiment 29. The method of Embodiment 24, comprising: (i) detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 in the blood sample, and (ii) diagnosing the patient as having a high risk for lymph node metastasis when the blood sample has an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411, relative to the control.

[0157] Embodiment 30. The method of Embodiment 24, comprising: (i) detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411, in the blood sample, and (ii) diagnosing the patient as having a high risk for lymph node metastasis when the blood sample has an elevated expression level of e exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411, relative to the control.

[0158] Embodiment 31. A method of monitoring a patient having colorectal cancer for an increased risk of lymph node metastasis, the method comprising: (i) detecting the expression level of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR- 411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient at a first time point; and (ii) detecting the expression level of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR- 411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient at a second time point later than the first time point; and (iii) diagnosing the patient: (a) as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level of the RNA at the first time point; or (b) as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level of the RNA at the first time point

[0159] Embodiment 32. The method of Embodiment 31, comprising detecting the expression level of miR-181b, miR-193b, miR-195, and miR-411 at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the miR- 181b, miR-193b, miR-195, and miR-411 compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of miR-181b, miR-193b, miR-195, and miR-411 compared to the expression level at the first time point.

[0160] Embodiment 33. The method of Embodiment 31, comprising detecting the expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level at the first time point.

[0161] Embodiment 34. The method of Embodiment 31, comprising detecting the expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level at the first time point.

[0162] Embodiment 35. The method of any one of Embodiments 31 to 34, wherein the RNA is exosomal RNA

[0163] Embodiment 36. The method of Embodiment 31, comprising detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 compared to the expression level at the first time point.

[0164] Embodiment 37. The method of Embodiment 31, comprising detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell- free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 compared to the expression level at the first time point.

[0165] Embodiment 38. The method of any one of Embodiments 31 to 37, wherein the expression level of the RNA in the blood sample obtained from the patient at the first time point is not elevated, and wherein the expression level of the RNA in the blood sample obtained from the patient at the second time point is elevated, thereby diagnosing an increased risk of lymph node metastasis in the patient having colorectal cancer.

[0166] Embodiment 39. The method of any one of Embodiments 31 to 37, wherein the expression level of the RNA in the blood sample obtained from the patient at the first time point is not elevated, and wherein the expression level of the RNA in the blood sample obtained from the patient at the second time point is not elevated, thereby diagnosing no increased risk of lymph node metastasis in the patient having colorectal cancer.

[0167] Embodiment 40. A method of monitoring a patient having colorectal cancer for an increased risk of lymph node metastasis, the method comprising: (i) detecting the expression level of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR- 411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient at a first time point; and (ii) detecting the expression level of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR- 411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient at a second time point later than the first time point; and (iii) diagnosing the patient as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level of the RNA at the first time point.

[0168] Embodiment 41. The method of Embodiment 40, comprising detecting the expression level of miR-181b, miR-193b, miR-195, and miR-411 at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the miR- 181b, miR-193b, miR-195, and miR-411 compared to the expression level at the first time point.

[0169] Embodiment 42. The method of Embodiment 40, comprising detecting the expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level at the first time point.

[0170] Embodiment 43. The method of Embodiment 40, comprising detecting the expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level at the first time point.

[0171] Embodiment 44. The method of any one of Embodiments 40 to 43, wherein the RNA is exosomal RNA

[0172] Embodiment 45. The method of Embodiment 40, comprising detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 compared to the expression level at the first time point.

[0173] Embodiment 46. The method of Embodiment 40, comprising detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell- free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 compared to the expression level at the first time point.

[0174] Embodiment 47. The method of any one of Embodiments 1 to 46, wherein the blood sample is a serum sample.

[0175] Embodiment 48. The method of any one of Embodiments 1 to 46, wherein the blood sample is a plasma sample.

[0176] Embodiment 49. The method of any one of Embodiments 1 to 48, wherein the colorectal cancer is invasive submucosal colorectal cancer.

[0177] Embodiment 50. The method of any one of Embodiments 1 to 48, wherein the colorectal cancer is colorectal cancer with lymph node metastasis.

[0178] Embodiment 51. The method of any one of Embodiments 1 to 48, wherein the colorectal cancer is invasive submucosal colorectal cancer with lymph node metastasis.

[0179] Embodiment 52. The method of any one of Embodiments 8 to 51, further comprising administering to the patient an effective amount of an anti-cancer agent.

[0180] Embodiment 53. The method of any one of Embodiments 1 to 7, comprising administering to the patient the effective amount of the anti-cancer agent.

[0181] Embodiment 54. The method of any one of Embodiments 1 to 7, comprising administering to the patient the effective amount of the anti-cancer agent and surgically removing all or a portion of the colon of the patient.

[0182] Embodiment 55. The method of any one of Embodiments 1-7 and 52-54, wherein the anti-cancer agent is a chemotherapeutic agent.

[0183] Embodiment 56. The method of Embodiment 55, wherein the chemotherapeutic agent comprises 5-fluorouracil, leucovorin, oxaliplatin, irinotecan, capecitabine, or a combination of two or more thereof.

[0184] Embodiment 57. The method of Embodiment 55, 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.

[0185] Embodiment 58. The method of Embodiment 57, wherein the alkylating agent is carboplatin, chlorambucil, cyclophosphamide, melphalan, mechlorethamine, procarbazine, or thiotepa; the antimetabolite compound is azacitidine, capecitabine, cytarabine, gemcitabine, doxifluridine, hydroxyurea, methotrexate, pemetrexed, 6-thioguanine, 5-fluorouracil, or 6- mercaptopurine; the anthracycline compound is daunorubicin, doxorubicin, idarubicin, epirubicin, or mitoxantrone; the antitumor antibiotic is actinomycin, bleomycin, mitomycin, or valrubicin; the platinum compound is cisplatin or oxaliplatin; the topoisomerase inhibitor is irinotecan, topotecan, amscarine, etoposide, teniposide, or eribulin; the vinca alkaloid is vincristine, vinblastine, vinorelbine, or vindesine; the taxane compound is paclitaxel or docetaxel; and the epothiolone compound is epithilone, ixabepilone patupilone, or sagopilone.

[0186] Embodiment 59. The method of any one of Embodiments 1 to 7, comprising surgically removing all or a portion of the colon of the patient.

[0187] Embodiment 60. The method of any one of Embodiments 8 to 58, further comprising surgically removing all or a portion of the colon of the patient.

[0188] Embodiment 61. The method of any one of Embodiments 1 to 60, wherein the method does not comprise detecting the expression level of miR-32, LYZ mRNA, C2CD4A mRNA, and RCC1 mRNA.

[0189] Embodiment 62. A kit comprising reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is selected from the group consisting of: (i) miR- 181b, miR-193b, miR-195, and miR-411; (ii) miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA; or (iii) AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

[0190] Embodiment 63. The kit of Embodiment 62, wherein the RNA is selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411.

[0191] Embodiment 64. A kit comprising reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411.

[0192] Embodiment 65. A kit comprising reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is: (i) exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411; or (ii) exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR- 195, and cell-free miR-411.

[0193] Embodiments P1-P10

[0194] Embodiment PI. A method of detecting an RNA biomarker in a subject, wherein the subject has, or is suspected of having, a colorectal cancer, the method comprising determining the presence of one or more biomarkers in a biological sample from the subject, wherein the one or more biomarker are miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, MMP9 mRNA, or a combination of two or more thereof.

[0195] Embodiment P2. The method of Embodiment PI, wherein the colorectal cancer is invasive submucosal colorectal cancer (T1 CRC).

[0196] Embodiment P3. The method of Embodiment PI or P2, wherein the biological sample is a liquid biological sample.

[0197] Embodiment P4. The method of Embodiment P3, wherein the liquid biological sample is blood. [0198] Embodiment P5. The method of any one of Embodiments PI to P4, wherein the determining is detecting the presence of one or more of the biomarkers.

[0199] Embodiment P6. The method of Embodiment P5, further comprising surgically removing a portion of the colon of the subject.

[0200] Embodiment P7. The method of any one of Embodiments PI to P4, wherein the determining is detecting the absence of the biomarkers.

[0201] Embodiment P8. The method of Embodiment P7, wherein the method further comprises monitoring the patient for the presence or progression of the colorectal cancer.

[0202] Embodiment P9. The method of any one of Embodiments P5 to P8, further comprising administering to the subject an effective amount of an anti cancer treatment.

[0203] Embodiment P10. The method of Embodiment P9, wherein the anticancer treatment comprises radiation therapy, chemotherapy, immunotherapy, or a combination of two or more thereof.

