METHODS FOR DIAGNOSING AND TREATING COLORECTAL CANCER USING snoRNA

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/143,647, filed April 6, 2015, and U.S. Provisional Patent Application No. 62/308,724, filed March 15, 2016, both of which are hereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

[0002] This invention was made with government support under Grant Nos. R01 CA72851, CA181572, CA184792, and U01 CA187956 awarded by the National Institute of Health and National Cancer Institute. The government has certain rights in the invention.

1. Field of the Invention

[0003] The present invention relates generally to the fields of molecular biology and oncology. More particularly, it concerns methods and compositions involving small nucleolar RNAs (snoRNAs) molecules and cancer prognosis, diagnosis, and treatment.

2. Description of Related Art

[0004] 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.

[0005] Colorectal cancer (CRC) remains one of the most common and lethal malignancies worldwide, and is the second leading cause of cancer-related deaths in the United States. At the time of initial diagnosis, a large majority of patients have already reached an advanced stage where tumor cell spreading has occurred, and approximately 50% of CRC patients will die from the development of distant metastases. Although progress in treatment options, such as development of novel chemotherapeutic drugs and technical advances in invasive treatment for metastatic lesion, have somewhat improved the prognosis of advanced CRC patients, there still is a clear need for prognostic biomarkers that can help identify high-risk patients who can benefit from intensive post-treatment surveillance protocols for early detection of recurrence. SUMMARY OF THE INVENTION

[0006] 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 snoRNA 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 snoRNA expression in a biological sample from the patient relative to the expression level of snoRNA in a non-cancerous biological sample. In some embodiments, the method further comprises measuring the expression level of snoRNA in a biological sample from the patient. [0007] Small nucleolar RNAs (snoRNAs) are a class of small RNA molecules that primarily guide chemical modifications of other RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs. There are two main classes of snoRNA, the C/D box snoRNAs, which are associated with methylation, and the H/ACA box snoRNAs, which are associated with pseudouridylation. SnoRNAs may be referred to as guide RNAs, but are distinct from guide RNAs that direct RNA editing in trypanosomes. In some embodiments, the snoRNA is selected from SNORD76, SNORD78, ACA11, SNORA42, SNORA21, SNORA34, and SNORD66.

[0008] SNORD76, small nucleolar RNA, C/D box 76, (also known as U76) has the sequence: tgccacaatg atgacagttt atttgctact cttgagtgct agaatgatga ggatcttaac caccattatc ttaactgagg c (SEQ ID NCv l).

[0009] SNORD78, small nucleolar RNA, C/D box 78, (also known as U78) has the sequence: gtgtaatgat gttgatcaaa tgtctgacct gaaatgagca tgtagacaaa ggta (SEQ ID NO: 2).

[0010] ACA11, also referred to as Homo sapiens small Cajal body-specific RNA 22 (SCARNA22), has the sequence: gcgctcacta aggctcggtc ctctccacgt ggtcctgacc tgtcctctgt gagcaagaga aacaggactg gtttgggggt gtcctgtctc agtggacaca ggacaccacg gttttcagta caaca (SEQ ID NO:3).

[0011] SNORA42, also referred to as Homo sapiens small nucleolar RNA, H/ACA box 80E (SNORA80E), has the sequence: tggtaatgga tttatggtgg gtccttctct gtgggcctct catagtgtac ccatgccata gcaaatggca gcctcgaacc attgcccagt ccccttacct gtgggctgtg agcactgaag ggggttgcac agtg (SEQ ID NO:4). [0012] SNORA21, also referred to as Homo sapiens small nucleolar RNA, H/ACA box 21 and ACA21, has the sequence: ccccctttta aaagcactca atgggcctgt ggctaatgac ctattgagcc gtcaagaaag gggagagtga aaacatcgct tttgggtgaa gtggcaacat gtgttgtttg cttcaatcgg tggtgtgaca agg (SEQ ID NO:5). [0013] SNORA34, also referred to as Homo sapiens small nucleolar RNA, H/ACA box 2C, SNORA2C, ACA34, and MIR1291, has the sequence: gtggccctga ctgaagacca gcagttgtac tgtggctgtt ggtttcaagc agaggcctaa aggactgtct tcctgtggtc tgttggctgt tctgggacct cagtagggaa tggctatttc atttggaaga aacaacc (SEQ ID NO:6).

[0014] SNORD66, also referred to as Homo sapiens small nucleolar RNA, C/D box 66 and HBII-142, has the sequence: ttcctctgat gacttcctgt tagtgccacg tgtctgggcc actgagacac catgatggaa ctgaggatct gaggaa (SEQ ID NO:7).

[0015] In some aspects of the methods described herein, the elevated level is a high elevated level.

[0016] In some aspects, the method further comprises comparing the expression level of snoRNA in the biological sample from the patient to the expression level of snoRNA 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 patient has or is determined to have Stage IV colorectal cancer. In some embodiments, the patient is determined to have Stage IV colorectal cancer based on the elevated snoRNA level. In some embodiments, the patient is determined to have distant metastasis based on the elevated snoRNA level. In some embodiments, the distant metastasis are liver metastasis.

[0017] 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.

[0018] 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 snoRNA 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 snoRNA expression in a biological sample from the patient. [0019] In some embodiments, the method further comprises measuring the expression level of snoRNA 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 snoRNA in the biological sample from the patient to the expression level of snoRNA from a noncancerous biological sample.

[0020] 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 snoRNA in a biological sample from the patient is determined to be not significantly different than the expression level of the snoRNA of a non-cancerous biological sample or when the expression level is determined to be a low elevated level of expression compared to the expression level of the snoRNA in a non-cancerous biological sample; or predicting that the patient is not likely to survive or have disease free survival when the expression level of snoRNA in the biological sample from the patient is determined to be an elevated level of expression compared to the expression level of the snoRNA in a non-cancerous biological sample or is determined to be a high elevated level of expression compared to the expression level of the snoRNA in a non-cancerous biological sample. In some embodiments, the method further comprises determining the expression level of snoRNA in a biological sample from the patient. In some embodiments, the method further comprises comparing the expression level of snoRNA in the biological sample from the patient to the expression level of snoRNA in a non-cancerous biological sample. In some embodiments, the patient is predicted to likely survive and or have disease free survival when the expression level of snoRNA in the biological sample from the patient is determined to be a low elevated level of expression.

[0021] 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 snoRNA. In some embodiments, a high expression of snoRNA indicates that the cells are colorectal cancer cells. In some embodiments, the elevated level of expression of snoRNA is relative to a non- cancerous tissue. In some embodiments, a high elevated level of expression of snoRNA is a biomarker for advanced colorectal cancer and a low elevated level of expression of snoRNA is a biomarker for early colorectal cancer. [0022] A further aspect of the disclosure relates to a method for treating colorectal cancer in a patient comprising: determining the expression level of snoRNA 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 snoRNA in tissue and or lymph node of the patient is determined to be higher than the expression level of snoRNA 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.

