WO2016160882A1

WO2016160882A1 - Methods for diagnosing and treating colorectal cancer using mirna549a

Info

Publication number: WO2016160882A1
Application number: PCT/US2016/024842
Authority: WO
Inventors: Ajay Goel; Yoshinaga OKUGAWA
Original assignee: Baylor Research Institute
Priority date: 2015-04-01
Filing date: 2016-03-30
Publication date: 2016-10-06

Abstract

The current disclosure relates to therapeutic treatments and diagnostic methods for colorectal cancer based on the expression level of miR-549a expression. Aspects of the current disclosure relate to a method for treating a patient for early or advanced colorectal cancer comprising: treating the patient for advanced colorectal cancer after the patient is determined to have a high elevated level of miR-549a expression in a biological sample from the patient; and treating the patient for early colorectal cancer after the patient is determined to have a low elevated level of miR-549a expression in a biological sample from the patient.

Description

METHODS FOR DIAGNOSING AND TREATING COLORECTAL CANCER USING miRNA549a

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/141,546, filed April 1, 2015, which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

[0002] The present invention relates generally to the fields of molecular biology and oncology. More particularly, it concerns methods and compositions involving microRNA (miRNAs) molecules and cancer prognosis, diagnosis, and treatment.

2. Description of Related Art

[0003] Colorectal cancer (CRC) is cancer of the large intestine that forms in the lining of the colon. Most cases of colon cancer begin as small clumps of cells called colon polyps. While these polyps start out as benign, if not discovered and removed, usually during a colonoscopy, they can become cancerous and develop into colon cancer.

[0004] Despite progresses made with regards to earlier diagnosis and treatment, prognosis in patients with colorectal cancer remains poor and CRC still ranks as the second leading cause of cancer-related deaths in the US. A better understanding of the mechanisms underlying CRC pathogenesis is required for developing clinically actionable diagnostic and prognostic biomarkers for CRC. Furthermore, more accurate diagnosis of the CRC stage will lead to novel and more effective therapeutic methods for treating CRC.

SUMMARY OF THE INVENTION

[0005] The current disclosure fulfills a need in the art by providing more effective therapeutic treatments and diagnostic methods for colorectal cancer based on the expression level of miR-549a expression. Aspects of the disclosure relate to a method for treating a patient determined to have colorectal cancer comprising: administering a colorectal cancer treatment to the patient, wherein the patient was determined to have an elevated level of miR- 549a expression in a biological sample from the patient relative to the expression level of miR-549a in a conrol. In some embodiments, the control sample is a non-cancerous biological sample. In some embodiments, the method further comprises measuring the expression level of miR-549a in a biological sample from the patient.

[0006] In some aspects, the method further comprises comparing the expression level of miR-549a in the biological sample from the patient to the expression level of miR-549a from a non-cancerous biological sample. In some embodiments, the patient has or is determined to have Stage I, II, III, or IV colorectal cancer. In some embodiments, the biological sample from the patient is a sample from a primary colorectal cancer tumor. In some embodiments, the colorectal cancer treatment comprises cetuximab, fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumuman, afibercept, leucovorin, and/or radiotherapy. In some embodiments, the colorectal cancer treatment comprises surgical incision of the primary tumor or a secondary tumor.

[0007] Aspects of the current disclosure relate to a method for treating a patient for early or advanced colorectal cancer comprising: treating the patient for advanced colorectal cancer after the patient is determined to have a high elevated level of miR-549a expression in a biological sample from the patient compared to a non-cancerous biological sample; or treating the patient for early colorectal cancer after the patient is determined to have a low elevated level of miR-549a expression in a biological sample from the patient compared to a non-cancerous biological sample.

[0008] In some embodiments, the method further comprises measuring the expression level of miR-549a in a biological sample from the patient. In some embodiments, the expression level is measured by a method for measuring nucleic acids described herein. In some embodiments, the method further comprises comparing the expression level of miR- 549a in the biological sample from the patient to the expression level of miR-549a in a noncancerous biological sample. [0009] A further aspect of the disclosure relates to a method for predicting a patient's prognosis for survival and/or disease free survival of colorectal cancer comprising: predicting that the patient is likely to survive and or have disease free survival when the expression level of miR-549a in a biological sample from the patient is determined to be not significantly different than the expression level of the non-cancerous biological sample or when the expression level is determined to be a low elevated level of expression compared to a noncancerous sample; or predicting that the patient is not likely to survive or have disease free survival when the expression level of miR-549a in a biological sample from the patient is determined to have a high elevated level of expression compared to a non-cancerous sample. In some embodiments, the patient is predicted to likely survive and or have disease free survival when the expression level of miR-549a in the biological sample from the patient is determined to be a low elevated level of expression. In some embodiments, the method further comprises determining the expression level of miR-549a in a biological sample from the patient. In some embodiments, the method further comprises comparing the expression level of miR-549a in the biological sample from the patient to the expression level of miR- 549a to a control. In some embodiments, the control is a non-cancerous biological sample.

[0010] A further aspect of the current disclosure relates to a biomarker for colorectal cancer cells, wherein the biomarker is an elevated level of expression of miR-549a. In some embodiments, a high expression of miR-549a indicates that the cells are colorectal cancer cells. In some embodiments, the elevated level of expression of miR-549a is relative to a non-cancerous tissue. In some embodiments, a high elevated level of expression of miR- 549a is a biomarker for advanced colorectal cancer and a low elevated level of expression of miR-549a is a biomarker for early colorectal cancer.

[0011] A further aspect of the disclosure relates to a method for treating colorectal cancer in a patient comprising: determining the expression level of miR-549a in tissue adjacent to a surgical site in a colorectal cancer patient and/or in a lymph node of the patient; treating the patient for colorectal cancer after the expression level of miR-549a in tissue and or lymph node of the patient is determined to be higher than the expression level of miR-549a in a noncancerous tissue. In some embodiments, the colorectal cancer comprises advanced colorectal cancer. In some embodiments, the colorectal cancer is early colorectal cancer. In some embodiments, the treatment for advanced colorectal cancer comprises cetuximab, fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumuman, afibercept, leucovorin, and/or radiotherapy. In some embodiments, In some embodiments, the treatment for the advanced colorectal cancer comprises surgical removal of one or more secondary tumors. In some embodiments, the secondary tumor is a distant liver metastasis. In some embodiments, the secondary tumor is a lung metastasis.

[0012] A further aspect of the disclosure relates to a method for diagnosing a patient with advanced or early colorectal cancer comprising: diagnosing the patient as having or likely to have advanced colorectal cancer or providing an analysis or report that the patient has or likely has advanced colorectal cancer when the expression level of miR-549a in the biological sample from the patient is determined to be a high elevated level of expression compared to the expression level in a non-cancerous biological sample; or diagnosing the patient as having or likely to have early colorectal cancer or providing an analysis or report that the patient has or likely has early colorectal cancer when the expression level of miR- 549a in a biological sample from the patient is determined to be a low elevated level of expression compared to the expression level in a non-cancerous biological sample. In some embodiments, the control is a cut-off value. In some embodiments, the method further comprises measuring the expression level of miR-549a in a biological sample from the patient. In some embodiments, the method further comprises comparing the expression level of miR-549a in the biological sample from the patient to a control level of expression. [0013] A further aspect of the disclosure relates to a method for determining whether a biological sample comprises colorectal cancer cells comprising: measuring the expression level of miR-549a in the biological sample; comparing the expression level of miR-549a in the biological sample to the expression level of miR-549a in a non-cancerous biological sample; determining that the biological sample comprises colorectal cancer cells when the expression level of miR-549a in the biological sample is higher than the expression level of miR-549a in the non-cancerous biological sample; and determining that the biological sample is non-cancerous when the expression level of miR-549a in the biological sample not significantly different than the expression level of miR-549a in the non-cancerous biological sample. In some embodiments, the biological sample is tissue adjacent to a surgical site of a colorectal cancer patient. In some embodiments, the biological sample comprises lymph node tissue.

[0014] A further aspect of the disclosure relates to a method for treating colorectal cancer in a subject in need thereof comprising: administering a therapeutically effective amount of an antagonist of miR-549a that reduces or inhibits the expression or activity of miR-549a. In some embodiments, the antagonist is an antagomir of miR-549a. In some embodiments, the subject is one that has been determined to have an increased level of expression of miR-549a in a biological sample from the patient compared to a control level of expression of miR-549a in a non-cancerous biological sample. In some embodiments, the subject has advanced colorectal cancer. In some embodiments, the method further comprises administration of one or more of cetuximab, fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumuman, afibercept, leucovorin, and radiotherapy. In some embodiments, the antagonist is linked to a targeting moiety. In some embodiments, the targeting moiety is an aptamer. In some embodiments, the targeting moiety delivers the antagonist to a specific cell type or tissue. In some embodiments, the cell or tissue is colorectal cells or colorectal tissues. In some embodiments, the cell or tissue is cancerous. In some embodiments, the cell or tissue is metastatic.

[0015] In some embodiments of the above-disclosed aspects, the treatment for advanced colorectal cancer comprises cetuximab, fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumuman, afibercept, leucovorin, and/or radiotherapy. In some embodiments of the above-disclosed aspects, the treatment for early colorectal cancer comprises surgical incision of the primary tumor. In some embodiments of the above-disclosed aspects, the treatment for early colorectal cancer excludes chemotherapy. In some embodiments of the above-disclosed aspects, the treatment for early colorectal cancer excludes one or more of cetuximab, fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumuman, afibercept, leucovorin, and/or radiotherapy. In some embodiments of the above-disclosed aspects, the treatment for the advanced colorectal cancer comprises surgical removal of one or more secondary tumors. In some embodiments of the above-disclosed aspects, the secondary tumor is a distant liver metastasis. In some embodiments of the above-disclosed aspects, the early colorectal cancer treatment excludes surgical removal of one or more secondary tumors.

[0016] In some embodiments of the above-disclosed aspects, a high or low elevated level of expression of miR-549a is determined from a cut-off value, above which defines a high elevated level of expression and below which defines a low level of expression. The cut-off value may be a pre-determined value based on a level of expression of miR-549a that is statistically determined to be associated with a threshold, above which values are associated with advanced colorectal cancer and below which values are associated with early colorectal cancer. Methods of statistically categorizing data based on two variables (high or low elevated expression in association with early or advanced cancer) are known in the art. In some embodiments, the cut-off value is determined by a ROC analysis. In statistics, a receiver operating characteristic (ROC), or ROC curve, is a graphical plot that illustrates the performance of a binary classifier system as its discrimination threshold is varied. ROC curves are further described in the detailed description of the disclosure.

[0017] In some embodiments of the above disclosed aspects, the advanced colorectal cancer comprises category T3 or T4 colorectal cancer. In some embodiments of the above disclosed aspects, the advanced colorectal cancer comprises lymph node metastasis. In some embodiments of the above disclosed aspects, the advanced colorectal cancer comprises category Nl and/or N2 colorectal cancer. In some embodiments of the above disclosed aspects, the advanced colorectal cancer comprises distant metastasis. In some embodiments of the above disclosed aspects, the distant metastasis is liver metastasis. In some embodiments, the distant metastasis is lung metastasis. In some embodiments of the above disclosed aspects, the advanced colorectal cancer comprises category Ml colorectal cancer. In some embodiments of the above disclosed aspects, the advanced colorectal cancer comprises Stage II, Stage III, and/or Stage IV colorectal cancer.

[0018] In some embodiments of the above disclosed aspects, early colorectal cancer comprises Stage I colorectal cancer. In some embodiments of the above disclosed aspects, early colorectal cancer excludes Stage II, III, and IV colorectal cancer. In some embodiments of the above disclosed aspects, early colorectal cancer excludes Stage III and IV colorectal cancer. In some embodiments of the above disclosed aspects, early colorectal cancer comprises category Tl or T2 colorectal cancer. In some embodiments of the above disclosed aspects, early colorectal cancer excludes category T3 and/or T4 colorectal cancer. In some embodiments of the above disclosed aspects, early colorectal cancer comprises category NO colorectal cancer. In some embodiments of the above disclosed aspects, early colorectal cancer comprises category M0 colorectal cancer. In some embodiments of the above disclosed aspects, early colorectal cancer excludes category Ml colorectal cancer.

[0019] In some embodiments of the method aspects described herein, the biological sample from the patient is a sample from a primary colorectal cancer tumor. In some embodiments of the method aspects described herein, the non-cancerous biological sample is a non-cancerous sample from the patient. In some embodiments of the method aspects described herein, the non-cancerous biological sample is normal mucosal tissue. In some embodiments of the method aspects described herein, the biological sample from the patient or the non-cancerous biological sample is from a biological source as described herein. [0020] In further embodiments, the method may comprise determining the expression level of the miRNA molecule. In still further embodiments, the method may comprise obtaining a sample of the subject or patient or obtaining a sample from the subject or patient. Non-limiting examples of the sample include a tissue sample, a whole blood sample, a urine sample, a saliva sample, a serum sample or a fecal sample. In particular embodiments, the sample is a rectum sample, a colon sample or a cecum sample.

