WO2010004562A2 - Procédés et compositions permettant de détecter un cancer colorectal - Google Patents

Procédés et compositions permettant de détecter un cancer colorectal Download PDF

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WO2010004562A2
WO2010004562A2 PCT/IL2009/000683 IL2009000683W WO2010004562A2 WO 2010004562 A2 WO2010004562 A2 WO 2010004562A2 IL 2009000683 W IL2009000683 W IL 2009000683W WO 2010004562 A2 WO2010004562 A2 WO 2010004562A2
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sequence
nucleic acid
seq
mirna
fragment
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PCT/IL2009/000683
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WO2010004562A3 (fr
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Baruch Brenner
Shlomit Gilad
Yaron Goren
Ayelet Chajut
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Rosetta Genomics Ltd.
Mor Research Applications Ltd.
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Publication of WO2010004562A2 publication Critical patent/WO2010004562A2/fr
Publication of WO2010004562A3 publication Critical patent/WO2010004562A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention relates in general to microRNA molecules associated with colorectal cancer, as well as various nucleic acid molecules relating thereto or derived thereof.
  • miRs microRNAs
  • Colorectal cancer is the third most frequently diagnosed malignancy in the United States and the second most common cause of cancer death.
  • the prognosis of CRC is directly related to the stage of the disease.
  • the five-year survival rate for patients with an early stage is approximately 90% but only one third of patients are diagnosed at this stage.
  • the survival rate drops to 70-80% in case of deep tumor penetration into the bowel wall, 50-70% in case of regional lymph node involvement and 5-7% in patients with distant metastases.
  • the prognosis of colorectal cancer is directly related to the degree of penetration of the tumor through the bowel wall and the presence or absence of nodal involvement; consequently early detection and treatment are crucial.
  • Circulating nucleic acids in body fluids offer unique opportunities for early diagnosis of colorectal cancer.
  • the present invention provides specific nucleic acid sequences for use in the identification, early detection and diagnosis of colorectal cancer.
  • the nucleic acid sequences can also be used as prognostic markers for prognostic evaluation of a subject based on their expression pattern in a biological sample.
  • the invention further provides a method of minimally-invasive early detection of colorectal cancer and/or of colorectal cancer precursor cells.
  • the invention further provides a method for the detection of colorectal cancer, the method comprising: obtaining a biological sample from a subject; determining an expression profile in said sample of nucleic acid sequences selected from the group consisting of SEQ ID NOS: 1-24, 33-34, 81-84, 86-105, 107-129, 132-155, 157-177; a fragment thereof or a sequence having at least about 80% identity thereto; and comparing said expression profile to a reference expression profile representing the expression levels of said nucleic acid in healthy controls; whereby an altered expression level of the nucleic acid sequence allows the detection of said colorectal cancer.
  • said altered expression level is a change in a score based on a combination of expression level of said nucleic acid sequences.
  • relatively high expression levels of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 4, 5, 9-12, 17, 18, 22-24, 33-34, 81-84, 86-104, 124, 128-129, 132-154, 174; a fragment thereof and a sequence having at least about 80% identity thereto is indicative of the presence of colorectal cancer.
  • relatively low expression levels of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 2, 3, 6-8, 13-16, 19-21, 105, 107-123, 125-127, 155, 157-173, 175-177; a fragment thereof and a sequence having at least about 80% identity thereto is indicative of the presence of colorectal cancer.
  • said method further comprising managing subject treatment based on the colorectal cancer status.
  • managing subject treatment is selected from ordering further diagnostic tests, administering at least one therapeutic agent, administering radiation therapy, immunotherapy, hyperthermia, surgery, surgery followed or preceded by chemotherapy and/or radiation therapy, biotherapy, and taking no further action.
  • said biological sample is selected from the group consisting of bodily fluid, a cell line and a tissue sample.
  • the bodily fluid sample is a serum sample.
  • said bodily fluid sample is a urine sample.
