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    • 专利摘要:
    methods for diagnosing or detecting colorectal cancer, biomarker for colorectal cancer disease progression, kit for a diagnosis of colorectal cancer, method for selecting a cancer therapy and method of conducting a clinical trial. the present invention relates in general to the field of detection of colorectal cancer, and more particularly, to plasma microwaves for the detection of early colorectal cancer. specifically, the present invention includes methods, kits and biomarkers for diagnosing or detecting colorectal neoplasia in a human subject comprising the tapas of: a method for diagnosing or detecting colorectal neoplasia in a human subject comprising the steps of: obtaining one or more biological samples from the subject suspected of suffering from colorectal neoplasia; measure a global expression level or pattern of one or more microwaves obtained from one or more biological samples from the subject; and to compare the global expression pattern of one or more microwaves from the biological sample of the subject suspected of suffering from colorectal neoplasia with the global expression pattern of the one or more microwaves from a biological sample of a normal subject, where the normal subject is a healthy subject who does not suffer from colorectal neoplasia, in which the overexpression of a combination of mir19a mir19b, or mir19a and mir19b and mir15b is indicative of colorectal cancer.
    公开号:BR112014009643B1
    申请号:R112014009643-0
    申请日:2012-10-20
    公开日:2020-12-15
    发明作者:Maria Dolores Giraldez；I Cos Maritxell Gironella；I Garangou Antoni Castells；Juan Jose Lozano Salvatella
    申请人:Centro De Investigación Biomédica En Red De Enfermedades Hepáticas Y Digestivas；Hospital Clinic De Barcelona；
    IPC主号:
    专利说明:

    CROSS REFERENCE TO RELATED ORDERS
    [001] This application claims priority of the North American provisional application serial number 61 / 550,148, filed on October 21, 2011, the complete contents of which are incorporated in this document as a reference. FIELD OF THE INVENTION
    [002] The present invention relates in general to the field of colorectal cancer detection, and more particularly, to plasma microRNAs for the detection of early colorectal cancer. DECLARATION OF RESEARCH FUNDED BY THE FEDERAL GOVERNMENT
    [003] None. INCORPORATION AS A REFERENCE OF MATERIALS DISPOSED ON COMPACT DISKS
    [004] None. BACKGROUND OF THE INVENTION
    [005] Without limiting the scope of the invention, its history is described in relation to colorectal cancers.
    [006] US patent application No. 20100317533 (Lou et al. 2010) provides a panel of tumor metastasis biomarkers that comprises either of the two of carbonic anhydrase-9 (CAIX), vascular endothelial growth factor C (VEGF-C), ephrin A5 (EFNA5), eph B2 receptor (EPHB2), transforming growth factor beta 3 (TGF-13), pyruvate dehydrogenase kinase, isoenzyme-3 (PDK3), carbonic anhydrase-12 (CAXII) , keratin 14 (KRT14), alpha subunit of the hypoxia-inducible factor 1 (HIF-I) or tenascin C (TNC). CAIX, VEGF-C, EFNA5, EPHB2, TGF-3 or PDK3 can be indicators of moderate metastatic potential, while CAXII, KRT14, HIF-1D, or TNC can be indicators of high metastatic potential. A method of determining the risk of tumor metastasis using the biomarkers mentioned above is also provided. Biomarkers can be used in diagnosis, prognosis, treatment selection or to test putative therapies. Biomarkers can be used to assess malignancies or cancers that have hypoxic regions, such as breast cancer.
    [007] US patent application No. 20100120898 (Croce et al.2010) reveals methods and compositions for the diagnosis, prognosis and treatment of hepatocellular carcinoma (HCC). Methods of identifying anti-HCC agents are also provided. Croce's request provides a method that diagnoses whether a subject has, or is at risk of developing, hepatocellular carcinoma (HCC), which comprises measuring the level of at least one miR gene product in a subject's test sample, in which a change in the level of the miR gene product in the test sample, in relation to the level of a corresponding miR gene product in a control sample, is indicative of the subject or having, or being at risk of developing, HCC.
    [008] US patent No. 7,939,255, granted to Chung refers to methods of diagnosis for colorectal cancer. In summary, the patent discloses a diagnostic method and a kit for the assessment of colorectal cancer (CRC) prognosis with a tumor suppressor gene to be used for the diagnosis of colorectal cancer (CRC), in which the method comprises: identifying recurrently altered regions (RAR) on a chromosome; and detect genomic changes in RAR. It is said that the invention makes it possible to perform an early diagnosis as well as assess the prognosis for various cancers and tumors including colorectal cancer (CRC).
    [009] Publication No. WO2011076147, titled, Plasma-Based Micro-RNA Biomarkers And Methods For Early Detection of Colorectal, deposited by Li, reveals a diagnostic kit for molecular markers in blood to diagnose colorectal cancer and / or monitor the therapeutic effect to treat colorectal cancer. The kit is said to comprise a plurality of nucleic acid molecules, and each nucleic acid molecule encodes a microRNA biomarker, where one or more of the plurality of nucleic acid molecules are differentially expressed in patient plasma and healthy control , and one or more nucleic acid molecules differentially expressed together represent a nucleic acid expression biomarker that is indicative of the presence of colorectal cancer. The invention is said to further provide corresponding methods that use such nucleic acid expression biomarkers to identify colorectal cancer as well as to prevent or treat such a condition. Finally, the invention provides a pharmaceutical composition for the prevention and / or treatment of colorectal cancer.
    [010] Publication No. WO2011076142, entitled, Compositions And Methods For MicroRNAs Expression Profiling in Plasma of Colorectal, also deposited by Li, is said to teach compositions and methods for obtaining the microRNAs (miRNA) expression profile in colorectal cancer plasma. In particular, it is said that the invention relates to a diagnostic kit for molecular markers in blood to diagnose colorectal cancer, monitor cancer therapy and / or treat colorectal cancer that includes a plurality of nucleic acid molecules, encoding each molecule nucleic acid to a microRNA sequence, in which one or more of the plurality of nucleic acid molecules are differentially expressed in colorectal cancer plasma and healthy control plasma, and in which one or more nucleic acid molecules expressed differentially together represent a nucleic acid expression signature that is indicative of the presence of colorectal cancer. The invention is said to further relate to corresponding methods of using such nucleic acid expression signatures to identify colorectal cancer as well as to prevent or treat such a condition. Finally, the invention relates to a pharmaceutical composition for the prevention and / or treatment of colorectal cancer.
    [011] Publication No. WO2011088226, titled, Detection Of Gastrointestinal Disorders, deposited by Christine, is said to teach methods and systems to characterize a phenotype by detecting microRNAs, vesicles or biomarkers that are indicative of disease or disease progression. The disease can be a gastrointestinal disorder, such as colorectal cancer. MicroRNAs, vesicles or biomarkers can be detected in a body fluid.
    [012] Publication No. WO2010004562, entitled, Methods And Compositions For Detecting Colorectal Cancer, deposited by Baruch, is said to teach a method for performing minimally invasive early detection of colorectal cancer and / or colorectal cancer precursor cells , using microRNA molecules associated with colorectal cancer, as well as several nucleic acid molecules related to or derived from it.
    [013] Finally, publication No. WO2011012136, entitled, A Method For Classifying A Human Cell Sample As Cancerous, filed by Fog, et al, is said to teach a method for discriminating between samples of cancer and non-cancer. It is said that the method comprises detecting the level of at least one microRNA (miR) selected from Mir's group I which consists of: miR-21, miR-34a and miR-141, and detecting the level of at least one miR selected from Mir II group consisting of: miR-126, miR-143 and miR-145 in a test cell sample and, compare the expression level of said miR selected from the test cell sample with the expression level of the same miR selected from a set of tests previously registered. BRIEF DESCRIPTION OF THE INVENTION
    [014] In one embodiment the present invention includes a method for diagnosing or detecting colorectal cancer in a human subject comprising the steps of: obtaining one or more biological samples from the subject suspected of suffering from colorectal cancer; measure a level or pattern of global expression of one or more microRNAs obtained from one or more biological samples from the subject; and to compare the global expression pattern of one or more microRNAs in the biological sample of the subject suspected of having colorectal neoplasia with the global expression pattern of the one or more microRNAs in a biological sample of a normal subject, where the normal subject is a healthy subject who does not suffer from colorectal neoplasia, in which the overexpression of a combination of miR19a and miR19b, or miR19a and miR19b and miR15b is indicative of colorectal cancer. In one aspect, the method further comprises the analysis of at least one of miR18a, miR29a or miR335 in comparison with the expression of the normal subject is indicative of colorectal neoplasia. In another aspect, the method further comprises the analysis of at least one of miR29a, miR92a or miR141. In another aspect, the one or more biological samples are selected from the group consisting of one or more biological fluids, a plasma sample, a serum sample, a blood sample, a tissue sample or a fecal sample. In another aspect, the method can detect early CRC (I-II) as precisely as advanced CRC (stage II-III), tumors on the right side and lesions on the left side. In another aspect, the method comprises a confidence interval that is 90, 91, 92, 93, 94 or 95% or greater. In another aspect, the method also comprises determining the level of expression of microRNAs that are underexpressed in colorectal neoplasia and are selected from:

    [015] In another aspect, the method also comprises determining the level of expression of microRNAs that are underexpressed in colorectal neoplasia and are selected from:

    [016] In another aspect, the expression level of one or more microRNAs is measured by obtaining the microarray expression profile, PCR, reverse transcriptase PCR, reverse transcriptase real-time PCR, quantitative real-time PCR, end-PCR point, multiplex end-point PCR, cold PCR, ice-cold PCR, mass spectrometry, in situ hybridization (ISH), multiplex in situ hybridization or nucleic acid sequencing. In another aspect, the method is used to treat a patient who is at risk or suffering from colorectal neoplasia, to select a therapy with antineoplastic agent for a patient who is at risk or suffering from colorectal neoplasia, to stratify a patient into a subgroup of colorectal neoplasia or to a clinical trial of colorectal cancer therapy, determining resistance or response to a colorectal cancer therapeutic regimen, developing a kit for the diagnosis of colorectal cancer or any combination thereof. In another aspect, the global expression level or pattern of 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 microRNAs selected at from tables 2, 3, 4 and 5, in which the microRNAs increase the specificity of the determination, diagnosis or detection of colorectal neoplasia. In another aspect, the method also comprises the stage of using the level or pattern of global expression of microRNAs for prognosis, treatment guide or monitoring response to the treatment of colorectal neoplasia.
    [017] Yet another embodiment of the present invention includes a biomarker for progression of colorectal neoplasia disease, metastasis or both in which the biomarker comprises one or more microRNAs and a change in the overall expression of one or more microRNAs in colorectal cancer cells obtained from a patient is indicative of colorectal cancer disease progression when compared to the global expression of one or more microRNAs in normal colorectal cancer cells or colorectal cancer cells obtained at an early point in time from the same patient, in which the overexpression of combination of miR19a and miR19b, or miR19a and miR19b and miR15b, is indicative of colorectal cancer. In one aspect, the method further comprises analyzing one or more of the following microRNAs miR29a, miR92a, miR141, miR18a, miR19a, miR19b, miR15b, miR29a or miR335. In another aspect, the biomarker further comprises microRNAs that are underexpressed in colorectal neoplasia and selected from:

    [018] In another aspect, the biomarker also comprises microRNAs that are underexpressed in colorectal neoplasia and selected from:

    [019] In another aspect, the biomarker also comprises microRNAs that are underexpressed in colorectal neoplasia and are selected from:

    [020] In another aspect, the biomarker comprises indamicroRNAs that are underexpressed in colorectal neoplasia and are selected from:


    [021] The technician will recognize that most of the time a bio-signature (assay) will include the combination of microRNAs both over and underexpressed. As such, the present invention also includes in one aspect the combination of microRNAs both over and underexpressed from the respective microRNAs. In another aspect, biological samples are selected from the group consisting of one or more biological fluids, a plasma sample, a serum sample, a blood sample, a tissue sample or a fecal sample. In another aspect, the method can detect early CRC (I-II) as precisely as advanced CRC (stage II-III), tumors on the right side and lesions on the left side. In another aspect, the global expression level or pattern of 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 microRNAs selected at from tables 2, 3, 4 and 5, in which the microRNAs increase the specificity of the determination, diagnosis or detection of colorectal neoplasia.
    [022] Yet another embodiment of the present invention includes a kit for a diagnosis of colorectal neoplasia comprising: biomarker detector reagents to determine a differential expression level of microRNAs miR19a and miR19b, or miR19a and miR19b and miR15b, in which overexpression of a combination of miR19a and miR19b, or miR19a and miR19b and miR15b is indicative of colorectal neoplasia, in which a confidence interval for colorectal cancer is 90% or greater. In one aspect, the kit further comprises reagents for the detection and analysis of at least one of miR18a, miR29a or miR335. In one aspect, the kit further comprises reagents for the detection and analysis of at least one of miR29a, miR92a or miR141. In another aspect, the kit also includes instructions for its use in diagnosing the risk of colorectal neoplasia, in which the instruction comprises step-by-step directions for comparing the level of expression of microRNAs, when measuring the expression of a sample obtained from a subject suspected of having colorectal cancer with the level of expression of a sample obtained from a normal subject, in which the normal subject is a healthy subject who does not suffer from colorectal cancer. In another aspect, the kit also includes instruments, containers and reagents necessary to obtain samples from a subject selected from the group consisting of one or more biological fluids, a plasma sample, a serum sample, a blood sample, a tissue sample or a fecal sample. In another aspect, the kit also includes reagents for the analysis of microRNAs that are underexpressed in colorectal neoplasia and are selected from:


    [023] In another aspect, the kit also includes reagents for the analysis of microRNAs that are underexpressed in colorectal neoplasia and are selected from:

    [024] In another aspect, the kit also includes reagents for the detection and analysis of the expression pattern or the expression level for 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30 , 35, 40, 45, 50, 55 or 60 microRNAs are determined to diagnose or detect colorectal neoplasia selected from the microRNAs in tables 2, 3, 4 and 5.
    [025] Yet another embodiment of the present invention includes a method for selecting cancer therapy for a patient diagnosed with colorectal cancer, comprising the method: obtaining a sample from a subject who has a colorectal cancer; and determine the expression level of miR18a, miR19a, miR19b, miR15b, miR29a and miR335 compared to the expression level of a biological sample from a normal subject, where the normal subject is a healthy subject who does not suffer from colorectal cancer, where overexpression of microRNAs is indicative of colorectal cancer; and select cancer therapy based on the determination of colorectal neoplasia in the patient.
    [026] Another embodiment of the present invention includes a method of conducting a clinical trial to evaluate a candidate drug that is believed to be useful in the treatment of a pathological condition, comprising the method: (a) measuring the level of microRNAs obtained from a set from patients, in which microRNAs are selected from one or more microRNAs selected from microRNAs miR19a and miR19b, or miR19a and miR19b and miR15b; (b) administering a drug candidate to a first subset of patients, and a placebo to a second subset of patients; a drug comparator to a second subset of patients; or a combination of drugs from the candidate drug and another active agent to a second subset of patients; (c) repeating step (a) after the administration of the candidate drug or the placebo, the comparator drug or the drug combination; and (d) determining whether the candidate drug reduces the number of colorectal neoplastic cells that have a change in microRNA expression that is statistically significant compared to any change that occurs in the second subset of patients, where a statistically significant reduction indicates that the candidate drug is useful in the treatment of said pathological state.
    [027] Yet another embodiment of the present invention includes a method for diagnosing or detecting colorectal neoplasia in a human subject comprising the steps of: identifying the human subject suffering from or suspected of suffering from colorectal neoplasia; obtaining one or more biological samples from the subject, in which biological samples are selected from one or more biological fluids, a plasma sample, a serum sample, a blood sample, a tissue sample or a fecal sample; measure a global expression level or pattern of miR18a, miR19a, miR19b, miR15b, miR29a and miR335; and to compare the global expression pattern of one or more microRNAs in the biological sample of the subject suspected of having colorectal neoplasia with the global expression pattern of the one or more microRNAs in a biological sample of a normal subject, where the normal subject is a healthy subject who does not suffer from colorectal neoplasia, in which the overexpression of microRNAs: miR18a, miR19a, miR19b, miR15b, miR29a and miR335, is indicative of colorectal cancer.
    [028] Yet another embodiment of the present invention includes a method for diagnosing or detecting colorectal neoplasia in a human subject comprising the steps of: identifying the human subject suffering from or suspected of suffering from colorectal neoplasia; obtaining one or more biological samples from the subject, in which biological samples are selected from one or more biological fluids, a plasma sample, a serum sample, a blood sample, a tissue sample or a fecal sample; measure a global expression level or pattern of one or more microRNAs selected from:

    where ROC are the area under the curve (AUC) parameters, CI is a 95% confidence interval, S is the sensitivity, and Sp is the specificity; and to compare the global expression pattern of the one or more microRNAs obtained from the biological sample of the subject suspected of suffering from colorectal neoplasia with the global expression pattern of the one or more microRNAs from a biological sample of a normal subject, where the normal subject is a healthy subject who does not suffer from colorectal neoplasia, in which a change in the global expression pattern of one or more microRNAs in the subject's biological sample is indicative of colorectal neoplasia. In one aspect, microRNAs are under-expressed in colorectal neoplasia and are selected from:

    [029] In another aspect, microRNAs are overexpressed in colorectal neoplasia and are selected from:

    [030] In another aspect, the level of expression of one or more microRNAs is measured by obtaining the microarray expression profile, PCR, reverse transcriptase PCR, reverse transcriptase real-time PCR, quantitative real-time PCR, end PCR -point, multiplex PCR endpoint, cold PCR, icy PCR, mass spectrometry, in situ hybridization (ISH), multiplex in situ hybridization or nucleic acid sequencing. In another aspect, the method is used to treat a patient at risk or suffering from colorectal neoplasia, to select a therapy with antineoplastic agent (for example, nucleic acid crosslinking agents, small molecules, biological agents such as monoclonal antibodies with or without cell destruction payloads, both directed and undirected) for a patient at risk or suffering from colorectal neoplasia, stratifying a patient into a colorectal cancer subgroup or for a colorectal cancer therapy clinical trial, determining resistance or response to a therapeutic regimen of colorectal neoplasia, develop a kit for the diagnosis of colorectal neoplasia or any combination thereof. In another aspect, the global expression level or pattern of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 microRNAs are determined to diagnose or detect colorectal neoplasia. BRIEF DESCRIPTION OF THE DRAWINGS
    [031] For a more complete understanding of the characteristics and advantages of the present invention, reference is now made to the detailed description of the invention together with the accompanying figures and in which: Figures 1A and 1B show the expression of differential miRNA through microarrays between patients with CRC and controls in set 1 (figure 1A), and between patients with AA and controls (figure 1B). The heat map shows the 50 miRNA significantly unregulated with the highest HR. The red pixels correspond to an increasing abundance of miRNA in the indicated plasma sample, while the green pixels indicate decreased miRNA levels.
    [032] Figure 2 is a graph of analysis between groups (BGA) that shows the agglomeration of samples based on obtaining the miRNA expression profile. Healthy controls (C); colorectal cancer (CRC) patients; patients with advanced adenomas (AA).
    [033] Figure 3 shows box diagrams showing plasma miRNA expression in the CRC 2 set determined by qRT-PCR. The levels of miRNA expression are normalized to miR16 and represented as -dCt values. The lines inside the boxes indicate the medians. The boxes mark the interval between the 25th and 75th percentiles.
    [034] Figures 4A and 4B are analyzes of receptor operating characteristics (ROC) for the signature of two plasma miRNAs: miR19a + miR19b (figure 4A) and the signature of three plasma miRNA: miR19a + miR19b + miR15b (figure 4B) according to the results obtained from obtaining the microarray profile in the CRC 1 set and qRT-PCR data in the CRC 2 set. DETAILED DESCRIPTION OF THE INVENTION
    [035] While the realization and use of the various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be incorporated in a wide variety of specific contexts. The specific modalities discussed in this document are merely illustrative of specific ways to carry out and use the invention and not to limit the scope of the invention.
    [036] To facilitate the understanding of this invention, several terms are defined below. The terms defined in this document have meanings as commonly understood by a person skilled in the art in the areas relevant to the present invention. The terms such as "one", "one" and "o / a" are not intended to refer to just a single entity, but include the general class of which a specific example can be used for illustration. The terminology in this document is used to describe specific embodiments of the invention, but its use does not limit the invention, except as mentioned in the claims.
    [037] Abbreviations: advanced adenomas, AA; area under the curve ROC, AUC; group analysis, BGA; colorectal cancer, CRC; change in times, FC; linear models for microarray data, LIMMA; microRNAs, miRNA; receiver operating characteristic curve, ROC curve.
    [038] As used herein, the terms "colorectal cancer" and "colorectal cancer" include the well-accepted medical definition that defines colorectal cancer as a medical condition characterized by cancer of cells of the digestive tract below the small intestine (ie ie, the large intestine (cervix), including the cecum, ascending colon, transverse colon, descending colon, sigmoid colon and rectum) and includes pre-cancer (also referred to herein as advanced adenomas), early-stage and late-stage cancer . Additionally, as used in this document, the term "colorectal cancer" also includes medical conditions that are characterized by cancer of the cells of the duodenum and small intestine (jejunum and ileum).
    [039] As used herein, the term "tissue sample" (the term "tissue" is used interchangeably with the term "tissue sample") refers to include any material composed of one or more cells , either individually or in complex with any matrix or in association with any chemical product. The definition will include any biological or organic material and any cell sub-portion, product or by-product thereof. The definition of “tissue sample” should be understood to include without limitation sperm, eggs, embryos and blood components. Also included within the definition of "tissue" for the purposes of this invention are certain defined acellular structures such as dermal layers of the skin that have an acellular origin, but are no longer characterized as cellular. The term “feces” as used in this document is a clinical term that refers to feces excreted by humans.
    [040] As used herein, the term "gene" refers to a unit that codes for a peptide, polypeptide or functional protein. As will be understood by those skilled in the art, this functional term includes genomic sequences, cDNA sequences, or fragments or combinations thereof, as well as gene products, including those that may have been altered by the hand of man. Purified genes, nucleic acids, protein and the like are used to refer to these entities when identified and separated from at least one contaminating protein or nucleic acid with which it is ordinarily associated. The term "allele" or "allelic form" refers to an alternative version of a gene that codes for the same functional protein, but which contains differences in the nucleotide sequence relative to another version of the same gene.
    [041] As used herein, the term “microRNAs” (“miRNA” or “miR”) refers to an RNA (or RNA analog) that comprises the product of a non-coding, endogenous gene whose transcripts of Precursor RNA can form small stem and loop structures from which mature "miRNA" are cleaved by, for example, Dicer. "MiRNA" are encoded in genes distinct from the mRNAs whose expression they control.
    [042] As used herein, “nucleic acid” or “nucleic acid molecule” refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the chain reaction of polymerase (PCR), and fragments generated by any binding, fission, endonuclease action and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or nucleotide analogs that naturally occur (for example, □ -enantiomeric forms of nucleotides that are produced in natural way), or a combination of both. The modified nucleotides may have changes in the remains of sugar and / or in the remains of pyrimidine or purine base. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. In addition, the rest of the whole sugar can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications to a base remnant include alkylated pyrimidines and purines, acylated pyrimidines or purines, or other well-known heterocyclic substitutes. The nucleic acid monomers can be joined by phosphodiester bonds or analogues of such bonds. Phosphodiester link analogs include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoranilothioate, phosphoranilidate, phosphoramidate and the like. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids", which comprise modified nucleic acid bases or which are naturally produced together with a polyamide backbone. Nucleic acids can be either single-stranded or double-stranded.
    [043] The term “biomarker” as used in this document in various modalities refers to a specific biochemist in the body that has a particular molecular characteristic to make it useful for diagnosing and measuring disease progress or treatment effects . For example, the common biomarkers or metabolites found in a person's breath, and the respective diagnostic status of the person providing that metabolite include, but are not limited to, acetaldehyde (source: ethanol, X-threonine; diagnosis: intoxication) , acetone (source: acetoacetate; diagnosis: diet / diabetes), ammonia (source: amino acid deamination; diagnosis: uremia and liver disease), CO (carbon monoxide) (source: CH2Cl2,% high COHb; diagnosis: pollution indoor air), chloroform (source: halogenated compounds), dichlorobencene (source: halogenated compounds), diethylamine (source: choline; diagnosis: intestinal bacterial overgrowth), H (hydrogen) (source: intestines; diagnosis: lactose intolerance), isoprene (source: fatty acid; diagnosis: metabolic stress), methanethiol (source: methionine; diagnosis: bacterial-intestinal overgrowth), methyl ethyl ketone (source: fatty acid; diagnosis: indoor air pollution / diet), O-toluidi na (source: carcinoma metabolite; diagnosis: bronchogenic carcinoma), sulphides and pentane sulphides (source: lipid peroxidation; diagnosis: myocardial infarction), H2S (source: metabolism; diagnosis: periodontal disease / ovulation), MeS (source: metabolism; diagnosis: cirrhosis) and Me2S (source: infection; diagnosis: necrotizing gingivitis).
    [044] The term “statistically significant” differences between the groups studied refers to the state when using the appropriate statistical analysis (eg Chi-square test, t test) the probability of the groups being equal is less than 5%, for example, p <0.05. In other words, the probability of getting the same results on a completely random basis is less than 5 out of 100 attempts.
    [045] The term “kit” or “test kit” indicates combinations of reagents and adjuvants required for an analysis. Although a test kit in many cases consists of several units, elements of analysis of a part are also available, which should also be considered as test kits.
    [046] The term “polymerase chain reaction” (PCR) as used in this document refers to the method of KB Mullis, US patents 4,683,195, 4,683,202 and 4,965,188, incorporated by this document as a reference, which describes a method to increase the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the bicatenary target sequence. To perform the amplification, the mixture is denatured and the primers then hybridize in their complementary sequences within the target molecule. After hybridization, the primers are extended with a polymerase so that it forms a new pair of complementary strands. The denaturation, primer hybridization and polymerase extension steps can be repeated many times (i.e. denaturation, hybridization and extension constitute a "cycle"; they can have numerous "cycles") to obtain a high concentration of an amplified segment of the sequence desired target. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. In view of the process repetition aspect, the method is referred to as the “polymerase chain reaction” (hereinafter referred to as PCR).
    [047] As used herein, one or more microRNAs can be measured by obtaining the microarray expression profile, PCR, reverse transcriptase PCR, reverse transcriptase real-time PCR, quantitative real-time PCR, end PCR -point, multiplex PCR endpoint, cold PCR, mass spectrometry, in situ hybridization (ISH), multiplex in situ hybridization or nucleic acid sequencing. However, other techniques for determining the expression of microRNAs can be used such as surface plasmon resonance, fluorescence resonance effects (transfer, extinction and variants thereof), or the next generation of any of the previously listed techniques and combinations of the all of which are within the scope of the present invention. The level of global expression of one or more microRNAs can be used to increase the sensitivity and quality of determining the presence of colorectal neoplasia. For example, it is normal for an increase in sensitivity to be accompanied by an increase in the number of microRNAs measured, for example, how many more microRNAs are measured (for example, 2 against 8 or 15 against 30) there is a concomitant increase in the quality of the determination as is well known to those skilled in the field of expression levels. The technician will recognize that most of the time a bio-signature (assay) will include the combination of both over- and under-expressed microRNAs. As such, the present invention also includes in one aspect the combination of both the over and under expressed microRNAs of the respective microRNAs.
    [048] The present invention can be used for the diagnosis and treatment of patients, which includes or can be extended to the prognosis, treatment guide, response to treatment monitoring, use in clinical trials, research and combinations thereof. The technician will recognize that the detection of the microRNAs identified in this document can be used for any of these uses.
    [049] MicroRNAs (miRNA) are small, endogenous non-coding RNA molecules, conserved in an evolutionary manner of 20-22 nucleotides that function as regulators of gene expression. Recent evidence shows that miRNA regulates several crucial cellular processes such as development, differentiation, proliferation and apoptosis. They are thought to play an important role in the initiation and progression of human cancer, acting as oncogenes or tumor suppressors [1].
    [050] Changes in miRNA expression profiles have been observed in various tissues in a variety of human pathologies including cancer. To date, each type of tumor analyzed by obtaining the miRNA tissue profile has shown significantly different miRNA profiles compared to normal cells in the same tissue [2-4]. In addition, some recent reports have shown that miRNA is present in the serum and plasma of humans and other animals [5-7]. The circulating levels of miRNA are quite stable, reproducible and consistent between individuals of the same species [5]. Therefore, obtaining the expression profile of circulating miRNA shows great promise as a new non-invasive strategy for the diagnosis of cancer and other diseases.
    [051] Colorectal cancer (CRC) is the second most common cancer in Western countries, and represents the second leading cause of cancer-related death [8]. Fortunately, there is evidence that examining individuals at medium risk can result in reduced mortality and incidence by detecting early cancer and removing precursor cancer lesions [9]. In fact, the aim of the screening programs is to detect early-stage CRC and advanced adenomas (AA) that are pre-malignant lesions associated with a high risk of progression to an invasive lesion.
    [052] Currently, there is no optimal and universally accepted strategy for the CRC exam [10, 11] and fecal based tests are hampered by its limited sensitivity, colonoscopy is an invasive approach [12]. Therefore, new approaches that can complement and improve current strategies are urgently needed. In this sense, previous studies have found that some miRNA are increased in plasma of patients with CRC, which suggests a potential role as non-invasive biomarkers (13-16). However, all of them were limited to the analysis of a small number of miRNA. Consequently, it is mandatory to further characterize the obtaining of the plasma miRNA profile using high-throughput techniques and to assess their functioning characteristics in detecting individuals who harbor CRC and / or cancer-stricken lesions. In the present study, the plasma miRNA profile was obtained using microarrays in a group of patients with CRC or AA, and healthy individuals, identifying a group of miRNA that can detect patients with colorectal neoplasia with a high discriminative capacity. Validation in an independent cohort of individuals and the use of a different technology allows 6 plasma miRNAs to be confirmed as very promising biomarkers for the non-invasive diagnosis of CRC. Unfortunately, the discriminative capacity of these miRNAs was limited in the detection of cancer-stricken lesions.
    [053] Circulating miRNAs show great promise as new biomarkers for the diagnosis of cancer and other diseases. New non-invasive approaches that can complement and improve current strategies for the examination of colorectal cancer (CRC) are urgently needed. Obtaining the plasma genome miRNA expression profile was performed by microarrays in a set of individuals (n = 61 ) including patients with CRC or pre-malignant lesions such as advanced colorectal adenomas (AA), and healthy subjects. qRT-PCR in real time was used to validate the expression of selected miRNA from an independent cohort of patients from another hospital (n = 135). CRC or AA patients showed significantly different plasma miRNA expression profiles compared to controls. A group of 13 miRNA was selected to be validated in an independent cohort of patients, and 6 of them were confirmed to be significantly overexpressed in the CRC group, showing a high discriminative precision. One of these 6 miRNA was confirmed to also be significantly overexpressed in patients with AA, with a moderate discriminative capacity.
    [054] A total of 273 subjects from two hospitals (Hospital Clinic de Barcelona, Catalonia, Spain and Hospital Donostia, Guipúscoa, Spain) were prospectively included in this study. Of these, 77 were excluded because they met any of the following criteria: clinical diagnosis of familial adenomatous polyposis or Lynch syndrome, presence of more than 10 colorectal adenomas, diagnosis of cancer elsewhere at the time of selection, having inflammatory bowel disease , try chemotherapy or radiation therapy at the time of blood collection, incomplete bowel examination, inadequate bowel preparation for diagnostic colonoscopy, or presence of hemolysis in plasma samples. Finally, 196 individuals were included: 123 newly diagnosed patients with sporadic colorectal neoplasia (63 with CRC and 40 with AA) and 73 healthy individuals with no personal history of any cancer and with a recent colonoscopy that confirms the lack of colorectal neoplastic lesions. AA patients were those with adenomas that have a length of at least 10mm or that have histologically high-grade dysplasia or> 20% villous component. These individuals were divided into two different and unrelated sets: set 1, 61 subjects from the Hospital Clinic of Barcelona, who were employed to obtain the expression profile of plasma microRNAs of the complete genome; and set 2, 135 subjects from Hospital Donostia, who were recruited to further validate the results obtained in set 1. The characteristics of participants are shown in table 1. Blood samples were collected before endoscopy or surgery in all individuals. 1 PATIENTS CHARACTERISTICS.