[0021] The term subject or patient may refer to an animal (for example a mammal), including but not limited to humans, non-human primates, rodents, dogs, or pigs. The methods of obtaining provided herein include methods of biopsy such as fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy.

[0022] In certain embodiments the sample is obtained from a biopsy from rectal, cecum, or colon tissue by any of the biopsy methods previously mentioned. In other embodiments the sample may be obtained from any of the tissues provided herein that include but are not limited to gall bladder, skin, heart, lung, breast, pancreas, liver, muscle, kidney, smooth muscle, bladder, intestine, brain, prostate, esophagus, or thyroid tissue.

[0023] Alternatively, the sample may include but not be limited to blood, serum, sweat, hair follicle, buccal tissue, tears, menses, urine, feces, or saliva. In particular embodiments, the sample may be a tissue sample, a whole blood sample, a urine sample, a saliva sample, a serum sample, a plasma sample or a fecal sample.

[0024] In certain aspects the sample is obtained from cystic fluid or fluid derived from a tumor or neoplasm. In yet other embodiments the cyst, tumor or neoplasm is in the digestive system. In certain aspects of the current methods, any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing. In further aspects of the current methods, the patient or subject may obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample. [0025] In further embodiments, the sample may be a fresh, frozen or preserved sample or a fine needle aspirate. In particular embodiments, the sample is a formalin-fixed, paraffin- embedded (FFPE) sample. An acquired sample may be placed in short term or long term storage by placing in a suitable medium, excipient, solution, or container. In certain cases storage may require keeping the sample in a refrigerated, or frozen environment. The sample may be quickly frozen prior to storage in a frozen environment. In certain instances the frozen sample may be contacted with a suitable cryopreservation medium or compound. Examples of cryopreservation mediums or compounds include but are not limited to: glycerol, ethylene glycol, sucrose, or glucose.

[0026] Some embodiments further involve isolating nucleic acids such as ribonucleic or RNA from a biological sample or in a sample of the patient. Other steps may or may not include amplifying a nucleic acid in a sample and/or hybridizing one or more probes to an amplified or non-amplified nucleic acid. The methods may further comprise assaying nucleic acids in a sample. In certain embodiments, a microarray may be used to measure or assay the level of miRNA expression in a sample. The methods may further comprise recording the miRNA expression level in a tangible medium or reporting the expression level to the patient, a health care payer, a physician, an insurance agent, or an electronic system. [0027] In some embodiments, methods will involve determining or calculating a prognosis score based on data concerning the expression level of the miRNA, meaning that the expression level of the miRNA is at least one of the factors on which the score is based. A prognosis score will provide information about the patient, such as the general probability whether the patient is sensitive to a particular therapy or has poor survival or high chances of recurrence. In certain embodiments, a prognosis value is expressed as a numerical integer or number that represents a probability of 0% likelihood to 100% likelihood that a patient has a chance of poor survival or cancer recurrence or poor response to a particular treatment.

[0028] In some embodiments, the prognosis scare is expressed as a number that represents a probability of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% likelihood (or any range derivable therein) that a patient has a chance of poor survival or cancer recurrence or poor response to a particular treatment. Alternatively, the probability may be expressed generally in percentiles, quartiles, or deciles.

[0029] A difference between or among weighted coefficients ore expression levels or between or among the weighted comparisons may be, be at least or be at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0. 19.5, 20.0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 410, 420, 425, 430, 440, 441, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000 times or -fold (or any range derivable therein). [0030] In some embodiments, determination of calculation of a diagnostic, prognostic, or risk score is performed by applying classification algorithms based on the expression values of biomarkers with differential expression p values of about, between about, or at most about 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.020, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.03, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.040, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.050, 0.051, 0.052, 0.053, 0.054, 0.055, 0.056, 0.057, 0.058, 0.059, 0.060, 0.061, 0.062, 0.063, 0.064, 0.065, 0.066, 0.067, 0.068, 0.069, 0.070, 0.071, 0.072, 0.073, 0.074, 0.075, 0.076, 0.077, 0.078, 0.079, 0.080, 0.081, 0.082, 0.083, 0.084, 0.085, 0.086, 0.087, 0.088, 0.089, 0.090, 0.091, 0.092, 0.093, 0.094, 0.095, 0.096, 0.097, 0.098, 0.099, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or higher (or any range derivable therein). In certain embodiments, the prognosis score is calculated using one or more statistically significantly differentially expressed biomarkers (either individually or as difference pairs), including expression levels in a gene encoding miR-549a.

[0031] Further aspects relate to kits comprising an agent for detecting miR-549a expression. In some embodiments, the agent comprises one or more nucleic acid probes for amplification of a miR-549a nucleic acid from a biological sample. In some embodiments, the agent is labeled. In some embodiments, the kit further comprises instructions for use.

[0032] Embodiments concern determining that the level of expression of miR-549a. In some embodiments, that level is compared to a control in order to determine whether the expression level or activity of the miR-549a is elevated as compared to the level in noncancerous biological sample. The control may be a non-cancerous colorectal tissue or it may be a cancerous colorectal tissue. If the control is a cancerous tissue, a sample may be determined to have an elevated level of snoRNA because the levels in the control and the patient sample are similar, such as within, at least or at most 1, 2, 3, or 4 standard deviations (or any range derivable therein) of one another.

[0033] Any of the methods described herein may be implemented on tangible computer- readable medium comprising computer-readable code that, when executed by a computer, causes the computer to perform one or more operations. In some embodiments, there is a tangible computer-readable medium comprising computer-readable code that, when executed by a computer, causes the computer to perform operations comprising: a) receiving information corresponding to an expression level of a gene encoding miR-549a in a sample from a patient; and b) determining a difference value in the expression levels using the information corresponding to the expression levels in the sample compared to a control or reference expression level for the gene.

[0034] In other aspects, tangible computer-readable medium further comprise computer- readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising making recommendations comprising: wherein the patient in the step a) is under or after a first treatment for colorectal cancer, administering the same treatment as the first treatment to the patient if the patient does not have increased expression level; administering a different treatment from the first treatment to the patient if the patient has increased expression level. [0035] In some embodiments, receiving information comprises receiving from a tangible data storage device information corresponding to the expression levels from a tangible storage device. In additional embodiments the medium further comprises computer-readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising: sending information corresponding to the difference value to a tangible data storage device, calculating a prognosis score for the patient, treating the patient with a traditional colorectal therapy if the patient does not have expression levels, and/or or treating the patient with an alternative colorectal therapy if the patient has increased expression levels.

[0036] The tangible, computer-readable medium further comprise computer-readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising calculating a prognosis score for the patient. The operations may further comprise making recommendations comprising: administering a treatment comprising a thymidylate synthase inhibitor to a patient that is determined to have a decreased expression level. [0037] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. [0038] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more. [0039] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0040] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS [0041] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0042] FIG. 1: miR expression in :Atsushi cohort." FIG. 1 shows miR-490-3p, miR- 549a, miR-1248, and miR-1273 expression in cancerous tissues and in adjacent normal mucosa. miR-549a and miR-1248 by quantitative real-time PCR (qRT-PCR). The qRT-PCR analyses revealed that miR-549a and miR-1248 expression were significantly up-regulated in CRC tissues (PO.001, Wilcoxon rank test), compared to NM tissues.

[0043] FIG. 2A-2B: miR-549a testing and validation set. Receiver operating characteristic (ROC) curves were used to evaluate the sensitivity and specificity of each miRNA expression in distinguishing CRC from normal tissues. Notably, expression of miR- 549a displayed robust predictive significance, with an area under curve (AUC) values of 0.92 (FIG. 2A) and 0.93 (FIG. 2B).

[0044] FIG. 3A-3B: Overall survival and disease free survival. The expression patterns of miR-549a were analyzed with various clinicopathological factors to determine whether the expression status has any prognostic significance in CRC patients. The expression cut-off thresholds for miR-549a were determined according to receiver operating characteristic analyses with Youden's index to determine overall survival (OS; FIG. 3A) and disease free survival (DFS; FIG. 3B) of CRC patients respectively. MiR-549a high elevated expression was significantly correlated with advanced T-category, lymphatic invasion, lymph node metastasis, distant metastasis, and poor prognosis compared to CRCs in the low elevated expression group in terms of OS and DFS (p=0.0003, 0.022, respectively, log rank test).

[0045] FIG. 4: Overall survival and disease free survival. There was significant correlation of elevated miR-549a expression in UICC stage I, II, III, and IV cancer compared to normal mucosa

[0046] FIG. 5: miR-549a expression analysis in colon cancer cell lines. To investigate whether miR-549a alters the biological characteristics of colon cancer cells, the expression levels of miR-549a were assessed in a panel of colon cancer cell lines (Caco2, HCT116, HT29, LoVo, SW480 and SW620) by real-time PCR. [0047] FIG. 6A-6E: In vitro analysis using miR-549a inhibitor. FIG 6 shows the knock-down (KD) effect (FIG. 6A), proliferation assay - MTT assay (FIG 6B), invasion/migration assay (FIG. 6C), migration scratch assay (FIG. 6D), and anoikis assay (FIG. 6E) on cells treated with a miR-549a inhibitor.

[0048] FIG. 7A-7E: In vitro analysis using miR-549a mimic. FIG 7 shows the overespression (KD) effect (FIG. 7A), proliferation assay - MTT assay (FIG 7B), invasion/migration assay (FIG. 7C), migration scratch assay (FIG. 7D), and anoikis assay (FIG. 7E) on cells treated with a miR-549a mimic.

[0049] FIG. 8: Effect of miR-549a ectopic expression in Caco2 cells on the xenograft model was assessed by evaluating tumour volume. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0050] Certain aspects of the invention provide a test that could assist physicians to select the optimal therapy for a patient from several alternative treatment options. A major clinical challenge in cancer treatment is to identify the subset of patients who will benefit from a therapeutic regimen, both in metastatic and adjuvant settings. The number of anti-cancer drugs and multi-drug combinations has increased substantially in the past decade, however, treatments continue to be applied empirically using a trial-and-error approach. Here methods and compositions are provided to determine the optimal treatment option for cancer patients.

Definitions

[0051] "Prognosis" refers to as a prediction of how a patient will progress, and whether there is a chance of recovery. "Cancer prognosis" generally refers to a forecast or prediction of the probable course or outcome of the cancer, with or without a treatment. As used herein, cancer prognosis includes the forecast or prediction of any one or more of the following: duration of survival of a patient susceptible to or diagnosed with a cancer, duration of recurrence-free survival, duration of progression free survival of a patient susceptible to or diagnosed with a cancer, response rate in a group of patients susceptible to or diagnosed with a cancer, duration of response in a patient or a group of patients susceptible to or diagnosed with a cancer, and/or likelihood of metastasis in a patient susceptible to or diagnosed with a cancer. Prognosis also includes prediction of favorable responses to cancer treatments, such as a conventional cancer therapy. A response may be either a therapeutic response (sensitivity or recurrence-free survival) or a lack of therapeutic response (residual disease, which may indicate resistance or recurrence).

[0052] The term substantially the same or not significantly different refers to a level of expression that is not significantly different than what it is compared to. Alternatively, or in conjunction, the term substantially the same refers to a level of expression that is less than 2, 1.5, or 1.25 fold different than the expression level it is compared to.

[0053] By "subject" or "patient" is meant any single subject for which therapy is desired, including humans, cattle, dogs, guinea pigs, rabbits, chickens, and so on. Also intended to be included as a subject are any subjects involved in clinical research trials not showing any clinical sign of disease, or subjects involved in epidemiological studies, or subjects used as controls.

[0054] The term "disease free survival" is a clinical endpoint and is usually used to analyze the results of the treatment for the localized disease which renders the patient apparently disease free, such as surgery or surgery plus adjuvant therapy. In the disease-free survival, the event is relapse rather than death. The people who relapse are still surviving but they are no longer disease-free. Just as in the survival curves not all patients die, in "disease- free survival curves" not all patients relapse and the curve may have a final plateau representing the patients who didn't relapse after the study's maximum follow-up. Because the patients survive for at least some time after the relapse, the curve for the actual survival would look better than disease free survival curve.

[0055] The term "primer," as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded and/or single-stranded form, although the single-stranded form is preferred.