  • the method comprises determining the expression of at least two nucleic acid sequences. According to some embodiments the method further comprising combining one or more expression ratios. According to some embodiments, the expression levels are determined by a method selected from the group consisting of nucleic acid hybridization, nucleic acid amplification, and a combination thereof.
  • the nucleic acid amplification method is real-time PCR (RT-PCR). According to one embodiment, said real-time PCR is quantitative real-time PCR (qRT- PCR). According to some embodiments, the RT-PCR method comprises forward and reverse primers.
  • the forward primer comprises a sequence selected from the group consisting of SEQ ID NOS: 36-46, 66-67, 225-265, 267-271; a fragment thereof and a sequence having at least about 80% identity thereto.
  • the real-time PCR method further comprises hybridization with a probe.
  • the probe comprises a nucleic acid sequence that is complementary to a sequence selected from the group consisting of any one of SEQ ID NOS: 1-24, 33-34, 81-84, 86-105, 107-129, 132-155, 157-177; a fragment thereof and sequences at least about 80% identical thereto.
  • the probe comprises a sequence selected from the group consisting of any one of SEQ ID NOS: 47-57, 77-78,178-203, 205-218, 220-224; a fragment thereof and a sequence having at least about 80% identity thereto.
  • the invention further provides a kit for the detection of colorectal cancer; said kit comprises a probe comprising a nucleic acid sequence that is complementary to a sequence selected from the group consisting of any one of SEQ ID NOS: 1-24, 33-34, 81-84, 86-105,
  • said probe comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 47-57, 77-78,178-203, 205- 218, 220-224; a fragment thereof and sequences having at least about 80% identity thereto.
  • the kit further comprises a forward primer comprising a sequence selected from the group consisting of SEQ ID NOS: 36-46, 66-67, 225-265, 267-
  • the kit further comprises a reverse primer comprising SEQ ID NO: 80, a fragment thereof and sequences having at least about 80% identity thereto.
  • Figures IA- ID are plots showing the detection of microRNAs in serum. MicroRNA profile is relatively constant in the population.
  • Figures IA- IB show the microRNA levels in serum samples taken from ten healthy individuals. Signals (CT values) from two samples are shown (One sample C T , vertical axis) relative to the median of all ten samples (horizontal axis). The levels of 363 different microRNAs were similar across the 10 samples with correlation coefficient above 0.91 for most pairs of samples.
  • Figure 1C is a histogram of standard deviations (horizontal axis) for detection level (C T ) of the microRNAs shown in figures IA- IB (vertical axis indicates the number of miRs with the specified standard deviation).
  • FIG. 1 demonstrates box plots showing the signals (C T values, vertical axis) of 11 microRNAs from Table 3 in serum samples taken from 74 healthy individuals. Boxplots show the median (horizontal line), 25 to 75 percentile (box), extent of data up to 1.5 times the interquartile range ("whiskers”), and outliers (crosses).
  • Figures 2A-2C demonstrate the different levels of circulating microRNAs in the sera of healthy individuals and CRC patients.
  • Figures 2A-2B demonstrate that the amounts of circulating lisa-miR-16 (SEQ ID NO: 2) (Fig. 2A) and hsa-miR-125b (SEQ ID NO: 3) (Fig. 2B) are lower (higher C ⁇ ) in sera of CRC patients (boxes 2, 4) compared to healthy individuals (boxes I 5 3).
  • Boxplots show the median (horizontal line), 25 to 75 percentile (box), and extent of data ("whiskers").
  • Figure 2C demonstrates the classification of CRC based on microRNA profile in serum.
  • Each of the 74 healthy controls and 44 CRC patients is represented by a marker. Squares represent healthy controls, and circles (of increasing darkness) represent CRC patients (with increasing stages of disease).
  • the horizontal axis shows the normalized C T values of hsa- miR-16 (C T 16 ), where increasing values of C T 16 indicate lower levels of hsa-miR-16.