[0204] Advantages

[0205] The present methods have significant advantages over other methods in the art, including, for example, robust detection/diagnostic accuracy (AUC = 0.90) by RT-qPCR (normalized to the expression of miR-16 for miRNA and B-actin for mRNA as internal controls), non-invasive, inexpensive and easy diagnostic prior to surgery (i.e., not using tissue samples from cancerous tissues). The methods allow for classification of patients into low or high-risk groups (Youden’s index, for LNM risk) and will prevent unnecessary treatments and/or surgeries by identifying true high-risk patients.

EXAMPLES

[0206] We recently reported use of tissue-based transcriptomic biomarkers (miRNA or mRNA) for identification of lymph node metastasis (LNM) in patients with invasive submucosal colorectal cancers (T1 CRC). In this study, we translated our tissue-based biomarkers into a blood-based liquid biopsy assay for noninvasive detection of LNM in patients with high-risk T1 CRC. The term “liquid biopsy” refers to a blood sample.

[0207] We analyzed 330 specimens from patients with high-risk T1 CRC, which included 188 serum samples from two clinical cohorts (training cohort: n=46, validation cohort: n=142) and matched FFPE samples (n=142). We performed RT-qPCR followed by logistic regression analysis to develop an integrated transcriptomic panel and establish a risk-stratification model, combined with clinical risk factors. [0208] We used comprehensive expression profiling of a training cohort of LNM-positive and -negative serum specimens to identify an optimized transcriptomic panel of four miRNAs (miR-181b, miR-193b, miR-195, miR-411) and five mRNAs (AMT, FOXA1, PIGR, MMP1, MMP9), which robustly identified patients with LNM (area under the curve [AUC]=0.86, 95% 0=0.72-0.94). We validated panel performance in an independent validation cohort (AUC=0.82, 95% 0=0.74-0.88). Our risk-stratification model was more accurate than the panel and an independent predictor for identification of LNM (AUC=0.90, Univariate: odds ratio [OR]=37.17, 95% 0=4.48-308.35, P< 001; Multivariate: OR=17.28, 95% 0=1.82-164.07, P=.013). The model limited potential overtreatment to only 18% of all patients, which is dramatically superior to currently used pathological features (92%).

[0209] Our novel risk-stratification model for noninvasive, liquid biopsy-based identification of T1 CRC in pre-surgical settings will allow for the prevention of unnecessary surgeries for patients classified as high-risk by conventional risk-classification criteria.

[0210] Example 1

[0211] Accumulating evidence indicates that the expression pattern of microRNAs (miRNAs) reflects the physiological and pathological status of cancer patients. In fact, several studies have identified the differential expression of specific miRNAs to be directly involved in CRC pathogenesis, as well as emphasized their potential as circulating biomarkers for CRC. (Refs. 26-30). Although considerable advances have been made in exploiting miRNAs as noninvasive diagnostic biomarkers, using circulating miRNAs to identify high-risk T1 CRCs clinically has thus far not been attempted. (Refs 31-33). We previously described a panel of tissue-based miRNAs and gene expression biomarkers that allowed robust detection of LNM in patients with T1 CRC. (Ref 34, 35). However, an ideal clinical application of these biomarkers would be to use them to diagnose patients with high-risk T1 CRC prior to surgery, before such tissue specimens are readily available. Therefore, translating these biomarkers into a “liquid biopsy” assay is attractive, as this would allow a noninvasive, facile, and inexpensive diagnostic assay for LNM in patients with high-risk T1 CRC. To address this gap in knowledge, we evaluated the feasibility of translating our previously reported transcriptomic biomarkers (miRNAs and mRNAs), into a blood-based, noninvasive assay by systematically analyzing blood specimens from multiple cohorts of patients with T1 CRC. As a result, we successfully established a novel, blood-based, transcriptomic signature that robustly identified the presence of LNM in patients with T1 CRC, with an area under the curve (AUC) value of 0.90. This assay allowed reclassification of 75% of high-risk T1 CRCs into the low-risk group, which would obviate the need for unnecessary surgeries in this significant majority of patients who would have otherwise been subjected to radical surgeries based upon conventional pathological risk-assessment criteria.

[0212] Materials and Methods [0213] Patient cohorts

[0214] We analyzed a total of 330 patient samples, which included 188 serum specimens from patients with high-risk T1 CRCs comprised of 2 independent clinical cohorts: a training cohort (n=46; 5 LNM-positive (LNP) and 41 LNM-negative (LNN) patients) from the National Cancer Center Hospital, Japan; and a validation cohort (n=142; 12 LNP and 130 LNN patients) from the National Cancer Center Hospital East, Japan (FIG. 4). Matched formalin-fixed paraffin- embedded (FFPE; n=142) specimens, which were obtained following endoscopic or surgical tumor resection, were also obtained from patients within the validation cohort. All patients were diagnosed as “high-risk” pathologically. The pathological criteria included depth of submucosal invasion (>1000 pm), presence of lymphatic or vascular invasion, high-grade tumor budding, and poorly differentiated histology. All patients underwent radical surgeries between January 2017 and December 2017 in the training cohort, and between January 2011 and December 2017 in the validation cohort. Exclusion criteria were: synchronous advanced CRCs, presence of distant metastases, hereditary or inflammation-associated CRC, non-adenocarcinoma, or non availability of serum specimens.

[0215] All patients underwent standard endoscopic and surgical procedures (resection of affected segment of colon or rectum and regional lymphadenectomy), and all specimens were evaluated by pathologists at each participating institution, according to the 7th edition of the American Joint Committee on Cancer TNM grading system. The study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients, and the study was approved by the institutional review boards of all participating institutions.

[0216] RNA extraction from serum and FFPE specimens

[0217] Total RNA extraction from all serum specimens was performed using the Qiagen miRNeasy Kit (Qiagen, Hilden, Germany). Briefly, 200 pL of serum was thawed on ice and centrifuged at 3000 xg for 5 min to remove cell debris. Next, 200 pL of the supernatant was lysed in 5 times the volume of Qiazol solution. To normalize any inadvertent sample-to-sample variations during the RNA isolation procedure, 3.5 pL of synthetic Caenorhabditis elegans miRNA (cel-miR-39) was spiked into each denatured sample. Total RNA was subsequently enriched and purified following the manufacturer’s protocol. For FFPE specimens, 10 pm thick sections were manually microdissected to enrich for cancer cells (>75% of tumor cells), and the RNA was extracted using the AllPrep DNA/RNA FFPE Kit (Qiagen). Extracted RNA from serum and FFPE specimens was then processed for the generation of complementary DNA (cDNA) prior to polymerase chain reaction (PCR) assays.

[0218] Real-time quantitative reverse transcription PCR (RT-qPCR) assays

[0219] For miRNA, synthesis of cDNA from total RNA was performed using the TaqMan microRNA Reverse Transcription Kit (ThermoFisher Scientific, Waltham, MA, USA). For mRNA, a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) was used to convert RNA into cDNA. RT-qPCR analysis was performed using the SensiFAST™ probe Lo-ROX Kit (Bioline, London, UK) on the QuantStudio 7 Flex Real Time PCR System (Applied Biosystems, Foster City, CA), and expression levels were evaluated using the corresponding software system. The relative abundance of target transcripts was evaluated and normalized to the expression of miR-16 for miRNA and b-actin for mRNA as internal controls, using the 2 ^Doa method. DCt represents the difference of Ct values between the miRNA of interest and the internal normalizing gene. Normalized expression values were loglO transformed, before downstream statistical analysis. (Ref 36). All primers for miRNAs analyzed in this study were purchased from ThermoFisher Scientific. The catalogue number for all miRNA primers was 4427975, and the assay IDs of individual miRNAs were as follows: Hsa- miR-16: 391, Hsa-miR-32-5p: 2109, Hsa-miR-181b: 1098, Hsa-miR-193b-3p: 2367, Hsa-miR- 195-5p: 494, Hsa-miR-411-5p: 1610. The primer sequences for the target genes used in the present study are shown in Table 4.