[0056] As used herein, "increased expression" or "elevated expression" or "decreased expression" refers to an expression level of a biomarker in the subject's sample as compared to a reference level representing the same biomarker or a different biomarker. In certain aspects, the reference level may be a reference level of expression from a non-cancerous tissue from the same subject. Alternatively, the reference level may be a reference level of expression from a different subject or group of subjects. For example, the reference level of expression may be an expression level obtained from a sample (e.g., a tissue, fluid or cell sample) of a subject or group of subjects without cancer, or an expression level obtained from a non-cancerous tissue of a subject or group of subjects with cancer. The reference level may be a single value or may be a range of values. The reference level of expression can be determined using any method known to those of ordinary skill in the art. In some embodiments, the reference level is an average level of expression determined from a cohort of subjects with cancer or without cancer. The reference level may also be depicted graphically as an area on a graph. In certain embodiments, a reference level is a normalized level, while in other embodiments, it may be a level that is not stable with respect to the tissue or biological sample being tested.

[0057] As used herein, "high elevated expression" or "low elevated expression" refers to a level of expression that is elevated as compared to normal non-cancerous tissue, but also refers to a level of elevated expression relative to a control amount. The control amount may be a cut-off value or range, above which defines a high elevated expression level and below which defines a low elevated expression level.

[0058] "About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms "about" and "approximately" may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. In some embodiments it is contemplated that an numerical value discussed herein may be used with the term "about" or "approximately." [0059] The term "aptamer" refers to a synthetic oligonucleotides or peptide molecules that bind to a specific target molecule.

[0060] As used herein, the term "antagomir" refers to a synthetic oligonucleotide or oligonucleotide mimetic having complementarity to a specific microRNA, and which inhibits the activity of that miRNA. In certain embodiments, the antagomir has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide differences (or any derivable range therein) from the miRNA that it inhibits. Further, antagomirs can have the same length, a longer length or a shorter length than the miRNA that it inhibits. In certain embodiments, the antagomir hybridizes to 6-8 nucleotides at the 5' end of the miRNA it inhibits. In other embodiments, an antagomir can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length (or any derivable range therein). In other embodiments, an antagomir can be 5-10, 6-8, 10-20, 10-15 or 5-500 (or any derivable range therein) nucleotides in length. In certain embodiments, antagomirs include nucleotides that are complementary to a miRNA described herein, such as miR549a. The antagomirs are synthetic reverse complements that tightly bind to and inactivate a specific miRNA. Various chemical modifications are used to improve nuclease resistance and binding affinity. The most commonly used modifications to increase potency include various 2 'sugar modifications, such as 2'-0-Me, 2'-0-methoxyethyl (2'-MOE), or 2'- fluoro(2'-F). The nucleic acid structure of the miRNA can also be modified into a locked nucleic acid (LNA) with a methylene bridge between the 2' oxygen and the 4' carbon to lock the ribose in the 3'-endo

[0061] (North) conformation in the A-type conformation of nucleic acids (Lennox KA et al.. Gene Ther. Dec 2011;18(12): 1111-1 120; Bader AG et al. Gene Ther. Dec 2011;18(12): 1121-1126). This modification significantly increases both target specificity and hybridization properties of the molecules. [0062] As used herein, the term "aptamir" refers to the combination of an aptamer (oligonucleic acid or peptide molecule that bind to a specific target molecule) and an antagomir as defined above, which allows cell or tissue-specific delivery of the miRNA agents.

II. MiRNA

[0063] Certain aspects are based, in part, on the systematic discovery and validation of prognostic/predictive miRNA(s) biomarkers in two independent clinical-based cohorts of CRC patients treated by a uniform 5-FU chemotherapy regimen. In certain embodiments, microRNAs (abbreviated miRNAs) may be used in methods and compositions for determining the prognosis, such as response to a particular cancer treatment, of a particular patient. [0064] MiRNAs may be naturally occurring, small non-coding RNAs that are about 17 to about 25 nucleotide bases (nt) in length in their biologically active form. miRNAs post- transcriptionally regulate gene expression by repressing target mRNA translation. It is thought that miRNAs function as negative regulators, i.e. greater amounts of a specific miRNA will correlate with lower levels of target gene expression. [0065] There may be three forms of miRNAs existing in vivo, primary miRNAs (pri- miRNAs), premature miRNAs (pre-miRNAs), and mature miRNAs. Primary miRNAs (pri- miRNAs) are expressed as stem-loop structured transcripts of about a few hundred bases to over 1 kb. The pri -miRNA transcripts are cleaved in the nucleus by an RNase II endonuclease called Drosha that cleaves both strands of the stem near the base of the stem loop. Drosha cleaves the RNA duplex with staggered cuts, leaving a 5' phosphate and 2 nt overhang at the 3' end.

[0066] The cleavage product, the premature miRNA (pre-miRNA) may be about 60 to about 110 nt long with a hairpin structure formed in a fold-back manner. Pre-miRNA is transported from the nucleus to the cytoplasm by Ran-GTP and Exportin-5. Pre-miRNAs are processed further in the cytoplasm by another RNase II endonuclease called Dicer. Dicer recognizes the 5' phosphate and 3' overhang, and cleaves the loop off at the stem-loop junction to form miRNA duplexes. The miRNA duplex binds to the RNA-induced silencing complex (RISC), where the antisense strand is preferentially degraded and the sense strand mature miRNA directs RISC to its target site. It is the mature miRNA that is the biologically active form of the miRNA and is about 17 to about 25 nt in length.

[0067] MicroRNAs function by engaging in base pairing (perfect or imperfect) with specific sequences in their target genes' messages (mRNA). The miRNA degrades or represses translation of the mRNA, causing the target genes' expression to be post- transcriptionally down-regulated, repressed, or silenced. In animals, miRNAs do not necessarily have perfect homologies to their target sites, and partial homologies lead to translational repression, whereas in plants, where miRNAs tend to show complete homologies to the target sites, degradation of the message (mRNA) prevails.

[0068] MicroRNAs are widely distributed in the genome, dominate gene regulation, and actively participate in many physiological and pathological processes. For example, the regulatory modality of certain miRNAs is found to control cell proliferation, differentiation, and apoptosis; and abnormal miRNA profiles are associated with oncogenesis. Additionally, it is suggested that viral infection causes an increase in miRNAs targeted to silence "pro-cell survival" genes, and a decrease in miRNAs repressing genes associated with apoptosis (programmed cell death), thus tilting the balance toward gaining apoptosis signaling.

[0069] In other embodiments of the invention, there are synthetic nucleic acids that are miRNA inhibitors or antagonists. In some embodiments, the miRNA inhibitor or antagonist is an antagomir. A miRNA inhibitor is between about 17 to 25 nucleotides in length and comprises a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of a mature miRNA. In certain embodiments, a miRNA inhibitor molecule is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. Moreover, a miRNA inhibitor has a sequence (from 5' to 3') that is or is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary, or any range derivable therein, to the 5' to 3' sequence of a mature miRNA, particularly a mature, naturally occurring miRNA. One of skill in the art could use a portion of the probe sequence that is complementary to the sequence of a mature miRNA as the sequence for a miRNA inhibitor. Moreover, that portion of the probe sequence can be altered so that it is still 90% complementary to the sequence of a mature miRNA.

[0070] In certain embodiments, a synthetic miRNA has one or more modified nucleic acid residues. In certain embodiments, the sugar modification is a 2'O-Me modification, a 2'F modification , a 2Ή modification, a 2'amino modification, a 4'ribose modification, or a phosphorothioate modification on the carboxy group linked to the carbon at position 6. In further embodiments, there is one or more sugar modifications in the first or last 2 to 4 residues of the complementary region or the first or last 4 to 6 residues of the complementary region. [0071] Yet further, the nucleic acid structure of the miRNA can also be modified into a locked nucleic acid (LNA) with a methylene bridge between the 2 Oxygen and the 4' carbon to lock the ribose in the 3'-endo (North) conformation in the A- type conformation of nucleic acids (Lennox, et al, 2011; Bader, et al 2011). This modification significantly increases both target specificity and hybridization properties of the molecules.

[0072] The miRNA region and the complementary region may be on the same or separate polynucleotides. In cases in which they are contained on or in the same polynucleotide, the miRNA molecule will be considered a single polynucleotide. In embodiments in which the different regions are on separate polynucleotides, the synthetic miRNA will be considered to be comprised of two polynucleotides.

[0073] When the RNA molecule is a single polynucleotide, there is a linker region between the miRNA region and the complementary region. In some embodiments, the single polynucleotide is capable of forming a hairpin loop structure as a result of bonding between the miRNA region and the complementary region. The linker constitutes the hairpin loop. It is contemplated that in some embodiments, the linker region is, is at least, or is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 residues in length, or any range derivable therein. In certain embodiments, the linker is between 3 and 30 residues (inclusive) in length.

[0074] In addition to having a miRNA region and a complementary region, there may be flanking sequences as well at either the 5' or 3' end of the region. In some embodiments, there is or is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides or more, or any range derivable therein, flanking one or both sides of these regions.

[0075] Other miRNA-based therapies that negatively manipulate oncogenic miRNAs', may include further include miRNA sponges, miRNA masks or locked nucleic acid (LNA). As used herein, the term "miRNA sponge" refers to a synthetic nucleic acid (e.g. a mRNA transcript) that contains multiple tandem-binding sites for a miRNA of interest, and that serves to titrate out the endogenous miRNA of interest, thus inhibiting the binding of the miRNA of interest to its endogenous targets.

[0076] Methods in certain aspects include reducing, eliminating, or inhibiting activity and/or expression of one or more miRNAs in a cell comprising introducing into a cell a miRNA inhibitor, antagonist, or antagomir; or supplying or enhancing the activity of one or more miRNAs in a cell. Certain embodiments also concern inducing certain cellular characteristics by providing to a cell a particular nucleic acid, such as a specific synthetic miRNA molecule or a synthetic miRNA inhibitor molecule. However, in methods of the invention, the miRNA molecule or miRNA inhibitor need not be synthetic. They may have a sequence that is identical to a naturally occurring miRNA or they may not have any design modifications. In certain embodiments, the miRNA molecule and/or a miRNA inhibitor are synthetic, as discussed above.

III. Colorectal Cancer Staging and Treatments

[0077] Methods and compositions may be provided for treating colorectal cancer with particular applications of miRNA expression levels. Based on a profile of miRNA expression levels, for example, the miR549a, different treatments may be prescribed or recommended for different cancer patients.

Cancer staging

[0078] Colorectal cancer, also known as colon cancer, rectal cancer, or bowel cancer, is a cancer from uncontrolled cell growth in the colon or rectum (parts of the large intestine), or in the appendix. Certain aspects of the methods are provided for patients that are stage II-IV colorectal cancer patients. In particular aspects, the patient is a stage IV patient.

[0079] The most common staging system is the TNM (for tumors/nodes/metastases) system, from the American Joint Committee on Cancer (AJCC). The TNM system assigns a number based on three categories. "T" denotes the degree of invasion of the intestinal wall, "N" the degree of lymphatic node involvement, and "M" the degree of metastasis. The broader stage of a cancer is usually quoted as a number I, II, III, IV derived from the TNM value grouped by prognosis; a higher number indicates a more advanced cancer and likely a worse outcome. Details of this system are in the graph below:

AJCC TNM stage TNM stage criteria for colorectal cancer stage

Stage 0 Tis NO MO Tis: Tumor confined to mucosa; cancer-in-situ

Stage I Tl NO MO Tl : Tumor invades submucosa

Stage I T2 NO MO T2: Tumor invades muscularis propria

Stage II-A T3 NO MO T3 : Tumor invades subserosa or beyond (without other organs involved)

Stage II-B T4 NO MO T4: Tumor invades adjacent organs or perforates the visceral peritoneum

Stage III-A T1-2 N1 MO Nl : Metastasis to 1 to 3 regional lymph nodes. Tl or T2.

Stage III-B T3-4 Nl MO Nl : Metastasis to 1 to 3 regional lymph nodes. T3 or T4.

Stage III-C any T, N2 MO N2: Metastasis to 4 or more regional lymph nodes. Any T.

Stage IV any T, any N, Ml : Distant metastases present. Any T, any N.

Ml

B. Therapy

[0080] For people with localized and/or early colorectal cancer, the preferred treatment is complete surgical removal with adequate margins, with the attempt of achieving a cure. This can either be done by an open laparotomy or sometimes laparoscopically. Sometimes chemotherapy is used before surgery to shrink the cancer before attempting to remove it (neoadjuvant therapy). The two most common sites of recurrence of colorectal cancer is in the liver and lungs. In some embodiments, the treatment of early colorectal cancer excludes chemotherapy. In further embodiments, the treatment of early colorectal cancer includes neoadjuvant therapy (chemotherapy or radiotherapy before the surgical removal of the primary tumor), but excludes adjuvant therapy (chemotherapy and/or radiotherapy after surgical removal of the primary tumor.