[0023] 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 snoRNA in a biological sample from the patient is determined to be a high elevated level; and 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 snoRNA in a biological sample from the patient is determined to be a low elevated level. In some embodiments, the control is a cut-off value. In some embodiments, the method further comprises measuring the expression level of snoRNA in the biological sample from the patient. In some embodiments, the method further comprises comparing the expression level of snoRNA in the biological sample from the patient to a control level of expression;

[0024] Embodiments concern determining that the level of expression of one or more snoRNAs. In some embodiments, that level is compared to a control in order to determine whether the expression level or activity of the snoRNA is elevated as compared to the level in non-cancerous colorectal tissue. The control may be a non-cancerous colorectal tissue or it may be a cancerous colorectal tissue. If the control is a cancerous colorectal 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. [0025] 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 snoRNA in the biological sample; comparing the expression level of snoRNA in the biological sample to the expression level of snoRNA in a non-cancerous biological sample; determining that the biological sample comprises colorectal cancer cells when the expression level of snoRNA in the biological sample is higher than the expression level of snoRNA in the non-cancerous biological sample; and determining that the biological sample is noncancerous when the expression level of snoRNA in the biological sample not significantly different than the expression level of snoRNA 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.

[0026] 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 snoRNA that reduces or inhibits the expression or activity of snoRNA. In some embodiments, the antagonist is an antagomir of snoRNA. In some embodiments, the subject is one that has been determined to have an increased level of expression of snoRNA in a biological sample from the patient compared to a control level of expression of snoRNA 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.

[0027] In some embodiments, the cancer is a recurrent cancer. In some embodiments, the patient or subject has previously been treated for the cancer.

[0028] 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.

[0029] In some embodiments of the above-disclosed aspects, a high or low elevated level of expression of snoRNA 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 snoRNA 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.

[0030] 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.

[0031] 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 MO colorectal cancer. In some embodiments of the above disclosed aspects, early colorectal cancer excludes category Ml colorectal cancer. [0032] 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 noncancerous 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 noncancerous biological sample is from a biological source as described herein.

[0033] In further embodiments, the method may comprise determining the expression level of the snoRNA 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.

[0034] 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.

[0035] 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. [0036] 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. [0037] 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.

[0038] 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.

[0039] 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. In certain embodiments, RNA may be isolated from exosomes. In some embodiments, the exosomes are serum exosomes. 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 snoRNA expression in a sample. Other methods to measure or assay the level of snoRNA expression may include, but not limited to RNAseq and Next-Gen sequencing. The methods may further comprise recording the snoRNA 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. [0040] In some embodiments, methods will involve determining or calculating a prognosis score based on data concerning the expression level of the snoRNA, meaning that the expression level of the snoRNA 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.

[0041] 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.

[0042] 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 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). [0043] 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 snoRNA. In some embodiments, the methods described herein further comprise determining a level of expression of a snoRNA, wherein the level of expression is determined using a kit described herein. In some embodiments of the methods described herein, the patient is one that has been determined to have an elevated or high elevated level of expression using a kit described herein. [0044] 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 snoRNA 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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. [0050] 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.

[0051] It is specifically contemplated that the embodiments described above may be used in combination with any of the methods descried herein. It is further contemplated that any of the above method steps, embodiments, reagents, snoRNAs, etc. may be specifically excluded from a claimed method, kit, or composition. [0052] 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

[0053] 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.

[0054] FIGs. 1A-B: Expression status of oncogenic-candidate snoRNAs in CRC tissues and adjacent normal mucosa in the screening and clinical validation cohorts, (a) Box plots show expression levels of four snoRNAs (SNORD76, SNORD78, ACA11, and SNORA42) in primary tumor tissues (CRC) and corresponding matched normal mucosa (NM) from eight patients enrolled in the screening cohort. The expression of all snoRNAs was significantly higher in CRC tissues than in adjacent normal mucosa. (p<0.01, Wilcoxon rank correlation test), (b) Box plots show expression levels of four snoRNAs (SNORD76, SNORD78, ACA11, and SNORA42) in CRC (n=192) and NM (n=16) tissues in the clinical validation cohort. Boxes represent interquartile ranges, and the horizontal line across each box indicates median value. The y-axis represents relative expression of four snoRNA, and data were normalized to miR-16 expression. Statistical analysis was performed using Mann-Whitney U tests. All statistical tests were two-sided. **p <0.01; ***p < 0.001

[0055] FIGs. 2A-D: Prognostic impact of snoRNA expression status in CRC patients, (a) Kaplan-Meier survival curves for overall survival (OS) in CRC patients based on the expression of four snoRNAs in the clinical validation cohort (n=192). The OS rate in CRC patients (n=96) with high-SNORA42 expression in tumor tissue was significantly lower than that for those with low-SNORA42 expression (cut-off threshold was median value in this cohort; p=0.018; log-rank test), (b) Disease-free survival analyses based on SNORA42 expression status in CRC tissue cohort (cut-off threshold was median value in stage I-III CRC patients; p=0.029; log-rank test), (c, d) Overall survival analysis based on SNORA42 expression status in performance evaluation cohort (c) (n=50) and total cohort (d) (n=250) (cut-off threshold was median value in each cohort; p=0.026, 0.002, respectively; log-rank test). All statistical tests were two-sided.

[0056] FIGs. 3A-I: Functional analysis of SNORA42 in cultured cells and an animal model of colorectal cancer, (a) SNORA42 expression status in colon cancer cell lines, (b) Ectopic SNORA42 expression in the colon cancer cell lines Caco2 and SW480. (c) Effect of SNORA42 over-expression on Caco2 and SW480 cell proliferation as assessed by MTT assay, (d) Colony formation assay. The number of colonies with >50 cells were counted after 10 day incubation, (e) Cell invasion and migration assay using Matrigel-coated transwell membranes (upper panel illustrates representative image for invasion, bottom panel depicts an image from migrated cells; average counts from five random microscopic fields)), (f) Anoikis assay to investigate anoikis resistance in Caco2 and SW480 cells with or without SNORA42 overexpression. After anoikis induction for 48 h, the number of viable floating cancer cells in low attachment plates was calculated by MTT assay, (g, h) Effect of SNORA42 ectopic expression in SW480 cells on the xenograft model was assessed by evaluating tumor volume (g) and weight (h) compared to controls, (i) SNORA42 expression levels in xenograft tumors, which were treated with pCDH-SNORA42 or pCDH-control. Statistical analysis was performed using one way ANOVA and Mann-Whitney U tests appropriately. All statistical tests were two-sided. *P < 0.05, **P <0.01, ***P < 0.001.

[0057] FIG. 4: ROC curve analysis for expression levels ofSNORD76, SNORD78, ACA11, and SNORA42 in distinguishing colorectal cancer from normal colonic mucosa. ROC curve analysis demonstrated that expression levels of all 4 snoRNAs could considerably distinguish CRC tissues from normal colonic mucosa, with AUC values of 0.79 (95% CI: 0.73-0.82), 0.78(95% CI: 0.72-0.84), 0.88 (95% CI: 0.83-0.92), 0.75 (95% CI: 0.69-0.81), respectively.

