WO2022235701A1 - Gene expression profiling methods and compositions for determining cancer and treatment thereof - Google Patents

Abstract

The present application provides gene expression profiling methods and compositions for determining cancer recurrence following hepatectomy in patients with colorectal liver metastasis (CRLM) tumor.

Description

GENE EXPRESSION PROFILING METHODS AND COMPOSITIONS FOR DETERMINING CANCER AND TREATMENT THEREOF

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/183,627, filed May 3, 2021, the content of which is incorporated herein by reference in its entirety and for all purposes.

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

[0002] The Sequence Listing written in file 048440-806001WO_SL_ST25.TXT, created May 3, 2022, 3,076 bytes in size, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference.

BACKGROUND

[0003] Cancer recurrence is an important predictor of survival outcomes in patients with CRLM who undergo radical hepatectomy. Therefore, identification of patients with the greatest risk of recurrence is critical for developing a precision oncology strategy that might include frequent surveillance (in low-risk patients) or a more aggressive treatment approach (in high-risk patients).

[0004] Colorectal cancer (CRC) is the second most common cause of cancer-related deaths worldwide ². Management of patients with distant metastases is a major challenge: 20-30% of patients with CRC have distant metastatic disease at the time of diagnosis, and 5 -year overall survival (OS) in these patients with metastatic CRC is less than 10% ^{3 5}. Approximately 50% of patients with CRC develop liver metastases after treatment of the primary tumor, and liver metastases is the most frequent cause of mortality in these patients ^{6 10}. Hepatectomy is the current treatment standard for colorectal liver metastases (CRLM) and can offer lead to prolonged survival, with a 5-year OS of 30-50% ^{n 14}. However, in spite of advances in surgical techniques, imaging modalities, and post-operative management, 50-70% of patients still experience cancer recurrence following hepatectomy, and most such recurrences occur within first 2 years of surgery ⁸- ^{15 18}. Consequently, due to these frequent recurrence events, the post operative treatment options available to these patients are often complicated. The National Comprehensive Cancer Network guidelines recommend several treatment options including post-surgical surveillance with or without adjuvant chemotherapy in patients with resectable CRLM. Unfortunately, there are no well-defined randomized studies available that compared recurrence in patients subjected to surgery alone or together with adjuvant chemotherapy; underscoring the need for precise biomarkers that can enable a more personalized treatment strategy.

[0005] Several clinical trials have demonstrated that the chemotherapy improves survival outcomes after hepatectomy in patients with CRLM. For example, perioperative FOLFOX (fluorouracil, leucovorin, and oxaliplatin) chemotherapy led to a 7% improvement in progression-free survival (PFS) when followed after hepatectomy for CRLM ¹⁹· ²⁰. Likewise, other studies evaluating the addition of targeted therapies such as anti-EGFR antibodies in metastatic CRC also noticed improved outcomes ²¹·²². On the same lines, inclusion of anti- VEGF antibodies to a modified FOLFOX or FOLFIXIRI regimen (fluorouracil, leucovorin, oxaliplatin, and irinotecan) associated with improved response and resection rates and prolonged PFS in patients with CRLM ^{23 25}. These findings indicate that patients with CRLM who have a higher risk of recurrence benefit from such interventions and should be offered such intensive treatments following hepatectomy. Regarding post-surgical surveillance alone, serological carcinoembryonic antigen (CEA) level measurements, and the use of computed tomography (CT) imaging of the chest, abdomen, and pelvis every 3-6 months for the first 2 years and thereafter every 6 months for 3 more years are recommended. However, these criteria are often inadequate in high-risk patients, given that some patients require a more aggressive surveillance approach and potentially chemotherapy considering that more than half of patients experience tumor recurrence within 2 years of their surgical treatment.

[0006] In recent years, several clinicopathological prognostic variables have been identified in patients with CRLM that predict the risk of cancer recurrence following hepatectomy ^{26 30}. However, instead of or in addition to clinical factors, unravelling the molecular properties that characterize tumors may be pivotal for tailoring individualized therapies based on molecular predictors of survival outcomes. To this end, various groups have reported molecular subtypes in primary cancers with distinct biological properties and corresponding predictive and prognostic values ¹⁸· ^{26 34}. There is a growing consensus that molecular markers may improve patient selection for chemotherapy and other treatments in addition to hepatectomy or intensive surveillance schemes. The ability to analyze tumors at the RNA level promises to revolutionize our understanding of the malignant disease process, and hopefully this will herald novel biomarkers. [0007] The present disclosure addresses these and other problems in the art and includes, for example, methods to identify biomarkers, metastatic risks, and appropriate treatment plans for subjects having or suspected of having an increased risk of cancer recurrence.

BRIEF SUMMARY

[0008] Provided herein are, for example, methods of treating colorectal liver metastases (CRLM) in a subject in need thereof, by surgically removing all or a portion of the liver of thee subject; detecting an elevated expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3 in a biological sample obtained from the subject, and administering to the subject radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof.

[0009] Provided herein are also methods of identifying an increased risk of developing colorectal liver metastasis in a subject with colorectal cancer or detecting a colorectal liver metastasis in a subject with colorectal cancer by detecting an elevated expression level, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3 in a biological sample obtained from the subject; wherein the elevated expression level of the one or more genes indicates an increased risk for the subject of developing colorectal liver metastasis or the presence of a colorectal liver metastasis in the subject.

[0010] In aspects, provided herein are methods of treating a distant metastasis tumor in a subject in need thereof. The disclosed methods comprise surgically removing all or a portion of the subject’s distant metastasis tumor; detecting an elevated expression level in a sample obtained from the subject, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and a combination thereof; and administering to the subject radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof.

[0011] In additional aspects, provided herein are methods of identifying an increased risk of developing a colorectal liver metastasis tumor in a subject with colorectal cancer and methods of detecting a colorectal liver metastasis tumor in a subject with colorectal cancer. These methods comprise detecting an elevated expression level, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and a combination thereof in a sample obtained from the subject. The elevated expression level of one or more of these genes or of a combination thereof indicates an increased risk of developing a colorectal liver metastasis tumor or the presence of a colorectal liver metastasis tumor.

[0012] In other aspects, provided herein are methods of diagnosing a subject having colorectal cancer as being at an increased risk for developing colorectal liver metastasis. The disclosed methods comprise detecting the expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3, in a sample obtained from the subject. The subject has an increased risk for colorectal liver metastasis tumor when an elevated expression level, relative to a control, of one or more of these genes is detected in the sample.

[0013] In further aspects, provided herein are methods of monitoring a subject having colorectal cancer for an increased risk of colorectal liver metastasis tumor. The disclosed methods comprise detecting the expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3 in a sample obtained from the subject at a first time point, and detecting the expression level of one or more of the same genes in a sample obtained from the subject at a second time point later than the first time point. An elevated expression level of one or more of these genes detected in the sample at the second time point compared to the expression level of the same genes at the first time point is indicative of an increased risk for colorectal liver metastasis tumor.

[0014] In additional aspects, provided herein are methods of detecting gene expression in a subject that has or is suspected of having cancer or metastasis tumors. The disclosed methods comprise measuring the level of expression of one or more genes selected from COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof in a sample obtained from the subject in comparison to a control.

[0015] In aspects, provided herein is a kit comprising reagents capable of detecting an expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, and TMTC3 in a sample obtained from a subject. These and other embodiments of the disclosure are provided in more detail herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGS. 1A-1D show training and validation of a transcriptomic panel for identifying cancer recurrence in patients with CRLM. (1A) ROC curve for the transcriptomic panel in tissue specimens from a training cohort of 169 patients (with recurrence = 119, non-recurrence = 50, AUC = 0.83). (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. (1C) ROC curve for the transcriptomic panel in tissue specimens from a validation cohort of 151 patients (with recurrence = 102, non-recurrence = 49, AUC = 0.81). (ID) Risk score distribution plot in validation cohort patients.

[0017] FIGS. 2A-2D show prognostic potential of the transcriptomic panel in patients with CRLM. (2A-2B) A comparison of (2A) RFS and (2B) OS between high- and low-risk groups estimated by the panel in the training cohort. (2C-2D) A comparison of (2C) RFS and (2D) OS between high- and low-risk groups estimated by the panel in the validation cohort.

[0018] FIGS. 3A-3E show clinical validation of the risk-stratification model in patients with CRLM. (3A) ROC curves for the risk-stratification model, which combines the transcriptomic panel and clinical risk factors, vs. the transcriptomic panel or indicated risk factors alone in tissue specimens from validation cohort patients. (3B-3C) Forest plot showing HRs of indicated clinicopathological variables, the transcriptomic panel, and the risk-stratification model in univariate (3B) and multivariate (3C) Cox’s proportional hazard regression analysis in validation cohort patients. (3D) Comparison of RFS between high- (Red) and low- (Blue) risk groups estimated by the risk-stratification model in the validation cohort. (3E) Bar graphs shows 61.9% of low-risk patients with CRLM who would have experienced non-recurrence, and 88.6% of high-risk patients with CRLM who would have experienced recurrence.

[0019] FIG. 4 shows an overview of the study described herein.

[0020] FIGS. 5A-5B show the genome-wide discovery of a novel gene panel to predict recurrence in patients with CRLM. (5A) ROC curve for the diagnostic performance of the 6- gene panel for distinguishing patients with cancer recurrence in GSE81423 (AUC = 0.90). (5B) Risk score distribution plot in GSE81423. Modified risk scores were obtained from individual risk scores by using Youden’s index values from the risk model.

[0021] FIGS. 6A-6B show the prediction of recurrence in training and validation cohorts without the transcriptomic panel. (6A) ROC curve for combined clinical risk factors for recurrence (CA19-9, CEA, synchronous, and tumor number) without the transcriptomic panel in the training cohort (AUC = 0.68). (6B) ROC curve for the combined clinical risk factors for recurrence without the transcriptomic panel in the validation cohort (AUC = 0.68).

[0022] FIG. 7 shows clinicopathological characteristics of clinical cohorts. [0023] FIG. 8 shows the primer sequences for B-actin, COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3.

[0024] FIG. 9 shows univariate and multivariate Cox proportional hazard regression analysis for recurrence free survival.

[0025] FIG. 10 shows model performance in estimating the risk of recurrence.

DETAILED DESCRIPTION

[0026] The present disclosure may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a part of this disclosure. It is to be understood that this invention is not limited to the specific devices, methods, applications, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention. Also, as used in the specification including the appended claims, the singular forms “a,” “an,” and “the” include the plural, and reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.

Definitions

[0027] The abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0028] As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer. Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.

[0029] A “gene,” or a “sequence which encodes” a particular protein, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the gene are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A gene can include, but is not limited to, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the gene sequence. Typically, polyadenylation signal is provided to terminate transcription of genes inserted into a recombinant virus.

[0030] As used herein, the term “expression” is used in accordance with its plain ordinary meaning and refers to any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0031] “Nucleic acid” refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. 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 nucleic acids 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 acids contemplated herein include any types of RNA (e.g., antisense RNA, mRNA, siRNA, miRNA, shRNA, guide RNA, dicer substrate RNA, dicer substrate siRNAs (dsiRNAs) (dsiRNA are cleaved by the RNase III class endoribonuclease dicer into 21-23 base duplexes having 2-base 3 ’-overhangs siRNA), and any type of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term “duplex” in the context of nucleic acids 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.