  • the vertical axis shows the normalized C T values of hsa-miR-125b (C ⁇ 125b ), where increasing value of C ⁇ 125b indicate lower levels of hsa-miR-125b.
  • CRC patients have lower levels of hsa-miR-16 and hsa-miR-125b (higher values of Or 16 and C ⁇ 125b ).
  • Two possible thresholds for classifications are shown.
  • the results are based on RT-PCR analysis, and show the median of the normalized signal of each microRNA (represented by crosses) for each of the two groups (the horizontal/vertical axes).
  • the parallel lines describe a fold change of 1.5 in either direction between the groups.
  • Statistically significant microRNAs are marked with circles (see details in Table 6).
  • P- values are calculated by two sided Student t-test, and significance is adjusted using FDR (false discovery rate) of 0.1.
  • Figures 4A-4H are boxplots presentations comparing distributions of the presence of exemplified statistically significant microRNAs: hsa-miR-211 (SEQ ID NO: 94) (4A), hsa- miR-451 (SEQ ID NO: 6) (4B), hsa-miR-107 (SEQ ID NO: 111) (4C), hsa-miR-15a (SEQ ID NO: 117) (4D), hsa-miR-622 (SEQ ID NO: 82) (4E), hsa-miR-658 (SEQ ID NO: 4) (4F), hsa-miR-501-5p (SEQ ID NO: 88) (4G) and hsa-miR-500* (SEQ ID NO: 92) (4H) in serum samples obtained from CRC patients or healthy subjects.
  • the results are based on Real time PCR, and a higher normalized signal indicates higher amounts of microRNA present in the sample or samples.
  • the normalized C T signal (vertical axis) is calculated as follows: for each sample, the sample-average-C ⁇ is calculated by taking the average C T of all probes tested, for this sample. The overall-average-C ⁇ is calculated by taking the mean of the sample-average-C ⁇ over all samples. For each sample, the rescaling-number is calculated by subtracting the overall-average-C ⁇ from the sample-average-C ⁇ . The rescaled-signals (for each probe) are calculated for each sample by subtracting the rescaling-number from the original C T of each probe.
  • the data is translated from the C ⁇ -space which is logarithmic in the amounts measured to a linear measurement space by taking the exponent (base 2). For each miR two boxes are shown, the left box is for the group of serum samples obtained from CRC patients and the right box is for the group of serum samples obtained from healthy samples.
  • the line in the box indicates the median value.
  • the box top and bottom boundaries indicate the 25 and 75 percentile.
  • the horizontal lines and crosses (outliers whose distance from top or bottom box boundary is more than 1.5 times the height of the box) show the full range of signals in this group.
  • the invention is based on the discovery that specific biomarker sequences (SEQ ID NOS: 1-271) can be used for the identification, early detection and diagnosis of colorectal cancers.
  • Biomarkers have the potential to revolutionize diagnosis and treatment of various medical conditions.
  • a theme of current cancer research is the quest for sensitive biomarkers that can be exploited to detect early neoplastic changes.
  • biomarkers should be sampled in a minimal-invasive way. Therefore the challenge of diverse biomedical research fields has been to identify biomarkers in body fluids, such as serum or urine.
  • body fluids such as serum or urine.
  • both cell-free DNA and mRNA are present in serum, as well as in other body fluids, and represent potential biomarkers.
  • monitoring the typically small amounts of these nucleic acids in body fluids requires sensitive detection methods, which are not currently clinically applicable.
  • the present invention provides a sensitive, specific and accurate method which can be used for conducting in a minimally-invasive early detection of colorectal cancer and/or of colorectal cancer precursor cells.
  • the methods of the present invention have high sensitivity and specificity.
  • the above method allows simple minimally-invasive test, for easy detection of colorectal cancer and/or colorectal cancer precursor cells at a very early stage with higher reliability and effectiveness, saving time, material and operating steps, as well as saving cost and fine chemicals difficult to obtain.