[0058] FIGs. 5A-C: Kaplan-Meier survival analysis in stage II CRC patients based on SNORA42 expression status in primary CRC tissues. Stage II CRC Patients with high expression of SNORA42 had significantly poor prognosis compared with those in the low- expression group in overall survival (a, p=0.048; log-rank test) and disease free survival (b, p=0.049; log-rank test) in clinical validation cohort, (c) Overall survival analyses of stage II CRC patients based on SNORA42 expression status in total cohort (p=0.039; log-rank test). [0059] FIG. 6: In situ hybridization analysis of SNORA42 in colorectal cancer tissues and corresponding normal mucosa. Positive control :U6 snRNA, Negative control: scramble control. [0060] FIGs. 7A-D: snoRNA in CRC analysis. Shown in (A) are boxplots of the normalized expression levels of SNORA21, SNORA34, SNORD66, and SNORD96A in normal and cancer tissue. Shown in (B) are boxplots of the normalized expression levls of SNORA21, SNORA34, and SNORD66 in normal and cancer tissue. Shown in (C) are boxplots of the normalized expression levels of SNORA21 in normal and Stage I, II, III, or IV colorectal cancer. Shown in (E) is a graphical representation of the overall survival in low or high SNORA21 expressing patients with colorectal cancer.

[0061] FIGs. 8A-B: SNORA21 analysis. Shown in (A) is the overall probability of cancer specific survival in low or high SNORA21 expressing patients with colorectal cancer. Shown in (B) is the overall probability of cancer specific survival in low or high SNORA21 expressing patients with Stage I, II, III, or IV colorectal cancer

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0062] 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

[0063] "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).

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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. [0069] 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. [0070] "About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements.

[0071] The term "aptamer" refers to a synthetic oligonucleotides or peptide molecules that bind to a specific target molecule.

[0072] As used herein, the term "antagomir" refers to a synthetic oligonucleotide or oligonucleotide mimetic having complementarity to a specific snoRNA, and which inhibits the activity of that snoRNA. 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 snoRNA that it inhibits. Further, antagomirs can have the same length, a longer length or a shorter length than the snoRNA that it inhibits. In certain embodiments, the antagomir hybridizes to 6-8 nucleotides at the 5' end of the snoRNA 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 snoRNA described herein, such as miR549a. The antagomirs are synthetic reverse complements that tightly bind to and inactivate a specific snoRNA. 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 snoRNA 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

[0073] (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. [0074] 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 snoRNA agents.

II. SnoRNA

[0075] Certain aspects are based, in part, on the systematic discovery and validation of prognostic/predictive snoRNA (s) biomarkers. In certain embodiments, snoRNAs may be used in methods and compositions for determining the prognosis, such as response to a particular cancer treatment, of a particular patient.

[0076] Small nucleolar RNAs (snoRNAs) are a class of small RNA molecules that primarily guide chemical modifications of other RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs. There are two main classes of snoRNA, the C/D box snoRNAs, which are associated with methylation, and the H/ACA box snoRNAs, which are associated with pseudouridylation. SnoRNAs are commonly referred to as guide RNAs but should not be confused with the guide RNAs that direct RNA editing in trypanosomes. [0077] After transcription, nascent rRNA molecules (termed pre-rRNA) undergo a series of processing steps to generate the mature rRNA molecule. Prior to cleavage by exo- and endonucleases, the pre-rRNA undergoes a complex pattern of nucleoside modifications. These include methylations and pseudouridylations, guided by snoRNAs. Methylation is the attachment or substitution of a methyl group onto various substrates. The rRNA of humans contain approximately 115 methyl group modifications. The majority of these are 2'0-ribose- methylations (where the methyl group is attached to the ribose group). Pseudouridylation is the conversion (isomerisation) of the nucleoside uridine to a different isomeric form pseudouridine (Ψ). Mature human rRNAs contain approximately 95 Ψ modifications. Each snoRNA molecule acts as a guide for only one (or two) individual modifications in a target RNA. In order to carry out modification, each snoRNA associates with at least four protein molecules in an RNA/protein complex referred to as a small nucleolar ribonucleoprotein (snoRNP). The proteins associated with each RNA depend on the type of snoRNA molecule (see snoRNA guide families below). The snoRNA molecule contains an antisense element (a stretch of 10-20 nucleotides), which are base complementary to the sequence surrounding the base (nucleotide) targeted for modification in the pre-RNA molecule. This enables the snoRNP to recognise and bind to the target RNA. Once the snoRNP has bound to the target site, the associated proteins are in the correct physical location to catalyse the chemical modification of the target base.

[0078] The two different types of rRNA modification (methylation and pseudouridylation) are directed by two different families of snoRNPs. These families of snoRNAs are referred to as antisense C/D box and H/ACA box snoRNAs based on the presence of conserved sequence motifs in the snoRNA. There are exceptions, but as a general rule C/D box members guide methylation and H/ACA members guide pseudouridylation. The members of each family may vary in biogenesis, structure, and function, but each family is classified by the following generalised characteristics. SnoRNAs are classified under small nuclear RNA in MeSH. The HGNC, in collaboration with snoRNABase and experts in the field, has approved unique names for human genes that encode snoRNAs.

[0079] C/D box snoRNAs contain two short conserved sequence motifs, C (RUGAUGA) and D (CUGA), located near the 5' and 3' ends of the snoRNA, respectively. Short regions (~ 5 nucleotides) located upstream of the C box and downstream of the D box are usually base complementary and form a stem-box structure, which brings the C and D box motifs into close proximity. This stem-box structure has been shown to be essential for correct snoRNA synthesis and nucleolar localization. Many C/D box snoRNA also contain an additional less- well-conserved copy of the C and D motifs (referred to as C and D') located in the central portion of the snoRNA molecule. A conserved region of 10-21 nucleotides upstream of the D box is complementary to the methylation site of the target RNA and enables the snoRNA to form an RNA duplex with the RNA. The nucleotide to be modified in the target RNA is usually located at the 5th position upstream from the D box (or D' box). C/D box snoRNAs associate with four evolutionary conserved and essential proteins— fibrillarin (Noplp), NOP56, NOP58, and Snul3 (15.5-kD protein in eukaryotes; its archaeal homolog is L7Ae)— which make up the core C/D box snoRNP. There exists a eukaryotic C/D box snoRNA (snoRNA U3) that has not been shown to guide 2'-0-methylation. Instead, it functions in rRNA processing by directing pre-rRNA cleavage.