[0032] The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, include, without limitation, phosphodiester derivatives including, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate (also known as phosphorothioate having double bonded sulfur replacing oxygen in the phosphate), phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press) as well as modifications to the nucleotide bases such as 2’0-methyl, 2 O-methoxy ethoxy, 2’fluoro, 5-methyl cytidine or pseudouridine; and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, modified sugars (e.g., deoxyribose), and non-ribose backbones (e.g. phosphorodiamidate morpholino oligos or locked nucleic acids (LNA) as known in the art), including those described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In aspects, the intemucleotide linkages in DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

[0033] The terms “activation,” “activate,” “activating” and the like are used in accordance with its plain ordinary meaning and refer to an interaction that positively affects (e.g. increasing) the activity or function of a protein or cell relative to the activity or function of the protein or cell in the absence of the activator. In embodiments, activation means positively affecting (e.g. increasing) the concentration or levels of the protein relative to the concentration or level of the protein in the absence of the activator. The terms may reference activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein decreased in a disease. Thus, activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up- regulating signal transduction or enzymatic activity or the amount of a protein associated with a disease (e.g., a protein that is decreased in a disease relative to a non-diseased control). Activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein

[0034] The terms “inhibition,” “inhibit,” “inhibiting” and the like are used in accordance with its plain ordinary meaning and refer to an interaction with in inhibitor that negatively affects (e.g. decreases) the activity or function of the protein or cell relative to the activity or function of the protein or cell in the absence of the inhibitor. In embodiments, inhibition means negatively affecting (e.g. decreasing) the concentration or levels of the protein relative to the concentration or level of the protein in the absence of the inhibitor. In embodiments, inhibition refers to reduction of a disease or symptoms of disease. In embodiments, inhibition refers to a reduction in the activity of a particular protein target. Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. In embodiments, inhibition refers to a reduction of activity of a target protein resulting from a direct interaction (e.g. an inhibitor binds to the target protein). In embodiments, inhibition refers to a reduction of activity of a target protein or cell from an indirect interaction (e.g. an inhibitor binds to a protein that activates the target protein, thereby preventing target protein activation or cell activations).

[0035] As used herein, the terms “inhibitor,” “repressor” or “antagonist” or “downregulator” are used in accordance with its plain ordinary meaning and refer to a substance capable of detectably decreasing the expression or activity of a given gene or protein. The antagonist can decrease expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the antagonist. In instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or lower than the expression or activity in the absence of the antagonist.

[0036] A “primer” refers to a short, single-stranded DNA sequence used in the polymerase chain reaction (PCR) technique. In the PCR method, a pair of primers is used to hybridize with the sample DNA and define the region of the DNA that will be amplified. Primers are also referred to as oligonucleotides. [0037] 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., NCBI web site www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then said 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.

[0038] The term “promoter” refers to a nucleic acid sequence that regulates, either directly or indirectly, the transcription of a corresponding nucleic acid coding sequence to which it is operably linked. The promoter may function alone to regulate transcription, or, in some cases, may act in concert with one or more other regulatory sequences such as an enhancer or silencer to regulate transcription of the transgene. The promoter comprises a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene, which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence.

[0039] As described herein, the complementarity of sequences may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing. Thus, two sequences that are complementary to each other, may have a specified percentage of 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).

[0040] “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.

[0041] 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.

[0042] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may In aspects 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. A “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0043] 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., hydroxy proline, 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. [0044] 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.

[0045] 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)).

[0046] The terms “treating” or “treatment” refers to any indicia of success in the therapy or amelioration of a disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient’s physical well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination. The term “treating” and conjugations thereof, may include prevention of a pathology, condition, or disease. In aspects, treating is preventing.

In aspects, treating does not include preventing.

[0047] The term “COX6A1” and the like refers, in the usual and customary sense, to the cytochrome c oxidase subunit 6A1, Cytochrome c oxidase 6A1 is a subunit of the cytochrome c oxidase complex, also known as Complex IV, which is the last enzyme in the mitochondrial electron transport chain.

[0048] The term “ERNl” and the like refers, in the usual and customary sense, to the serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1 a (IRE la). Without being bound to a theory, ERNl has been shown to interact with Heat shock protein 90ka alpha (cystosolic), member Al. [0049] The term “IFITM2” and the like refers, in the usual and customary sense, to interferon- induced transmembrane protein 2.

[0050] The term “SI OOP” and the like refers, in the usual and customary sense, to SI 00 calcium-binding protein P. Without being bound to a theory, SI OOP has been shown to interact with EZR and RAGE.

[0051] The term “STK24” and the like refers, in the usual and customary sense, to serine/threonine-protein kinase 24.

[0052] The term “TMTC3” and the like refers, in the usual and customary sense, to Transmembrane O-Mannosyltransferase Targeting Cadherins 3.

[0053] The terms "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 a selected gene or selected group of genes, the level or concentration of which is associated with a particular biological state, particularly a state associated with colorectal cancer and colorectal cancer liver metastasis. 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 markers described herein (e.g., COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3). In embodiments, no additional biomarkers are analyzed and/or detected in the methods described herein, other that the markers provided herein (e.g., COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3).

[0054] The term “antibody” is used in the broadest sense and includes fully assembled antibodies, tetrameric antibodies, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments that can bind an antigen (e.g., Fab', F'(ab)2, Fv, single chain antibodies, diabodies), and recombinant peptides comprising the forgoing as long as they exhibit the desired biological activity. An “immunoglobulin” or “tetrameric antibody” is a tetrameric glycoprotein that consists of two heavy chains and two light chains, each comprising a variable region and a constant region. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antibody fragments or antigen-binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibody (dAb), complementarity determining region (CDR) fragments, CDR-grafted antibodies, single-chain antibodies (scFv), single chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibody, linear antibody; chelating recombinant antibody, a tribody or bibody, an intrabody, a nanobody, a small modular immunopharmaceutical (SMIP), an antigen-binding-domain immunoglobulin fusion protein, a camelized antibody, a VHH containing antibody, or a variant or a derivative thereof, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide, such as one, two, three, four, five or six CDR sequences, as long as the antibody retains the desired biological activity.

[0055] “Monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0056] “Antibody variant” as used herein refers to an antibody polypeptide sequence that contains at least one amino acid substitution, deletion, or insertion in the variable region of the reference antibody variable region domains. Variants may be substantially homologous or substantially identical to the unmodified antibody.

[0057] As used herein, the term “cancer” is used in accordance with its plain ordinary meaning and refers to all types of cancer, neoplasm or malignant tumors found in mammals (e.g. humans), including leukemias, lymphomas, carcinomas and sarcomas. Exemplary types of cancer include melanoma, ovarian cancer, colon cancer, brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, lung cancer, cancer of the head, Hodgkin's disease, and non-Hodgkin's lymphoma, thyroid carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, skin cutaneous melanoma, colon adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer. In embodiments, the cancer is glioblastoma, ovarian cancer, pancreatic cancer, myeloma, leukemia, or lymphoma. In embodiments, the cancer is melanoma, ovarian cancer, or colon cancer. In embodiments, the cancer is metastatic cancer. In embodiments, the cancer is metastatic melanoma, metastatic ovarian cancer, or metastatic colon cancer.

[0058] The term “distant metastasis” or “distant metastasis tumor” refers to a cancer that has spread from the original (primary) tumor to distant organs or distant lymph nodes.

[0059] The term “radiation therapy” or “radiotherapy” refers to a therapy that uses ionizing radiation, and is generally provided as part of cancer treatment to control or kill malignant cells. Radiation therapy is normally delivered by a linear accelerator. Radiation therapy may be curative in a number of types of cancer if they are localized to one area of the body. It may also be used as part of adjuvant therapy, to prevent tumor recurrence after surgery that removes a primary malignant tumor. Radiation therapy is synergistic with chemotherapy, and has been used before, during, and after chemotherapy in susceptible cancers.

[0060] The term “chemotherapy” refers to a type of cancer treatment that uses one or more anti-cancer drugs (chemotherapeutic agents) as part of a standardized chemotherapy regimen. Chemotherapy may be given with a curative intent (which almost always involves combinations of drugs), or it may aim to prolong life or to reduce symptoms (palliative chemotherapy). Chemotherapy drugs include, but are not limited to, alkylating agents, nitrosoureas, antimetabolites, alkaloids, antitumor antibiotics, hormonal agents and biological response modifiers, Traditional chemotherapeutic agents are cytotoxic by means of interfering with cell division (mitosis) but cancer cells vary widely in their susceptibility to these agents. Many of the side effects of chemotherapy can be traced to damage to normal cells that divide rapidly and are thus sensitive to anti-mitotic drugs such as, but not limited to, cells in the bone marrow, digestive tract and hair follicles. In embodiments, the chemotherapy includes administration of an effective amount of an anticancer agent as set forth herein.

[0061] The term “targeted therapy” refers to a method for treating cancer that blocks the growth of cancer cells by interfering with specific targeted molecules needed for carcinogenesis and tumor growth, rather than by simply interfering with all rapidly dividing cells. Exemplary forms of targeted therapy include, but are not limited to, antibody-drug conjugates, nano- engineered enzymes that bind to a tumor cell, and chemical entities that target or preferentially target a protein or enzyme that carries a mutation or other genetic alteration that is specific to cancer cells and is not found in normal host tissue. In embodiments, the targeted therapy includes administration of an effective amount of an anticancer agent as set forth herein. [0062] The term “immunotherapy” refers to methods of treating cancer that are based on the stimulation of the patient’s immune system. Cancer immunotherapy exploits the fact that cancer cells often have tumor antigens that can be detected and bound by the antibodies of the immune system. Clinical success of cancer immunotherapy is highly variable between different forms of cancer. Examples of immunotherapy include, but are not limited to, therapeutic cancer vaccines, CAR-T cell, and targeted antibody therapies. In embodiments, the immunotherapy includes administration of an effective amount of an anticancer agent as set forth herein.

[0063] The term “hormonal therapy” refers to a type of a cancer treatment that slows or stops the growth of cancer that uses hormones to grow. Hormonal therapy may be used alone as the main treatment or with other treatments. It may be used before surgery or radiation therapy to shrink the tumor. Hormonal therapy may be given in addition to main treatments such as surgery, radiation therapy or chemotherapy to lower the risk of cancer recurrence. Hormonal therapy includes, but is not limited to, removing the gland or organ that makes the hormone, irradiating the gland or organ to destroy hormone-producing cells, and administration of drugs that suppress hormonal production. In embodiments, the hormonal therapy includes administration of an effective amount of an anticancer agent as set forth herein.