[0081] In both cancer of the colon and rectum, chemotherapy may be used in addition to surgery in certain cases. In rectal cancer, chemotherapy may be used in the neoadjuvant setting.

[0082] In certain embodiments, there may be a decision regarding the therapeutic treatment based on the miRNA expression. Chemotherapy based on antimetabolites or thymidylate synthase inhibitors such as fluorouracil (5-FU) have been the main treatment for metastatic colorectal cancer. Major progress has been made by the introduction of regimens containing new cytotoxic drugs, such as irinotecan or oxaliplatin. The combinations commonly used, e.g., irinotecan, fluorouracil, and Jeucovorin (FOLFIRI) and oxaliplatin, fluorouracil, and leucovorin (FOLFOX) can reach an objective response rate of about 50% . However, these new combinations remain inactive in one half of the patients and, in addition, resistance to treatment appear in almost all patients who were initially responders. More recently, two monoclonal antibodies targeting vascular endothelial growth factor Avastin® (bevacizumab) (Genentech Inc., South San Francisco CA) and epidermal growth factor receptor Erbitux®(cetuximab) (Imclone Inc. New York City) have been approved for treatment of metastatic colorectal cancer but are always used in combination with standard chemotherapy regimens. In some embodiments, the cancer therapy may include one or more of the chemical therapeutic agents including thymidylate synthase inhibitors or antimetabolites such as fluorouracil (5-FU), alone or in combination with other therapeutic agents.

[0083] For example, in some embodiments, the first treatment to be tested for response therapy may be antimetabolites or thymidylate synthase inhibitors, prodrugs, or salts thereof. In some embodiments, this treatment regimen is for advanced cancer. In some embodiments, this treatment regimen is excluded for early cancer. [0084] Antimetabolites can be used in cancer treatment, as they interfere with DNA production and therefore cell division and the growth of tumors. Because cancer cells spend more time dividing than other cells, inhibiting cell division harms tumor cells more than other cells. Anti-metabolites masquerade as a purine (azathioprine, mercaptopurine) or a pyrimidine, chemicals that become the building-blocks of DNA. They prevent these substances becoming incorporated in to DNA during the S phase (of the cell cycle), stopping normal development and division. They also affect RNA synthesis. However, because thymidine is used in DNA but not in RNA (where uracil is used instead), inhibition of thymidine synthesis via thymidylate synthase selectively inhibits DNA synthesis over RNA synthesis. Due to their efficiency, these drugs are the most widely used cytostatics. In the ATC system, they are classified under L01B. In some embodiments, this treatment regimen is for advanced cancer. In some embodiments, this treatment regimen is excluded for early cancer.

[0085] Thymidylate synthase inhibitors are chemical agents which inhibit the enzyme thymidylate synthase and have potential as an anticancer chemotherapy. As an anti-cancer chemotherapy target, thymidylate synthetase can be inhibited by the thymidylate synthase inhibitors such as fluorinated pyrimidine fluorouracil, or certain folate analogues, the most notable one being raltitrexed (trade name Tomudex). Five agents were in clinical trials in 2002: raltitrexed, pemetrexed, nolatrexed, ZD9331, and GS7904L. Additional non-limiting examples include: Raltitrexed, used for colorectal cancer since 1998; Fluorouracil, used for colorectal cancer; BGC 945; OSI-7904L. In some embodiments, this treatment regimen is for advanced cancer. In some embodiments, this treatment regimen is excluded for early cancer. [0086] In further embodiments, there may be involved prodrugs that can be converted to thymidylate synthase inhibitors in the body, such as Capecitabine (INN), an orally- administered chemotherapeutic agent used in the treatment of numerous cancers. Capecitabine is a prodrug, that is enzymatically converted to 5-fluorouracil in the body. In some embodiments, this treatment regimen is for advanced cancer. In some embodiments, this treatment regimen is excluded for early cancer.

[0087] If cancer has entered the lymph nodes, adding the chemotherapy agents fluorouracil or capecitabine increases life expectancy. If the lymph nodes do not contain cancer, the benefits of chemotherapy are controversial. If the cancer is widely metastatic or unresectable, treatment is then palliative. For example, a number of different chemotherapy medications may be used. Chemotherapy agents for this condition may include capecitabine, fluorouracil, irinotecan, leucovorin, oxaliplatin and UFT. Another type of agent that is sometimes used are the epidermal growth factor receptor inhibitors. In some embodiments, this treatment regimen is for advanced cancer. In some embodiments, this treatment regimen is excluded for early cancer. [0088] In certain embodiments, alternative treatments may be prescribed or recommended based on the biomarker profile. In addition to traditional chemotherapy for colorectal cancer patients, cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments. Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing. In some embodiments, treatment with one or more of the compounds described herein is for advanced cancer. In some embodiments, treatment with one or more of the compounds described herein is excluded for early cancer. [0089] While a combination of radiation and chemotherapy may be useful for rectal cancer, its use in colon cancer is not routine due to the sensitivity of the bowels to radiation. Just as for chemotherapy, radiotherapy can be used in the neoadjuvant and adjuvant setting for some stages of rectal cancer. . In some embodiments, this treatment regimen is for advanced cancer. In some embodiments, this treatment regimen is excluded for early cancer.

[0090] In people with incurable colorectal cancer, treatment options including palliative care can be considered for improving quality of life. Surgical options may include noncurative surgical removal of some of the cancer tissue, bypassing part of the intestines, or stent placement. These procedures can be considered to improve symptoms and reduce complications such as bleeding from the tumor, abdominal pain and intestinal obstruction. Non-operative methods of symptomatic treatment include radiation therapy to decrease tumor size as well as pain medications. In some embodiments, this treatment regimen is for advanced cancer. In some embodiments, this treatment regimen is excluded for early cancer.

[0091] Immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. In some embodiments, this treatment regimen is for advanced cancer. In some embodiments, this treatment regimen is excluded for early cancer.

[0092] Generally, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55. Markers described herein may be used in the context of the current claims for the purposes of developing a targeting moiety. For example, the targeting moiety may be one that binds the tumor marker. In some embodiments, the targeting moiety is an antibody. In further embodiments, the targeting moiety is an aptamer or aptamir. [0093] In yet another embodiment, the treatment is a gene therapy. In certain embodiments, the therapeutic gene is a tumor suppressor gene. A tumor suppressor gene is a gene that, when present in a cell, reduces the tumorigenicity, malignancy, or hyperproliferative phenotype of the cell. This definition includes both the full length nucleic acid sequence of the tumor suppressor gene, as well as non-full length sequences of any length derived from the full length sequences. It being further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell. Examples of tumor suppressor nucleic acids within this definition include, but are not limited to APC, CYLD, HIN-I, KRAS2b, plo, pl9, p21, p27, p27mt, p53, p57, p73, PTEN, Rb, Uteroglobin, Skp2, BRCA-I, BRCA-2, CHK2, CDKN2A, DCC, DPC4, MADR2/JV18, MEM, MEN2, MTS1, NF1, F2, VHL, WRN, WTl, CFTR, C-CAM, CTS-I, zacl, scFV, MMACl, FCC, MCC, Gene 26 (CACNA2D2), PL6, Beta* (BUT), Luca-1 (HYALl), Luca-2 (HYAL2), 123F2 (RASSF1), 101F6, Gene 21 (NPRL2), or a gene encoding a SEM A3 polypeptide and FUS1. Other exemplary tumor suppressor genes are described in a database of tumor suppressor genes at www.cise.ufl.edu/~yyl/HTML-TSGDB/Homepage.litml. This database is herein specifically incorporated by reference into this and all other sections of the present application. Nucleic acids encoding tumor suppressor genes, as discussed above, include tumor suppressor genes, or nucleic acids derived therefrom {e.g., cDNAs, cRNAs, mRNAs, and subsequences thereof encoding active fragments of the respective tumor suppressor amino acid sequences), as well as vectors comprising these sequences. One of ordinary skill in the art would be familiar with tumor suppressor genes that can be applied.

C. ROC analysis

[0094] In statistics, a receiver operating characteristic (ROC), or ROC curve, is a graphical plot that illustrates the performance of a binary classifier system as its discrimination threshold is varied. The curve is created by plotting the true positive rate against the false positive rate at various threshold settings. (The true-positive rate is also known as sensitivity in biomedical informatics, or recall in machine learning. The false- positive rate is also known as the fall-out and can be calculated as 1 - specificity). The ROC curve is thus the sensitivity as a function of fall-out. In general, if the probability distributions for both detection and false alarm are known, the ROC curve can be generated by plotting the cumulative distribution function (area under the probability distribution from -infinity to + infinity) of the detection probability in the y-axis versus the cumulative distribution function of the false-alarm probability in x-axis.

[0095] ROC analysis provides tools to select possibly optimal models and to discard suboptimal ones independently from (and prior to specifying) the cost context or the class distribution. ROC analysis is related in a direct and natural way to cost/benefit analysis of diagnostic decision making.

[0096] The ROC curve was first developed by electrical engineers and radar engineers during World War II for detecting enemy objects in battlefields and was soon introduced to psychology to account for perceptual detection of stimuli. ROC analysis since then has been used in medicine, radiology, biometrics, and other areas for many decades and is increasingly used in machine learning and data mining research.

[0097] The ROC is also known as a relative operating characteristic curve, because it is a comparison of two operating characteristics (TPR and FPR) as the criterion changes. ROC analysis curves are known in the art and described in Metz CE (1978) Basic principles of ROC analysis. Seminars in Nuclear Medicine 8:283-298; Youden WJ (1950) An index for rating diagnostic tests. Cancer 3 :32-35; Zweig MH, Campbell G (1993) Receiver-operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine. Clinical Chemistry 39:561-577; and Greiner M, Pfeiffer D, Smith RD (2000) Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests. Preventive Veterinary Medicine 45:23-41, which are herein incorporated by reference in their entirety.

D. Monitoring

[0098] In certain aspects, the biomarker-based method may be combined with one or more other colon cancer diagnosis or screening tests at increased frequency if the patient is determined to be at high risk for recurrence or have a poor prognosis based on the biomarker described above.

[0099] The colon monitoring may include any methods known in the art. In particular, the monitoring include obtaining a sample and testing the sample for diagnosis. For example, the colon monitoring may include colonoscopy or coloscopy, which is the endoscopic examination of the large bowel and the distal part of the small bowel with a CCD camera or a fiber optic camera on a flexible tube passed through the anus. It can provide a visual diagnosis (e.g. ulceration, polyps) and grants the opportunity for biopsy or removal of suspected colorectal cancer lesions. Thus, colonoscopy or coloscopy can be used for treatment.

[0100] In further aspects, the monitoring diagnosis may include sigmoidoscopy, which is similar to colonoscopy— the difference being related to which parts of the colon each can examine. A colonoscopy allows an examination of the entire colon (1200-1500 mm in length). A sigmoidoscopy allows an examination of the distal portion (about 600 mm) of the colon, which may be sufficient because benefits to cancer survival of colonoscopy have been limited to the detection of lesions in the distal portion of the colon. A sigmoidoscopy is often used as a screening procedure for a full colonoscopy, often done in conjunction with a fecal occult blood test (FOBT). About 5% of these screened patients are referred to colonoscopy.

[0101] In additional aspects, the monitoring diagnosis may include virtual colonoscopy, which uses 2D and 3D imagery reconstructed from computed tomography (CT) scans or from nuclear magnetic resonance (MR) scans, as a totally non-invasive medical test.

[0102] The monitoring include the use of one or more screening tests for colon cancer including, but not limited to fecal occult blood testing, flexible sigmoidoscopy and colonoscopy. Of the three, only sigmoidoscopy cannot screen the right side of the colon where 42% of malignancies are found. Virtual colonoscopy via a CT scan appears as good as standard colonoscopy for detecting cancers and large adenomas but is expensive, associated with radiation exposure, and cannot remove any detected abnormal growths like standard colonoscopy can. Fecal occult blood testing (FOBT) of the stool is typically recommended every two years and can be either guaiac based or immunochemical. Annual FOBT screening results in a 16% relative risk reduction in colorectal cancer mortality, but no difference in all- cause mortality. The M2-PK test identifies an enzyme in colorectal cancers and polyps rather than blood in the stool. It does not require any special preparation prior to testing. M2-PK is sensitive for colorectal cancer and polyps and is able to detect bleeding and non-bleeding colorectal cancer and polyps. In the event of a positive result people would be asked to undergo further examination e.g. colonoscopy.