[0080] H/ACA box snoRNAs have a common secondary structure consisting of a two hairpins and two single-stranded regions termed a hairpin-hinge-hairpin-tail structure. H/ACA snoRNAs also contain conserved sequence motifs known as H box (consensus ANANNA) and the ACA box (ACA). Both motifs are usually located in the single-stranded regions of the secondary structure. The H motif is located in the hinge and the ACA motif is located in the tail region; 3 nucleotides from the 3' end of the sequence. The hairpin regions contain internal bulges known as recognition loops in which the antisense guide sequences (bases complementary to the target sequence) are located. This recognition sequence is bipartite (constructed from the two different arms of the loop region) and forms complex pseudo-knots with the target RNA. H/ACA box snoRNAs associate with four evolutionary conserved and essential proteins— dyskerin (Cbf5p), GARl, NHP2, and NOP10— which make up the core of the H/ACA box snoRNP. However, in lower eukaryotic cells such as trypanosomes, similar RNAs exist in the form of single hairpin structure and an AGA box instead of ACA box at the 3' end of the RNA.

[0081] The RNA component of human telomerase (hTERC) contains an H/ACA domain for pre-RNP formation and nucleolar localization of the telomerase RNP itself. The H/ACA snoRNP has been implicated in the rare genetic disease dyskeratosis congenita (DKC) due to its affiliation with human telomerase. Mutations in the protein component of the H/ACA snoRNP result in a reduction in physiological TERC levels. This has been strongly correlated with the pathology behind DKC, which seems to be primarily a disease of poor telomere maintenance. [0082] An unusual guide snoRNA U85 that functions in both 2'-0-ribose methylation and pseudouridylation of small nuclear RNA (snRNA) U5 has been identified. This composite snoRNA contains both C/D and H/ACA box domains and associates with the proteins specific to each class of snoRNA (fibrillarin and Garlp, respectively). More composite snoRNAs have now been characterised. [0083] These composite snoRNAs have been found to accumulate in a subnuclear organelle called the Cajal body and are referred to as small Cajal body-specific RNAs. This is in contrast to the majority of C/D box or H/ACA box snoRNAs, which localise to the nucleolus. These Cajal body specific RNAs are proposed to be involved in the modification of RNA polymerase II transcribed spliceosomal RNAs Ul, U2, U4, U5 and U12. Not all snoRNAs that have been localised to Cajal bodies are composite C/D and H/ACA box snoRNAs.

[0084] The targets for newly identified snoRNAs are predicted on the basis of sequence complementarity between putative target RNAs and the antisense elements or recognition loops in the snoRNA sequence. However, there are increasing numbers of Orphan' guides without any known RNA targets, which suggests that there might be more proteins or transcripts involved in rRNA than previously and/or that some snoRNAs have different functions not concerning rRNA. There is evidence that some of these orphan snoRNAs regulate alternatively spliced transcripts. For example, it appears that the C/D box snoRNA SNORD1 15 regulates the alternative splicing of the serotonin 2C receptor mRNA via a conserved region of complementarity. Another C/D box snoRNA, SNORD116, that resides in the same cluster as SNORD115 has been predicted to have 23 possible targets within protein coding genes using a bioinformatic approach. Of these, a large fraction were found to be alternatively spliced, suggesting a role of SNORD116 in the regulation of alternative splicing.

[0085] In the human genome, there are at least two examples where C/D box snoRNAs are found in tandem repeats within imprinted loci. These two loci (14q32 on chromosome 14 and 15ql lql3 on chromosome 15) have been extensively characterised, and in both regions multiple snoRNAs have been found located within introns in clusters of closely related copies.

[0086] In 15ql lql3, five different snoRNAs have been identified (SNORD64, SNORD107, SNORD108, SNORD109 (two copies), SNORD116 (29 copies) and SNORD115 (48 copies). Loss of the 29 copies of SNORD116 (HBII-85) from this region has been identified as a cause of Prader-Willi syndrome whereas gain of additional copies of SNORD115 has been linked to autism.

[0087] Region 14q32 contains repeats of two snoRNAs SNORD113 (9 copies) and SNORD114 (31 copies) within the introns of a tissue-specific ncRNA transcript (MEG8). The 14q32 domain has been shown to share common genomic features with the imprinted 15ql l-ql3 loci and a possible role for tandem repeats of C/D box snoRNAs in the evolution or mechanism of imprinted loci has been suggested. [0088] In some embodiments of the invention, there are synthetic nucleic acids that are snoRNA inhibitors or antagonists. In some embodiments, the snoRNA inhibitor or antagonist is an antagomir. A snoRNA 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 snoRNA. In certain embodiments, a snoRNA inhibitor molecule is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. Moreover, a snoRNA 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 snoRNA, particularly a mature, naturally occurring snoRNA. One of skill in the art could use a portion of the probe sequence that is complementary to the sequence of a snoRNA as the sequence for a snoRNA inhibitor. Moreover, that portion of the probe sequence can be altered so that it is still 90% complementary to the sequence of a snoRNA.

[0089] In certain embodiments, a synthetic snoRNA 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.

[0090] Yet further, the nucleic acid structure of the snoRNA 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.

[0091] The snoRNA 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 snoRNA molecule will be considered a single polynucleotide. In embodiments in which the different regions are on separate polynucleotides, the synthetic snoRNA will be considered to be comprised of two polynucleotides.

[0092] When the RNA molecule is a single polynucleotide, there is a linker region between the snoRNA 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 snoRNA 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.

[0093] In addition to having a snoRNA 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.

[0094] Other snoRNA-based therapies that negatively manipulate oncogenic snoRNAs', may include further include RNA sponges, RNA masks or locked nucleic acid (LNA). As used herein, the term "RNA sponge" refers to a synthetic nucleic acid (e.g. a mRNA transcript) that contains multiple tandem-binding sites for a RNA of interest, and that serves to titrate out the endogenous RNA of interest, thus inhibiting the binding of the RNA of interest to its endogenous targets.

[0095] Methods in certain aspects include reducing, eliminating, or inhibiting activity and/or expression of one or more snoRNAs in a cell comprising introducing into a cell a snoRNA inhibitor, antagonist, or antagomir; or supplying or enhancing the activity of one or more snoRNAs 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 snoRNA molecule or a synthetic snoRNA inhibitor molecule. However, in methods of the invention, the snoRNA molecule or snoRNA inhibitor need not be synthetic. They may have a sequence that is identical to a naturally occurring snoRNA or they may not have any design modifications. In certain embodiments, the snoRNA molecule and/or a snoRNA inhibitor are synthetic, as discussed above.

III. Colorectal Cancer Staging and Treatments

[0096] Methods and compositions may be provided for treating colorectal cancer with particular applications of snoRNA expression levels. Based on a profile of snoRNA expression levels, for example, the miR549a, different treatments may be prescribed or recommended for different cancer patients. Cancer staging

[0097] 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.

[0098] 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 M0 Tis: Tumor confined to mucosa; cancer-in-situ

Stage I Tl NO M0 Tl : Tumor invades submucosa

Stage I T2 NO M0 T2: Tumor invades muscularis propria

Stage II-A T3 NO M0 T3 : Tumor invades subserosa or beyond (without other organs involved)

Stage II-B T4 NO M0 T4: Tumor invades adjacent organs or perforates the visceral peritoneum

Stage III-A T1-2 N1 M0 Nl : Metastasis to 1 to 3 regional lymph nodes. Tl or T2.

Stage III-B T3-4 Nl M0 Nl : Metastasis to 1 to 3 regional lymph nodes. T3 or T4.