[0064] The term “angiogenesis inhibitor administration therapy” refers to methods of treating cancer that block the growth of blood vessels that support tumor growth rather than blocking the growth of tumor cells themselves. Exemplary angiogenesis inhibitors include, but are not limited to, monoclonal antibodies that specifically recognize and bind to vascular endothelial growth factor (VEGF) and thus block activation of the VEGF receptor, and immunomodulatory drugs that stimulate or suppress the immune system. For some cancers, angiogenesis inhibitors are most effective when combined with additional therapies. The term “synthetic lethality therapy” refers to a type of cancer treatment in which the simultaneous mutation of two genes leads to cell death, whereas mutation of only one of the genes is not lethal. For most cancer mutations caused by a loss-of-function, there are no targeted therapies available, and synthetic lethal therapy provides additional opportunities. It requires identification of inactive genes in a cancer and the targeting of their synthetic lethal partner genes. Examples of targeted therapies exploiting the synthetic lethality (SL) principle include, but are not limited to, the use of poly- ADP ribose polymerase (PARP) inhibitors in breast and ovarian cancer that harbor mutations in the breast cancer gene (BRCA). Treatment of BRC A-deficient tumors with PARP inhibitors generally selectively kills the cancer cells in breast and ovarian cancer. In embodiments, the angiogenesis inhibitor administration therapy includes administration of an effective amount of an anti cancer agent as set forth herein.

[0065] “Anti-cancer agent” and “anticancer agent” are used in accordance with their 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 some embodiments, an anti-cancer agent is an agent identified herein having utility in methods of treating cancer. In some 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., hexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, semustine, streptozocin), triazenes (decarbazine)), anti-metabolites (e.g., 5- azathioprine, leucovorin, capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid analog (e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil, floxouridine, Cytarabine), 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 (VP16), 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, Syk inhibitors, mTOR inhibitors, antibodies (e.g., rituxan), gossyphol, genasense, polyphenol E, Chlorofusin, all trans-retinoic acid (ATRA), bryostatin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), 5-aza-2'-deoxycytidine, all trans retinoic acid, doxorubicin, vincristine, etoposide, gemcitabine, imatinib (Gleevec.RTM.), geldanamycin, 17-N-Allylamino-17- Demethoxygeldanamycin (17-AAG), flavopiridol, LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, PD184352, 20-epi-l, 25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; anti estrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5 -azacyti dine; 9-dioxamycin; diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflomithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 -based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliphne; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum- triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists; raltitrexed; ramosetron; ras famesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone Bl; ruboxyl; safmgol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen-binding protein; sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfmosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfm; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfm; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatin stimalamer, Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin, acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefmgol; chlorambucil; cirolemycin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; iimofosine; interleukin II (including recombinant interleukin II, or rlL.sub.2), interferon alfa-2a; interferon alfa-2b; interferon alfa- nl; interferon alfa-n3; interferon beta-la; interferon gamma-lb; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie; nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safmgol; safmgol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfm; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfm; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride, agents that arrest cells in the G2-M phases and/or modulate the formation or stability of microtubules, (e.g. Taxol.TM (i.e. paclitaxel), Taxotere.TM, compounds comprising the taxane skeleton, Erbulozole (i.e. R-55104), Dolastatin 10 (i.e. DLS-10 and NSC-376128), Mivobulin isethionate (i.e. as CI-980), Vincristine, NSC-639829, Discodermolide (i.e. as NVP- XX-A-296), ABT-751 (Abbott, i.e. E-7010), Altorhyrtins (e.g. Altorhyrtin A and Altorhyrtin C), Spongistatins (e.g. Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4, Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, and Spongistatin 9), Cemadotin hydrochloride (i.e. LU-103793 and NSC-D-669356), Epothilones (e.g. Epothilone A, Epothilone B, Epothilone C (i.e. desoxyepothilone A or dEpoA), Epothilone D (i.e. KOS-862, dEpoB, and desoxyepothilone B), Epothilone E, Epothilone F, Epothilone B N-oxide, Epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B (i.e. BMS-310705), 21 -hydroxy epothilone D (i.e. Desoxyepothilone F and dEpoF), 26-fluoroepothilone, Auristatin PE (i.e. NSC-654663), Soblidotin (i.e. TZT-1027), LS-4559-P (Pharmacia, i.e. LS-4577), LS-4578 (Pharmacia, i.e. LS- 477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis), Vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, i.e. WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungarian Academy of Sciences), BSF-223651 (BASF, i.e. ILX-651 and LU-223651), SAH-49960 (Lilly/Novartis), SDZ-268970 (Lilly/Novartis), AM-97 (Armad/Kyowa Hakko), AM-132 (Armad), AM-138 (Armad/Kyowa Hakko), IDN-5005 (Indena), Cryptophycin 52 (i.e. LY-355703), AC-7739 (Ajinomoto, i.e. AVE-8063A and CS- 39.HC1), AC-7700 (Ajinomoto, i.e. AVE-8062, AVE-8062A, CS-39-L-Ser.HCl, and RPR- 258062A), Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (i.e. NSC-106969), T-138067 (Tularik, i.e. T-67, TL-138067 and TI-138067), COBRA-1 (Parker Hughes Institute, i.e. DDE- 261 and WHI-261), H10 (Kansas State University), HI 6 (Kansas State University), Oncocidin A1 (i.e. BTO-956 and DIME), DDE-313 (Parker Hughes Institute), Fijianolide B, Laulimalide, SPA-2 (Parker Hughes Institute), SPA-1 (Parker Hughes Institute, i.e. SPIKET-P), 3-IAABU (Cytoskeleton/Mt. Sinai School of Medicine, i.e. MF-569), Narcosine (also known as NSC- 5366), Nascapine, D-24851 (Asta Medica), A-105972 (Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton/Mt. Sinai School of Medicine, i.e. MF-191), TMPN (Arizona State University), Vanadocene acetylacetonate, T-138026 (Tularik), Monsatrol, lnanocine (i.e. NSC-698666), 3- IAABE (Cytoskeleton/Mt. Sinai School of Medicine), A-204197 (Abbott), T-607 (Tuiarik, i.e. T-900607), RPR-115781 (Aventis), Eleutherobins (such as Desmethyleleutherobin, Desaetyleleutherobin, lsoeleutherobin A, and Z-Eleutherobin), Caribaeoside, Caribaeolin, Halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica), Diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), Taccalonolide A, TUB-245 (Aventis), A-259754 (Abbott), Diozostatin, (-)-Phenylahistin (i.e. NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica), Myoseverin B, D-43411 (Zentaris, i.e. D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286 (i.e. SPA-110, trifluoroacetate salt) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCI), Resverastatin phosphate sodium, BPR-OY-007 (National Health Research Institutes), and SSR-250411 (Sanofi)), 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.), immunotherapy (e.g., cellular immunotherapy, antibody therapy, cytokine therapy, combination immunotherapy, etc.), radioimmunotherapy (e.g., anti-CD20 monoclonal antibody conjugated to lllln, 90Y, or 1311, etc.), immune checkpoint inhibitors (e.g., CTLA4 blockade, PD-1 inhibitors, PD-L1 inhibitors, 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.

[0066] “Remission” means that the clinical signs and symptoms of cancer have been significantly diminished or have disappeared entirely based on clinical diagnostics, although cancerous cells can still exist in the body. Thus, it is contemplated that remission encompasses partial and complete remission. Remission can occur for any period of time, such as from one month to several years or more.

[0067] “Relapse” or “Recurrence” refers to the clinical diagnosis of a return of cancer after a period of remission.

[0068] “Relapse-free survival” or “Recurrence-free survival” or “RFS” refers to the time from the date of diagnosis of cancer to the date of relapse.

[0069] “Good prognosis” refers to a normal risk of relapse, a reduced risk of relapse, an increased chance for remission, an increased relapse-free survival time, or a high survival rate.

In embodiments, a “good prognosis” refers to a reduced risk of relapse, an increased chance for remission, an increased relapse-free survival time, or a high survival rate. In embodiments, a “good prognosis” refers to a reduced risk of relapse. In embodiments, a “good prognosis” refers to an increased chance for remission. In embodiments, a “good prognosis” refers to an increased relapse-free survival time. In embodiments, a “good prognosis” refers to a high survival rate. In embodiments, a high survival rate refers to a 5-year survival rate greater than 50%. In embodiments, a high survival rate refers to a 5-year survival rate greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In embodiments, “good prognosis” is an increased likelihood of a good prognosis.

[0070] “Biological sample" refers to a material of biological origin (e.g., blood, plasma, cells, tissues, organs, fluids). In embodiments, biological sample is blood. In embodiments, the biological sample is a tumor. In embodiments, the biological sample is tumor tissue. In embodiments, the biological sample is tumor cells.

[0071] “Peripheral blood” refers to blood circulating throughout the body. The components of peripheral blood include red blood cells (erythrocytes), white blood cells (leukocytes), and platelets.

[0072] “Peripheral blood mononuclear cell” or “PBMC” refers to cells in peripheral blood that have a nucleus, generally a round nucleus. Exemplary peripheral blood mononuclear cells include lymphocytes and monocytes. Exemplary lymphocytes are T cells, B cells, and NK cells.

[0073] “Gene expression” refers to the conversion of genetic information from genes via messenger RNA (mRNA) to proteins. The genetic information (base sequence) on DNA is copied to a molecule of mRNA (transcription). The mRNA molecules then leave the cell nucleus and enter the cytoplasm, where they participate in protein synthesis by specifying the particular amino acids that make up individual proteins (translation).

[0074] The terms an “elevated level” or an “increased level” or a “high level” of gene expression is an expression level of the gene or protein that is higher than the expression level of the gene or protein in a standard control or in a control with noor very low risk of recurrence (e.g. a control biological sample derived from a subject or subjects with no or low risk of recurrence). The standard control may be any suitable control, examples of which are described herein. The control with no risk of recurrence may be a patient or subject who has undergone hepatectomy for treatment of colorectal liver matastases (CRLM) and is at no risk or very low risk of developing cancer recurrence within the first 5 years after surgery, examples of which are described herein.

[0075] The terms a “reduced level” or a “decreased expression level” or a “low level” of gene expression is an expression level of the gene or protein that is lower than the expression level of the gene or protein in a standard control or in a control with no risk of recurrence.. The standard control may be any suitable control, examples of which are described herein. The control with no risk of recurrence is a patient or subject who has undergone hepatectomy for treatment of colorectal liver matastases (CRLM) and is at no risk or very low risk of developing cancer recurrence within the first 5 years after surgery, examples of which are described herein.

[0076] “Pathway” refers to a set of system components involved in two or more sequential molecular interactions that result in the production of a product or activity. A pathway can produce a variety of products or activities that can include, for example, intermolecular interactions, changes in expression of a nucleic acid or polypeptide, the formation or dissociation of a complex between two or more molecules, accumulation or destruction of a metabolic product, activation or deactivation of an enzyme or binding activity. Thus, the term "pathway" includes a variety of pathway types, such as, for example, a biochemical pathway, a gene expression pathway, and a regulatory pathway. Similarly, a pathway can include a combination of these exemplary pathway types.