[0220] Statistical analysis

[0221] Clinicopathologic characteristics of the patient cohorts are shown as patient number and ratio except for age (median and range) (Table 1). The cutoff thresholds for continuous variables were divided by the median value in the total participants. Several clinicopathologic characteristics were compared between LNP and LNN patients, using the Chi-Square test or Mann-Whitney U test for categorical data. Binary logistic regression was used to train a classifier based on the expression of four miRNAs and five mRNAs. Of note, once the model was trained in the training cohort, the same statistical model variables (weights and cutoff thresholds) were applied in the validation cohort. The LNM risk score for all patients was calculated based on the individual biomarker coefficients derived from the training cohort as follows: Logit (P) = (-0.318*MIR181b) + (-0.762*MIR193b) + (-1.019*MIR195) + (- 0.627 *MIR411) + (-0.135*AMT) + (-0.010*FOXA1) + (0.241 *MMP1) + (-0.776*MMP9) + (0.231*PIGR) - 8.363. The cutoff threshold for the LNM risk score was chosen as 0.08, which was determined by Youden’s index. For all cohorts, receiver operator characteristic curves and AUC values were used to evaluate the performance of the panel for LNM detection in patients with T1 CRC. A P value <0.05 was considered statistically significant. Statistical analyses were performed using JMP Genomics V 9.0 statistical software (SAS Institute Japan, Tokyo, Japan), Medcalc statistical software V.16.2.0 (Medcalc Software bvba, Ostend, Belgium), GraphPad Prism V7.0 (GraphPad Software, San Diego, CA, USA), and R (3.5.0, R Development Core T earn, https : //cran. r-proj ect. org/).

[0222] Table 1 - Clinicopathological characteristics of clinical cohorts

[0223] Results

[0224] Patients within the training and validation cohorts exhibited similar clinicopathological features, including the rate of LNM

[0225] To develop a blood-based, noninvasive assay, we first confirmed that both cohorts of patients with T1 CRC showed similar clinicopathologic characteristics. The training cohort was comprised of 46 patients with T1 CRC, which included 5 patients with LNM (11%); the median age of patients in this cohort was 70 years. The validation was comprised of 142 patients with T1 CRC, which included 12 patients with LNM (8%); the median age of patients in this cohort was 67 years. The detailed clinicopathological characteristics of patients within these cohorts are provided in Table 1. We observed no statistically significant difference in the prevalence of LNM rates, nor any other clinicopathological characteristic, between the two cohorts, which eliminates any inadvertent bias between the patient cohorts examined in our study.

[0226] A noninvasive transcriptomic risk-assessment model identifies LNM in patients with T1 CRC

[0227] By undertaking a systematic and comprehensive biomarker discovery and validation effort, we previously reported tissue-based miRNA and mRNA signatures for the identification of LNM in patients with T1 CRC. (Ref 34, 35). In these studies, we described a panel of five miRNAs (miR-32, miR-181b, miR-193b-3p, miR-195-5p, and miR-411-5p) and eight genes (AMT, FOXA1, MMP1, MMP9, LYZ, C2CD4A, RCC1, and PIGR), which identify LNM in patients with T1 CRC. However, an ideal clinical application of these biomarker signatures would be in a noninvasive, liquid biopsy, diagnostic platform. Such a platform would obviate the need for analysis of tissue specimens, which are generally not available from most patients in pre-operative settings. Therefore, in this study we focused on translating the tissue-based biomarkers into a blood-based assay, which could yield a clinically attractive assay for noninvasive diagnosis of LNM in patients with T1 CRC.

[0228] Accordingly, we evaluated the expression of our transcriptomic biomarkers in two independent cohorts of patients with T1 CRCs. First, we evaluated the feasibility of detecting the miRNA and mRNA markers using RT-qPCR in serum specimens from the training cohort of patients (5 LNP and 41 LNN). One miRNA (miR-32-5p) and 3 mRNAs (LYZ, C2CD4A,

RCC1) were not detectable in serum specimens. In view thereof, we established a panel of four miRNAs (miR-181b, miR-193b-3p, miR-195-5p, and miR-411-5p) and five mRNAs (AMT, FOXA1, MMP1, MMP9, and PIGR) that were detectable in blood. Next, we systematically interrogated the diagnostic accuracy of our transcriptomic panel for its ability to detect LNM in patients with T1 CRC. Using logistic regression analysis, we trained a risk-assessment model in the training cohort of patients that allowed robust identification of LNM in patients with T1 CRC using the four miRNAs (AUC = 0.78, 95% Cl = 0.64-0.89) or the five mRNAs (AUC= 0.77, 95% Cl = 0.62-0.88) (FIGS. 1A-1B and Table 5). Identification of LNM was notably superior when we used all four miRNAs and five mRNAs to establish a combined transcriptomic panel (AUC = 0.86, 95% Cl = 0.72-0.94). [0229] We performed univariate analysis to confirm that each of the biomarker panels was quite robust individually (miRNA panel: odds ratio [OR] = 8.62, P = .06; mRNA panel: OR = 8.44, P = .05; FIG. 1C, Table 6). However, the combined panel was significantly superior in diagnosing the presence of LNM in patients with T1 CRC (OR = 14.22; P = .02). We developed this risk-assessment scoring model based on the coefficients derived from individual markers by using the logistic regression analysis as following model parameters: Logit (P) =

(-0.318 *MIR181b) + (-0.762*MIR193b) + (-1.019*MIR195) + (-0.627 *MIR411) +

(-0.135* AMT) + (-0.010*FOXA1) + (0.241*MMP1) + (-0.776*MMP9) + (0.231*PIGR) - 8.363. Taken together, these data show we successfully established a novel transcriptomic panel for noninvasive identification of LNM in patients with T1 CRC.

[0230] Transcriptomic biomarkers and a risk nomogram identify LNM in patients with T1 CRC in an independent validation cohort

[0231] Following the results of our blood-based transcriptomic panel for the noninvasive detection of LNM, we next confirmed its robustness and accuracy by applying the same statistical coefficients in serum specimens from a large, independent, validation cohort (LNP = 12, LNN =130 cases). To further assess our ability to assay transcriptomic biomarkers shed by the primary cancer into the systemic circulation, we also used matched endoscopically or surgically resected FFPE tissue specimens from patients within this cohort. We first evaluated the diagnostic accuracy of our transcriptomic panel in these FFPE surgical specimens, and observed that its diagnostic performance was comparable to that observed in serum specimens in the training cohort (AUC = 0.83, 95% Cl = 0.75-0.89; FIG. 2A). When we evaluated the performance of the signature in matched blood serum specimens, the diagnostic accuracy was in-line with the findings from tissue specimens (AUC = 0.82, 95% Cl = 0.74-0.88, FIGS. 2B- 2C). This highlights the clinical significance of our transcriptomic panel in identifying presence of LNM in patients with T1 CRC.

[0232] To ease the translation of our panel into the clinic, we evaluated its performance along with other pathological risk features (i.e. lymphatic or vascular invasion, high-grade tumor budding), and established a nomogram. We incorporated all pathological and molecular risk features and determined that although other pathological risk-assessment features added some weight to the model, our panel had the highest weight in this model and was an independent and the most significant predictor for the presence of LNM in patients with T1 CRC (FIG. 2D).

[0233] A risk-stratification model that combines transcriptomic biomarkers and current risk- assessment features significantly improves diagnosis of LNM in patients with T1 CRC [0234] Considering the current landscape of widely used clinical risk factors for identifying patients with T1 CRC, we asked whether a risk-stratification model that includes some of the currently used pathological risk features (i.e., lymphatic and vascular invasion, tumor budding grade, and depth of tumor invasion) along with our transcriptomic biomarkers might further improve diagnostic accuracy in detecting LNM in patients with T1 CRC. As 12 patients were lack of clinical information, totally 130 patients were included in risk-stratification model. When we performed such an analysis in the patients within the serum specimens of validation cohort, this led to a significant improvement in its diagnostic sensitivity and specificity for the identification of LNM (AUC = 0.90, 95% Cl = 0.83-0.95, FIG. 3A and Table 2).

[0235] We next determined specific diagnostic correlates for our combined biomarker panel in blood samples from the validation cohort; its sensitivity, specificity, PPV, and negative predictive value (NPV) were 83.3%, 76.2%, 24.4%, and 98.0%, respectively (Table 2). When we performed a similar analysis of the newly established risk-stratification model that also included pathological risk features, its performance was significantly superior; its sensitivity, specificity, PPV, and NPV were 90.0%, 81.4%, 29.0%, and 99.0%, respectively. This highlights the superiority of the risk-stratification model for identifying LNM in patients with T1 CRC.

[0236] Table 2 - Model performance in estimating the risk of LNM

[0237] In Table 2: AUC, area under the curve; PPV, positive predictive value; NPV, negative predictive value; FFPE, formalin-fixed paraffin-embedded; LNM, lymph node metastasis; Cl, confidence interval.