  • the method according to the invention combines the advantages of easy sample collection and the option of diagnosing colorectal cancer or colorectal cancer precursor cells at an early stage. Being a minimally-invasive method, in which e.g. delivering a sample of serum, the method has a good potential to achieve high acceptance among subjects, which subjects can be humans or animals, for example. Therefore, the method can be used in routine tests, but also in prophylactic medical examinations. Definitions
  • aberrant proliferation means cell proliferation that deviates from the normal, proper, or expected course.
  • aberrant cell proliferation may include inappropriate proliferation of cells whose DNA or other cellular components have become damaged or defective.
  • Aberrant cell proliferation may include cell proliferation whose characteristics are associated with an indication caused by, mediated by, or resulting in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both.
  • Such indications may be characterized, for example, by single or multiple local abnormal proliferations of cells, groups of cells, or tissue(s), whether cancerous or non-cancerous, benign or malignant. about
  • antisense refers to nucleotide sequences which are complementary to a specific DNA or RNA sequence.
  • antisense strand is used in reference to a nucleic acid strand that is complementary to the "sense" strand.
  • Antisense molecules may be produced by any method, including synthesis by ligating the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a complementary strand. Once introduced into a cell, this transcribed strand combines with natural sequences produced by the cell to form duplexes. These duplexes then block either the further transcription or translation. In this manner, mutant phenotypes may be generated. attached
  • “Attached” or “immobilized” as used herein refer to a probe and a solid support and may mean that the binding between the probe and the solid support is sufficient to be stable under conditions of binding, washing, analysis, and removal.
  • the binding may be covalent or non-covalent. Covalent bonds may be formed directly between the probe and the solid support or may be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe, or both.
  • Non-covalent binding may be one or more of electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as streptavidin, to the support and the non- covalent binding of a biotinylated probe to the streptavidin. Immobilization may also involve a combination of covalent and non-covalent interactions.
  • biological sample such as streptavidin
  • Bio sample as used herein means a sample of biological tissue or fluid that comprises nucleic acids. Such samples include, but are not limited to, tissue or fluid isolated from subjects. Biological samples may also include sections of tissues such as biopsy and autopsy samples, FFPE samples, frozen sections taken for histological purposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, and skin. Biological samples also include explants and primary and/or transformed cell cultures derived from animal or patient tissues.
  • Biological samples may also be blood, a blood fraction, urine, effusions, ascitic fluid, saliva, cerebrospinal fluid, cervical secretions, vaginal secretions, endometrial secretions, gastrointestinal secretions, bronchial secretions, sputum, cell line, tissue sample, or secretions from the breast.
  • a biological sample may be provided by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods described herein in vivo.
  • Archival tissues such as those having treatment or outcome history, may also be used.
  • cancer is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • cancers include but are nor limited to solid tumors and leukemias, including: apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, small cell lung, non-small cell lung (e.g., lung squamous cell carcinoma, lung adenocarcinoma and lung undifferentiated large cell carcinoma), oat cell, papillary, bronchiolar, bronchogenic, squamous cell, and transitional cell), histiocytic disorders, leukemia (e.g., B cell, mixed cell, null cell, T cell, T-cell chronic, HTLV
  • classification refers to a procedure and/or algorithm in which individual items are placed into groups or classes based on quantitative information on one or more characteristics inherent in the items (referred to as traits, variables, characters, features, etc) and based on a statistical model and/or a training set of previously labeled items. According to one embodiment, classification means determination of the type of colorectal cancer. colorectal cancer status The term “colorectal cancer status” refers to the status of the disease in the patient.
  • colorectal cancer statuses include, but are not limited to, the subject's risk of cancer, including colorectal carcinoma, the presence or absence of disease (e.g., carcinoma), the stage of disease in a patient (e.g., carcinoma), and the effectiveness of treatment of disease.
  • Other statuses and degrees of each status are known in the art complement
  • Complement or “complementary” as used herein means Watson-Crick (e.g., A- TYU and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.