[0077] “Control,” “standard control,” or “control experiment” is used in accordance with its plain ordinary meaning and refers to an 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 of a protein in the absence of a compound as described herein (including embodiments and examples). For example, a test sample can be taken from a patient suspected of having a given disease (cancer) and compared to samples from a known cancer patient 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, same 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 expression level 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 10 or more, 25 of more, 50 or more, 100 or more, 1000 or more, or 5000 or more. In embodiments, the control is a population of cancer subjects. 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. In embodiments, the standard control is a CD8+ T cell population from a healthy subject, a population of healthy subjects, a cancer patient who is responsive to treatment with a non-chemotherapeutic therapy (e.g., hormone therapy), or a population of breast cancer patients who are responsive to treatment with a non- chemotherapeutic therapy (e.g., hormone therapy). In embodiments, the standard control is a CD26+CD4+ T cell population from a healthy subject, a population of healthy subjects, or a cancer patient who is responsive to surgery and can be spared intensive chemotherapy treatment. In embodiments, the standard control is a CD26+CD4+ T cell population from a healthy subject. In embodiments, the standard control is a population of healthy subjects. In embodiments, the standard control is a cancer patient who is responsive to surgery and can be spared intensive chemotherapy treatment. In embodiments, the control is a cancer patient or a population of cancer patients with no risk of cancer recurrence. In embodiments, the control is a cancer patient or a population of cancer patients with a low (e.g. very low) risk of cancer recurrence. In embodiments, the control is a patient or subject who has undergone hepatectomy for treatment of colorectal liver metastases (CRLM) and is at no risk or very low risk of developing cancer recurrence within the first 5 years after surgery. In embodiments, the subject has a risk of developing cancer recurrence within the first 5 years after surgery below 10%. In embodiments, the subject has a risk of developing cancer recurrence within the first 5 years after surgery below 8%. In embodiments, the subject has a risk of developing cancer recurrence within the first 5 years after surgery below 5%. In embodiments, the subject has a risk of developing cancer recurrence within the first 5 years after surgery below 2%. In embodiments, the subject has a risk of developing cancer recurrence within the first 5 years after surgery below 1%. In embodiments, the subject has a risk of developing cancer recurrence within the first 5 years after surgery below 0.5%. In embodiments, the subject has a risk of developing cancer recurrence within the first 5 years after surgery below 0.1%. In embodiments, the control is a sample from a subject with no elevated expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and a combination thereof. In embodiments, the control is a sample obtained from a healthy subject, or a sample from a subject with colorectal cancer who has undergone surgical removal of all or a portion of the liver and has a low risk of developing colorectal liver metastases. In embodiments, the control is a sample obtained from a healthy subject. In embodiments, the standard control is a sample from a subject with colorectal cancer who has undergone surgical removal of all or a portion of the liver and has a low risk of developing colorectal liver metastases.

[0078] The terms “treating” and “treatment” refers to any indicia of success in the therapy or amelioration of an injury, disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination. Treating does not include preventing.

[0079] “Treating” or “treatment” as used herein (and as well-understood in the art) also broadly includes any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, 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 or amelioration of a disease. Treatment may inhibit the disease’s spread; relieve the disease’s symptoms, fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.

[0080] “Treating” and “treatment” as used herein 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 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 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, the treating or treatment is not prophylactic treatment.

[0081] Cancer treatment refers to, but are not limited to, surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, and synthetic lethality therapy.

[0082] It is contemplated that the methods herein reduce tumor size or tumor burden in the subject, and/or reduce metastasis in the subject. In various embodiments, the methods reduce the tumor size by 10%, 20%, 30% or more. In various embodiments, the methods reduce tumor size by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0083] “Patient” or “subject” in need thereof refers to a living organism suffering from or prone to a disease or condition. In embodiments, a patient is human. In embodiments, a patient is a human with cancer. In embodiments, a patient is a human with colorectal cancer. In embodiments, a patient is a human with colorectal liver metastasis.

[0084] The term “metastasis” or the plural form “metastases” refers to the development of secondary malignant growths at a distance from a primary site of cancer. The condition refers to when cancer cells break away from the main tumor and enter the bloodstream or lymphatic system. 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.

[0085] An “effective amount,” as used herein, is an amount sufficient for a compound to accomplish a stated purpose relative to the absence of the compound (e.g. achieve the effect for which it is administered, treat a disease, reduce a signaling pathway, or reduce one or more symptoms of a disease or condition). In these methods, the effective amount of the active agent (e.g., oncolytic virus, viral vector) described herein is an amount effective to accomplish the stated purpose of the method. An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0086] The term “therapeutically effective amount,” as used herein, refers to that amount of the therapeutic agent sufficient to ameliorate the disorder, as described above. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.

Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. 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. As is 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.

[0087] 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.

[0088] As used herein, the term "administering" is used according to its plain and ordinary meeting and includes 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.

[0089] The term “administering” includes intranasal administration, inhalation administration, 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 the use of lipid nanoparticles, aerosols, liposomal formulations, intravenous infusion, transdermal patches, and the like.

Methods of Treatment

[0090] Provided herein are methods of treating a distant metastasis tumor in a subject in need thereof. The disclosed methods comprise surgically removing all or a portion of the subject’s distant metastasis tumor; detecting an elevated expression level in a sample obtained from the subject, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and a combination thereof; and administering to the subject radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof. The disclosed methods may also comprise surgically removing all or a portion of the subject’s distant metastasis tumor; detecting an elevated expression level in a sample obtained from the subject, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24,

TMTC3, and a combination thereof; and administering to the an effective amount of an anticancer agent as set forth herein. In embodiments, the distant metastasis tumor is a colorectal liver metastasis (CRLM) tumor. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof

[0091] In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof, three months after surgical removal of the tumor. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof, six months after surgical removal of the tumor. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof, nine months after surgical removal of the tumor. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof, 12 months after surgical removal of the tumor. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof, 15 months after surgical removal of the tumor. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof, 18 months after surgical removal of the tumor. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof, 24 months after surgical removal of the tumor.

[0092] Additionally provided herein are methods of treating a subject who has or is suspected of having cancer. The disclosed methods comprise: (i) detecting an elevated expression level, relative to a control, of one or more genes, wherein the one or more genes are selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and a combination thereof in a sample obtained from the subject; and (ii) administering to the subject radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof. The disclosed methods may comprise: (i) detecting an elevated expression level, relative to a control, of one or more genes, wherein the one or more genes are selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and a combination thereof in a sample obtained from the subject; and (ii) administering to the an effective amount of an anticancer agent as set forth herein. In embodiments, the subject has previously undergone surgical removal of all or a portion of the cancer. In embodiments, the disclosed method comprise surgically removing all or a portion of the cancer after detecting an elevated expression level, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and a combination thereof. In embodiments, the cancer is a liver metastasis. In embodiments, the liver metastasis is a colorectal liver metastasis. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof.

[0093] In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof, three months after surgical removal of the cancer. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof, six months after surgical removal of the cancer. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof, nine months after surgical removal of the cancer. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof, 12 months after surgical removal of the cancer. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof, 15 months after surgical removal of the cancer. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof, 18 months after surgical removal of the cancer. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof, 24 months after surgical removal of the cancer.

[0094] In embodiments, the sample is a liquid biological sample. In embodiments, the liquid biological sample is blood, plasma, urine, or saliva. In embodiments, the liquid biological sample is blood. In embodiments, the liquid biological sample is plasma. In embodiments, the liquid biological sample is urine. In embodiments, the liquid biological sample is saliva. In embodiments, the control is a sample from a subject with no elevated expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and a combination thereof. In embodiments, the control is a sample obtained from a healthy subject. In embodiments, the control is a subject with a tumor who has undergone surgical removal of all or a portion of the tumor and has a low risk of developing tumor metastases.

[0095] In embodiments, the methods of treatment provided herein comprise administering to the subject chemotherapy in an effective amount to treat the tumor or cancer. In embodiments, the chemotherapy is a perioperative FOLFOX chemotherapy. In embodiments, the methods of treatment provided herein include administering an effective amount of one or more anticancer agents as described herein. In embodiments, the methods of treatment provided herein include administering an effective amount of an anticancer agents as described herein.

[0096] In embodiments, the methods of treatment provided herein comprise administering to the subject targeted therapy in an effective amount to treat the tumor or cancer. In embodiments, the targeted therapy comprises the use of an anti-EGFR antibody or antibodies. In embodiments, the targeted therapy comprises the use of an anti-VEGF antibody or antibodies combined to a modified FOLFOX or FOLFIXIRI regimen.

[0097] In embodiments, the subject has an increased risk of cancer recurrence.

Methods of Detecting CRLM or Identifying Increased Risk of CRLM

[0098] Provided herein are methods of identifying an increased risk of developing a colorectal liver metastasis tumor in a subject with colorectal cancer or detecting a colorectal liver metastasis tumor in a subject with colorectal cancer. The disclosed methods comprise detecting an elevated expression level, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and a combination thereof in a sample obtained from the subject; wherein the elevated expression level of the one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and a combination thereof, indicates an increased risk of developing a colorectal liver metastasis tumor or the presence of a colorectal liver metastasis tumor. In embodiments, the methods comprise identifying an increased risk of developing a colorectal liver metastasis tumor in a subject with colorectal cancer. In embodiments, the methods comprise detecting a colorectal liver metastasis tumor in a subject with colorectal cancer. In embodiments, the disclosed methods comprise detecting an elevated expression level, relative to a control, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof, in a sample obtained from the subject.

[0099] Also provided herein are methods of diagnosing a subject having colorectal cancer as being at an increased risk for developing a colorectal liver metastasis tumor, the method comprising detecting the expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3, in a sample obtained from the subject; wherein the subject has an increased risk for a colorectal liver metastasis tumor when an elevated expression level, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, and TMTC3 is detected in the sample.

[0100] Additionally provided herein are methods of detecting gene expression in a subject that has or is suspected of having a cancer. The disclosed methods comprise measuring the level of expression of one or more genes in a sample obtained from the subject in comparison to a control, wherein the subject has, or is suspected of having, a metastasis tumor, and wherein the one or more genes are COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, or any combination thereof. In embodiments, the metastasis tumor is a distant metastasis tumor. In embodiments, the distant metastasis tumor is a distant colorectal cancer metastasis tumor. In embodiments, the distant colorectal cancer metastasis tumor is a colorectal liver metastasis (CRLM) tumor.

[0101] In embodiments, the subject has previously undergone surgical removal of all or a portion of the cancer. In embodiments, the disclosed method comprise surgically removing all or a portion of the cancer after detecting an elevated expression level, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and a combination thereof. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof.

[0102] In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof, three months after surgical removal of the cancer. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof, six months after surgical removal of the cancer. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof, nine months after surgical removal of the cancer. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof, 12 months after surgical removal of the cancer. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof, 15 months after surgical removal of the cancer. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof, 18 months after surgical removal of the cancer. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a control, of the genes COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof, 24 months after surgical removal of the cancer.

[0103] In embodiments, the sample is a liquid biological sample. In embodiments, the liquid biological sample is blood, plasma, urine, or saliva. In embodiments, the control is a sample from a subject with no elevated expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and a combination thereof. In embodiments, the control is a sample obtained from a healthy subject. In embodiments, the control is a subject with a tumor who has undergone surgical removal of all or a portion of the tumor and has a low risk of developing tumor metastases.