    [055] The study was approved by the Institutional Ethics Committee of the Hospital Clinic de Barcelona (approval date: 03/26/2009), and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki.
    [056] RNA extraction from plasma samples. Twenty ml of whole blood from each participant was collected in EDTA tubes. Blood samples were placed at 4 ° C until plasma separation, and the plasma was frozen within 6 hours of blood collection. In summary, the samples were centrifuged at 1,600 x g for 10 min. at 4 ° C to pellet blood cells, and the plasma was transferred to new tubes, followed by additional centrifugation at 16,000 x g for 10 minutes at 4 ° C to completely remove cell components. Then the plasma was aliquoted and stored at -80 ° C until use. Total RNA containing small RNAs was isolated from 550 l of plasma using Trizol LS reagent (Invitrogen, Carlsbad, CA) and miRNeasy Mini kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol with the following modifications. The Trizol LS reagent was added to the plasma samples in a 2: 1 volumetric ratio. After separating the stages by adding chloroform and centrifuging, the aqueous stage was separated into a new tube and a volume of Trizol LS was added additionally. After the second stage separation 1.5 volumes of 100% ethanol were added to the aqueous stage and the mixture was loaded onto a miRNeasy column, according to the manufacturer's instructions. DNase treatment (Qiagen) was carried out to remove any contaminating DNA. The final elution volume was 30 Dl. The concentration of RNA was quantified using NanoDrop 1000 (Nanodrop, Wilmington, DE) in all samples and ranged between 3 and 35 ng / nl. The extraction process was repeated for each sample until sufficient RNA was obtained for the next steps.
    [057] Obtaining the plasma genome miRNA profile of the complete genome by microarray. Obtaining the miRNA expression profile was performed on all samples in set 1 using the microRNA expression profile assay based on the SAM-Bead Array platform (Illumina, Inc. San Diego, CA). This microarray contains 1,146 probes, including 743 validated miRNA, which detect around 97% of human miRNA recorded in the miRBase Sanger v.12.0 database. The miRNA microarray assay was performed using 200 ng of total RNA per sample. All steps were performed according to the manufacturer's protocol, as previously described [13,14]. The data were extracted using the data analysis software BeadStudio and transformed on the log base scale 2. The microarray data of all samples were normalized by quantiles using the Lumi bioconductor package [15].
    [058] Analysis of miRNA expression using real-time qRT-PCR. MiRNA expression was analyzed using real-time qRT-PCR with a previous multiplex preamplification process. In summary, 21 ng of plasma RNA was retrotranscribed with a mixture of Megaplex RT primers (Applied Biosystems Inc., Foster City, CA) and pre-amplified with Megaplex PreAmp primers and TaqMan PreAmp mother mix (Applied Biosystems Inc.) for 14 cycles . The expression of each miRNA was assessed using qPCR using TaqMan miRNA assays (Applied Biosystems Inc.) in a Viia7 real-time PCR system (Applied Biosystems Inc.). Several small putative maintenance nuclear RNAs were analyzed in order to determine the most suitable sample (RNU6B, miR16, miR423-5p, RNU48, miR544, miR103, miR525, miR451). MiR16 showed the highest abundance and stability and, therefore, the levels of miRNA expression were normalized to miR16 as an internal control according to other publications [16, 17]. Ct values were calculated from the automatic threshold. No model control showed amplification. Three technical repetitions were included for each qPCR point. The relative expression levels of the selected miRNA were calculated for each sample as ΔCt values [ΔCt = target miRNA Ct - internal control miRNA Ct].
    [059] Statistical analysis. A linear model for microarray data (LIMMA) was used to identify miRNA expressed differently between groups. LIMMA uses linear models and F statistics and tmoderated statistics matched with empirical Bayes [18]. The most significant miRNAs that use F statistics were used to perform correspondence analysis as implemented in the group analysis function (BGA) included in the made4 package [19]. This method can visualize highly dimensional data (such as multiple expression measurements) on a 2D graph in which the areas enclosed by the ellipses represent 95% of the estimated binormal distribution of the sample scores on the first and second axes [20]. The Venn diagrams were made considering only a miRNA hit with a change in absolute times greater than 1.5 and a moderate p value <0.05 (VennCounts and VennDiagram from the LIMMA package). Quantitative variables were analyzed using the Student's t test. A two-sided p-value <0.05 was considered significant. The predictability assessment of individual miRNA and different miRNA combinations, adjusted for age and gender, were calculated using logistic regression (GLM binomial distribution). The ROC analysis graphs and derived cut points, as well as global discriminative precision parameters, were computed using the DiagnosisMed R package. Sensitivity and specificity were calculated from the optimal cut point associated with the minimum distance between the ROC and the upper left corner.
    [060] Obtaining the complete genome miRNA profile in plasma samples from patients with colorectal cancer. Plasma miRNA expression discriminates patients with colorectal cancer from healthy individuals. To assess the achievement of the differential circulating miRNA expression profile between patients with CRC and healthy individuals, the miRNA microarray experiments were performed on total RNA obtained from plasma samples from 21 patients with CRC and 20 healthy controls. To further investigate whether altered miRNA expression patterns were found in patients with colorectal cancer-stricken lesions, miRNA microarray experiments on plasma RNA of 20 patients with advanced colorectal adenomas (AA) were also performed.
    [061] An initial comparative statistical analysis using LIMMA produced a total of 93 significantly unregulated miRNA (p <0.05) in patients with CRC compared to healthy subjects, and 125 miRNA when AA patients were compared with healthy controls. All microarray data is available at GEO (accession number: GSE 25609). Figures 1A and 1B show heat maps including 50 miRNA with the highest significant change in times between patients with CRC and controls (figure 1A), and between patients with AA and controls (figure 1B). The changing differences in times and p values, as well as the corresponding predictability parameters for these miRNA in CRC or AA, are shown in tables 2-3 and 4-5, respectively. Then the BGA graph was performed to visually represent the proximity between patients harboring CRC or AA, and controls according to plasma miRNA expression. As shown in figure 2, patients with CRC or AA, and healthy individuals appeared as three clearly separate groups. The specificities of miRNA expression for each type of neoplastic lesion were also analyzed, that is, CRC and AA, in comparison with control samples using Venn analysis (figure 2). It was found that a subset of 21 and 28 miRNA were exclusively and significantly regulated by increment in patients with CRC and AA, respectively, while both types of neoplastic patients shared 24 miRNA significantly regulated by increment. Therefore, each colorectal neoplastic lesion has a particular miRNA expression profile, but both of them also share an important number of unregulated miRNA, which could allow the identification of both lesions using a simple test based on a common plasma miRNA signature. TABLE 2. PARAMETERS OF CHANGE IN TIMES AND P VALUE FOR THE MAIN UNREGULATED 50MIRNS IN CRC THAT SHOW CHANGES HIGHER TIMES.