[0238] We next categorized all patients into high- and low-risk groups using cutoff thresholds derived from Youden’s index for the nine miRNA and mRNA biomarkers. Accordingly, we performed univariate logistic regression analysis, which revealed that our transcriptomic panel emerged as an independent predictor for LNM in patients with T1 CRC in both clinical cohorts, compared to any singular clinical risk factor (training cohort: OR = 14.22, 95% Cl = 1.41- 143.68, P = .025; validation cohort: OR = 15.97, 95% Cl = 3.32-76.82, P < .001; Table 3A). Further, univariate and multivariate logistic regression analysis revealed that our novel risk- stratification model was a superior than the panel and independent predictor of LNM (Univariate: OR = 37.17, 95% Cl = 4.48-308.35, P < .001; Multivariate: OR = 17.28, 95% Cl = 1.82-164.07, P = .013) in the validation cohort of patients (FIGS. 3B-3C and Table 3B). Collectively, these data highlight the potential clinical significance of our risk-stratification model for diagnosis and risk assessment in the identification of LNM.

[0239] Table 3A - Training Cohort - Univariate Logistic Regression Analysis for LNM

[0240] Table 3B - Validation Cohort - Univariate Logistic Regression Analysis for LNM

[0241] Table 3C - Validation Cohort - Multivariate Logistic Regression Analysis for LNM

[0242] In Tables 3A-3C: OR, odds ratio; Cl, confidence interval; LNM, lymph node metastasis. MSI refers to microsatellite instability; MSI-H refers to high-frequency microsatellite instability; MSI-L refers to low-frequency microsatellite instability; MSS refers to microsatellite stable.

[0243] Our noninvasive risk-assessment model is significantly superior to currently used pathological risk factors for identifying patients with high-risk T1 CRC and reducing the burden of unnecessary surgical treatments

[0244] The ultimate goal of our study was to determine the clinical usefulness of our transcriptomic panel in noninvasively identifying patients who truly have LNM and sparing the rest from unnecessary surgeries. In this study, we only enrolled patients who were deemed high- risk based upon the currently used pathological risk factors. However, only 8% of “high-risk” patients (12 of 142) were actually high risk, indicating that 92% of patients (130 of 142) were erroneously categorized as high risk and underwent unnecessary radical surgeries (FIG. 3D, left panel). In contrast, when we analyzed the same patients using our transcriptomic classifier and divided into high and low risk by Youden’s index, it stratified 29% of patients into the high-risk category (41 of 142). Among these, 10 patients had LNM (7%), indicating that only 22% of the entire cohort (31 of 142) received overtreatment, which is notably superior to potential overtreatment compared with the currently used pathological features (92% vs 22%; FIG. 3D, middle panel). Our newly established risk model was even more accurate than the panel, as it stratified only 25% of patients into the high-risk group (32 of the 130), and the remaining 75% (98 of the 130) of patients were deemed as low risk. Of the 32 patients who were classified as high risk, 9 patients had LNM (7%), indicating that only 18% (23 of 130) of all patients with T1 CRC were potentially overtreated, which is dramatically superior compared with currently used pathological features (92% vs 18%; FIG. 3D, right panel). This highlights the urgency and need to use our liquid biopsy-based risk-assessment model in patients with high-risk T1 CRC.

[0245] Discussion

[0246] The presence of LNM is an important risk factor for additional surgery following curative endoscopic treatment in patients with T1 CRC. Our present study overcomes the inadequacy of clinicopathologic risk features that are currently used in the clinic to identify LNM in “high-risk” subsets of patients with T1 CRC. Our data demonstrate that a blood-based, transcriptomic assay accurately estimates risk in pre-operative settings, has robust risk- stratification for the identification of LNM, and will lead to a dramatic reduction in the number of unnecessary surgeries that are currently being performed in these patients. Identifying true high-risk patients and saving others from such unnecessary treatment will reduce patient complications, physician burdens, and associated healthcare costs. (Refs. 37-39)

[0247] In this study, our newly established noninvasive risk model exhibited a significantly superior diagnostic accuracy for LNM (AUC= 0.90) vs. the currently used clinical risk models (AUC= 0.73 [training] and 0.76 [validation]) (FIGS. 5A-5B). Although all patients enrolled in our study were deemed to be high-risk for LNM and received radical surgery, post-surgical pathological analyses identified that only 9% (17 of 188 (46 in training cohort and 142 in validation cohort)) of patients were LNP and 91% of patients underwent unnecessary surgeries. Our newly established diagnostic signature revealed that only 18% were overtreated, which is dramatically better for identification of LNM.

[0248] Several reports have indicated the potential of ESD for diagnosing LNM in patients with T1 CRC; however, others suggest its diagnostic accuracy for LNM is still inadequate.

(Refs. 40-42). Furthermore, because current clinical guidelines consider the presence of LNM an important risk factor for classifying a patient with T1 CRC as high risk, this highlights the need to develop robust biomarkers for LNM prior to treatment, which would be clinically transformative in selecting patients who truly require such invasive and radical surgical treatments. Our ability to successfully validate our signature in pre-treatment serum samples underscores its clinical significance for improved treatment strategies in patients with T1 CRC, especially the ones who truly have LNM. Our previous studies similarly highlighted the clinical use of pre-treatment serum samples for diagnostic purposes in patients with CRC; however, none of the previous studies used these samples directly for diagnosing LNM status, which could have a profound impact in the selection of treatment strategies. (Refs 31-33, 43). Pre operative application of our transcriptomic biomarkers as a robust, facile, and inexpensive assay will lead to minimized risks from surgical procedures, including perforation or bleeding, and a reduction in overall healthcare burden from such expensive surgical procedures.

[0249] In conclusion, we have identified and developed a novel risk-stratification model that allows identification of LNM in a liquid biopsy assay for more robust and accurate identification of patients with high-risk T1 CRC. Our findings highlight the clinical impact our model will have for improved selection of patients with high-risk T1 CRC, which will reduce the overall burden of unnecessary surgeries and expense associated with these procedures, and improve the overall management of patients with this malignancy.

[0250] Table 4 - Primer Sequences (-F = Forward; -R = Reverse)

[0251] Table 5 - Model performance in estimating the risk of LNM in the training cohort using each panel

[0252] In Table 5: AUC = area under the curve; PPV = positive predictive value; NPV = negative predictive value; LNM = lymph node metastasis; Cl = confidence interval.

[0253] Table 6 - Univariate logistic regression analysis for LNM

[0254] In Table 6: OR = odds ratio; LNM = lymph node metastasis; Cl = confidence interval.

[0255] Example 2

[0256] Background & Aims: Patients with risk factors of lymph node metastases (LNM) invasive submucosal colorectal cancers (T1 CRC) are recommended radical operation. However, the current pathologic criteria are inadequate, and operation is often considered as overtreatment. In Example 1, we describe a blood-based transcriptomic panel which can robustly identify LNM among patients with T1 CRC. To further improve our panel, we evaluated the exosomal and cell-free miRNA (cf-miRNA) signature.

[0257] Methods: We analyzed a total of 200 patients with high risk T1 CRC from two independent cohorts: a training cohort (n=58) and a validation cohort (n=142). We extracted exosomal and cell-free RNA from pre-operative blood samples and performed quantitative reverse-transcription polymerase chain reaction for the four miRNAs (miR-181b, miR-193b, miR-195, and miR-411). Next, we analyzed the predictive accuracy of the combined exosomal and cell-free miRNA signature for its ability to detect LNM in preoperative settings in T1 CRC patients.

[0258] Results: In a training cohort, all four exosomal miRNAs showed slightly higher accuracy of LNM detection than cell-free miRNAs. And combination panel robustly identified patients with LNM (AUC, 0.905; 95% Cl, 0.803-1.000). We validated our panel’s performance in a validation cohort (AUC, 0.844; 95% Cl 0.700-0.979). Moreover, when we develop risk- stratification model to combine pathological features, the accuracy of LNM detection was more improved (AUC, 0.933; 95% Cl, 0.876-0.989), and 76% unnecessary operations can be reduced.

[0259] Conclusions: Herein, we report a novel exosomal miRNA-based liquid biopsy signature that robustly identifies T1 CRC patients at risk of LNM in pre-operative settings.

[0260] Materials and Methods

[0261] Patient cohorts

[0262] This retrospective cohort study included totally 200 high risk T1 CRC patients, who were pathologically diagnosed as high-risk of LNM and underwent radical surgery, from 2 independent institutes: a training cohort of 58 patients with 7 LNM-positive (LNP) and 51 LNM-negative (LNN) patients from Tokyo Medical and Dental University Hospital, Japan, and a validation cohort of 142 patients with 12 LNP and 130 LNN patients from the National Cancer Center Hospital East, Japan. Radical surgeries were performed during the period between January 2012 and November 2014 in the training cohort, and January 2011 and December 2017 in the validation cohort. Preoperative blood samples were obtained and used for analysis.