[0104] In embodiments, the subject has an increased risk of cancer recurrence if the expression level of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, or any combination thereof is above the expression level of the same genes in the control. In embodiments, the subject having an increased risk of cancer recurrence is proposed further treatment or treatments. In embodiments, the treatment or treatments comprise one or more of radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof. In embodiments, the treatment or treatments includes administration of an effective amount of an anticancer agent as described herein. In embodiments, the treatment or treatments comprise administering to the subject chemotherapy in an effective amount to treat the tumor or cancer. In embodiments, the chemotherapy is a perioperative FOLFOX chemotherapy. In embodiments, the treatment or treatments comprise administering to the subject targeted therapy in an effective amount to treat the tumor or cancer. In embodiments, the targeted therapy comprises the use of an anti-EGFR antibody or antibodies. In embodiments, the targeted therapy comprises the use of an anti-VEGF antibody or antibodies combined to a modified FOLFOX or FOLFIXIRI regimen.

[0105] In embodiments, the subject has a low risk of cancer recurrence if the expression level of COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, or any combination thereof is equal or below the expression level of these genes in the control. In embodiments, the subject at low risk of cancer recurrence is periodically monitored for cancer development. Monitoring may comprise serological carcinoembryonic antigen (CEA) level measurements or the use of computed tomography (CT) imaging of the chest, abdomen, and pelvis. In embodiments, the subject having a low risk of cancer recurrence is monitored every 3 to 6 months for two years, and thereafter every 6 months for three additional years.

Methods of Monitoring

[0106] Provided herein are methods of monitoring a subject having colorectal cancer for an increased risk of colorectal liver metastasis tumor. The disclosed methods comprise detecting the expression level of one or more genes selected from the group consisting of COX6A1,

ERN1, IFITM2, SI OOP, STK24, and TMTC3 in a sample obtained from the subject at a first time point; and detecting the expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3 in a sample obtained from the subject at a second time point later than the first time point. In embodiments, an elevated expression level of COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, or any combination thereof detected in the sample at the second time point compared to the expression level of these genes at the first time point is indicative of an increased risk for colorectal liver metastasis tumor. In embodiments, anon-elevated expression level of COX6A1, ERN1,

IFITM2, SI OOP, STK24, TMTC3, or any combination thereof detected in the sample at the second time point compared to the expression level of these genes at the first time point is indicative of no increased risk for colorectal liver metastasis tumor. [0107] In embodiments, the subject has previously undergone surgical removal of all or a portion of the liver. In embodiments, the disclosed method comprise surgically removing all or a portion of the liver after detecting an elevated expression level, relative to a first time point, of the genes s COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, or any combination thereof

[0108] In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a first time point, of the genes COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof, three months after surgical removal of the liver. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a first time point, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof, six months after surgical removal of the liver. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a first time point, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof, nine months after surgical removal of the liver. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a first time point, of the genes COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof, 12 months after surgical removal of the liver. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a first time point, of the genes COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and any combination thereof, 15 months after surgical removal of the liver. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a first time point, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof, 18 months after surgical removal of the liver. In embodiments, the disclosed methods comprise detecting an elevated expression level in a sample obtained from the subject, relative to a first time point, of the genes COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and any combination thereof, 24 months after surgical removal of the liver.

[0109] In embodiments, the sample is a liquid biological sample. In embodiments, the liquid biological sample is blood, plasma, urine, or saliva.

[0110] In embodiments, the subject has an increased risk of cancer recurrence if the expression level of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, or any combination thereof at the second time point is above the expression level of the same genes at the first time point. In embodiments, the subject having an increased risk of cancer recurrence is proposed further treatment or treatments. In embodiments, the treatment or treatments comprise one or more of radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof. In embodiments, the treatment or treatments includes administration of an effective amount of an anticancer agent as described herein. In embodiments, the treatment or treatments comprise administering to the subject chemotherapy in an effective amount to treat the tumor or cancer. In embodiments, the chemotherapy is a perioperative FOLFOX chemotherapy. In embodiments, the treatment or treatments comprise administering to the subject targeted therapy in an effective amount to treat the tumor or cancer. In embodiments, the targeted therapy comprises the use of an anti-EGFR antibody or antibodies. In embodiments, the targeted therapy comprises the use of an anti-VEGF antibody or antibodies combined to a modified FOLFOX or FOLFIXIRI regimen.

[0111] In embodiments, the subject has a low risk of cancer recurrence if the expression level of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, or any combination thereof at the second time point is equal or below the expression level of these genes at the first time point. In embodiments, the subject at low risk of cancer recurrence is periodically monitored for cancer development. Monitoring may comprise serological carcinoembryonic antigen (CEA) level measurements or the use of computed tomography (CT) imaging of the chest, abdomen, and pelvis. In embodiments, the subject having a low risk of cancer recurrence is monitored every 3 to 6 months for two years, and thereafter every 6 months for three additional years. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer. In embodiments, the colorectal cancer is colorectal cancer with colorectal liver metastasis. In embodiments, the colorectal cancer is invasive submucosal colorectal cancer with colorectal liver metastasis.

Kits

[0112] Provided here are kits comprising components, such as reagents and reaction mixtures, to conduct tests of assays to detect the genes as described herein. As part of the kit, materials and instruction are provided, e.g., for storage and use of kit components.

[0113] “Assaying" or “detecting” means using an analytic procedure to qualitatively assess or quantitatively measure the presence or amount or the functional activity of the genes as described herein such as, for example, detecting the expression level of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, or any combination thereof, using an analytic procedure (such as an in vitro procedure) to qualitatively assess or quantitatively measure the presence or amount of the selected gene. In embodiments, raw expression values are normalized by performing quantile normalization relative to the reference distribution and subsequent log 10- transformation. In embodiments, the kit comprises reagents capable of detecting an expression level of COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, or any combination thereof in a sample obtained from a subject. In embodiments, the kit comprises one or more of a probe that can hybridize to a biomarker, pairs of primers for PCR amplification, instructions on how to use the kit, and a label or insert indicating regulatory approval for diagnostic use.

Embodiments 1-79

[0114] Embodiment 1 : A method of treating a distant metastasis tumor in a subject in need thereof, the method comprising surgically removing all or a portion of the subject’s distant metastasis tumor; detecting an elevated expression level in a sample obtained from the subject, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and a combination thereof; and administering to the subject radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof.

[0115] Embodiment 2: The method of Embodiment 1, wherein the distant metastasis tumor is a colorectal liver metastasis (CRLM) tumor.

[0116] Embodiment 3: The method of Embodiment 1 or Embodiment 2, wherein the sample is a liquid biological sample.

[0117] Embodiment 4: The method of Embodiment 3, wherein the liquid biological sample is blood, plasma, urine, or saliva.

[0118] Embodiment 5: The method of anyone of Embodiments 1-4, wherein the chemotherapy is a perioperative FOLFOX chemotherapy.

[0119] Embodiment 6: The method of anyone of Embodiments 1-4, wherein the targeted therapy comprises the use of an anti-EGFR antibody or antibodies.

[0120] Embodiment 7: The method of anyone of Embodiments 1-6, wherein the targeted therapy comprises the use of an anti-VEGF antibody or antibodies combined to a modified FOLFOX or FOLFIXIRI regimen.

[0121] Embodiment 8: The method of anyone of Embodiments 1-7, wherein the control is a sample from a subject with no elevated expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and a combination thereof.

[0122] Embodiment 9: The method of Embodiment 8, wherein the control is a sample obtained from a healthy subject or from a subject with colorectal cancer who has undergone surgical removal of all or a portion of the liver and has a low risk of developing colorectal liver metastases.

[0123] Embodiment 10: A method of treating a subject who has or is suspected of having cancer, the method comprising: (i) detecting an elevated expression level, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and a combination thereof in a sample obtained from the subject; and (ii) administering to the subject radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof.

[0124] Embodiment 11: The method of Embodiment 10, wherein the subject had previously undergone surgical removal of all or a portion of the liver.

[0125] Embodiment 12: The method of Embodiment 10 or Embodiment 11, wherein the cancer is a cancer metastasis.

[0126] Embodiment 13: The method of anyone of Embodiments 10-12, wherein the cancer metastasis is a liver metastasuis.

[0127] Embodiment 14: The method of Embodiment 12, wherein the liver metastasis is a colorectal liver metastasis.

[0128] Embodiment 15: The method of anyone of Embodiments 10-14, wherein the sample is a liquid biological sample.

[0129] Embodiment 16: The method of Embodiment 15, wherein the liquid biological sample is blood, plasma, urine, or saliva.

[0130] Embodiment 17: The method of anyone of Embodiments 10-16, wherein the chemotherapy is a perioperative FOLFOX chemotherapy.

[0131] Embodiment 18: The method of anyone of Embodiments 10-15, wherein the targeted therapy comprises the use of an anti-EGFR antibody or antibodies, or the use of an anti-VEGF antibody or antibodies combined to a modified FOLFOX or FOLFIXIRI regimen. [0132] Embodiment 19: The method of anyone of Embodiments 10-18, wherein the control is a sample from a subject with no elevated expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and a combination thereof.

[0133] Embodiment 20: The method of Embodiment 19, wherein the control is a sample obtained from a healthy subject or from a subject with cancer who has undergone surgical removal of all or a portion of the liver and has a low risk of developing colorectal liver metastases.

[0134] Embodiment 21: The method of anyone of Embodiments 10-20, wherein the subject has an increased risk of cancer recurrence.

[0135] Embodiment 22: A method of identifying an increased risk of developing colorectal liver metastasis in a subject with colorectal cancer or detecting a colorectal liver metastasis in a subject with colorectal cancer, the method comprising detecting an elevated expression level, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and a combination thereof in a sample obtained from the subject; wherein the elevated expression level of the one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and a combination thereof, indicates an increased risk of developing colorectal liver metastasis or the presence of a colorectal liver metastasis.

[0136] Embodiment 23: The method of Embodiment 22, wherein the method comprises identifying an increased risk of developing colorectal liver metastasis in a subject with colorectal cancer.

[0137] Embodiment 24: The method of Embodiment 22, wherein the method comprises detecting a colorectal liver metastasis in a subject with colorectal cancer.

[0138] Embodiment 25: The method of anyone of Embodiments 22-24, wherein the sample is a liquid biological sample.

[0139] Embodiment 26: The method of Embodiment 25, wherein the liquid biological sample is blood, plasma, urine, or saliva.

[0140] Embodiment 27: The method of anyone of Embodiments 22-26, wherein the subject had previously undergone surgical removal of all or a portion of the liver. [0141] Embodiment 28: The method of anyone of Embodiments 22-27, wherein the method further comprises proposing further treatment or treatments to a subject with an increased risk of developing colorectal liver metastasis or having a colorectal liver metastasis.

[0142] Embodiment 29: The method of Embodiment 28, wherein the treatment or treatments comprise one or more of radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof.

[0143] Embodiment 30: The method of Embodiment 29, wherein the chemotherapy is a perioperative FOLFOX chemotherapy.