    TABLE 3. PREDICTABILITY OF EACH INDIVIDUAL MIRNA AMONG THE 50 MAIN REGULAR MIRNADES IN CRC. THE PARAMETERS OF THE ROC CURVE (AREA B TO CURVE (AUC) AND A 95% CONFIDENCE INTERVAL (CI), AND SENSITIVITY (S) AND SPECIFICITY (SP) CORRESPONDING TO AN OPTIMAL DECOR POINT ARE SHOWN.

    TABLE 4. PARAMETERS OF CHANGE IN TIMES AND P VALUE FOR THE MAIN UNREGULATED 50MIRNS IN AA THAT SHOW THE CHANGES HIGHER TIMES.

    Table 5. Predictability Of Each Individual Mirna Among The Top 50 Deregulated Mirna In AA. The ROC curve parameters (area under the curve (AUC) and a 95% confidence interval (CI), and sensitivity (S) and specificity (Sp) corresponding to an optimum cutoff point are shown.



    [062] Plasma miRNA expression validation using real-time qRT-PCR. The results of microarray-based plasma miRNA expression are technically reproducible. Initially, a real-time qRT-PCR was performed to confirm microarray results in 28 samples selected at random from set 1 (19 patients with colorectal neoplasms and 9 healthy controls). For these studies, a total of 14 miRNA candidates were selected. Twelve miRNA candidates (miR17-5p, miR92a, miR19b, miR18a, miR29a, miR302a, miR23a, miR27a, miR24, miR335, miR424 and miR15b) were elected to be present in the top 50 unregulated miRNA in CRC and / or AA and to have one base log microarray intensity 2> 8. Two additional miRNAs (miR19a and miR20a) were also selected to form part of the miR17-92 cluster, one of the best characterized oncogenic miRNA clusters, despite not meeting the previous criteria. In general, the results of qRT-PCR and microarray were correlated (data not shown) except for miR424 which did not show any amplification using qRT-PCR and, therefore, was excluded from further analysis.
    [063] Six plasma microRNAs have been confirmed to be overexpressed in CRC patients from an independent cohort. Second, the 13 miRNA candidates that showed adequate amplification in the previous stage were analyzed (miR92a, miR17-5p, miR18a, miR19a, miR19b, miR20a, miR15b, miR29a, miR302a, miR23a, miR27a, miR24 and miR335) in plasma from one independent set of 42 CRC patients and 53 healthy controls, to validate the results using real-time qRT-PCR.
    [064] Interestingly, miR18a, miR19a, miR19b, miR15b, miR29a and miR335 have been confirmed to be significantly regulated by increment in patients with CRC (figure 3). In addition, miR-24 was also overexpressed in this group of patients, but without reaching statistical significance (p = 0.08). Notably, the miRNA validated in this second set also demonstrated high accuracy in the CRC discrimination of healthy controls with areas under the ROC curve (AUC) ranging from 0.8 (95% CI: 0.71-0.89) and 0.7 (95% CI: 0.59-0.80). Next, we sought to see if any combination of these miRNA could improve the discriminative accuracy in the detection of CRC in relation to each of them alone. Among the combinations that show the best discriminative capacity, the signatures miR19a + miR19b, and miR19a + miR19b + miR15b (table 6; figure 4) were highlighted. Finally, the predictive capacity of these signatures in patients with early CRC (TNM I-II) was explored. and advanced (TNM III-IV). As shown in Table 2, both signatures showed high discriminative precision in both early and advanced cases. Similarly, it was examined whether these signatures could also detect tumors on the right side as accurately as those on the left side, and this was the case for both (table 2). TABLE 6. PREVISIBILITY OF THE BEST PLASMATIC MIRNA SIGNATURES IN PATIENTS WITH CRC FROM SET 2.