[0263] Pathological high-risk LNM patients were diagnosed according to Japanese Society for Cancer of the Colon and Rectum guidelines 2019 for the treatment of colorectal cancer; pTlb (depth of submucosal invasion > 1000 pm), lympho vascular invasion positive, histology (poorly differentiated adenocarcinoma, signet-ring carcinoma, or mucinous carcinoma), and high grade of tumor budding at the site of deepest invasion. (Ref 8). Radical surgery was performed by standard procedure as intestinal resection with lymph node dissection. All surgical specimens were evaluated by pathologists at each institute and pathologically diagnosed the presence of LNM. We excluded patients with synchronous advanced CRC, distant metastasis, non adenocarcinoma, and hereditary or inflammation related CRC.

[0264] Exosome isolation from serum and plasma

[0265] Total exosome isolation was performed by using Total Exosome Isolation Kit (from serum) for serum samples or Total Exosome Isolation Kit (from plasma) for plasma samples according to manufacturer’s recommendation protocol (ThermoFisher Scientific, Waltham, MA,

USA). Briefly, for serum specimens, 200 pL of serum was thawed on ice and centrifuged at

2000 xg for 30 min at room temperature to remove cells and debris. After that we added 40 pL of the Total Exosome Isolation (from serum) regent and incubated it for 30 min at 4 degree.

After incubation, samples were centrifuged 10000 xg for 10 min at room temperature and discard the supernatant. Exosomes were contained in the pellet. We added 200 pL of phosphate- buffered saline (PBS) and resuspended the pellet.

[0266] For plasma specimens, 200 pL of plasma was thawed on ice and centrifuged at 10000 xg for 20 min twice at room temperature to remove cells and debris. After that we added 60 pL of the Total Exosome Isolation (from plasma) regent and 100 pL of (PBS). After 10 min incubation at room temperature, samples were centrifuged 10000 xg for 5 min at room temperature and discard the supernatant. Exosomes were contained in the pellet. We added 200 pL of phosphate-buffered saline (PBS) and resuspended the pellet. Isolated exosomes are preserved at 4 degree until RNA extraction.

[0267] RNA extraction from exosome, serum, and plasma

[0268] The procedures of RNA extraction were those reported previously in Example 1. The procedures are the same for exosome, serum, and plasma. Total RNA extraction was performed using the Qiagen miRNeasy Kit (Qiagen, Hilden, Germany). Briefly, 200 pL of samples was lysed in 1000 pL of Qiazol solution. To normalize any inadvertent sample-to-sample variations during the RNA isolation procedure, 3.5 pL of synthetic Caenorhabditis elegans miRNA (cel- miR-39) was spiked into each denatured sample. After that, by using QIAcube System, automated machinery for purification of DNA, RNA, or proteins (Qiagen, Hilden, Germany), total RNA was subsequently enriched and purified. Extracted RNA was then converted to complementary DNA (cDNA) prior to polymerase chain reaction (PCR) assays. And conversion of cDNA was performed by using the TaqMan microRNA Reverse Transcription Kit (ThermoFisher Scientific, Waltham, MA, USA).

[0269] Real-time quantitative reverse transcription PCR (RT-qPCR) assays

[0270] The procedures of RT-qPCR were also those reported in Example 1. The following probes were used for TaqMan miRNA assays (Thermo Fisher Scientific, Inc., Waltham, MA, USA): Has-miR-181b (ID, 001098), Has-miR-193b-3p (ID, 002367). Has-miR-195-5p (ID, 000494), Has-miR-411-5p (ID, 001610), and Has-miR-16 (ID, 000391). Real-time reverse transcription quantitative PCR analysis was performed by using the QuantStudio 7 Flex Real Time PCR System (Applied Biosystems, Foster City, CA) and the expression of the target miRNA was normalized to that of miR-16.

[0271] Statistical analysis

[0272] All statistical analyses were performed by using JMP 8.0.1 (SAS Institute Inc., Cary,

NC) or EZR 1.54 (Saitama Medical Center, Jichi Medical University, Saitama, Japan). In all tests, P values less than 0.05 were considered significant. [0273] Receiver operator characteristics curves were used to evaluate the accuracy for LNM detection in each miRNAs and panels. And cut-off values were determined by Youden’s index. Binary logistic regression model was used to train a classifier based on the expression of exosomal miRNA and cell-free miRNA (cf-miRNA). Once the model was made in the training cohort, the same statistical model was applied in the validation cohort and the performance evaluation cohort. Decision curve analysis for net benefit (detection of LNM) and net benefit untreated (avoidance of unnecessary surgery) were performed by following formulas. (Ref 30).

[0274] net benefit= (true positive; TP)/n-(false positive; FP)/n ^c (Pt/l-Pt)

[0275] net benefit untreated= (true negative; TN)/n-(false negative; FN)/n ^c (1-Pt/Pt)

[0276] And decision curve plotting was made between 0-15% risk threshold probability.

[0277] Results

[0278] Clinicopathological factors in two clinical cohort

[0279] Table 7 shows the clinicopathological factors in the two independent clinical cohorts. In training cohort, 7 out of 58 T1 CRC patients (12%) were LNP patients and, 12 out of 142 (8%) T1 CRC patients in validation cohort were LNP patients. The two clinical cohorts can be considered almost clinicopathologically equivalent.

[0280] Table 7

[0281] Exosomal and cell-free miRNA panels for identification of lymph node metastasis in patients with T1 colorectal cancer in a training cohort

[0282] We analyzed diagnostic accuracy of each miRNAs for detecting LNM in T1 CRC patients. FIGS. 6A-6H show the ROC curves and AUC values of each miRNAs in both exosomal and cf-miRNA. Exosomal miRNAs tended to have slightly higher AUC values than cf-miRNAs in all 4 miRNAs. Combination panel of 4 miRNAs in both exosomal and cf-miRNA showed high accurate ability of LNM detection (FIGS. 61-6 J). The exosomal miRNA panel (AUC, 0.860; 95% Cl, 0.701-1.000) also tended to have higher AUC values than the cf-miRNA panel (AUC, 0.824; 95% Cl 0.664-0.983). From these results, exosomal miRNA is a better cancer biomarker than cf-miRNA.

[0283] The accuracy of LNM detection was higher when a combined panel of exosomal miRNA and cf-miRNA was used (FIG. 6K, AUC, 0.905; 95% Cl, 0.803-1.000). FIG. 6L showed waterfall plot of modified risk score in combination panel. By using a combination panel, all LNP patients in the training cohort were identified as LNM high risk. Taken together, these results show that exosomal miRNAs are superior cancer biomarkers to cf-miRNAs. In addition, exosomal miRNAs and cf-miRNAs might be different biomarker that provide an excellent combination panel.

[0284] We developed a risk-prediction model by using the logistic regression analysis in this combination panel; Logit (P) = (0.468 ^c exosomal miR-181b) + (0.293 x exosomal miR-193b) + (9.573 x exosomal miR-195) + (1.310 ^c exosomal miR-411) + (-0.233 ^c cf-miR-181b) + (- 1.239 x cf-miR-193b) + (15.043 x cf-miR-195b) + ( 1.005 cf-mir-411) + 45.908.

[0285] Exosomal and cell-free miRNA combination panel and risk-stratification model for identification of lymph node metastasis in patients with T1 colorectal cancer in an independent validation cohort

[0286] We applied the above formula to independent validation cohort of 142 CRC patients (FIG. 7A). Our combination panel robustly identified LNP patients (AUC, 0.844; 95% Cl 0.700-0.979). Moreover, we developed risk-stratification model by combine pathological features to our exosome and cell-free combination panel. FIG. 7B showed ROC curves and AUC values of risk-stratification model, combination panel, and pathological features. Combination panel had higher AUC value than all pathological features. Moreover, when we developed a risk-stratification model, the accuracy of LNM detection was improved (AUC, 0.933; 95% Cl, 0.876-0.989).

[0287] We performed univariate and multivariate analyses to confirm which biomarkers had better ability to detect LNM (Tables 8A-8B, FIGS. 7C-7D). In the univariate analysis, high tumor budding grade (OR, 3.889; 95% Cl, 10.85-13.945) and lymphatic invasion (OR, 3.778; 95% Cl, 1.079-13.231) were significant predictive factors of LNM. Combination panel (OR, 18.667; 95% Cl, 4.611-75.563) and risk-stratification model (OR, 56.111; 95% Cl 6.763- 465.559) were also significant predictors of LNM and their ORs were higher than pathological factors. Moreover, in multivariate analysis, only risk-stratification model (OR, 21.133; 95% Cl, 1.990-224.425) became an independent predictive factor of LNM (Tables 8A-8B).