[0144] Embodiment 31 : The method of Embodiment 29, wherein the targeted therapy comprises the use of an anti-EGFR antibody or antibodies.

[0145] Embodiment 32: The method of Embodiment 29, wherein the targeted therapy comprises the use of an anti-VEGF antibody or antibodies combined to a modified FOLFOX or FOLFIXIRI regimen.

[0146] Embodiment 33: The method of anyone of Embodiments 22-32, wherein the control is a sample from a subject with no elevated expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and a combination thereof.

[0147] Embodiment 34: The method of Embodiment 33, wherein the control is a sample obtained from a healthy subject or from a subject with colorectal cancer who has undergone surgical removal of all or a portion of the liver and has a low risk of developing colorectal liver metastases.

[0148] Embodiment 35: A method of diagnosing a subject having colorectal cancer as being at high risk for developing colorectal liver metastasis, the method comprising detecting the expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3, in a sample obtained from the subject; wherein the subject has an increased risk for colorectal liver metastasis when an elevated expression level, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, and TMTC3 is detected in the sample.

[0149] Embodiment 36: The method of Embodiment 35, wherein the sample is a liquid biological sample. [0150] Embodiment 37: The method of Embodiment 36, wherein the liquid biological sample is blood, plasma, urine, or saliva.

[0151] Embodiment 38: The method of anyone of Embodiments 35-37, wherein the subject had previously undergone surgical removal of all or a portion of the liver.

[0152] Embodiment 39: The method of anyone of Embodiments 35-38, wherein the method further comprises proposing further treatment or treatments to a subject diagnosed with an increased risk of developing colorectal liver metastasis.

[0153] Embodiment 40: The method of Embodiment 39, wherein the treatment or treatments comprise one or more of radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof.

[0154] Embodiment 41: The method of Embodiment 40, wherein the chemotherapy is a perioperative FOLFOX chemotherapy.

[0155] Embodiment 42: The method of Embodiment 40, wherein the targeted therapy comprises the use of an anti-EGFR antibody or antibodies.

[0156] Embodiment 43: The method of Embodiment 40, wherein the targeted therapy comprises the use of an anti-VEGF antibody or antibodies combined to a modified FOLFOX or FOLFIXIRI regimen.

[0157] Embodiment 44: The method of anyone of Embodiments 35-43, wherein the control is a sample from a subject with no elevated expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, and TMTC3.

[0158] Embodiment 45: The method of Embodiment 44, wherein the control is a sample obtained from a healthy subject or from a subject with colorectal cancer who has undergone surgical removal of all or a portion of the liver and has a low risk of developing colorectal liver metastases.

[0159] Embodiment 46: A method of monitoring a subject having colorectal cancer for an increased risk of colorectal liver metastasis, the method comprising: (i) detecting the expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3 in a sample obtained from the subject at a first time point; (ii) detecting the expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, and TMTC3 in a sample obtained from the subject at a second time point later than the first time point; wherein an elevated expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3 detected in the sample at the second time point compared to the expression level of the genes at the first time point is indicative of an increased risk for colorectal metastasis.

[0160] Embodiment 47: The method of Embodiment 46, wherein anon-elevated expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2,

SI OOP, STK24, and TMTC3 detected in the sample at the second time point compared to the expression level of the genes at the first time point is indicative of no increased risk for colorectal metastasis.

[0161] Embodiment 48: The method of Embodiment 46, wherein the sample is a liquid biological sample, and wherein the liquid biological sample is blood, plasma, urine, or saliva.

[0162] Embodiment 49: The method of anyone of Embodiments 46-48, wherein the subject had previously undergone surgical removal of all or a portion of the liver.

[0163] Embodiment 50: The method of anyone of Embodiments 46-49, wherein the method further comprises proposing further treatment or treatments to a subject diagnosed as being at an increased risk of developing colorectal liver metastasis.

[0164] Embodiment 51 : The method of Embodiment 50, wherein the treatment or treatments comprise one or more of radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof.

[0165] Embodiment 52: The method of Embodiment 51, wherein the chemotherapy is a perioperative FOLFOX chemotherapy.

[0166] Embodiment 53: The method of Embodiment 51, wherein the targeted therapy comprises the use of an anti-EGFR antibody or antibodies.

[0167] Embodiment 54: The method of Embodiment 51, wherein the targeted therapy comprises the use of an anti-VEGF antibody or antibodies combined to a modified FOLFOX or FOLFIXIRI regimen.

[0168] Embodiment 55: The method of any one of Embodiments 46-54, wherein the expression level of the one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3 in the sample obtained from the subject at the first time point is not elevated, and wherein the expression level of the one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, and TMTC3 in the sample obtained from the subject at the second time point is elevated, thereby diagnosing an increased risk of colorectal liver metastasis in the subject having colorectal cancer.

[0169] Embodiment 56: The method of any one of Embodiments 46-54, wherein the expression level of the one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3 in the sample obtained from the subject at the first time point is not elevated, and wherein the expression level of the one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, and TMTC3 in the sample obtained from the subject at the second time point is not elevated, thereby diagnosing no increased risk of colorectal liver metastasis in the subject having colorectal cancer.

[0170] Embodiment 57: The method of anyone of Embodiments 46-56, wherein an increased risk of colorectal liver metastasis is predictive of cancer recurrence.

[0171] Embodiment 58: The method of anyone of Embodiments 46-57, wherein the monitoring comprises serological carcinoembryonic antigen (CEA) level measurements or the use of computed tomography (CT) imaging of the chest, abdomen, and pelvis.

[0172] Embodiment 59: The method of Embodiment 58, wherein the subject with no increased risk of colorectal liver metastasis is monitored every 3 to 6 months for two years, and thereafter every six months for three additional years.

[0173] Embodiment 60: The method of anyone of Embodiments 2-59, wherein the colorectal cancer is invasive submucosal colorectal cancer.

[0174] Embodiment 61: The method of anyone of Embodiments 2-59, wherein the colorectal cancer is colorectal cancer with colorectal liver metastasis.

[0175] Embodiment 62: The method of anyone of Embodiments 2-59, wherein the colorectal cancer is invasive submucosal colorectal cancer with colorectal liver metastasis.

[0176] Embodiment 63: A method of detecting gene expression in a subject that has or is suspected of having cancer, wherein the method comprises measuring the level of expression of one or more genes in a sample obtained from the subject in comparison to a control, wherein the subject has, or is suspected of having, metastases, and wherein the one or more genes are COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, or a combination of two or more thereof.

[0177] Embodiment 64: The method of Embodiment 63, wherein the control is a healthy subject. [0178] Embodiment 65: The method of Embodiment 63, wherein the subject had previously undergone a hepatectomy.

[0179] Embodiment 66: The method of Embodiment 63, wherein the metastases are liver metastases.

[0180] Embodiment 67: The method of Embodiment 66, wherein the liver metastases are colorectal liver metastases.

[0181] Embodiment 68: The method of Embodiment 63, wherein the sample is a liquid biological sample.

[0182] Embodiment 69: The method of Embodiment 68, wherein the liquid biological sample is blood, plasma, urine, or saliva.

[0183] Embodiment 70: The method of Embodiment 63, wherein the subject is at high risk of cancer recurrence if the expression level of the one or more genes are above that of said control.

[0184] Embodiment 71: The method of Embodiment 70, wherein the subject at high risk of cancer recurrence is proposed further treatment or treatments.

[0185] Embodiment 72: The method of Embodiment 71, wherein the treatment or treatments are surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof.

[0186] Embodiment 73: The method of Embodiment 72, wherein the chemotherapy is a perioperative FOLFOX chemotherapy.

[0187] Embodiment 74: The method of Embodiment 72, wherein the targeted therapy comprises the use of an anti-EGFR antibody or antibodies.

[0188] Embodiment 75: The method of Embodiment 72, wherein the targeted therapy comprises the use of an anti-VEGF antibody or antibodies combined to a modified FOLFOX or FOLFIXIRI regimen.

[0189] Embodiment 76: The method of Embodiment 63, wherein the subject is at low risk of cancer recurrence if the expression level of the one or more genes are equal or below that of a control.

[0190] Embodiment 77: The method of Embodiment 76, wherein the subject at low risk of cancer recurrence is periodically monitored for the development of cancer. [0191] Embodiment 78: The method of Embodiment 77, wherein the monitoring comprises serological carcinoembryonic antigen (CEA) level measurements or the use of computed tomography (CT) imaging of the chest, abdomen, and pelvis.

[0192] Embodiment 79: The method of Embodiment 78, wherein the subject at low risk of cancer recurrence is monitored every 3 to 6 months for the first 2 years, and thereafter every 6 months for 3 additional years.

[0193] Embodiment 80: The method of Embodiment 63, wherein the metastases are distant metastases.

[0194] Embodiment 81: The method of Embodiment 80, wherein the distant metastases are distant colorectal metastases.

[0195] Embodiment 82: The method of Embodiment 81, wherein the distant colorectal metastases are colorectal liver metastases.

[0196] Embodiment 83: The method of anyone of Embodiments 63-82, wherein the subject had previously undergone surgical removal of all or a portion of the liver.

[0197] Embodiment 84: The method of Embodiment 63, wherein the control is a sample obtained from a subject with a cancer who has undergone surgical removal of all or a portion of the liver and has a low risk of developing colorectal liver metastases.

[0198] Embodiment 85: The method of anyone of Embodiments 63-84, wherein the subject has a decreased risk of cancer recurrence if the expression level of the one or more genes is equal or below the expression level of the one or more genes in the control.

[0199] Embodiment 86: The method of Embodiment 85, wherein the subject having a decreased risk of cancer recurrence is periodically monitored for cancer development.

[0200] Embodiment 87: The method of Embodiment 86, wherein the monitoring comprises serological carcinoembryonic antigen (CEA) level measurements or the use of computed tomography (CT) imaging of the chest, abdomen, and pelvis.

[0201] Embodiment 88: The method of Embodiment 87, wherein the subject having a decreased risk of cancer recurrence is monitored every 3 to 6 months for two years, and thereafter every 6 months for three additional years. [0202] Embodiment 89: A kit comprising reagents capable of detecting an expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3 in a sample.

EXAMPLES

[0203] The following examples are for purposes of illustration only and are not intended to limit the scope of the disclosure or claims.

[0204] In the present study we performed a genome-wide, systematic, and comprehensive biomarker discovery effort to identify a gene expression signature for predicting cancer recurrence following hepatectomy in patients with CRLM. We initially identified this signature in a genome wide expression profiling dataset, following by its rigorous validation and performance evaluation in large, independent clinical cohorts. Our final risk-stratification model allows robust identification of patients with CRLM who are at high risk of cancer recurrence; which will improve overall management of patients by recommending more intensive treatment selection in high-risk patients and sparing other low-risk patients from the unnecessary toxicity and expense associated with adjuvant treatments.