Stage III-C any T, N2 M0 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

[0099] 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.

[0100] 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.

[0101] In certain embodiments, there may be a decision regarding the therapeutic treatment based on the snoRNA 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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. [0107] 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.

[0108] 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.

[0109] In people with incurable colorectal cancer, treatment options including palliative care can be considered for improving quality of life. Surgical options may include non-curative 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.

[0110] 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.

[0111] 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.

[0112] 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, WT1, CFTR, C-CAM, CTS-I, zacl, scFV, MMAC1, FCC, MCC, Gene 26 (CACNA2D2), PL6, Beta* (BLU), Luca-1 (HYALl), Luca-2 (HYAL2), 123F2 (RASSF1), 101F6, Gene 21 ( PRL2), 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

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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. [0121] 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

[0122] 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, serum, plasma, 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. [0123] A sample may include but is not limited to, tissue, cells, exosomes 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. [0124] 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.

[0125] 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.

[0126] 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.

[0127] 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. [0128] 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.

[0129] 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. [0130] 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

[0131] 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 snoRNA 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, snoRNA 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.

[0132] 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.

[0133] In addition to the use of arrays and microarrays, it is contemplated that a number of difference assays could be employed to analyze snoRNAs, 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), RNAseq, Next-Generation Sequencing, and/or Bridge Litigation Assay (Genaco).

VI. Pharmaceutical Compositions

[0134] In certain aspects, the compositions or agents for use in the methods, such as chemotherapeutic agents or snoRNA 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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. [0142] 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.

[0143] 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.

[0144] 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.

[0145] 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

[0146] 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 snoRNA molecules. In certain embodiments, a kit contains, contains at least or contains at most 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, 100, 500, 1,000 or more snoRNA probes, synthetic snoRNA molecules or snoRNA inhibitors, or any value or range and combination derivable therein. In some embodiments, there are kits for evaluating snoRNA activity in a cell. In some embodiments, the kits comprise one or more detecting agents wherein the detecting agents detect a snoRNA. In some embodiments, the detecting agents consist of a detecting agent for detecting SNORD76, SNORD78, AC Al l, SNORA42, SNORA21, SNORA34, or SNORD66. In some embodiments, the detecting agents consist of a detecting agent for detecting SNORD76, SNORD78, ACA11, SNORA42, SNORA21, SNORA34, or SNORD66 and a detecting agent control. In some embodiments, the detecting agents consist of a detecting agent for detecting SNORA42. In some embodiments, the detecting agents consist of a detecting agent for detecting SNORA21. In some embodiments, the detecting agents consist of a detecting agent for detecting SNORA42 and a control. In some embodiments, the detecting agents consist of a detecting agent for detecting SNORA21 and a control.

[0147] 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.

[0148] 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.

[0149] Kits for using snoRNA probes, synthetic snoRNAs, nonsynthetic snoRNAs, and/or snoRNA 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 snoRNA identified herein.

[0150] In certain aspects, negative and/or positive control synthetic snoRNAs and/or snoRNA 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.

[0151] 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 snoRNA molecules or snoRNA may be implemented with respect to synthetic snoRNAs to the extent the synthetic snoRNA is exposed to the proper conditions to allow it to become a mature snoRNA 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. [0152] Any embodiment of the invention involving specific snoRNAs by name is contemplated also to cover embodiments involving snoRNAs 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 snoRNA.

[0153] Embodiments of the invention include kits for analysis of a pathological sample by assessing snoRNA profile for a sample comprising, in suitable container means, two or more snoRNA probes, wherein the snoRNA probes detect one or more of the snoRNA identified herein. The kit can further comprise reagents for labeling snoRNA 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. The kits may also include labeled snoRNA probes/detection molecules. The labeled snoRNA may be a snoRNA described herein.

VIII. Examples

[0154] 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 - Overexpression of snoRNAs in colorectal cancer during the screening phase

[0155] In the screening step of this study, expression levels of four snoRNAs (SNORD76, SNORD78, ACA11, and SNORA42) were examined in a subset of 16 CRCs and paired adjacent normal mucosa by quantitative real-time PCR. The expression levels of all for snoRNAs were significantly higher in CRC tissues compared to matched normal mucosa tissue specimens (SNORD76, p<0.01; SNORD78, p<0.01; ACAl l, p<0.01; SNORA42, p<0.01; FIG. la).

Example 2 - High SNORA42 expression was associated with distant metastasis and poor outcome in colorectal cancer patients during the clinical validation phase

[0156] Next, the expression patterns for all four snoRNAs with various clinicopathological factors was analyzed to determine whether their expression status has any prognostic significance in CRC patients by analyzing an independent, large cohort of CRC patients (Supplementary Table 1). In line with our screening phase results, the expression levels of all snoRNAs in CRC tissues were significantly up-regulated compared in tumor vs. normal mucosa tissues in the clinical evaluation cohort (SNORD76, pO.001; SNORD78, pO.001; ACAl l, pO.001; SNORA42, p<0.01; FIG. lb). Receiver operating characteristic (ROC) curves were used to evaluate the sensitivity and specificity of each snoRNA expression in distinguishing CRC from normal tissues. Notably, expression of all snoRNAs displayed considerable predictive significance, with an area under curve (AUC) values of 0.79 (95% CI: 0.73-0.82), 0.78 (95% CI: 0.72-0.84), 0.88 (95% CI: 0.83-0.92), 0.75 (95% CI: 0.69- 0.81), respectively (FIG. 4).

[0157] Further, to perform the time-to-event analysis in order to evaluate the prognostic impact of these snoRNAs, median value of each snoRNA expression in all patients and curatively resected patients with stage I/II/III disease were used as cut-off thresholds for analyzing prognostic impact of overall survival (OS) and disease free survival (DFS) respectively. CRC patients with expression values higher than the median for each snoRNA were assigned to a high-expression group, and the others to a low-expression group. Although expression status of three snoRNAs (SNORD76, SNORD78, and ACAl l) did not significantly correlate with survival or any other clinicopathological factors, high SNORA42 expression associated with venous invasion, lymphatic invasion, distant metastasis, UICC classification (Table 1, Supplementary Table 2) and poor prognosis compared to CRCs in the low-expression group in terms of OS and DFS (p=0.018, 0.029, respectively, log rank test, FIG. 2a and b). Multivariate Cox's regression analysis showed high SNORA42 expression was an independent prognostic factor for OS (HR:2.11, 95%CI: 1.12-3.98, p=0.021) and DFS (HR:3.17, 95%CI: 1.32-7.65, p=0.011) in CRC patients (Table 2a, 2b). Furthermore, multivariate logistic regression analysis revealed that high expression of SNORA42 was also an independent predictor of distant metastasis in CRC patients (OR:2.66, 95%CI: 1.14-6.21, p=0.023, Table 2c). Example 3 -Prognostic impact of SNORA42 expression status in colorectal cancer patients during the performance evaluation phase

[0158] To further confirm the prognostic impact of SNORA42 expression in CRC patients, results were validated in an additional independent CRC cohort. Intriguingly, high SNORA42 expression, seen with the survival outcomes in the clinical validation cohort, was associated with poor prognosis with regards to OS (p=0.026, log rank test, Figure 2c). Furthermore, multivariate Cox's regression analysis revealed that high SNORA42 expression was an independent predictor for poor prognosis in the performance evaluation cohort, as well as total cohort (HR:3.00, 95%CI: 1.10-8.19, p=0.033, HR:2.5, 95%CI: 1.51-4.14, p=0.0004, respectively, Table 3a and b).