[0288] Table 8A - Univariate analysis for lymph node metastasis detection (Validation Cohort)

[0289] Table 8B - Multivariate analysis for lymph node metastasis detection (Validation Cohort)

[0290] In Tables 8A-8B, data are shown as n (%) unless indicated otherwise. MSI, microsatellite instability; MSI-H, high-frequency microsatellite instability; MSI-L, low- frequency microsatellite instability; MSS, microsatellite stable.

[0291] Performance of Exosomal and cell-free miRNA combination panel and risk- stratification model

[0292] FIG. 8A shows radar chart plotting of combination panel, risk-stratification model, and pathological factors for accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Combination panel and risk-stratification model tended to have higher performance in parameters other than specificity and NPV. There were almost no differences in these two factors among combination panel, risk-stratification model and pathological factors. Combination panel showed comparable performance with risk-stratification model. However, sensitivity of combination panel (0.750; 95% Cl, 0.428-0.945) was lower than that of risk-stratification model (0.909; 95% Cl, 0.587-0.998) (Table 9). In the context of LNM prediction, low sensitivity means missing LNP patients. Among pathological factors, tumor size had higher sensitivity of LNM (0.917; 95% Cl, 0.615-0.998). Thus, tumor size seemed to complement lower sensitivity of combination panel in risk-stratification model. PPVs were almost two times higher in combination panel (0.333; 95% Cl, 0.165-0.540) and risk- stratification model (0.357; 95% Cl, 0.186-0.559) than budding grade (0.192; 95% Cl, 0.066- 0.394), which had highest PPV among pathological factors.

[0293] Table 9 - Performance of current guidelines, Exosome + cell-free panel and Risk- stratification model (NPV, negative predictive value; PPV, positive predictive value).

[0294] FIG. 8B shows decision curve plotting for LNM detection in T1 CRC patients. Yellow line is operation for all patients, which corresponded current guidelines’ strategy. Curve for both combination panel and risk-stratification model showed higher net benefit than for all between 0 to 15% threshold probability. Risk-stratification model also showed higher net benefit than combination panel between same range of threshold probability. Thus, both combination panel and risk-stratification model can be superior strategies than current guidelines’ strategy. And risk-stratification model can be better than combination panel. FIG. 8C shows decision curve plotting for avoidance of unnecessary operation. Between 0 to 15% threshold probability, risk- stratification model showed highest net benefit especially around small threshold probability. Taken together them, our developed risk-stratification model can be superior in both LNM detection and avoidance of unnecessary operation.

[0295] According to current guidelines, 92% patients in validation cohort underwent unnecessary operation (FIG. 9A, Table 9). In contrast, when we apply our developed risk- stratification model to same cohorts, we can reduce totally 76% operation with less than 1% of LNP patients missing from the diagnosis (FIG. 9B). Moreover, the percentages of LMP among high risk patients were 4 times higher in risk-stratification model than in current guidelines. Therefore, our newly developed risk-stratification model can divide T1 CRC patients, who have pathological high-risk factors of LMN, into real LMN high-risk and low-risk.

[0296] Discussion

[0297] In the present study, we developed an exosomal miRNA and cf-miRNA combination panel and risk-stratification model for pre-operative prediction of LNM among patients with T1 CRC. We also revealed exosomal miRNAs might be different biomarker from cf-miRNAs and become superior cancer biomarkers to cf-miRNAs. Moreover, their combination panel can be promising biomarker.

[0298] In an training cohort, all four miRNAs in exosome had better accuracy of LNM prediction than cf-miRNAs. Exosomal miRNA panel (ACU, 0.860; 95% Cl 0.701-1.000) also had higher AUC value than cf-miRNA panel (AUC, 0.824; 95% Cl 0.664-0.983). Moreover, a combined panel of exosomal and cf-miRNA further improved the diagnostic accuracy for LNM detection (AUC, 0.905; 95% Cl 0.803-1.000) than exosomal miRNA panel or cf-miRNA panel. Our candidate four miRNAs, miR-181b, miR-193b, miR-195, and miR-411, were identified from cancer tissue-based study. (Ref 27). Therefore, the origin of exosomal miRNAs seemed to be more cancer specific than that of cf-miRNAs. And then, exosomal miRNAs might become a superior source of cancer biomarker to whole serum or plasma. Endzelins et al. reported that expression levels of exosomal miRNAs and cf-miRNAs showed poor correlation. (Ref 26). Chevillet et al. also reported only a small minority of miRNAs in plasma was contained in exosome. (Ref 33). In our present study, both exosomal miRNA panel and cf-miRNA panel had good ability to detect LNM, and their combination panel robustly improve the ability than either alone. miRNA and cf-miRNA may be different biomarkers, and they can complement each other.

[0299] In the present study, the same clinical cohort as in Example 1 was used for validation to compare the performance of our newly developed panel. In the same validation cohorts, exosome and cf-miRNA panel (AUC, 0.844; specificity, 0.862; PPV, 0.333) showed higher AUC, specificity, and PPV than the transcriptomic (4 miRNAs and 5 mRNAs) panel in Example 1 (AUC, 0.815; specificity, 0.762; PPV, 0.244). Sensitivity was an only weak aspect of our combination panel inferior to transcriptomic panel (0.750 vs 0.833). However, low sensitivity can be complemented by pathological factors in the risk-stratification model. Thus, our newly developed combination panel can omit mRNA evaluation.

[0300] Our risk-stratification model included tumor size, lymphatic invasion, vascular invasion, and tumor budding grade from pathological factors. These factors have been reported as important predictor of LNM. (Refs 11, 12, 34). On the other hand, we excluded the depth of submucosal invasion. The depth of submucosal invasion (>1000 pm) is considered as one of the high-risk factors of LNM in current guidelines. (Ref 8). However, many recent studies suggested the depth of submucosal invasion may not be a decisive factor of LMN prediction. (Refs 11, 35, 36). In line with these studies, we excluded the depth of submucosal invasion from our risk- stratification model. Our risk-stratification model showed robust LNM detection ability (AUC, 0.933; sensitivity, 0.909; specificity 0.824; PPV, 0.323). According to current guidelines, in a validation cohort, only 8% patients gained the benefit of operation. And remaining 92% patients underwent unnecessary operation and gained no clinical benefit but the risks of surgery. By using our risk-stratification model, 76% patients can avoid unnecessary operations with less than 1% missing of LNP patients. In addition, overtreatment frequency can be reduced from 92% to 16%. This percentage was reduced by 2% compared to Example 1 (16% vs 18%). This risk- stratification model robustly divides T1 CRC patients into real LMN high-risk and low-risk.

[0301] While various embodiments and aspects of the disclosure are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein might be employed in practicing the disclosure.

[0302] 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 including, without limitation, patents, patent applications, articles, books, manuals, and treatises are incorporated by reference in their entirety for any purpose. The abbreviations used herein have their conventional meaning within the chemical and biological arts. [0303] References for Example 1

[0304] 1. Stamos et al, Clin Cancer Res 2007;13:6885s-9s. 2. Iida et al, World J Surg