Example 1: Gene biomarker discovery

[0205] To perform a comprehensive genome-wide biomarker discovery, gene expression profiling results from a dataset (GSE81423) were analyzed to identify and establish a biomarker panel for predicting recurrence following radical hepatectomy in patients with CRLM. The GSE81423 dataset (normalized gene profiling and clinical data) was downloaded from the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov). In total, gene expression profiling data was obtained from 63 patients with CRLM (33 recurrence and 30 non-recurrence), as illustrated in Fig. 4. To evaluate the diagnostic potential of the discovered gene signature, we first established a logistic regression model using selected biomarkers and subsequently determined area under the curve (AUC) values for each of the receiver operator characteristic (ROC) plots ³⁵· ³⁶.

Example 2: Patient cohorts [0206] To perform clinical training and validation of the identified gene panel, a total of 320 formalin-fixed paraffin-embedded (FFPE) specimens from two independent cohorts of patients with CRLM were analyzed. This included a training cohort (n = 169; 119 with recurrence and 50 non recurrence) of patients enrolled at Tokushima and Kyushu University, and a validation cohort (n = 151; 102 with recurrence and 49 non-recurrence) of patients enrolled at Kumamoto University. All patients underwent an initial radical hepatectomy between January 1995 and December 2017 in the training cohort, and between January 2001 and December 2016 in the validation cohort. All specimens were diagnosed as CRLM by pathologists at each participating institution. The follow-up protocol included a physical examination, evaluation of serum CEA and carbohydrate antigen 19-9 (CA19-9) levels, as well as imaging modalities such as ultrasonography, CT, and colonoscopy.

Only regions confirmed by imaging modalities were diagnosed as tumor recurrence sites. Exclusion criteria included macroscopically incomplete resection or a tumor histology other than diagnosis of CRLM. The study was conducted in accordance with the Declaration of Helsinki. A written informed consent was obtained from all patients, and the study was approved by the institutional review boards of all participating institutions.

Example 3: RNA extraction and RT-qPCR

[0207] Total RNA was isolated from 10 pm thick sections of FFPE surgical tissues by performing manual microdissection to enrich for cancer cells (>75% of tumor cells), followed by RNA extraction using an AllPrep DNA/RNA FFPE Kit (Qiagen, Hilden, Germany). Synthesis of complementary DNA (cDNA) was conducted with 500 ng of total RNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis was performed using the SensiFAST™ Lo-ROX Kit (Bioline, London, UK) on a Quantstudio 7 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA), and expression levels were evaluated using Applied Biosystems QuantStudio 7 Flex Real-Time PCR System Software. The relative abundance of target transcripts was evaluated and normalized to the expression of b-actin as an internal control using the 2 ^ADCt method. Normalized expression values were logio-transformed ³⁷. The primer sequences for the target genes used in the present study are shown in Fig. 8.

Example 4: Statistical analysis [0208] Clinicopathologic characteristics of the patient cohorts are shown in Fig. 7. The cutoff thresholds for continuous variables were divided by the median value in all participants. Several clinicopathologic characteristics were compared between recurrence and non-recurrence groups, using aChi-Square test or Mann-Whitney U test for categorical data. Cox’s proportional hazard regression analysis was used to train a classifier based on the expression of each gene. Of note, once the model was trained (in the training cohort), the same statistical model variables (weights and cutoff thresholds) were applied in the independent validation cohort, to better appreciate the translational potential of our risk-stratification model. The cutoff threshold for the recurrence risk scores was chosen as 1.44, which was determined by Youden’s index. For all cohorts, ROC curves and AUC values were used to evaluate the performance of the panel for recurrence prediction in patients with CRLM. OS and recurrence-free survival (RFS) times were calculated from the date of surgery to the date of death from any cause or recurrence, or last follow-up date, and estimated using the Kaplan-Meier method. Univariate and multivariate Cox’s proportional hazard regression models were established; a P value < 0.05 was considered statistically significant. Hazard ratios (HR) were calculated with 95% confidence intervals (Cl). Statistical analyses were performed using JMP Pro V 13.0 statistical software (SAS Institute Japan, Tokyo, Japan), Medcalc statistical software V.17.4.0 (Medcalc Software bvba, Ostend, Belgium), GraphPad Prism V8.0 (GraphPad Software, San Diego, CA, USA), and R (3.5.0, R Development Core Team, https://cran.r- project.org/).

Example 5: Genome-wide expression profiling identifies a 6-gene panel that predicts cancer recurrence in patients with CRLM

[0209] To identify a gene panel that can predict cancer recurrence in patients with CRLM, we first performed a genome-wide, unbiased, comprehensive biomarker discovery analysis in a public gene expression profiling dataset from a cohort of patients with CRLM (GSE81423). By comparing gene expression profiles between patients with and without recurrence after radical hepatectomy, we identified a panel of 18 candidate genes differentially expressed (P < 0.05, |log2 fold-change]

>1). During these analyses, we included genes that had available data in at least 50% of all cases and excluded highly correlated genes. Next, we performed lasso regression analysis with cross validation, which reduced the candidates to a panel of 6 genes: COX6A1, ERN1, IFITM2, SIOOP, STK24, and TMTC3. Applying the 6-gene panel to the GSE81423 data resulted in an AUC of 0.90 (95% Cl = 0.79-0.96; Fig. 5A), highlighting the diagnostic performance of this panel for identifying recurrence in patients with CRLM (Fig. 5B).

Example 6: Clinical training and validation established a transcriptomic panel that predicts cancer recurrence in patients with CRLM

[0210] To confirm the predictive robustness of our discovered 6-gene panel, we evaluated its performance in two clinical cohorts of patients with CRLM who underwent radical hepatectomy. The training cohort consisted of 169 patients, which included 119 patients with recurrence (70.4%); the median age of patients in this cohort was 65 years. The validation cohort consisted of 151 patients, which included 102 patients with recurrence (67.5%) with a median age of 64 years. We first confirmed that the patients within our training and validation cohorts possessed similar clinicopathologic characteristics (Fig. 7). We observed no statistically significant differences in the prevalence of recurrence rates, nor any other clinicopathological characteristics between the two cohorts, eliminating the possibility of any inadvertent bias.

[0211] Next, we systematically interrogated the diagnostic accuracy of our transcriptomic panel for its ability to predict recurrence in patients within the training cohort (119 with recurrence vs. 50 with non-recurrence). Using Cox’s proportional hazard regression analysis, we trained the panel that robustly identified recurrence in patients with CRLM (AUC = 0.83, 95% Cl = 0.76-0.88, Fig. 1A, B). Thereafter, we developed a risk-stratification panel based on the coefficients derived from individual markers by using the following model parameters: Logit (P) = (COX6A 1 *0.1442) + (E7riV7*-0.4654) + (ZF/7M2*-0.2096) + (SI OOP* -0.2334) + (STK24* -0.1346) + (ZM7 *-0.2320).

[0212] We next evaluated the robustness and accuracy of the 6-gene transcriptomic panel by applying the same statistical model (i.e., same coefficients and cutoff thresholds) to an independent validation cohort of patients with CRLM (102 with recurrence, 49 non-recurrence). The diagnostic performance of our panel in the validation cohort was comparable to that observed in the training cohort (AUC = 0.81, 95% Cl = 0.74-0.87; Fig. 1C, D). Taken together, our findings indicate that we successfully identified a novel transcriptomic panel for identifying recurrence in patients with CRLM.

Example 7: Recurrence prediction correlates with survival outcomes in patients with CRLM

[0213] Cancer recurrence is often associated with poor survival in patients with CRLM. Thus, to determine the prognostic potential of our gene panel, we performed survival analysis (RFS and OS) in the clinical training and validation cohorts. The median follow-up times after hepatectomy were 39.7 months (95% Cl = 34.5-44.4) in the training cohort and 37.1 months (95% Cl = 26.9-47.8) in the validation cohort. Patients categorized into the high-risk group demonstrated significantly worse prognosis in both, the training cohort (RFS [P < 0.01]; OS [P < 0.01]; Fig. 2A, B), and the validation cohort (RFS [P < 0.01]; OS [P < 0.01]; Fig. 2C, D). We thereafter performed a univariate Cox’s proportional hazard regression analysis, which revealed that our transcriptomic panel was an independent predictor of recurrence in both clinical cohorts, compared to any singular clinical risk factors (training cohort: HR = 3.18, 95% Cl = 2.03-4.98, P < 0.01; validation cohort: HR = 2.60, 95% Cl = 1.73-3.92, P < 0.01; Fig. 9).

Example 8: Risk-stratification model that combines transcriptomic biomarkers and specific clinicopathological factors improves recurrence prediction in patients with CRLM

[0214] Considering that some of the pathological risk factors currently used in the clinic offer some risk assessment potential in patients with CRLM, we asked whether a risk-stratification model that couples known risk factors with our transcriptomic panel might further improve its accuracy in predicting recurrence. Indeed, when we incorporated four classic clinicopathological factors (i.e., CA19-9, CEA, tumor number, and synchronous lesions) into such a risk-stratification model, the model exhibited a markedly superior diagnostic accuracy compared to the transcriptomic panel alone, or vs. other individual risk factors (AUC = 0.85, Fig. 3A).

[0215] We next determined specific diagnostic correlates for our 6-gene biomarker panel in the validation cohort. Herein, we observed that its sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV) were 59.8%, 83.7%, 88.4%, and 50.0%, respectively (Fig. 10). When we performed a similar analysis of the newly established risk-stratification model that also included risk factors, the model was significantly superior to the transcriptomic panel alone in predicting sensitivity, specificity, PPV, and NPV, with values of 77.2%, 81.6%, 89.7%, and 63.5%, respectively. Furthermore, univariate and multivariate Cox’s proportional hazard regression analysis revealed that our novel risk-stratification model was superior to the transcriptomic panel and various clinicopathological factors in terms of predicting recurrence (Univariate: HR = 4.34, 95% Cl = 2.71-6.93, P < 0.01, Fig. 3B; Multivariate: HR = 3.65, 95% Cl = 2.02-6.59, P < 0.01,

Fig. 3C) in patients within the validation cohort (Fig. 9). Collectively, these data highlight the potential clinical significance of our risk model for identifying recurrence in CRC patients with liver metastasis. Example 9: Risk-stratification model robustly identifies CRLM patients at high risk for cancer recurrence

[0216] The ultimate translational goal of our study was to determine the clinical usefulness of our risk-stratification model in identifying patients who actually require post-operative therapy and spare the rest (i.e. low-risk) from unnecessary treatments. To this end, we applied our final risk- stratification classifier to dichotomize all patients within the validation cohort into low- and high- risk groups of patients for their risk of developing cancer recurrence. We observed that the RFS was significantly lower in the high- vs. low-risk groups estimated by the risk-stratification model (P < 0.01; Fig. 3D). Of the 88 patients who were classified as high risk, 78 experienced tumor recurrence within 5 years (88.6%), indicating that these were indeed high-risk patients and would likely have benefited from post-treatment adjuvant chemotherapy (Fig. 3E). Likewise, of the 63 patients who were classified as low risk, 39 did not experience recurrence within 5 years (61.9%), again emphasizing that these low-risk patients could potentially avoid unnecessary toxicity and expense from adjuvant therapy. These data highlight the clinical potential of our risk-stratification model to inform personalize treatment recommendations in patients with high- and low-risk CRLM.