Example 4 -SNORA42 expression status identified high-risk stage II CRC patients

[0159] Next, to determine the clinical significance of SNORA42 expression as a predictive biomarker of recurrence and prognosis in stage II CRC patients, we evaluated survival outcomes in stage II patients subdivided on the basis of tissue SNORA42 expression (shown in Figure 5a, b, and c). Elevated SNORA42 expression was associated with poor OS and DFS in patients belonging to the clinical validation cohort (p=0.048 and 0.049, respectively, log-rank test). Furthermore, high expression status of SNORA42 was significantly associated with poor prognosis in patients with stage II disease in the total cohort (p=0.039, log rank test). Collectively, our data suggests that SNORA42 expression could identify high-risk patients with stage II CRC.

Example 5 -SNORA42 were highly expressed in cancer cells compared with cancer stroma or corresponding normal mucosa

[0160] To further confirm the pathological expression pattern of SNORA42 in clinical specimens, in situ hybridization (ISH) staining was performed on 10 primary CRC tissues and corresponding adjacent normal mucosa from the clinical validation cohort. The ISH experiments revealed nuclear staining for SNORA42 in CRC cells, an observation that is consistent with previous reports in non-small cell lung cancer[21]. Furthermore, SNORA42 expression was up-regulated in the primary CRC cells compared with the matched normal mucosa, confirming our qRT-PCR results for its expression in primary CRC and adjacent normal mucosa tissues (Figure 6). The results indicated that SNORA42 was overexpressed in CRC cells compared with normal mucosa, and its expression significantly correlated with disease progression in CRC patients. Therefore, SNORA42 was evaluated for further assessment of its biological function in colorectal neoplasia. Example 6 -Overexpression of SNORA42 results in increased cell proliferation, tumorigenicity, migration, invasion and anoikis resistance in colon cancer cells

[0161] To investigate whether SNORA42 alters the biological characteristics of colon cancer cells, expression levels of SNORA42 was first assessed in a panel of colon cancer cell lines (Caco2, HCT116, HT29, LoVo, SW480 and SW620) by real-time PCR. Caco2 and SW480 cell lines were selected for overexpression studies because both cell lines showed the lowest SNORA42 expression (FIG. 3a). The pCDH-SNORA42 and pCDH-controls were transfected into these cell lines, which facilitated significant over-expression of SNORA42 in these cells (FIG. 3b). [0162] In order to determine whether ectopic expression of SNORA42 resulted in enhanced cell proliferation in human cancer cell lines, the rate of cell proliferation was analyzed by MTT assays using transfected cell lines. Cell proliferation was significantly increased in both pCDH-SNORA42 transfected cell lines, compared with control transfected cell lines (FIG. 3c). Next, to examine the colony-forming ability of single cells overexpressing SNORA42, we performed colony formation assays. Both Caco2 and SW480 cells expressing pCDH- SNORA42 demonstrated significantly higher number of colonies compared to pCDH-control transfected cell lines (FIG. 3d).

[0163] To determine whether ectopic expression of SNORA42 altered cell migration and invasion, in vitro migration and invasion assays were performed. As shown in FIG. 3e, SNORA42 overexpressing CRC cell lines showed significantly enhanced invasive and migratory potential compared to cells transfected with pCDH-control.

[0164] Anoikis is a unique form of apoptosis that is induced by detachment of cells from the extracellular matrix [34 35]. Resistance to anoikis is recognized as one of the oncogenic hallmarks contributing to cancer metastasis [36-38]. Considering that the clinical data revealed SNORA42 overexpression as an independent risk factor for distant metastasis, it was hypothesized that SNORA42 enhances resistance to anoikis in CRC cells. To further confirm whether ectopic expression of SNORA42 increases anoikis resistance, anchorage independent cell viability was assessed after cells were incubated in an ultra-low attachment plate. After induction of anoikis, pCDH-SNORA42 transfected Caco2 and SW480 cells exhibited an increase in the number of viable cells compared with the pCDH-control cells (FIG. 3f). All of these in vitro results suggest that SNORA42 might be intimately involved in CRC pathogenesis by promoting cell growth, colonogenic survival and enhancing invasion, migration, and potential of anoikis resistance in colon cancer cells. Example 7 -High SNORA42 expression results in enhanced colorectal cancer growth in an animal model

[0165] To assess whether ectopic expression of SNORA42 promotes tumorigenicity in vivo, SW480 cells, transfected with pCDH-SNORA42 or pCDH control (3 l0 ⁶ cells per mouse), were subcutaneously injected into nude mice. During the initial 13 days post-injection, no significant difference in tumor size was observed between two groups. However, mice injected with SNORA42-expressing cells appear to accelerate tumor growth around 16 days post-injection compared to the control group (FIG. 3g). Tumor size and weight were significantly higher for SNORA42-expressing cells than those of control animals (FIG. 3h and i, Supplementary Table 3) at 28 days after injection. Collectively, these results clearly demonstrate that high SNORA42 expression enhanced tumor growth in vivo, which is consistent with our in vitro and clinical findings.

Example 8 -Materials and Methods

[0166] Patients and Sample Collection [0167] This study included examination of 274 tissue specimens including 250 formalin- fixed paraffin-embedded (FFPE) primary CRC (pCRC) tissues, and 24 matched corresponding normal mucosa (NM) tissues, from 3 different CRC patient cohorts that were enrolled at Mie University and Okayama University in Japan, as described in Supplemental Table SI. The study design included an initial screening phase, followed by a subsequent clinical validation phase and a performance evaluation phase.

[0168] SnoRNA expression by qRT-PCR and in-situ hybridization analysis

[0169] Total RNA were extracted from FFPE specimens using Total Nucleic Acid Isolation Kits for FFPE tissues (Ambion, Austin, TX, USA). Careful micro-dissection was performed on FFPE tissue slides to enrich for tumor cells. Expression of snoRNAs was analyzed using Custom TaqMan small RNA assays (Applied Biosystems, Foster City, CA, USA), and the average expression levels of snoRNAs were normalized against miR-16 [25 28 30 31].

[0170] For in-situ hybridization (ISH) analysis, five-micrometer-thick FFPE tissue sections were hybridized with the SNORA42 probe (LNA-modified and 5'- and 3 '-DIG labeled oligonucleotide; Exiqon, Woburn, MA), as described previously [27 28]. Positive (U6 snRNA, Exiqon) and negative controls (scrambled, Exiqon) were included in each hybridization experiment [27 28]. Cell Lines

[0171] 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 (EVIDM) (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS) and antibiotics at 37°C in a 5% humidified C0 ₂ atmosphere.