IV. Sample Preparation

[0103] In certain aspects, methods involve obtaining a sample from a subject. The methods of obtaining provided herein may include methods of biopsy such as fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy. In certain embodiments the sample is obtained from a biopsy from colorectal tissue by any of the biopsy methods previously mentioned. In other embodiments the sample may be obtained from any of the tissues provided herein that include but are not limited to non-cancerous or cancerous tissue and non-cancerous or cancerous tissue from the serum, gall bladder, mucosal, skin, heart, lung, breast, pancreas, blood, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, or thyroid tissue. Alternatively, the sample may be obtained from any other source including but not limited to blood, sweat, hair follicle, buccal tissue, tears, menses, feces, or saliva. In certain aspects the sample is obtained from cystic fluid or fluid derived from a tumor or neoplasm. In yet other embodiments the cyst, tumor or neoplasm is colorectal. In certain aspects of the current methods, any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing. Yet further, the biological sample can be obtained without the assistance of a medical professional.

[0104] A sample may include but is not limited to, tissue, cells, or biological material from cells or derived from cells of a subject. The biological sample may be a heterogeneous or homogeneous population of cells or tissues. The biological sample may be obtained using any method known to the art that can provide a sample suitable for the analytical methods described herein. The sample may be obtained by non-invasive methods including but not limited to: scraping of the skin or cervix, swabbing of the cheek, saliva collection, urine collection, feces collection, collection of menses, tears, or semen. [0105] The sample may be obtained by methods known in the art. In certain embodiments the samples are obtained by biopsy. In other embodiments the sample is obtained by swabbing, scraping, phlebotomy, or any other methods known in the art. In some cases, the sample may be obtained, stored, or transported using components of a kit of the present methods. In some cases, multiple samples, such as multiple colorectal samples may be obtained for diagnosis by the methods described herein. In other cases, multiple samples, such as one or more samples from one tissue type (for example colon) and one or more samples from another tissue (for example buccal) may be obtained for diagnosis by the methods. In some cases, multiple samples such as one or more samples from one tissue type (e.g. rectal) and one or more samples from another tissue (e.g. cecum) may be obtained at the same or different times. Samples may be obtained at different times are stored and/or analyzed by different methods. For example, a sample may be obtained and analyzed by routine staining methods or any other cytological analysis methods. [0106] In some embodiments the biological sample may be obtained by a physician, nurse, or other medical professional such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or a pulmonologist. The medical professional may indicate the appropriate test or assay to perform on the sample. In certain aspects a molecular profiling business may consult on which assays or tests are most appropriately indicated. In further aspects of the current methods, the patient or subject may obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.

[0107] In other cases, the sample is obtained by an invasive procedure including but not limited to: biopsy, needle aspiration, or phlebotomy. The method of needle aspiration may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy. In some embodiments, multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material.

[0108] General methods for obtaining biological samples are also known in the art. Publications such as Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001, which is herein incorporated by reference in its entirety, describes general methods for biopsy and cytological methods. In one embodiment, the sample is a fine needle aspirate of a colorectal or a suspected colorectal tumor or neoplasm. In some cases, the fine needle aspirate sampling procedure may be guided by the use of an ultrasound, X-ray, or other imaging device.

[0109] In some embodiments of the present methods, the molecular profiling business may obtain the biological sample from a subject directly, from a medical professional, from a third party, or from a kit provided by a molecular profiling business or a third party. In some cases, the biological sample may be obtained by the molecular profiling business after the subject, a medical professional, or a third party acquires and sends the biological sample to the molecular profiling business. In some cases, the molecular profiling business may provide suitable containers, and excipients for storage and transport of the biological sample to the molecular profiling business.

[0110] In some embodiments of the methods described herein, a medical professional need not be involved in the initial diagnosis or sample acquisition. An individual may alternatively obtain a sample through the use of an over the counter (OTC) kit. An OTC kit may contain a means for obtaining said sample as described herein, a means for storing said sample for inspection, and instructions for proper use of the kit. In some cases, molecular profiling services are included in the price for purchase of the kit. In other cases, the molecular profiling services are billed separately. A sample suitable for use by the molecular profiling business may be any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of an individual to be tested. Methods for determining sample suitability and/or adequacy are provided.

[0111] In some embodiments, the subject may be referred to a specialist such as an oncologist, surgeon, or endocrinologist. The specialist may likewise obtain a biological sample for testing or refer the individual to a testing center or laboratory for submission of the biological sample. In some cases the medical professional may refer the subject to a testing center or laboratory for submission of the biological sample. In other cases, the subject may provide the sample. In some cases, a molecular profiling business may obtain the sample. V. Nucleic Acid Assays

[0112] Aspects of the methods include assaying nucleic acids to determine expression levels. Arrays can be used to detect differences between two samples. Specifically contemplated applications include identifying and/or quantifying differences between miRNA from a sample that is normal and from a sample that is not normal, between a cancerous condition and a non-cancerous condition, or between two differently treated samples. Also, miRNA may be compared between a sample believed to be susceptible to a particular disease or condition and one believed to be not susceptible or resistant to that disease or condition. A sample that is not normal is one exhibiting phenotypic trait(s) of a disease or condition or one believed to be not normal with respect to that disease or condition. It may be compared to a cell that is normal with respect to that disease or condition. Phenotypic traits include symptoms of, or susceptibility to, a disease or condition of which a component is or may or may not be genetic or caused by a hyperproliferative or neoplastic cell or cells.

[0113] An array comprises a solid support with nucleic acid probes attached to the support. Arrays typically comprise a plurality of different nucleic acid probes that are coupled to a surface of a substrate in different, known locations. These arrays, also described as "microarrays" or colloquially "chips" have been generally described in the art, for example, U.S. Pat. Nos. 5, 143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424, 186 and Fodor et al., 1991), each of which is incorporated by reference in its entirety for all purposes. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261, incorporated herein by reference in its entirety for all purposes. Although a planar array surface is used in certain aspects, the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Pat. Nos. 5,770,358, 5,789, 162, 5,708,153, 6,040, 193 and 5,800,992, which are hereby incorporated in their entirety for all purposes. [0114] In addition to the use of arrays and microarrays, it is contemplated that a number of difference assays could be employed to analyze miRNAs, their activities, and their effects. Such assays include, but are not limited to, nucleic amplification, polymerase chain reaction, quantitative PCR, RT-PCR, in situ hybridization, Northern hybridization, hybridization protection assay (HPA)(GenProbe), branched DNA (bDNA) assay (Chiron), rolling circle amplification (RCA), single molecule hybridization detection (US Genomics), Invader assay (ThirdWave Technologies), and/or Bridge Litigation Assay (Genaco).

VI. Pharmaceutical Compositions

[0115] In certain aspects, the compositions or agents for use in the methods, such as chemotherapeutic agents or miR-549a inhibitors, are suitably contained in a pharmaceutically acceptable carrier. The carrier is non-toxic, biocompatible and is selected so as not to detrimentally affect the biological activity of the agent. The agents in some aspects of the invention may be formulated into preparations for local delivery (i.e. to a specific location of the body, such as skeletal muscle or other tissue) or systemic delivery, in solid, semi-solid, gel, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants and injections allowing for oral, parenteral or surgical administration. Certain aspects of the invention also contemplate local administration of the compositions by coating medical devices and the like.

[0116] Suitable carriers for parenteral delivery via injectable, infusion or irrigation and topical delivery include distilled water, physiological phosphate-buffered saline, normal or lactated Ringer's solutions, dextrose solution, Hank's solution, or propanediol. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose any biocompatible oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The carrier and agent may be compounded as a liquid, suspension, polymerizable or non-polymerizable gel, paste or salve.

[0117] The carrier may also comprise a delivery vehicle to sustain (i.e., extend, delay or regulate) the delivery of the agent(s) or to enhance the delivery, uptake, stability or pharmacokinetics of the therapeutic agent(s). Such a delivery vehicle may include, by way of non-limiting examples, microparticles, microspheres, nanospheres or nanoparticles composed of proteins, liposomes, carbohydrates, synthetic organic compounds, inorganic compounds, polymeric or copolymeric hydrogels and polymeric micelles. [0118] In certain aspects, the actual dosage amount of a composition administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0119] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active agent, such as an isolated exosome, a related lipid nanovesicle, or an exosome or nanovesicle loaded with therapeutic agents or diagnostic agents. In other embodiments, the active agent may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 microgram/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered. [0120] Solutions of pharmaceutical compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0121] In certain aspects, the pharmaceutical compositions are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg or less, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.

[0122] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antgifungal agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well-known parameters.

[0123] Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

[0124] In further aspects, the pharmaceutical compositions may include classic pharmaceutical preparations. Administration of pharmaceutical compositions according to certain aspects may be via any common route so long as the target tissue is available via that route. This may include oral, nasal, buccal, rectal, vaginal or topical. Topical administration may be particularly advantageous for the treatment of skin cancers, to prevent chemotherapy- induced alopecia or other dermal hyperproliferative disorder. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For treatment of conditions of the lungs, aerosol delivery can be used. Volume of the aerosol is between about 0.01 ml and 0.5 ml. [0125] An effective amount of the pharmaceutical composition is determined based on the intended goal. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the pharmaceutical composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.

[0126] Precise amounts of the pharmaceutical composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment {e.g. , alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.

VII. Kits

[0127] Certain aspects of the present invention also concern kits containing compositions of the invention or compositions to implement methods of the invention. In some embodiments, kits can be used to evaluate one or more miRNA molecules. In certain embodiments, a kit contains, contains at least or contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more miRNA probes, synthetic miRNA molecules or miRNA inhibitors, or any value or range and combination derivable therein. In some embodiments, there are kits for evaluating miRNA activity in a cell.

[0128] Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.

[0129] Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more. [0130] Kits for using miRNA probes, synthetic miRNAs, nonsynthetic miRNAs, and/or miRNA inhibitors of the invention for prognostic or diagnostic applications are included as part of the invention. Specifically contemplated are any such molecules corresponding to any miRNA identified herein. [0131] In certain aspects, negative and/or positive control synthetic miRNAs and/or miRNA inhibitors are included in some kit embodiments. The control molecules can be used to verify transfection efficiency and/or control for transfection-induced changes in cells.

[0132] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined. It is specifically contemplated that any methods and compositions discussed herein with respect to miRNA molecules or miRNA may be implemented with respect to synthetic miRNAs to the extent the synthetic miRNA is exposed to the proper conditions to allow it to become a mature miRNA under physiological circumstances. The claims originally filed are contemplated to cover claims that are multiply dependent on any filed claim or combination of filed claims.

[0133] Any embodiment of the invention involving specific miRNAs by name is contemplated also to cover embodiments involving miRNAs whose sequences are at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to the mature sequence of the specified miRNA. [0134] Embodiments of the invention include kits for analysis of a pathological sample by assessing miRNA profile for a sample comprising, in suitable container means, two or more miRNA probes, wherein the miRNA probes detect one or more of the miRNA identified herein. The kit can further comprise reagents for labeling miRNA in the sample. The kit may also include labeling reagents, including at least one of amine-modified nucleotide, poly(A) polymerase, and poly(A) polymerase buffer. Labeling reagents can include an amine-reactive dye.

VIII. Examples

[0135] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 - Identification of novel colorectal cancer-specific miRNA Biomarkers using next-generation sequencing

[0136] Despite progresses made with regards to earlier diagnosis and treatment, prognosis in patients with colorectal cancer (CRC) remains poor and CRC still ranks as the second leading cause of cancer-related deaths in the US. A better understanding of the mechanisms underlying CRC pathogenesis is required for developing clinically actionable diagnostic and prognostic biomarkers for CRC. MicroRNAs (miRNAs) are small non-coding RNAs, which play a central role in CRC pathogenesis. However, previous studies aimed at developing miRNA-based biomarkers, have unsuccessfully relied upon the use of microarray-based technologies. Next generation sequencing (NGS) has emerged as a powerful tool for enabling determination of the identity and abundance of whole transcripts, and has thus allowed discovery of novel, cancer-specific miRNA biomarkers.

[0137] The aim of this study was to perform a systematic and comprehensive analysis using NGS-based RNA-Seq to identify CRC-specific miRNAs which have the potential as diagnostic/prognostic biomarkers in CRC.

[0138] CRC-specific miRNA profiles were generated by NGS using 8 pairs of matched CRC and adjacent normal mucosa (NM) tissues. To further confirm the robustness of the miRNA signature discovered by NGS, these results were compared with an independent cohort of miRNA-microarray based expression profiles from 74 colorectal tissues (54 CRCs and 20 NMs). Quantitative RT-PCR analysis was performed for analytical and clinical validation of newly discovered miRNAs in an independent collection of 239 colorectal tissues from two independent cohorts. Knockdown or overexpression of miR-549 was performed for functional validation of miR-549 in CRC cells.