Discussion

[0217] Accumulating studies continue to emphasize the importance of identifying clinical predictive markers for recurrence in CRC patients with liver metastasis (CRLM) ^x- ^{15 18}. However, the specific clinical risk factors that can robustly predict cancer recurrence and long-term survival outcomes in patients with CRLM remains unclear. Clinically, CRLM rarely spreads via micro- metastases adjacent to the liver metastases. Thus, the width of a negative surgical margin following hepatectomy in patients with CRLM does not affect the risk of marginal recurrence or impact survival ^{38 40}. Furthermore, a recent study suggested that parenchyma-sparing hepatectomy did not increase recurrence in the remnant liver and improved salvageability for recurrent patients ⁴¹. Considering this evidence in the context of our findings about cancer outcomes, our transcriptomic panel could identify high-risk CRC patients who require close follow-up surveillance and intensive chemotherapy to prevent subsequent recurrence.

[0218] Response to perioperative chemotherapy is recognized as a strong predictor of survival after resection, and provides better prognostic information than many traditional clinicopathological factors ⁴². However, several issues complicate the inclusion of response to chemotherapy in a clinical scenario. For instance, the indications and timing of chemotherapy, agents used, and number of cycles varies widely between centers and countries. The optimal method of assessing treatment response also remains unclear. Although histopathological scoring of response to chemotherapy is clearly predictive of therapeutic outcome, such an assessment can only be made following surgery on a resected specimen, and different groups use different scoring systems ⁴³. Previous data indicated that CA19-9, CEA, synchronous lesions, and tumor number were associated with advanced tumor characteristics, a higher risk of tumor recurrence, and poor survival ^{44 47}. In the present study, our newly developed model exhibited notably superior diagnostic accuracy for recurrence (AUC = 0.85) vs. clinical risk factors (i.e., CA19-9, CEA, synchronous, and tumor number; AUC = 0.68 [training] and 0.68 [validation]; Fig. 6). In addition, our model robustly stratified patients into high-and low-risk groups, indicating that 88.6% of high-risk patients experienced tumor recurrence and 61.9% of low-risk patients did not experience recurrence. This is a dramatically superior performance compared to currently used clinicopathological risk factors in the clinic. Collectively, this highlights the potential significance of our findings for their clinical translation for recurrence identification and improving survival in patients with CRLM.

[0219] From a functional viewpoint, various genes in our biomarker panel have been shown to be bonafide candidates involved in cancer pathogenesis. For instance, IFITM2 is a member of IFN- inducible transmembrane gene family, which plays key roles in modulating the immune response and thus may control tumor development ^4X- ⁴⁹. IFITM2 has been reported to be highly specific to human colorectal carcinogenesis and overexpressed in CRC ⁵⁰. SI OOP is a member of the SI 00 family of proteins containing 2 EF-hand calcium-binding motifs, which regulates many intracellular and extracellular activities⁵¹ _’ ⁵². SI OOP is overexpressed in CRC and regulates the invasion and metastasis of CRC by promoting epithelial-mesenchymal transition⁵³. Furthermore, STK24, encoding a serine/threonine protein kinase, is a member of serine/threonine kinase family that functions upstream of mitogen-activated protein kinase (MAPK) signaling and positively regulates the cell cycle, cell growth, migration, and synapse development ⁵⁴. Similarly, the STK24 activity was increased in the immunoblot analyses of CRC, which revealed an increased amount of STK24 kinase in CRC⁵⁵.

[0220] In conclusion, using genome-wide gene expression profiling, we established a novel risk- stratification model that was successfully validated in clinical cohort for accurate identification of high-risk patients with CRLM. Our findings highlight the potential clinical impact of our model on accurate risk-stratification of patients with CRLM, which will improve personalized management of patients suffering from this malignancy.

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Claims

CLAIMS What is claimed is:

1. A method of treating a distant metastasis tumor in a subject in need thereof, the method comprising surgically removing all or a portion of the subject’s distant metastasis tumor; detecting an elevated expression level in a sample obtained from the subject, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, and a combination thereof; and administering to the subject radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof.

2. The method of claim 1, wherein the distant metastasis tumor is a colorectal liver metastasis (CRLM) tumor.

3. A method of treating a subject who has or is suspected of having cancer, the method comprising:

(i) detecting an elevated expression level, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2,

SI OOP, STK24, TMTC3, and a combination thereof in a sample obtained from the subject; and

(ii) administering to the subject radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof.

4. The method of claim 3, wherein the subject had previously undergone surgical removal of all or a portion of the cancer.

5. The method of claim 3 or claim 4, wherein the cancer is a liver metastasis tumor.

6. The method of claim 5, wherein the liver metastasis tumor is a colorectal liver metastasis tumor.

7. The method of anyone of claims 1-6, wherein the sample is a liquid biological sample.

8. The method of claim 7, wherein the liquid biological sample is blood, plasma, urine, or saliva.

9. The method of anyone of claims 1-8, wherein the chemotherapy is a perioperative FOLFOX chemotherapy.

10. The method of anyone of claims 1-8, wherein the targeted therapy comprises the use of an anti-EGFR antibody or antibodies.

11. The method of anyone of claims 1-10, wherein the targeted therapy comprises the use of an anti-VEGF antibody or antibodies combined to a modified FOLFOX or FOLFIXIRI regimen.

12. The method of anyone of claims 1-11, wherein the subject has an increased risk of cancer recurrence.

13. A method of identifying an increased risk of developing a colorectal liver metastasis tumor in a subject with colorectal cancer or detecting a colorectal liver metastasis tumor in a subject with colorectal cancer, the method comprising detecting an elevated expression level, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and a combination thereof in a sample obtained from the subject; wherein the elevated expression level of the one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, TMTC3, and a combination thereof, indicates an increased risk of developing a colorectal liver metastasis tumor or the presence of a colorectal liver metastasis tumor.

14. The method of claim 13, wherein the method comprises identifying an increased risk of developing a colorectal liver metastasis tumor in a subject with colorectal cancer.

15. The method of claim 13, wherein the method comprises detecting a colorectal liver metastasis tumor in a subject with colorectal cancer.

16. A method of diagnosing a subject having colorectal cancer as being at an increased risk for developing colorectal liver metastasis, the method comprising detecting the expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, S100P, STK24, and TMTC3, in a sample obtained from the subject; wherein the subject has an increased risk for colorectal liver metastasis tumor when an elevated expression level, relative to a control, of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3 is detected in the sample.

17. A method of monitoring a subject having colorectal cancer for an increased risk of colorectal liver metastasis tumor, the method comprising:

(i) detecting the expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3 in a sample obtained from the subject at a first time point; and

(ii) detecting the expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3 in a sample obtained from the subject at a second time point later than the first time point; wherein an elevated expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3 detected in the sample at the second time point compared to the expression level of the genes at the first time point is indicative of an increased risk for colorectal liver metastasis tumor.

18. The method of claim 38, wherein a non-elevated expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3 detected in the sample at the second time point compared to the expression level of the genes at the first time point is indicative of no increased risk for colorectal liver metastasis tumor.

19. The method of anyone of claims 13-18, wherein the sample is a liquid biological sample.

20. The method of claim 19, wherein the liquid biological sample is blood, plasma, urine, or saliva.

21. The method of anyone of claims 13-20, wherein the subject had previously undergone surgical removal of all or a portion of the liver.

22. The method of anyone of claims 13-21, wherein the method further comprises proposing further treatment or treatments to a subject with an increased risk of developing colorectal liver metastasis or having a colorectal liver metastasis.

23. The method of claim 22, wherein the treatment or treatments comprise one or more of radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof.

24. The method of claim 23, wherein the chemotherapy is a perioperative FOLFOX chemotherapy.

25. The method of claim 23, wherein the targeted therapy comprises the use of an anti-EGFR antibody or antibodies.

26. The method of claim 23, wherein the targeted therapy comprises the use of an anti-VEGF antibody or antibodies combined to a modified FOLFOX or FOLFIXIRI regimen.

27. The method of anyone of claims 1-26, wherein an increased risk of colorectal liver metastasis is predictive of cancer recurrence.

28. The method of claim 17 or 18, wherein the monitoring comprises serological carcinoembryonic antigen (CEA) level measurements or the use of computed tomography (CT) imaging of the chest, abdomen, and pelvis.

29. The method of claim 28, wherein the subject with no increased risk of colorectal liver metastasis tumor is monitored every 3 to 6 months for two years, and thereafter every six months for three additional years.

30. The method of anyone of claims 13-29, wherein the colorectal cancer is invasive submucosal colorectal cancer.

31. The method of anyone of claims 13-29, wherein the colorectal cancer is colorectal cancer with colorectal liver metastasis.

32. The method of anyone of claims 13-29, wherein the colorectal cancer is invasive submucosal colorectal cancer with colorectal liver metastasis.

33. A method of detecting gene expression in a subject that has or is suspected of having cancer, wherein the method comprises measuring the level of expression of one or more genes in a sample obtained from the subject in comparison to a control, wherein the subject has, or is suspected of having, metastasis tumors, and wherein the one or more genes are COX6A1, ERN1, IFITM2, SI OOP, STK24, TMTC3, or any combination thereof.

34. The method of claim 33, wherein the subject had previously undergone surgical removal of all or a portion of the cancer.

35. The method of claim 34, wherein the metastasis tumor is a distant metastasis tumor.

36. The method of claim 35, wherein the distant metastasis tumor is a distant colorectal cancer metastasis tumor.

37. The method of claim 36, wherein the distant colorectal cancer metastasis tumor is a colorectal liver metastasis (CRLM) tumor.

38. The method of anyone of claims 33-37, wherein the subject has an increased risk of cancer recurrence if the expression level of the one or more genes is above the expression level of the one or more genes in the control.

39. The method of claim 38, wherein the subject having an increased risk of cancer recurrence is proposed further treatment or treatments.

40. The method of claim 39, wherein the treatment or treatments comprise one or more of radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibitor administration therapy, synthetic lethality therapy, or a combination of two or more thereof.

41. The method of claim 40, wherein the chemotherapy is a perioperative FOLFOX chemotherapy.

42. The method of claim 40, wherein the targeted therapy comprises the use of an anti-EGFR antibody or antibodies.

43. The method of claim 40, wherein the targeted therapy comprises the use of an anti-VEGF antibody or antibodies combined to a modified FOLFOX or FOLFIXIRI regimen.

44. The method of anyone of claims 33-43, wherein the subject has a low risk of cancer recurrence if the expression level of the one or more genes are equal or below the expression level of the one or more genes in the control.

45. The method of claim 44, wherein the subject at low risk of cancer recurrence is periodically monitored for cancer development.

46. The method of claim 45, wherein the monitoring comprises serological carcinoembryonic antigen (CEA) level measurements or the use of computed tomography (CT) imaging of the chest, abdomen, and pelvis.

47. The method of claim 46, wherein the subject having a low risk of cancer recurrence is monitored every 3 to 6 months for two years, and thereafter every 6 months for three additional years.

48. A kit comprising reagents capable of detecting an expression level of one or more genes selected from the group consisting of COX6A1, ERN1, IFITM2, SI OOP, STK24, and TMTC3 in a sample.