Establishment of SNORA42 overexpressing cell lines

[0172] A pCDH vector (System biosciences, Mountain view, CA, USA) was used for ectopic SNORA42 expression. The pCDH vector encoding intact sequence of SNORA42 RNA or empty vector as a control was transfected into HEK293T cells together with pPACKHl Packaging Plasmid mix (System biosciences) for producing viral particles using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Culture supernatants were harvested 24 and 48 h after transfection, and centrifuged at 3000 rpm for 30 min. Caco2 and SW480 cells, which demonstrated weakest expression of SNORA42 in CRC cell lines, were infected with supernatants containing lentiviral particles in the presence of 6 ug/ml polybrene (Sigma- Aldrich, St. Louis, MO, USA). After 48 h of culture, lentiviral-infected cells were selected by 10 ug/ml puromycin (Life technologies), and the expression of SNORA42 was analyzed using TaqMan mRNA assays (Applied Biosystems), with GAPDH expression as an endogenous control.

Cell proliferation and colony formation assay

[0173] The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Sigma, USA) was used to determine the degree of cell proliferation as described previously[32] . For colony formation assay, the number of colonies with >50 cells were counted after seeding of colon cancer cell lines following manufacture's instruction.

Cell invasion, migration and anoikis assay

[0174] Invasion and migration assays were performed using Boyden chambers (Corning, Corning, NY, USA) using 8 um pore size membrane with Matrigel (for invasion assay) or without Matrigel (for migration assay). Anoikis assay was performed as described previously[33]. All experiments were conducted as three independent experiments. In vivo studies

[0175] 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). The animal protocol was approved by the Institutional Animal Care and Use Committee of the Baylor Research Institute. To establish a xenograft tumor model, cancer cells stably expressing SNORA42 or control cells were mixed with Matrigel (Corning) and implanted subcutaneously into the abdominal flanks of each mice (3 ^χ 10 ⁶ cells/animal). Nine mice were used in each group. The mice were monitored for 28 days following injection, and subcutaneous tumors were measured every three days. Tumor size was measured using calipers and the volume was calculated using the following formula: L x W x H, where L represents length, W width, and H height. At 28 days post-injection, all animals were sacrificed. Tumor samples were dissected and stored in RNA-later (Sigma-Aldrich), and the expression of SNORA42 in xenograft tissues was confirmed by qRT-PCR.

Statistical Analysis [0176] Results were expressed as means ± S.E, and all statistical analysis was performed using Medcalc version 12.3.0 (Broekstraat 52, 9030; Mariakerke, Belgium). Further information is provided in the Supplementary material and methods.

Example 9 -Supplementary Materials and Methods Patients and Sample Collection [0177] A total of 274 tissue specimens, including 250 resected formalin-fixed paraffin- embedded (FFPE) primary CRC (pCRC) tissues and 24 matched corresponding normal mucosa (NM) tissues from 3 different CRC patient cohorts, were analyzed in this study. The study design included an initial screening phase, followed by a subsequent clinical validation phase and a performance evaluation phase. During the screening phase, we quantified expression levels of four snoRNAs in CRC tissues and corresponding normal mucosa using TaqMan-based quantitative reverse transcription polymerase reaction (qRT-PCR). In the clinical evaluation phase, changes in snoRNA expression patterns in cancerous tissues from CRC patients were evaluated in a larger cohort of tumor tissues to determine its clinical significance in CRC patients. In the performance evaluation phase, we analyzed the SNORA42 expression status using an independent cohort of specimens to determine and validate its prognostic biomarker potential in CRC patients. The diagnosis of colorectal cancer was confirmed for all enrolled patients based on clinicopathological findings. The Tumor Node Metastasis (TNM) staging system from the American Joint Committee on Cancer was used for the pathological tumor staging of CRCs. All CRC patients who underwent surgery were followed up for tumor recurrence at regular intervals for up to five years. During each annual hospital visit, all patients underwent a chest x-ray, colonoscopy, and abdominal computed tomography. Patients treated with radiotherapy or chemotherapy before surgery were excluded from this study. All patients with stage III/IV disease received 5-fluorouracil-based chemotherapy, whereas no adjuvant chemotherapy was given to stage I & II patients.

Cell proliferation assay [0178] The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Sigma, USA) was used to determine the degree of cell proliferation following the manufacture's instruction. Colon cancer cells transfected with SNORA42-pCDH or control - pCDH cells were seeded at 5 ^χ 10 ³ cells per well in a 96-well flat-bottomed microtiter plates, in a final volume of 100 uL culture medium per well, and incubated in a humidified atmosphere. After 0-72 h incubation, cell proliferation was measured. Each independent experiment was performed three times in triplicates.

Colony formation assay

[0179] For colony formation assay, colon cancer cell lines transfected with SNORA42- pCDH or control-pCDH were seeded into six-well plates (500 cells/well) and were incubated for 10 days in medium with serum (10% FBS) at 37 °C. The number of colonies with >50 cells were counted, and the relative change of tumorigenicity in SNORA42-pCDH transfected vs. control-pCDH transfected cells was determined. Each independent experiment was performed three times.

Anoikis assay [0180] For anoikis assay, we used 6-well Costar Ultra Low Attachment Microplates (Corning). SNORA42-pCDH, or control-pCDH transfected colon cancer cells were suspended in EVIDM with 10% FBS at a concentration of 5 x 10 ⁵ 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% C0 ₂ ). After induction of anoikis, cells were seeded at 5 x 10 ³ cells/well in microtiter plates (96 wells, flat bottom) in a final volume of 100 uL culture medium/well, and analyzed using MTT assays.

Statistical analysis [0181] 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. The cut-off between high and low expression groups for SNORA42 were defined by the median value of each cohort in cancerous tissues. Overall survival (OS) was measured from the date the patient underwent surgery until the date of death resulting from any cause (i.e. cancer-unrelated deaths were not censored), or last known follow-up for patients that were still alive. Disease-free survival (DFS) 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.

[0182] Receiver operating characteristic (ROC) curve analysis was performed to determine the diagnostic performance of snoRNA expression levels in distinguishing CRC tissues from normal colonic mucosa. The Cox's proportional hazards models were used to estimate hazard ratios (EfRs) for recurrence or 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. Multivariate logistic regression models were used to predict factors influencing distant metastasis. 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.

Example 10 - Clinical significance of SNORA21, an H/ACA box snoRNA, as a

metastasis predicting and prognostic biomarker in colorectal cancer.

[0183] Colorectal cancer (CRC) remains a prevalent malignancy worldwide with a high mortality rate. Over the last decade, the roles of several types of noncoding RNAs in oncogenesis have been clarified. Emerging evidence indicates that not only microRNAs, but small nucleolar RNAs (snoRNAs) appear to be dysregulated in various malignancies and they play a critical role in modulating cell transformation, tumor progression and metastasis. Functionally, cancer associated snoRNAs are subdivided into four major categories based on their oncogenic functions: 1) an independent function from imprinting gene in cancer initiation and progression 2) Activation of human telomerase 3) Inhibition of ribosomal maturation 4) Act directly as oncogene or tumor suppressor. Despite these exciting evidences, the role of snoRNAs in CRC remains limited and their clinical significance remains largely unexplored in this malignancy.