2012;36:424-30. 3. Tanaka et al, Dig Endosc 2015;27:417-434. 4. Shimura et al, Clin Gastroenterol Hepatol 2014;12:662-8 el-2. 5. Vleugels et al, Gut 2020;69:977-980. 6. Muto et al, Dis Colon Rectum 2003;46:S89-93. 7. Tanaka et al, Oncol Rep 2000;7:783-8. 8. Nascimbeni et al, Dis Colon Rectum 2002;45:200-6. 9. Sohn et al, J Clin Pathol 2007;60:912- 5. 10. Suh et al, Endoscopy 2012;44:590-5. 11. Watanabe et al, Int J Clin Oncol 2015;20:207- 39. 12. Ueno et al, Gastroenterology 2004;127:385-94. 13. Choi et al, Dis Colon Rectum 2009;52:438-45. 14. Tateishi et al, Mod Pathol 2010;23:1068-72. 15. Ikematsu et al, Gastroenterology 2013;144:551-9; quiz el4. 16. Yoda et al, Endoscopy 2013;45:718-24. 17. Nakadoi et al, J Gastroenterol Hepatol 2012;27:1057-62. 18. Pimentel-Nunes et al, Endoscopy 2015;47:829-54. 19. Ha et al, Ann Surg Treat Res 2017;93:266-271. 20. Yamamoto et al, Hepatogastroenterology 2004;51:998-1000. 21. Son et al, Hepatogastroenterology 2008;55:1293-7. 22. Okabe et al, J Gastrointest Surg 2004;8:1032-9; discussion 1039-40. 23. Tamaru et al, J Gastroenterol 2017;52: 1169-1179. 24. Ricciardi et al, Clin Gastroenterol Hepatol 2006;4: 1522-7. 25. Brunner et al, Surg Endosc 2016;30:4405-15. 26. Nozawa et al, Surgery 2016;159:713-20. 27. Chen et al, Cell Res 2008;18:997-1006. 28. Huang et al, Int J Cancer 2010;127:118-26. 29. Kanaan et al. Plasma miR-21: a potential diagnostic marker of colorectal cancer. Ann Surg 2012;256:544-51. 30. Ng et al. Differential expression of microRNAs in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening. Gut 2009;58: 1375-81. 31. Toiyama et al. Serum miR-21 as a diagnostic and prognostic biomarker in colorectal cancer. J Natl Cancer Inst 2013;105:849-59. 32. Hur et al. Circulating microRNA-203 predicts prognosis and metastasis in human colorectal cancer. Gut 2017;66:654-665. 33. Toiyama et al. Serum miR-200c is a novel prognostic and metastasis- predictive biomarker in patients with colorectal cancer. Ann Surg 2014;259:735-43. 34. Ozawa et al. A MicroRNA Signature Associated With Metastasis of T1 Colorectal Cancers to Lymph Nodes. Gastroenterology 2018;154:844-848. e7. 35. Kandimalla et al. Gene Expression Signature in Surgical Tissues and Endoscopic Biopsies Identifies High-Risk T1 Colorectal Cancers. Gastroenterology 2019;156:2338-2341. e3. 36. Livak et al, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-8. 37. Jepsen et al. Intra-tumor heterogeneity of microRNA-92a, microRNA-375 and microRNA-424 in colorectal cancer. Exp Mol Pathol 2016;100:125-31. 38. Studdert et al, Jama 2005;293:2609-17. 39. Earner et al, Can J Surg 2018;61:19-27. 40. Kim et al, Medicine (Baltimore) 2016;95:e4373. 41. Overwater et al, Gut 2018;67:284-290. 42. Barel F, Cariou M, Saliou P, et al. Histopathological factors help to predict lymph node metastases more efficiently than extra-nodal recurrences in submucosa invading pTl colorectal cancer. Sci Rep 2019;9:8342. 43. Imaoka et al. Circulating microRNA-1290 as a novel diagnostic and prognostic biomarker in human colorectal cancer. Ann Oncol 2016;27: 1879-86.

[0305] References for Example 2

[0306] 1. Bretthauer et al, JAMA Intern Med 2016;176:894-902. 2. Moss et al, Gut 2017;66:1631-1644. 3. Kim et al, Dig Dis Sci 2015;60:2785-92. 4. Ikematsu et al, Gastroenterology 2013;144:551-9; uiz el4. 5. van de Ven et al, Gastrointest Endosc 2020;91:142-152.e3. 6. Jayanna et al, Clin Gastroenterol Hepatol 2016;14:271-8. el-2. 7. Haller et al, J Clin Oncol 2011;29: 1465-71. 8. Hashiguchi et al, Int J Clin Oncol 2020;25: 1-42. 9. Pimentel-Nunes et al, Endoscopy 2015;47:829-54. 10. Benson et al, J Natl Compr Cane Netw 2017;15:370-398. 11. Ronnow et al, Ann Surg 2020. 12. Kudo et al, Gastroenterology 2021;160:1075-1084. e2. 13. Heitzer E, Haque IS, Roberts CES, et al. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat Rev Genet 2019;20:71-88.

14. Alix-Panabieres C. The future of liquid biopsy. Nature 2020;579:S9. 15. Lucchetti D, Fattorossi A, Sgambato A. Extracellular Vesicles in Oncology: Progress and Pitfalls in the Methods of Isolation and Analysis. Biotechnol J 2019;14:el700716. 16. Hu W, Liu C, Bi ZY, et al. Comprehensive landscape of extracellular vesicle-derived RNAs in cancer initiation, progression, metastasis and cancer immunology. Mol Cancer 2020;19: 102. 17. Sequeira JP, Constancio V, Lobo J, et al. Unveiling the World of Circulating and Exosomal microRNAs in Renal Cell Carcinoma. Cancers (Basel) 2021;13. 18. Becker A, Thakur BK, Weiss JM, et al. Extracellular Vesicles in Cancer: Cell-to-Cell Mediators of Metastasis. Cancer Cell 2016;30:836-848. 19. Melo et al, Nature 2015;523:177-82. 20. Richards et al, Oncogene 2017;36:1770-1778. 21. Sun LH, Tian D, Yang ZC, et al. Exosomal miR-21 promotes proliferation, invasion and therapy resistance of colon adenocarcinoma cells through its target PDCD4. Sci Rep 2020;10:8271. 22. Tokuda A, Miyake T, Yasukawa D, et al. Cancer-derived Exosomes Activate Immune Surveillance and Suppress Peritoneal Metastasis of Murine Colonic Cancer. Anticancer Res 2021;41:1327-1339. 23. Lobb et al, Proteomics 2017;17. 24. Shiao MS, Chang JM, Lertkhachonsuk AA, et al. Circulating Exosomal miRNAs as Biomarkers in Epithelial Ovarian Cancer. Biomedicines 2021 ;9. 25. Nik Mohamed Kamal N, Shahidan WNS. Non-Exosomal and Exosomal Circulatory MicroRNAs: Which Are More Valid as Biomarkers? Front Pharmacol 2019; 10: 1500. 26. Endzelins E, Berger A, Melne V, et al. Detection of circulating miRNAs: comparative analysis of extracellular vesicle-incorporated miRNAs and cell-free miRNAs in whole plasma of prostate cancer patients. BMC Cancer 2017;17:730. 27. Ozawa T, Kandimalla R, Gao F, et al. A MicroRNA Signature Associated With Metastasis of T1 Colorectal Cancers to Lymph Nodes. Gastroenterology 2018;154:844-848. e7. 28. Kandimalla R, Ozawa T, Gao F, et al. Gene Expression Signature in Surgical Tissues and Endoscopic Biopsies Identifies High-Risk T1 Colorectal Cancers. Gastroenterology 2019;156:2338-2341. e3.

29. Wada Y, Shimada M, Murano T, et al. A Liquid Biopsy Assay for Noninvasive Identification of LymphNode Metastases in Tl Colorectal Cancer. Gastroenterology 2021;161:151-162. el.

30. Zhang et al, Ann Transl Med 2018;6:308. 31. Liu W, Hu J, Zhou K, et al. Serum exosomal miR-125b is a novel prognostic marker for hepatocellular carcinoma. Onco Targets Ther 2017;10:3843-3851. 32. Eichelser C, Stiickrath I, Miiller V, et al. Increased serum levels of circulating exosomal microRNA-373 in receptor-negative breast cancer patients. Oncotarget 2014;5:9650-63. 33. Chevillet et al, Proc Natl Acad Sci U S A2014;lll:14888-93. 34. Lee et al, Hum Pathol 2018;78:8-17. 35. Pai et al, Mod Pathol 2017;30:113-122. 36. Suh JH, Han KS, Kim BC, et al. Predictors for lymph node metastasis in T1 colorectal cancer. Endoscopy 2012;44:590-5. 37. Ueno H, Mochizuki H, Hashiguchi Y, et al. Risk factors for an adverse outcome in early invasive colorectal carcinoma. Gastroenterology 2004;127:385-94. 38. Miyo M, Kato T, Nakamura Y, et al. DENEB: Development of new criteria for curability after local excision of pathological T1 colorectal cancer using liquid biopsy. Cancer Sci 2021.

Claims

CLAIMS What is claimed is:

1. A method of treating colorectal cancer in a patient in need thereof, the method comprising administering to the patient an effective amount of an anti-cancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof; wherein a blood sample obtained from the patient comprises an elevated expression level, relative to a control, of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

2. A method of treating colorectal cancer in a patient in need thereof, the method comprising:

(i) detecting an elevated expression level, relative to a control, of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient; and

(ii) administering to the patient an effective amount of an anti-cancer agent, surgically removing all or a portion of the colon of the patient, or a combination thereof.

3. The method of claim 1, comprising detecting an elevated expression level, relative to the control, of miR-181b, miR-193b, miR-195, and miR-411.

4. The method of claim 1, comprising detecting an elevated expression level, relative to the control, of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

5. The method of claim 1, comprising detecting an elevated expression level, relative to the control, of AMT mRNA, FOXAl mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

6. The method of claim 1, wherein the RNA is exosomal RNA.

7. The method of claim 1, comprising detecting an elevated expression level, relative to the control, of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411.