[0139] NGS analysis combined with microarray-based profiles identified 7 CRC- specific miRNAs in the discovery phase. Among these, expression status of miR-549 was significantly elevated in CRC tissues and robustly discriminated CRC tissues from normal mucosa in the testing cohort (AUC = 0.92). In the validation set, expression of miR-549 was successfully validated with an even higher AUC value (AUC = 0.94). Furthermore, increased miR-549 expression was significantly correlated with poor overall and disease free survival and TNM classification for CRC. Functional analysis revealed that miR-549 was involved in cell proliferation, migration, invasion and anoikis resistance in CRC. Applicants are currently exploring novel downstream target genes of miR-549 to gain a better understanding of the role of miR-549 in CRC. Based upon this one of a kind, systematic and comprehensive biomarker study, Applicants successfully discovered a novel CRC-specific miRNA using NGS-based profiling. MiR-549 appears to play an important role in CRC development, and has the potential to serve as novel biomarker for the diagnosis and prognosis in CRC patients.

MATERIAL AND METHODS [0140] Cell Lines: Human colon cancer cell lines Caco2, HCT116, HT29, LoVo,

SW480, and SW620 were obtained from the American Type Culture Collection (ATCC, Rockville, Maryland, USA). These cell lines were tested and authenticated using a panel of genetic and epigenetic markers on a regular basis. All cell lines were maintained in Iscove's modified Dulbecco's medium (IMDM) (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) and antibiotics at 37°C in a 5% humidified C0₂ atmosphere.

[0141] Patients and Sample Collection: This study included examination of 321 tissue specimens including 257 primary CRC (pCRC) tissues, and 44 matched corresponding normal mucosa (NM) tissues, from 4 different CRC patient cohorts that were enrolled in this study. Twenty-normal colonic tissues in microarray analysis were obtained from individuals undergoing colonic surgery for reasons other than cancer (i.e., diverticulosis), showing microscopically normal mucosa. The study design included an initial discovery phase, followed by a subsequent validation phase and a clinical evaluation phase.

[0142] Discovery of dysregulated miRNAs in CRC tissues using NGS: TruSeq miRNA libraries generated from CRC and NM tissues were sequenced using an Illumina HiSeq 2000 sequencer with single end read length of 50 bases, following the manufacturer's instructions. For the computational analysis of Illumina' s small RNA-seq data, raw sequencing reads were subjected to quality filters as described previously (Fehniger TA, Wylie T, Germino E, et al. Next-generation sequencing identifies the natural killer cell microRNA transcriptome. Genome research 2010;20: 1590-604). Before alignment, raw reads were initially filtered for (1) quality, (2) presence of the 30 adapter, to ensure a small RNA was ligated and sequenced completely and (3) size of small RNA reads (17-27 nt). Alignment of reads was compared against human miRNA hairpin sequences in the miRBase V19 using Novoalign V2.08.01 (available on the world wide web at novocraft.com) with the following parameters: -m -r All 1 -1 18 -t 30 -h 90 -o SAM, default options. After alignment, the reads were further separated into two categories of mapped reads versus unmapped reads. For the mapped reads, reads containing more than two mismatches were filtered out. [0143] After raw reads were mapped and filtered, an expression value (abundance) for each miRNA across all samples was calculated. For all differential expression analyses, the number of reads for a given miRNA was normalized by dividing the total number of mapped reads in that sample. This resulted in the per cent normalized read count for each miRNA, which was then multiplied by 10⁶ to scale the expression value to an appropriate range. Principal component analysis was performed to identify possible outlier samples. Log transformation was applied to further scale the range of expression values. One-way analysis of variance (ANOVA) tests were performed between case and control groups in our cohort.

[0144] MiRNA microarray experiments: RNA processing. Global miRNA expression profiles were analyzed using the MicroRNA Expression Profiling Assay based on BeadArray v.2 (Illumina Inc.), which contains 1,146 probes including 743 validated miRNAs. The miRNA microarray analysis was carried out in collaboration with the Genomics Platform CICbioGUNE (Center for Cooperative Research in Biosciences, Derio, Spain). The assay was done following the manufacturer's instructions (Illumina, Inc.), as previously described in the art. [0145] Microarray data normalization: Data were extracted using Bead Studio data analysis software and transformed to a log base 2 scale. Microarray data from 74 samples (20 normal colonic tissues, 54 CRC tissues) were quantile normalized using the lumi bioconductor package. Next, Applicants employed a conservative probe-filtering step, excluding those probes not reaching a detection value of P < 0.05 in 90% of the samples, which resulted in the selection of a total of 891 probes of the original 1,146 set. Fold changes in miRNA expression in the microarray analyses were calculated on the basis of the difference of the group median values (2^{log2 difference}) All microarray data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus (GEO;accession number GSE30454) as described previously (Balaguer F, Moreira L, Lozano JJ, et al. Colorectal cancers with microsatellite instability display unique miRNA profiles. Clinical cancer research : an official journal of the American Association for Cancer Research 2011; 17:6239-49). [0146] RNA isolation and qRT-PCR: Total RNA was isolated from FFPE tissues using the RecoverAll Total Nucleic Acid Isolation Kit (Ambion Inc, Austin, TX). Briefly, tissue sections were microdissected to enrich for neoplastic cells, followed by deparaffinization and RNA extraction using the manufacturer's protocol. Total RNA was eluted in the appropriate buffer, and quantified using a NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE). Reverse transcription reactions were carried out using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) in a total reaction volume of 15 μL·. MiR-490-3p, 549a, 1248, 1273 and miR-16 were quantified in duplicate by qRT-PCR, using MicroRNA Assay Kits (Applied Biosystems). qRT-PCR was performed on an Applied Biosystems 7000 Sequence Detection System, with the following cycling conditions: 95°C for 10 min, followed by 45 cycles of 95°C for 15s and 60°C for 1 min. Cycle threshold (Ct) values were calculated with SDS 1.4 software (Applied Biosystems). Expression levels of tissue miRNAs were normalized against miR-16 using the 2-ACt method as previously described (Toiyama Y, Takahashi M, Hur K, et al. Serum miR- 21 as a diagnostic and prognostic biomarker in colorectal cancer. Journal of the National Cancer Institute 2013; 105:849-59 and Hur K, Toiyama Y, Takahashi M, et al. MicroRNA- 200c modulates epithelial-to-mesenchymal transition (EMT) in human colorectal cancer metastasis. Gut 2013;62: 1315-26). Differences between the groups were presented as ACt, indicating differences between Ct values of the miRNA of interest and those of the normalizer miRNA.

[0147] In situ hybridization: Five-micrometer-thick formalin fixed paraffin embedded tissue sections were hybridized with the miR-549a probe (LNA-modified and 5'- and 3'- DIG labeled oligonucleotide; Exiqon, Woburn, MA), followed by incubation with anti-DIG-AP Fab fragments conjugated to alkaline phosphatase, and the hybridization signal was detected by applying nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color substrate (Roche Applied Science, Mannheim, Germany). Positive controls (U6 snRNA, LNA-modified and 5'- and 3'-DIG-labeled oligonucleotide; Exiqon) and negative controls (scrambled, LNA-modified and 5 '-and 3'-DIG-labeled oligonucleotide; Exiqon) were included in each hybridization experiment. [0148] Transfection experiments using miR-549a mimic, and miR-549a inhibitor: In order to transiently induce or inhibit miR-549a expression, mirVana™ hsa-miR-549a mimics (Applied Biosystems) or mirVana™ hsa-miR-549a inhibitor (Applied Biosystems) was used to transfect CRC cells, as described previously (Han TS, Hur K. MicroRNA-29c mediates initiation of gastric carcinogenesis by directly targeting ITGB 1. 2014). Verification of transfection efficiency was conducted using the mirVana™ miRNA mimic Negative Control (Applied Biosystems) and mirVana™ miRNA Inhibitor Negative Control (Applied Biosystems), respectively. [0149] Cell proliferation and colony formation assay: The MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Sigma, USA) was used to determine the degree of cell proliferation. Colon cancer cells were seeded at 5 ^χ 10³ cells per well in a 96-well flat-bottomed microtiter plates, in a final volume of 100 μΙ_, culture medium per well, and incubated in a humidified atmosphere. After 0-72 h incubation, cell proliferation was measured as described previously (Okugawa Y, Toiyama Y, Hur K, et al. Metastasis-associated long non-coding RNA drives gastric cancer development and promotes peritoneal metastasis. Carcinogenesis 2014).

[0150] Cell invasion, migration, wound healing, and anoikis assay: The invasion assay was performed as described previously . Invasion and migration assays were performed using Boy den chambers (Corning, Corning, NY, USA) using (Okugawa Y, Toiyama Y, Hur K, et al. Metastasis-associated long non-coding RNA drives gastric cancer development and promotes peritoneal metastasis. Carcinogenesis 2014) μπι pore size membrane with Matrigel (for invasion assay) or without Matrigel (for migration assay). For wound healing assays, cell monolayers were scratched with a sterile 200 μΐ pipette tip, and cell migration was observed for up to 48 h. For anoikis assay, 6-well Costar Ultra Low Attachment Microplates (Corning) were used. Transfected colon cancer cells were suspended in IMDM with 10% FBS at a concentration of 5 ^χ 10⁵ cells/mL; and 2 mL of cell suspension were added to each well and incubated in the microtiter plates for 24 h in a humidified incubator (37 °C and 5% C0₂). After induction of anoikis, cells were seeded at 5 ^χ 10³ cells/well in microtiter plates (96 wells, flat bottom) in a final volume of 100 μΙ_, culture medium/well, and analyzed using MTT assays as described previously (Okugawa Y, Tanaka K, Inoue Y, et al. Brain-derived neurotrophic factor/tropomyosin-related kinase B pathway in gastric cancer. British journal of cancer 2013; 108: 121-30).

[0151] Statistical Analysis: Statistical analysis was performed using Medcalc version 12.3.0 (Broekstraat 52, 9030; Mariakerke, Belgium). Results were expressed as means ± S.E. Differences between groups were estimated by Wilcoxon's signed rank test, the χ test, and Mann-Whitney U test, and One-way ANOVA analysis, as appropriate. F-tests were used to assess the equality of variance for comparable groups. For time-to-event analyses, survival estimates were calculated using the Kaplan-Meier analysis, and groups were compared with the log-rank test. Receiver operating characteristic curves were established to discriminate the patients with or without death, and Youden's index was used to determine the optimal cutoff threshold of miRNA expression values from clinical evaluation cohort to predict the overall survival and disease free survival, respectively. Overall survival was measured from the date the patient underwent surgery until the date of death resulting from any cause, or last known follow-up for patients that were still alive. Disease-free survival analysis was measured from the date the patient underwent curative surgery to the date of disease recurrence, death from any cause (i.e. cancer unrelated deaths were not censored) or until last contact with the patient. Receiver operating characteristic (ROC) curve analysis was performed to determine the diagnostic performance of miR-549a expression levels in distinguishing CRC tissues from normal colonic mucosa. The Cox's proportional hazards models were used to estimate hazard ratios (HRs) for death. Assumption of proportionality were confirmed for the Cox proportional hazards analyses by generating Kaplan-Meier survival curves (e.g. high vs. low expression groups) and by ensuring that the two curves did not intersect each other. Forced-entry regression was used to include these variables in all multivariable equations in order to analyze whether each of the predictors affected the outcome after adjusting for known confounders. All P values were 2-sided, and those less than 0.05 were considered statistically significant. RESULTS

[0152] Next-generation sequencing (NGS) based discovery of dysregulated miRNAs in CRC tissues: To discover miRNA transcriptomes that distinguish CRC from noncancerous or normal colonic tissues, NGS of small RNAs was performed on four pairs of frozen CRCs and adjacent normal tissues. For all samples, the number of filtered reads for a given miRNA was normalized to the total mapped reads by scaling the expression values through logarithmic transformation. After filtering the low quality reads and 3' adapter sequences, 51 miRNAs were found to be differentially expressed between CRC and NM tissues using one-way ANOVA test, (P<0.05 with >2 fold change). In order to further confirm the robustness of our CRC-specific miRNA signature discovered by NGS, the miRNA sequencing results (NGS profile) were compared with independent miRNA expression profiles using miRNA microarray in 54 CRC tissues and 20 normal mucosal tissue specimens, as previously described (Balaguer F, Moreira L, Lozano JJ, et al. Colorectal cancers with microsatellite instability display unique miRNA profiles. Clinical cancer research : an official journal of the American Association for Cancer Research 2011; 17:6239- 49). By comparing the NGS-based miRNA sequencing results with this independent miRNA profiles, 7 miRNAs that were shared between these datasets and were differentially expressed in CRCs were identified. These seven miRNAs were miR-30a-3p, mi-133a, miR-139-5p, miR-490-3p, miR-549a, miR-1248, and miR-1273a.