[0184] The inventors aimed to clarify the clinical relevance of snoRNAs in tumors and evaluate their prognostic potential in CRC. Furthermore, the inventors investigated the functional role of snoRNAs in CRC tumorigenesis.

[0185] Initially, the inventors screened for the most commonly differentially expressed snoRNAs from publicly available microarray and RNA-sequence databases for other malignancies. Three clinical cohorts (a total 345 CRC tissues) were then used to select/validate the candidate snoRNAs. The prognostic potential of the candidate snoRNAs were evaluated and the association with clinicopathological features were assessed. CRISPR/Cas9 system was used to knockdown the target snoRNA in multiple CRC cell lines to determine the functional role of the target snoRNA in a series of in vitro assays.

[0186] In the discovery phase, the inventors identified four differentially overexpressed snoRNAs in human cancers. It was then confirmed that overexpression of three snoRNAs (SNORA21, SNORA34 and SNORD66) was present in CRC compared to adjacent normal tissues. Of these three snoRNAs, high SNORA21 expression in tumors resulted in poor prognosis (p=0.0086, Log-rank test). In an independent validation cohort, we confirmed high SNORA21 expression results in poor overall survival (p=0.0060), especially in stage IV CRC (p=0.0272). Clinicopathological analysis showed positive association between SNORA21 and vascular invasion and metastasis (p=0.030 and 0.011, by Kruskal-Wallis test and Mann- Whitney U test, respectively) indicating that SNORA21 could be involved in metastasis. Moreover, multivariate analysis revealed that increased SNORA21 expression was an independent prognostic factor for overall survival. Using CRISPR/Cas9 stable SNORA21 knocked-down cell lines, we demonstrated that SNORA21 suppression resulted in reduced cell proliferation and colony forming capacity.

[0187] In conclusion, the inventors first identified SNORA21 as a novel oncogene which could also be a potential prognostic marker in patients with CRC. Table 1 : Clinicopathological variables and SNORA42 expression in the clinical validation cohort

*p<0.05.

†Pearson's χ2 test. Table 2a: Multivariate analysis for predictors of overall survival in the clinical validation cohort

*p<0.05.

†The median age at surgery is 68 years in the clinical evaluation cohort.

JCut-off threshold of SNORA42 expression is median value in all patients in this cohort. TNM, tumour, node, metastasis.

Table 2b: Multivariate analysis for predictors of disease-free survival in the clinical validation cohort

*p<0.05.

†Cut-off threshold of SNORA42 expression is median value in curatively resected patients (stage I/II/III) in this cohort.

Table 2c: Multivariate analysis for distant metastasis in the clinical validation cohort

*p<0.05.

†Cut-off threshold of SNORA42 expression is median value in all patients in this cohort. Table 3a: Multivariate analysis for predictors of overall survival in the performance evaluation cohort

*p<0.05.

†The median age at surgery are 64 years in the performance evaluation cohort. TNM, tumour, node, metastasis.

Table 3b: Multivariate anal sis for redictors of overall survival in the total cohort

*p<0.05.

†The median age at surgery are 67 years in the total cohort.

TNM, tumour, node, metastasis. Supplementary Table 1: Characteristics of all patients from each CRC cohorts

CRC CRC CRC

Characteristics Screening set Clinical validation cohort Performance evaluation cohort

patients n=8 patients n=192 patients n=50

Gender Male 5 109 29

Female 3 83 21

Age (y) Mean (SD) 68.6 (13.1) 67.4 (10.3) 62.9 (10.1)

Median (Min-Max) 65 (54-92) 68 (37-89) 64 (32-88)

Tumor

Colon 4 120 24

location

Rectum 4 72 26

Histological

Differentiated 6 175 46

type

Undifferentiated 2 17 1

Unknown 0 0 3

TNM stage 1 44

II 4 60 7

III 1 43 15

IV 3 45 28

Supplementary Table 2: Clinicopathological Variables and SNORNA expression in Clinical validation cohort

SNORD76 SNORD78 ACA11 SNORA42

Variable n High Low P High Low P high low P high low P

(n=96) (n=96) (n=96) (n=96) (n=96) (n=96) (n=96) (n=96)

Gender Male 109 60 49 0.145 56 53 0.771 53 56 0.771 56 53 0.771 #

Female 83 36 47 40 43 43 40 40 43

Age (y) <68 (median) 99 43 56 0.083 47 52 0.564 51 48 0.773 49 50 1.000 #

≥68 93 53 40 49 44 45 48 47 46

Tumor location Colon 120 57 63 0.456 63 57 0.456 65 55 0.18 59 61 0.882 #

Rectum 72 39 33 33 39 31 41 37 35

Histological type Differentiated 175 91 84 0.127 90 85 0.31 89 86 0.61 89 86 0.611 #

Undifferentiated 17 5 12 6 11 7 10 7 10

Pathological T category 11/12 56 31 25 0.427 30 26 0.634 24 32 0.266 22 34 0.081 #

T3/T4 136 65 71 66 70 72 64 74 62

Venous invasion + 83 43 40 0.771 36 47 0.145 46 37 0.244 49 34 0.041* #

- 109 53 56 60 49 50 59 47 62

Lymphatic invasion + 143 74 69 0.508 71 72 1.000 74 69 0.508 78 65 0.047* #

- 49 22 27 25 24 22 27 18 31

Lymph node metastasis + 84 39 45 0.467 41 43 0.884 40 44 0.663 45 39 0.467 #

- 108 57 51 55 53 56 52 51 57

Distant metastasis + 45 21 24 0.733 25 20 0.496 26 19 0.307 30 15 0.017* #

- 147 75 72 71 76 70 77 66 81

UlCC stage classification Stage 1 44 26 18 0.206 23 21 0.842 21 23 0.549 17 27 0.028* #

Stage II 60 30 30 29 31 31 29 31 29

Stage III 43 19 24 19 24 18 25 18 25

Stage IV 45 21 24 25 20 26 19 30 15

#: Pearson's chi-square test * p<0.05

Supplementary Table 3

Time course Tumor size ( mean ± SE (mm ³ ))

P value

(Days) SNORA42 pCDH Control

4 142.0 ± 17.7 143.4117.4 0.956

7 175.1 ± 24.8 164.5117.5 0.731

10 211.0 ± 18.9 193.6113.8 0.467

13 280.4142.7 238.8129.7 0.435

16 546.71105.9 382.21109.0 0.293

19 997.91174.7 591.41176.0 0.118

22 1856.61281.2 926.91252.0 0.024

25 2158.91330.3 1085.51340.8 0.036

28 3225.41408.7 1553.71333.1 0.005

* * *

[0188] 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.

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