8 A method of identifying an increased risk of developing lymph node metastasis in a patient with colorectal cancer or detecting a lymph node metastasis in a patient with colorectal cancer, the method comprising detecting an elevated expression level, relative to a control, of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR- 411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient; wherein the elevated expression level of the RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, indicates an increased risk of developing lymph node metastasis or the presence of a lymph node metastasis.

9. The method of claim 8, wherein the method comprises identifying an increased risk of developing lymph node metastasis in a patient with colorectal cancer.

10. The method of claim 8, wherein the method comprises detecting a lymph node metastasis in a patient with colorectal cancer.

11. The method of any one of claims 8 to 10, comprising detecting an elevated expression level, relative to the control, of miR-181b, miR-193b, miR-195, and miR-411.

12. The method of any one of claims 8 to 10, comprising detecting an elevated expression level, relative to the control, of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

13. The method of any one of claims 8 to 10, comprising detecting an elevated expression level, relative to the control, of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

14. The method of any one of claims 8 to 13, wherein the RNA is exosomal RNA.

15. The method of any one of claims 8 to 10, comprising detecting an elevated expression level, relative to the control, of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal and miR-411.

16. The method of any one of claims 8 to 10, comprising detecting an elevated expression level, relative to the control, of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal and miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free and miR-411.

17. A method of diagnosing a patient having colorectal cancer as high risk for lymph node metastasis, the method comprising:

(i) detecting the expression level of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient; and

(ii) diagnosing the patient as having a high risk for lymph node metastasis when the blood sample has an elevated expression level, relative to a control, of the RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

18. The method of claim 17, comprising: (i) detecting the expression level of miR- 181b, miR-193b, miR-195, and miR-411 in the blood sample, and (ii) diagnosing the patient as having a high risk for lymph node metastasis when the blood sample has an elevated expression level of miR-181b, miR-193b, miR-195, and miR-411, relative to the control.

19. The method of claim 17, comprising: (i) detecting the expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in the blood sample, and (ii) diagnosing the patient as having a high risk for lymph node metastasis when the blood sample has an elevated expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, relative to the control.

20. The method of claim 17, comprising: (i) detecting the expression level of miR- 181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in the blood sample, and (ii) diagnosing the patient as having a high risk for lymph node metastasis when the blood sample has an elevated expression level of miR- 181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA, relative to the control.

21. The method of any one of claims 17 to 20, wherein the RNA is exosomal RNA.

22. The method of claim 17, comprising: (i) detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 in the blood sample, and (ii) diagnosing the patient as having a high risk for lymph node metastasis when the blood sample has an elevated expression level of exosomal miR-181b, exosomal miR- 193b, exosomal miR-195, and exosomal miR-411, relative to the control.

23. The method of claim 17, comprising: (i) detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411, in the blood sample, and (ii) diagnosing the patient as having a high risk for lymph node metastasis when the blood sample has an elevated expression level of e exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR- 195, and cell-free miR-411, relative to the control.

24. A method of monitoring a patient having colorectal cancer for an increased risk of lymph node metastasis, the method comprising:

(i) detecting the expression level of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient at a first time point; and

(ii) detecting the expression level of an RNA selected from the group consisting of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA in a blood sample obtained from the patient at a second time point later than the first time point; and

(iii) diagnosing the patient:

(a) as having an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the RNA selected from the group consisting of miR-181b, miR-193b, miR- 195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level of the RNA at the first time point; or

(b) as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of the RNA selected from the group consisting of miR-181b, miR- 193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level of the RNA at the first time point.

25. The method of claim 24, comprising detecting the expression level of miR-181b, miR-193b, miR-195, and miR-411 at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of the miR-181b, miR-193b, miR-195, and miR-411 compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of miR-181b, miR-193b, miR-195, and miR-411 compared to the expression level at the first time point.

26. The method of claim 24, comprising detecting the expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level at the first time point.

27. The method of claim 24, comprising detecting the expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA compared to the expression level at the first time point.

28. The method of any one of claims 24 to 27, wherein the RNA is exosomal RNA.

29. The method of claim 24, comprising detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411 compared to the expression level at the first time point.

30. The method of claim 24, comprising detecting the expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 at the first time point and at the second time point, and diagnosing the patient as having in an increased risk for lymph node metastasis when the blood sample at the second time point has an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 compared to the expression level at the first time point; or as not having an increased risk for lymph node metastasis when the blood sample at the second time point does not have an elevated expression level of exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR-411, cell- free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411 compared to the expression level at the first time point.

31. The method of any one of claims 24 to 30, wherein the expression level of the RNA in the blood sample obtained from the patient at the first time point is not elevated, and wherein the expression level of the RNA in the blood sample obtained from the patient at the second time point is elevated, thereby diagnosing an increased risk of lymph node metastasis in the patient having colorectal cancer.

32. The method of any one of claims 24 to 30, wherein the expression level of the RNA in the blood sample obtained from the patient at the first time point is not elevated, and wherein the expression level of the RNA in the blood sample obtained from the patient at the second time point is not elevated, thereby diagnosing no increased risk of lymph node metastasis in the patient having colorectal cancer.

33. The method of any one of claims 1 to 32, wherein the blood sample is a serum sample.

34. The method of any one of claims 1 to 32, wherein the blood sample is a plasma sample.

35. The method of any one of claims 1 to 34, wherein the colorectal cancer is invasive submucosal colorectal cancer.

36. The method of any one of claims 1 to 34, wherein the colorectal cancer is colorectal cancer with lymph node metastasis.

37. The method of any one of claims 1 to 34, wherein the colorectal cancer is invasive submucosal colorectal cancer with lymph node metastasis.

38. The method of any one of claims 8-37, further comprising administering to the patient an effective amount of an anti-cancer agent.

39. The method of any one of claims 1-7, comprising administering to the patient the effective amount of the anti -cancer agent.

40. The method of any one of claims 1-7, comprising administering to the patient the effective amount of the anti-cancer agent and surgically removing all or a portion of the colon of the patient.

41. The method of any one of claims 1-7 and 38-40, wherein the anti-cancer agent is a chemotherapeutic agent.

42. The method of claim 41, wherein the chemotherapeutic agent comprises 5- fluorouracil, leucovorin, oxaliplatin, irinotecan, capecitabine, or a combination of two or more thereof.

43. The method of claim 41, 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.

44. The method of claim 43, wherein the alkylating agent is carboplatin, chlorambucil, cyclophosphamide, melphalan, mechlorethamine, procarbazine, or thiotepa; the antimetabolite compound is azacitidine, capecitabine, cytarabine, gemcitabine, doxifluridine, hydroxyurea, methotrexate, pemetrexed, 6-thioguanine, 5-fluorouracil, or 6-mercaptopurine; the anthracycline compound is daunorubicin, doxorubicin, idarubicin, epirubicin, or mitoxantrone; the antitumor antibiotic is actinomycin, bleomycin, mitomycin, or valrubicin; the platinum compound is cisplatin or oxaliplatin; the topoisomerase inhibitor is irinotecan, topotecan, amscarine, etoposide, teniposide, or eribulin; the vinca alkaloid is vincristine, vinblastine, vinorelbine, or vindesine; the taxane compound is paclitaxel or docetaxel; and the epothiolone compound is epithilone, ixabepilone, patupilone, or sagopilone.

45. The method of any one of claims 1 to 7, comprising surgically removing all or a portion of the colon of the patient.

46. The method of any one of claims 8 to 44, further comprising surgically removing all or a portion of the colon of the patient.

47. The method of any one of claims 1 to 46, wherein the method does not comprise detecting the expression level of miR-32, LYZ mRNA, C2CD4A mRNA, and RCC1 mRNA.

48. A kit comprising reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is selected from the group consisting of:

(i) miR-181b, miR-193b, miR-195, and miR-411;

(ii) miR-181b, miR-193b, miR-195, miR-411, AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA; and (iii) AMT mRNA, FOXA1 mRNA, PIGR mRNA, MMP1 mRNA, and MMP9 mRNA.

49. The kit of claim 48, wherein the RNA is selected from the group consisting of miR-181b, miR-193b, miR-195, and miR-411.

50. A kit comprising reagents capable of detecting an expression level of RNA from a blood sample; wherein the RNA is:

(i) exosomal miR-181b, exosomal miR-193b, exosomal miR-195, and exosomal miR-411; or

(ii) exosomal miR-181b, exosomal miR-193b, exosomal miR-195, exosomal miR- 411, cell-free miR-181b, cell-free miR-193b, cell-free miR-195, and cell-free miR-411.