[0153] Although three-miRNAs (miR-30a-3p, mi-133a, and miR-139-5p) have already been published in connection with CRC, the other four-miRNAs have never been demonstrated to have an association with CRC development. These four miRNAs were selected for further validation in CRC tissues. In terms of validation phase, we quantified the expression of these four miRNAs in 24 pairs of CRCs and matched NMs. The qRT-PCR analyses revealed that miR-549a and miR-1248 expression were significantly up-regulated in CRC tissues (P<0.001), compared to NM tissues (FIG. 1). Receiver operating characteristic (ROC) curves were used to evaluate the sensitivity and specificity of each miRNA expression in distinguishing CRC from normal tissues (FIG. 2A-2B). Notably, expression of miR-549a displayed robust predictive significance, with an area under curve (AUC) values of 0.92 (FIG. 2A).

[0154] Next, the expression patterns for these two miRNAs were analyzed along with various clinicopathological factors to determine whether their expression status has any prognostic significance in CRC patients by analyzing an independent, large cohort of CRC patients. Although miR-1248 expression was not significantly dysregulated in this cohort, expression of miR-549a was remarkably up-regulated in CRC tissues compared with normal mucosa tissues which was consistent with previous cohort data. Receiver operating characteristic (ROC) curves were used to evaluate the sensitivity and specificity of miR-549a expression in distinguishing CRC from normal tissues. Notably, expression of miR-549a displayed remarkable predictive significance, with an area under curve (AUC) values of 0.93 (FIG. 2B).

[0155] Next, the expression patterns of miR-549a along with various clinicopathological factors were analyzed to determine whether the expression status has any prognostic significance in CRC patients. The expression cut-off thresholds for miR-549a were determined according to receiver operating characteristic analyses with Youden's index to determine overall survival (OS; FIG. 3A) and disease free survival (DFS; FIG. 3B) of CRC patients respectively. miR-549a low expression significantly correlated with overall survival and disease free survival (FIG. 3, Table 3). Furthermore, there was significant correlation of high miR-549a expression in UICC stage I, II, III, and IV cancer compared to normal mucosa (FIG. 4). Interestingly, miR-549a high expression were significantly correlated with advanced T-category, lymphatic invasion, lymph node metastasis, distant metastasis (Table 1), and poor prognosis compared to CRCs in the low-expression group in terms of OS (FIG. 3 A) and DFS (FIG. 3B) (p=0.0003, 0.022, respectively, log rank test) (FIG. 3, Table 3).

Table 1 : Clinicopathological variables and miR-549a expression in validation set

#: Pearson's chi-square test *p<0.5

[0156] Multivariate Cox's regression analysis showed high miR-549a expression was an independent prognostic factor for OS (HR: 1.94, 95%CI: 1.05-3.61, p=0.035) in CRC patients (Table 2). Table 2: Multivariate analysis for predictors of survival in validation set

HR: hazard ratio *p<0.5

Table 3 : Number at risk

[0157] MiR-549a were highly expressed in cancer cells compared with cancer stroma or corresponding normal mucosa. To further confirm the pathological expression pattern of miR-549a in clinical specimens, in situ hybridization (ISH) staining was performed on primary CRC tissue and corresponding adjacent normal mucosa from the clinical validation cohort. The ISH experiments revealed cytoplasmic staining for miR-549a in CRC cells specifically. Furthermore, miR-549a expression was up-regulated in the primary CRC cells compared with the matched normal mucosa, confirming the qRT-PCR results for its expression in primary CRC and adjacent normal mucosa tissues. Based on these results, miR-549a was overexpressed in CRC cells compared with normal mucosa, and its expression significantly correlated with disease progression in CRC patients. Therefore, Applicants decided to focus the rest of the study on miR-549a for further assessment of its biological function in colorectal neoplasia.

[0158] Inhibition of miR-549a expression suppress cell proliferation, invasion, migration and anoikis resistance in CRC cells. To investigate whether miR-549a alters the biological characteristics of colon cancer cells, expression levels of miR-549a in a panel of colon cancer cell lines (Caco2, HCT116, HT29, LoVo, SW480 and SW620) were first assessed by real-time PCR (FIG. 5). HT29 and SW620 cell lines were selected for knockdown studies, since both cell lines showed highest miR-549a expression (FIG. 6A-6E). Forty-eight hours after transfection of HT29 and SW620 cells with miR-inhibitor, its expression was considerably reduced in the miR-549a inhibitor transfected cells (FIG. 6A). In order to determine whether transfection of miR-549a inhibitor has biological consequences in controlling cell proliferation in human cancer cell lines, the effect of miR-549a inhibitor on cell proliferation by MTT assays was analyzed in transfected cell lines (FIG. 6B). Cell proliferation was significantly reduced following miR-549a knockdown, in comparison with negative control-transfected cells (Negative control inhibitor) as measured by a MTT proliferation assay (FIG. 6B).

[0159] Next, to determine whether silencing miR-549a inhibited cell migration and invasion, in vitro migration and invasion assays were performed (FIG. 6C-6D). First, a wound-healing scratch assay was performed to compare the migratory potential of CRC cells transfected with miR-549a inhibitor versus negative control inhibitor. As illustrated in FIGS. 6C-6D, miR-549a inhibitor transfection of HT29 and SW620 CRC cells resulted in significantly diminished invasive and migratory potential compared with cells transfected with negative control inhibitor. [0160] Furthermore, to interrogate whether miR-549a inhibition induces anoikis, we incubated CRC cells with or without miR-549a knockdown treatments in anchorage- independent culture using an ultra-low attachment plate. Given the importance of miR-549a function in CRC cells, we examined cell survival using MTT assay to evaluate the number of viable CRC cells floating in low-attachment plates. CRC cell lines with knockdown of miR- 549a showed a decreased number of viable CRC cells, which was significantly lower than observed in negative control inhibitor -transfected cells (FIG. 6E).

[0161] Overexpression of miR-549a results in increased cell proliferation, tumorigenicity, migration, invasion and anoikis resistance in colon cancer cells (FIG. 7A-E). To confirm whether ectopic miR-549a expression enhance the malignant characteristics of colon cancer cells, two CRC cell lines (Caco2 and HCT116) with lowest-miR-549a expression were selected for overexpression studies. The miRVana™ miR-549a mimic and mimic-negative control were transfected into these cell lines, which facilitated significant over-expression of miR-549a in these cells (FIG. 7A). [0162] In order to determine whether ectopic expression of miR-549a resulted in enhanced cell proliferation in human cancer cell lines, the rate of cell proliferation was analyzed by MTT assays using transfected cell lines (FIG. 7B). Cell proliferation was significantly increased in both miR-549a mimic transfected cell lines, compared with control transfected cell lines (FIG. 7B). Next, to determine whether ectopic expression of miR-549a altered cell migration and invasion, in vitro migration, invasion and wound healing assays were performed (FIG. 7C-7D). As shown in FIGS. 7C-7D, miR-549a overexpressing CRC cell lines showed significantly enhanced invasive and migratory potential compared to cells transfected with mimic-negative control. Furthermore, after induction of anoikis, miR-549a mimic transfected Caco2 and HCT116 cells exhibited a decrease in the number of viable cells compared with the mimic-negative control cells (FIG. 7E).

[0163] Collectively, all of these in vitro results suggest that miR-549a might be intimately involved in CRC pathogenesis by promoting cell growth, colonogenic survival and enhancing invasion and migratory potential of colon cancer cells.

Example 2 - Effect of miR-549a ectopic expression in Caco2 cells in a xenograft mouse model

[0164] Male athymic nude mice were obtained from Harlan Laboratories (Houston, TX, USA) at 5 weeks of age and kept under controlled conditions (12 h light and dark cycles). Inventors generated xenograft tumours using Caco2 cell line stably overexpressing miR-549a or its controls. These cancer cells were suspended in phosphate-buffered saline and Matrigel (Corning; 1 : 1 ratio) and 2x 106 cells were subcutaneously injected into the abdominal flanks of each mice. Matrigel was used to improve the attachment and differentiation of both miR- 549a stably overexpressed cells and control cells in athymic nude mice. Ten mice were used in each group, and subcutaneous tumours were monitored for 28 days following injection. As shown in FIG. 8, tumor growth, as measured by tumor volume, was increased in mice overexpressing miR-549a compared to the control. This provides further evidence that miR- 549a expression is involved in CRC pathogenesis and further validates the potential therapeutic effect of targeting miR-549a expression for the treatment of CRC.

* * *

[0165] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

The following references and the publications referred to throughout the specification, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

Balaguer F, Moreira L, Lozano JJ, et al. Colorectal cancers with microsatellite instability display unique miRNA profiles. Clinical cancer research : an official journal of the American Association for Cancer Research 2011; 17:6239-49.

Fehniger TA, Wylie T, Germino E, et al. Next-generation sequencing identifies the natural killer cell microRNA transcriptome. Genome research 2010;20: 1590-604.

Chen J, Lozach J, Garcia EW, et al. Highly sensitive and specific microRNA expression profiling using BeadArray technology. Nucleic acids research 2008;36:e87.

Cunningham JM, Oberg AL, Borralho PM, et al. Evaluation of a new high- dimensional miRNA profiling platform. BMC medical genomics 2009;2:57.

Du P, Kibbe WA, Lin SM. lumi: a pipeline for processing Illumina microarray. Bioinformatics 2008;24: 1547-8.

Toiyama Y, Takahashi M, Hur K, et al. Serum miR-21 as a diagnostic and prognostic biomarker in colorectal cancer. Journal of the National Cancer Institute 2013; 105:849-59.

Hur K, Toiyama Y, Takahashi M, et al. MicroRNA-200c modulates epithelial-to- mesenchymal transition (EMT) in human colorectal cancer metastasis. Gut 2013;62: 1315-26.

Han TS, Hur K. MicroRNA-29c mediates initiation of gastric carcinogenesis by directly targeting ITGB 1. 2014.

Okugawa Y, Toiyama Y, Hur K, et al. Metastasis-associated long non-coding RNA drives gastric cancer development and promotes peritoneal metastasis. Carcinogenesis 2014.

Okugawa Y, Tanaka K, Inoue Y, et al. Brain-derived neurotrophic factor/tropomyosin- related kinase B pathway in gastric cancer. British journal of cancer 2013; 108: 121-30.

Claims

1. A method for treating colorectal cancer in a subject in need thereof comprising:

administering a therapeutically effective amount of an antagonist of miR-549a that reduces or inhibits the expression or activity of miR-549a.

2. The method of claim 1, wherein the antagonist is an antagomir of miR-549a.

3. The method of claim 1 or 2, wherein the subject is one that has been determined to have an increased level of expression of miR-549a in a biological sample from the patient compared to a control level of expression of miR-549a in a non-cancerous biological sample.

4. The method of claim 3, wherein the non-cancerous biological sample is a noncancerous sample from the patient.

5. The method of claim 3 or 4, wherein the non-cancerous biological sample is normal mucosal tissue.

6. The method of any one of claims 3-5, wherein the biological sample from the patient is a sample from a primary colorectal cancer tumor.

7. The method of any one of claims 1-6, wherein the subject has advanced colorectal cancer.

8. The method of claim 7, wherein the advanced colorectal cancer comprises category T3 or T4 colorectal cancer.

9. The method of claim 7 or 8, wherein the advanced colorectal cancer comprises lymph node metastasis.

10. The method of any one of claims 7-9, wherein the advanced colorectal cancer comprises category Nl and/or N2 colorectal cancer.

11. The method of any one of claims 7-10, wherein the advanced colorectal cancer comprises distant metastasis.

12. The method of claim 12, wherein the distant metastasis is liver metastasis.

13. The method of any one of claims 7-12, wherein the advanced colorectal cancer comprises category Ml colorectal cancer.

14. The method of any one of claims 7-13, wherein the advanced colorectal cancer comprises Stage II, Stage III, and/or Stage IV colorectal cancer.

15. The method of any one of claims 1-14, further comprising administration of one or more of cetuximab, fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumuman, afibercept, leucovorin, and radiotherapy.

16. The method of any one of claims 1-15, wherein the antagonist is linked to a targeting moiety.

17. The method of claim 16, wherein the targeting moiety is an aptamer.

18. The method of claim 16 or 17, wherein the targeting moiety delivers the antagonist to a specific cell type or tissue.