    [065] The parameters of the ROC curve (area under the curve (AUC) and a 95% confidence interval (CI) are shown for all cases of CRC as well as for different tumor stages (I / II and III / IV) and locations (right and left, in relation to splenic flexure).
    [066] A plasma microRNA has been confirmed to be increased in patients with advanced colorectal adenomas. In order to assess whether any of the plasma miRNA found overexpressed in both sets of patients with CRC were also increased in patients harboring cancer-stricken lesions, which were analyzed using real-time qRT-PCR in plasma samples from a cohort independent of 40 patients with AA and 53 healthy controls. MiR18a was confirmed to be significantly overexpressed in this second set of patients with AA compared to controls, as it was in the first set (figure 1B). However, although this miRNA showed a good discriminative capacity in the first set of patients with AA (AUC: 0.84, 95% CI: 0.72-0.96, S: 80%, Sp: 80%), this parameter was lower in the second set of patients (AUC: 0.64, 95% CI: 0.52-0.75, S: 72%, Sp: 57%).
    [067] In this study, the inventors obtained a complete genome miRNA profile using microarrays in plasma samples from patients with CRC or AA, and healthy individuals. These results show that the expression of plasma miRNA can discriminate between patients with colorectal cancer and control subjects, which suggests its potential value for the noninvasive detection of these lesions. To our knowledge, this is the first report that analyzes the expression of the complete plasma miRNA genome in patients with CRC and AA using high-performance technology and then validating the results in an independent, external cohort. Based on this high-throughput analysis, a large number of putative plasma miRNA biomarkers useful for detecting patients harboring CRC or AA have been identified. In addition, the high capacity for discrimination between individuals with CRC and healthy individuals from six plasma microRNAs in a different cohort and using different technology was confirmed. It is worth mentioning that five of these miRNA (ie miR19a, miR19b, miR18a, miR15b and miR335) represent new biomarkers, not previously reported.
    [068] The recent discovery of stable miRNA in plasma and other fluids has opened up the possibility of using these molecules as disease biomarkers, and several studies have evaluated this strategy in different configurations. In human cancer, this approach is really promising because the analysis of tumor-related circulating miRNA could probably be able to detect neoplasia in a non-invasive way. However, while most studies of obtaining the miRNA expression profile in cancer have been carried out using tissue samples, only a limited number of them have been focused on the potential value of circulating miRNA in diagnosis and prognosis [21-23]. Notably, the use of miRNA as biomarkers seems to be a better strategy than RNA or protein markers due to the high stability of these molecules, even in the presence of RNAse [23].
    [069] So far, only a few studies have analyzed the expression of some candidate plasma miRNA in CRC. Initially, Ng et al. found by qRT-PCR analysis that two plasma miRNAs (i.e., miR17-3p and miR92a) were significantly increased in patients with CRC compared to control subjects. However, their initial analysis was limited to 95 miRNA in 10 plasma samples (five patients with CRC and five healthy individuals). Most importantly, this study only evaluated the potential usefulness of plasma miRNA in the diagnosis of CRC, but not in the identification of precursor lesions [24]. Shortly thereafter, Huang et al. reported the potential utility of 2 plasma miRNAs - miR29a and miR92a- to detect patients with both CRC and AA. However, this qRT-PCR analysis was restricted to 12 miRNA selected based on previous reports. Pu et al. analyzed using qRT-PCR the expression of three miRNA in plasma samples from patients with CRC who report a significant overexpression of miR221, but patients with AA were not included in this study [25]. Finally, Cheng et al. reported regulation by a significant increase in plasma levels of miR141 in patients with metastatic CRC after analyzing the expression of three miRNA [26].
    [070] The study was to obtain a complete profile of circulating miRNA using high-yield technology in patients with CRC and AA, in order to identify those plasma miRNA with potential utility as biomarkers for the diagnosis of patients with these lesions. Among the 743 miRNAs analyzed, they found 95 circulating miRNAs significantly unregulated in patients with CRC, and 125 in patients with AA, some of them showing good discriminatory capacity. It is important to mention that among those miRNAs regulated by a significant increase in microarray analysis, the present inventors not only confirmed the plasma miRNAs related to CRC in previous studies, such as miR29a, miR92a and miR141, but also reported new candidate miRNAs with potential implication in carcinogenesis. of CRC. In addition, among these overexpressed miRNAs, all members of the miR17-92 cluster (miR17, miR92a, miR19a, miR19b, miR18a and miR20a) and two members of the miR106b-25 (miR-25 and miR93) cluster were found, a characterized cluster less extensively similar to miR17-92 in mammals. These findings highlight the miR17-92 cluster as a central player in colorectal carcinogenesis. The miR17-92 cluster, also known as oncomiRl, is one of the best characterized oncogenic miRNA clusters [27,28]. In this sense, it has been suggested that the increase in chromosomal instability, responsible for the progression from adenoma to adenocarcinoma, not only leads to regulation by increment of oncogenes, but also causes overexpression of critical miRNA, including the miR17-92 cluster [29].
    [071] The present inventors demonstrated that some of the incrementally regulated miRNA in the first set of patients with CRC (i.e., miR18a, miR19a, miR19b, miR15b, miR29a and miR335) were also overexpressed in an independent cohort. Notably, these validated miRNAs showed a high discriminative accuracy for CRC and various combinations of these miRNA improved the discriminative ability of any of these miRNA alone. In addition, they were able to detect early CRC (I-II) as precisely as advanced CRC (stage II-III), as well as tumors on the right side as accurately as lesions on the left side. These results point to the potential utility of these plasma miRNAs as new non-invasive biomarkers for the diagnosis of CRC.
    [072] As for colorectal cancer-stricken lesions, it was found that only one of six miRNAs overexpressed in CRC (miR-18a) was confirmed to be significantly overexpressed in the two independent cohorts of patients with AA. Although miR-18a showed a very good discriminative capacity in the first set of patients, this capacity was moderate in the second cohort. Consequently, the results obtained in patients with AA are not conclusive and further studies would be needed to establish whether there is any miRNA alone or in combination that can accurately detect these premalignant lesions. In fact, in the cohort of patients with AA analyzed by Huang et al. two plasma miRs (miR-29a and miR-92a) showed fairly good accuracy in discriminating AA from controls [16]. These two plasma miRNAs were also significantly overexpressed in the first cohort of patients with AA, but not in the second set. A potential explanation for this discrepancy between both sets of patients could be the less advanced characteristics of AA in the second patient cohorts (Table 1). Together, these results indicate that the assessment of the usefulness of plasma microRNAs in patients with AA constitutes an interesting line of research and deserves further investigation. Currently, the detection of AA remains a major challenge for non-invasive CRC examination strategies. In fact, it is worth mentioning that the fecal immunochemical test, a strategy currently accepted for the examination of non-invasive CRC in the medium-risk population, shows a good performance for the diagnosis of CRC, but ignores almost 50% of AA, as it was recently demonstrated in a study of the present group [12].
    [073] Interestingly, it was previously reported that all miRNAs validated in this study are overexpressed in tissue samples from patients with CRC [30-34]. Consequently, it can be assumed that CRC is the source of these plasma miRNAs. Therefore, the results not only point to miR29a, miR15b, miR19a, miR-19b, miR-18a and miR-335 as powerful biomarkers for the diagnosis of CRC, but they also highlight their involvement in colorectal carcinogenesis. Furthermore, considering the potential oncogenic role of these miRNA, the results open the possibility of future design of new targeted treatments focused on the inhibition of these molecules.
    [074] In addition to the listed microRNAs and microRNA families, other microRNAs can be added to enhance the detection of colorectal cancer. For example, the method may also comprise determining the level of expression of microRNAs that are underexpressed in colorectal neoplasia and are selected from:

    [075] The method can also comprise determining the level of expression of microRNAs that are underexpressed in colorectal neoplasia and are selected from:

    [076] Yet another bio-signature or assay will include the combination of microRNAs both over and under-expressed, for example, those listed earlier in this document or revealed in this document. As such, the present invention also includes in the aspect the combination of microRNAs both over and underexpressed from the respective microRNAs, with or without the specific listed microRNAs and families of microRNAs (or co-expressed).
    [077] In summary, patients with CRC and AA show significantly different plasma miRNA profiles compared to healthy individuals. These circulating tumor-related miRNAs are new biomarkers and represent a potential strategy for an early, non-invasive diagnosis. six promising candidate plasma miRNAs have been identified for the detection of CRC. However, this approach must be further validated in larger cohorts of patients, especially those with AA, in order to assess its effectiveness and potential applicability in an examination setting.
    [078] It is contemplated that any modality discussed in this specification can be implemented in relation to any method, kit, reagent or composition of the invention, and vice versa. In addition, the compositions of the invention can be used to achieve methods of the invention.
    [079] It will be understood that particular embodiments described in this document are shown by way of illustration and not as limitations of the invention. The main features of this invention can be used in several modalities without departing from the scope of the invention. Those skilled in the art will recognize, or may determine using, no more than routine experimentation, numerous equivalents to the specific procedures described in this document. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
    [080] All publications and patent applications mentioned in the specification are indicative of the level of the technicians in the subject to which this invention belongs. All publications and patent applications are incorporated into this document by reference to the same degree as if each individual publication or patent application was specifically and individually indicated to be incorporated as a reference.
    [081] The use of the word "one" or "one" when used in conjunction with the term "that comprises" in the claims and / or the specification may mean "one", but is also consistent with the meaning of "one or more ”,“ At least one ”and“ one or more than one ”. 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 description supports a definition that refers to only alternatives and " and / or ”. Throughout this application, the term “approximately” is used to indicate that a value includes the inherent variation of error for the device, the method used to determine the value, or the variation that exists between the objectives of the study.
    [082] As used in this specification and claim / claims, the words "comprising" (and any form of understanding, such as "understanding" and "understands"), "having" (and any form of having, such as “Have” and “has”), “including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”) they are inclusive or open and do not exclude additional elements or steps of the method not mentioned. As used in this document, the expression “which essentially consists of” limits the scope of the claim to the specified steps or materials and those that do not materially affect the basic and new characteristic (s) of the claim. claimed invention. As used herein, the term “consisting of” excludes any element, step or component not specified in the claim except for, for example, impurities ordinarily associated with the element or limitation.
    [083] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items that precede the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC or CAB. Continuing with this example, combinations that contain repetitions of one or more items or terms, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. The coach will understand that there is usually no limit on the number of items or terms in any combination, unless the context is otherwise shown.
    [084] As used in this document, the approximation words such as, without limitation, "approximately", "substantial" or "substantially" refer to a condition which when so modified is understood to be not necessarily absolute or perfect , but it would be considered quite close for the technicians in the subject to justify the designation of the condition that is present. The degree to which the description can vary depends on how big a change can be instituted and still have one of the technicians on the subject who recognizes the modified feature as it still has the required features and characteristics of the unmodified feature. In general, but subject to the previous discussion, a numerical value in this document that is modified by an approximation word such as "approximately" may vary from the value indicated by at least ± 1,2, 3, 4, 5, 6 , 7, 10, 12 or 15%.
    [085] All of the compositions and / or methods disclosed and claimed in this document may be made and performed without undue experimentation in the light of this description. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions and / or methods and in the steps or sequence of steps of the method described in this document without departing from of the concept, spirit and scope of the invention. All such similar substitutes and modifications evident to those skilled in the art are considered to be within the spirit, scope and concept of the invention as defined by the attached claims. REFERENCES 1.Esquela-Kerscher, A. and Slack, F.J. (2006) Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer, 6, 259-69. 2.Calin, G.A. and Croce, CM. (2006) MicroRNAs signatures in human cancers. Nat Rev Cancer, 6, 857-66. 3. 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    权利要求:
    Claims (4)
    [0001]
    1. METHOD FOR DIAGNOSTICING OR COLORECTAL DETECTARNEOPLASIA, in a human subject, characterized by understanding the steps of: (a) measuring in a biological sample obtained from a subject suspected of suffering from colorectal neoplasia, selected from the list consisting of a sample plasma, a serum sample and a blood sample, an overall expression level or pattern of a microRNA bioassignature comprising at least miR15b microRNA; and (b) to compare the global expression pattern of the microRNA bioassignature of the biological sample of the subject suspected of suffering from colorectal neoplasia with the global expression pattern of the microRNA bioassignature of a biological sample of a normal subject, where the normal subject is a healthy subject who does not suffer from colorectal cancer, and in which overexpression of at least miR15b is indicative of colorectal cancer.
    [0002]
    2. METHOD according to claim 1, characterized by further comprising the analysis of at least one of miR29a, miR19a, miR19b or miR335 in comparison with the expression of the normal subject, wherein the overexpression of miRNAb miRNA in combination with at least one miR29a, miR19a, miR19b or miR335 is indicative of colorectal neoplasia.
    [0003]
    3. METHOD according to any one of claims 1 to 2, characterized by the method being able to detect early CRC (I-II) as accurately as advanced CRC (stage II-III), tumors on the right side and lesions on the side left.
    [0004]
    4. METHOD according to any one of claims 1 to 3, characterized in that the method is used to stratify a patient to a subgroup at risk of suffering from colorectal neoplasia and / or to determine resistance or response to a colorectal cancer therapeutic regimen.
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    法律状态:
    2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-06-04| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
    2019-12-17| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|
    2020-02-11| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-09-24| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-12-15| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 20/10/2012, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US201161550148P| true| 2011-10-21|2011-10-21|
    US61/550,148|2011-10-21|
    PCT/IB2012/003035|WO2013093635A2|2011-10-21|2012-10-20|Plasma micrornas for the detection of early colorectal cancer|BR122015004941-5A| BR122015004941B1|2011-10-21|2012-10-20|METHOD FOR DETECTING ADVANCED COLORECTAL ADENOMAS IN A HUMAN SUBJECT|
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