19. The method of claim 18, wherein the cell or tissue is colorectal cells or colorectal tissues.

20. The method of claim 18 or 19, wherein the cell or tissue is cancerous.

21. The method of claim 20, wherein the cell or tissue is metastatic.

22. A method for treating a patient determined to have colorectal cancer comprising: administering a colorectal cancer treatment to the patient, wherein the patient was determined to have an elevated level of miR-549a expression in a biological sample from the patient relative to the expression level of miR-549a in a control sample.

23. The method of claim 22, wherein the control sample is a non-cancerous biological sample.

24. The method of claim 22 or 23, wherein the method further comprises measuring the expression level of miR-549a in a biological sample from the patient.

25. The method of any one of claims 22-24, wherein the method further comprises comparing the expression level of miR-549a in the biological sample from the patient to the expression level of miR-549a from a non-cancerous biological sample.

26. The method of any one of claims 22-25, wherein the patient has or is determined to have Stage I, II, III, or IV colorectal cancer.

27. The method of any one of claims 22-26, wherein the biological sample from the patient is a sample from a primary colorectal cancer tumor.

28. The method of any one of claims 22-27, wherein the colorectal cancer treatment comprises cetuximab, fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumuman, afibercept, leucovorin, and/or radiotherapy.

29. The method of any one of claims 22-28, wherein the colorectal cancer treatment comprises surgical incision of the primary tumor.

30. A method for treating a patient for early or advanced colorectal cancer comprising: treating the patient for advanced colorectal cancer after the patient is determined to have a high elevated level of miR-549a expression in a biological sample from the patient compared to a non-cancerous biological sample; or

treating the patient for early colorectal cancer after the patient is determined to have a low elevated level of miR-549a expression in a biological sample from the patient, compared to a non-cancerous biological sample.

31. The method of claim 30, wherein the method further comprises measuring the expression level of miR-549a in a biological sample from the patient.

32. The method of claim 30 or 31, wherein the method further comprises comparing the expression level of miR-549a in the biological sample from the patient to the expression level of miR-549a in a non-cancerous biological sample.

33. The method of any one of claims 30-33, wherein the advanced colorectal cancer comprises category T3 or T4 colorectal cancer.

34. The method of any one of claims 30-33, wherein the advanced colorectal cancer comprises lymph node metastasis.

35. The method of any one of claims 30-34, wherein the advanced colorectal cancer comprises category Nl and/or N2 colorectal cancer.

36. The method of any one of claims 30-35, wherein the advanced colorectal cancer comprises distant metastasis.

37. The method of claim 36, wherein the distant metastasis is liver metastasis.

38. The method of any one of claims 30-37, wherein the advanced colorectal cancer comprises category Ml colorectal cancer.

39. The method of any one of claims 30-38, wherein the advanced colorectal cancer comprises Stage II, Stage III, and/or Stage IV colorectal cancer.

40. The method of any one of claims 30-39, wherein early colorectal cancer comprises Stage I colorectal cancer.

41. The method of any one of claims 30-40, wherein early colorectal cancer comprises category Tl or T2 colorectal cancer.

42. The method of any one of claims 30-41, wherein early colorectal cancer comprises category NO colorectal cancer.

43. The method of any one of claims 30-42, wherein early colorectal cancer comprises category M0 colorectal cancer.

44. The method of any one of claims 30-43, wherein the biological sample from the patient is a sample from a primary colorectal cancer tumor.

45. The method of any one of claims 30-44, wherein the high or low elevated level is determined from a cut-off value, above which defines a high elevated level of expression and below which defines a low level of expression.

46. The method of claim 45, wherein the cut-off value is determined by a ROC analysis.

47. The method of any one of claims 30-46, wherein the treatment for advanced colorectal cancer comprises cetuximab, fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumuman, afibercept, leucovorin, and/or radiotherapy.

48. The method of any one of claims 30-47, wherein the treatment for early colorectal cancer comprises surgical incision of the primary tumor.

49. The method of any one of claims 30-48, wherein the treatment for early colorectal cancer excludes chemotherapy.

50. The method of any one of claims 30-49, wherein the treatment for early colorectal cancer excludes one or more of cetuximab, fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumuman, afibercept, leucovorin, and/or radiotherapy.

51. The method of any one of claims 30-50, wherein the treatment for the advanced colorectal cancer comprises surgical removal of one or more secondary tumors.

52. The method of claim 51, wherein the secondary tumor is a distant liver metastasis.

53. The method of any one of claims 30-52, wherein the early colorectal cancer treatment excludes surgical removal of one or more secondary tumors.

54. A method for predicting a patient's prognosis for survival and/or disease free survival of colorectal cancer comprising:

predicting that the patient is likely to survive and or have disease free survival when the expression level of miR-549a in a biological sample from the patient is determined to be not significantly different than the expression level of a non-cancerous biological sample or when the expression level is determined to be a low elevated level of expression compared to a non-cancerous sample; or

predicting that the patient is not likely to survive or have disease free survival when the expression level of miR-549a in a biological sample from the patient is determined to have a high elevated level of expression compared to a non-cancerous sample.

55. The method of claim 54, wherein the method further comprises determining the expression level of miR-549a in a biological sample from the patient.

56. The method of claim 54 or 55, wherein the method further comprises comparing the expression level of miR-549a in the biological sample from the patient to the expression level of miR-549a to a control.

57. The method of claim 56, wherein the control is a non-cancerous biological sample.

58. The method of claim 54, wherein the biological sample from the patient is a sample from a primary colorectal cancer tumor.

59. The method of any one of claims 54-58, wherein the high or low elevated level is determined from a cut-off value, above which defines a high elevated level of expression and below which defines a low level of expression .

60. The method of claim 59, wherein cut-off value is determined by a ROC analysis.

61. The method of any one of claims 54-60, wherein the patient is predicted to likely survive and or have disease free survival when the expression level of miR-549a in the biological sample from the patient is determined to be a low elevated level of expression.

62. A biomarker for colorectal cancer cells, wherein the biomarker is an elevated level ofexpression of miR-549a.

63. The biomarker of claim 62, wherein the elevated level of expression of miR-549a indicates that the cells are colorectal cancer cells.

64. The biomarker of claim 62 or 63, wherein the elevated level of expression of miR- 549a is relative to a non-cancerous tissue.

65. A kit comprising an agent for detecting miR-549a expression.

66. The kit of claim 65, wherein the agent comprises one or more nucleic acid probes for amplification of a miR-549a nucleic acid from a biological sample.

67. The kit of claim 65 or 66, wherein the agent is labeled.

68. The kit of any one of claims 65-67, further comprising instructions for use.

69. A method for treating colorectal cancer in a patient comprising:

determining the expression level of miR-549a in tissue adjacent to a surgical site in a colorectal cancer patient and/or in a lymph node of the patient; and

treating the patient for colorectal cancer after the expression level of miR-549a in tissue and or lymph node of the patient is determined to be higher than the expression level of miR-549a in a non-cancerous tissue.

70. The method of claim 69, wherein the colorectal cancer comprises advanced colorectal cancer.

71. The method of claim 70, wherein the advanced colorectal cancer comprises category T3 or T4 colorectal cancer.

72. The method of claim 70 or 71, wherein the advanced colorectal cancer comprises lymph node metastasis.

73. The method of any one of claims 70-72, wherein the advanced colorectal cancer comprises category Nl and/or N2 colorectal cancer.

74. The method of any one of claims 70-73, wherein the advanced colorectal cancer comprises distant metastasis.

75. The method of claim 74, wherein the distant metastasis is liver metastasis.

76. The method of any one of claims 70-75, wherein the advanced colorectal cancer comprises category Ml colorectal cancer.

77. The method of any one of claims 70-76, wherein the advanced colorectal cancer comprises Stage II, Stage III, and/or Stage IV colorectal cancer.

78. The method of any one of claims 69-77, wherein the non-cancerous biological sample is a non-cancerous sample from the patient.

79. The method of any one of claims 69-78, wherein the non-cancerous biological sample is normal mucosal tissue.

80. The method of any one of claims 70-79, wherein the treatment for advanced colorectal cancer comprises cetuximab, fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumuman, afibercept, leucovorin, and/or radiotherapy.

81. The method of any one of claims 70-80, wherein the treatment for the advanced colorectal cancer comprises surgical removal of one or more secondary tumors.

82. The method of claim 81, wherein the secondary tumor is a distant liver metastasis.

83. The method of claim 69, wherein the colorectal cancer is early colorectal cancer.

84. A method for diagnosing a patient with advanced or early colorectal cancer comprising: diagnosing the patient as having or likely to have advanced colorectal cancer or providing an analysis or report that the patient has or likely has advanced colorectal cancer when the expression level of miR-549a in a biological sample from the patient is determined to be a high elevated level of expression compared to the expression level in a non-cancerous biological sample; or

diagnosing the patient as having or likely to have early colorectal cancer or providing an analysis or report that the patient has or likely has early colorectal cancer when the expression level of miR-549a in a biological sample from the patient is determined to be a low elevated level of expression compared to the expression level in a non-cancerous biological sample.

85. The method of claim 84, wherein the method further comprises measuring the expression level of miR-549a in a biological sample from the patient.

86. The method of claim 84 or 85, wherein the method further comprises comparing the expression level of miR-549a in the biological sample from the patient to a control level of expression.

87. The method of any one of claims 84-86, wherein the advanced colorectal cancer comprises category T3 or T4 colorectal cancer.

88. The method of any one of claims 84-87, wherein the advanced colorectal cancer comprises lymph node metastasis.

89. The method of any one of claims 84-88, wherein the advanced colorectal cancer comprises category Nl and/or N2 colorectal cancer.

90. The method of any one of claims 84-89, wherein the advanced colorectal cancer comprises distant metastasis.

91. The method of claim 90, wherein the distant metastasis is liver metastasis.

92. The method of any one of claims 84-91, wherein the advanced colorectal cancer comprises category Ml colorectal cancer.

93. The method of any one of claims 84-92, wherein the advanced colorectal cancer comprises Stage II, Stage III, and/or Stage IV colorectal cancer.

94. The method of any one of claims 84-93, wherein early colorectal cancer comprises Stage I colorectal cancer.

95. The method of any one of claims 84-94, wherein early colorectal cancer comprises category Tl or T2 colorectal cancer.

96. The method of any one of claims 84-95, wherein early colorectal cancer comprises category NO colorectal cancer.

97. The method of any one of claims 84-96, wherein early colorectal cancer comprises category MO colorectal cancer.

98. The method of any one of claims 84-97, wherein the biological sample from the patient is a sample from a primary colorectal cancer tumor.

99. The method of any one of claims 84-98, wherein the control is a cut-off value, above which defines a high elevated level of expression and below which defines a low level of expression.

100. The method of claim 99, wherein cut-off value is determined by a ROC analysis.

101. The method of any one of claims 84-100, wherein the method further comprises treating the patient for early or advanced colorectal cancer.

102. The method of claim 101, wherein the treatment for advanced colorectal cancer comprises cetuximab, fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumuman, afibercept, leucovorin, and/or radiotherapy.

103. The method of claim 101 or 102, wherein the treatment for early colorectal cancer comprises surgical incision of the primary tumor.

104. The method of any one of claims 101-103, wherein the treatment for early colorectal cancer excludes chemotherapy.

105. The method of any one of claims 101-104, wherein the treatment for early colorectal cancer excludes one or more of cetuximab, fluorouracil, oxaliplatin, irinotecan, bevacizumab, panitumuman, afibercept, leucovorin, and/or radiotherapy.

106. The method of any one of claims 101-105, wherein the treatment for the advanced colorectal cancer comprises surgical removal of one or more secondary tumors.

107. The method of claim 106, wherein the secondary tumor is a distant liver metastasis.

108. The method of any one of claims 101-108, wherein the early colorectal cancer treatment excludes surgical removal of one or more secondary tumors.

109. A method for determining whether a biological sample comprises colorectal cancer cells comprising:

measuring the expression level of miR-549a in the biological sample;

comparing the expression level of miR-549a in the biological sample to the expression level of miR-549a in a non-cancerous biological sample; and

determining that the biological sample comprises colorectal cancer cells when the expression level of miR-549a in the biological sample t is higher than the expression level of miR-549a in the non-cancerous biological sample; or

determining that the biological sample is non-cancerous when the expression level of miR-549a in the biological sample is not significantly different than the expression level of miR-549a in the non-cancerous biological sample.

110. The method of claim 109, wherein the biological sample is tissue adjacent to a surgical site of a colorectal cancer patient.

111. The method of claim 109, wherein the biological sample is comprises lymph node tissue.

112. The method of any one of claims 109-111, wherein the non-cancerous biological sample is normal mucosal tissue.

113. The method of any of claims 109-112, further comprising obtaining the biological sample from a patient.