  • a full complement or fully complementary may mean 100% complementary base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.
  • C T signals represent the first cycle of PCR where amplification crosses a threshold (cycle threshold) of fluorescence. Accordingly, low values of C T represent high abundance or expression levels of the microRNA.
  • the PCR C T signal is normalized such that the normalized CT remains inversed from the expression level, hi other embodiments the PCR C T signal may be normalized and then inverted such that low normalized-inverted C T represents low abundance or expression levels of the microRNA. detection
  • Detection means detecting the presence of a component in a sample. Detection also means detecting the absence of a component. Detection also means measuring the level of a component, either quantitatively or qualitatively. differential expression
  • differential expression means qualitative or quantitative differences in the temporal and/or cellular gene expression patterns within and among cells and tissue.
  • a differentially expressed gene may qualitatively have its expression altered, including an activation or inactivation, in, e.g., normal versus disease tissue. Genes may be turned on or turned off in a particular state, relative to another state thus permitting comparison of two or more states.
  • a qualitatively regulated gene may exhibit an expression pattern within a state or cell type which may be detectable by standard techniques. Some genes may be expressed in one state or cell type, but not in both.
  • the difference in expression may be quantitative, e.g., in that expression is modulated, either up-regulated, resulting in an increased amount of transcript, or down-regulated, resulting in a decreased amount of transcript.
  • the degree to which expression differs need only be large enough to quantify via standard characterization techniques such as expression arrays, quantitative reverse transcriptase PCR, northern analysis, real-time PCR, in situ hybridization and RNase protection.
  • expression profile is used broadly to include a genomic expression profile, e.g., an expression profile of microRNAs. Profiles may be generated by any convenient means for determining a level of a nucleic acid sequence e.g. quantitative hybridization of microRNA, labeled microRNA, amplified microRNA, cDNA, etc., quantitative PCR, ELISA for quantitation, and the like, and allow the analysis of differential gene expression between two samples.
  • a subject or patient tumor sample e.g., cells or collections thereof, e.g., tissues, is assayed. Samples are collected by any convenient method, as known in the art.
  • Nucleic acid sequences of interest are nucleic acid sequences that are found to be predictive, including the nucleic acid sequences provided above, where the expression profile may include expression data for 5, 10, 20, 25, 50, 100 or more of, including all of the listed nucleic acid sequences.
  • expression profile means measuring the abundance of the nucleic acid sequences in the measured samples. , expression ratio
  • “Expression ratio” as used herein refers to relative expression levels of two or more nucleic acids as determined by detecting the relative expression levels of the corresponding nucleic acids in a biological sample.
  • Fram is used herein to indicate a non-full length part of a nucleic acid or polypeptide.
  • a fragment is itself also a nucleic acid or polypeptide, respectively.
  • Gene as used herein may be a natural (e.g., genomic) or synthetic gene comprising transcriptional and/or translational regulatory sequences and/or a coding region and/or non- translated sequences (e.g., introns, 5'- and 3'-untranslated sequences).
  • the coding region of a gene may be a nucleotide sequence coding for an amino acid sequence or a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA or antisense RNA.
  • a gene may also be an niRNA or cDNA corresponding to the coding regions (e.g., exons and miRNA) optionally comprising 5'- or 3 '-untranslated sequences linked thereto.
  • a gene may also be an amplified nucleic acid molecule produced in vitro comprising all or a part of the coding region and/or 5'- or 3 '-untranslated sequences linked thereto.
  • “Groove binder” and/or “minor groove binder” may be used interchangeably and refer to small molecules that fit into the minor groove of double-stranded DNA, typically in a sequence-specific manner.
  • Minor groove binders may be long, flat molecules that can adopt a crescent-like shape and thus, fit snugly into the minor groove of a double helix, often displacing water.
  • Minor groove binding molecules may typically comprise several aromatic rings connected by bonds with torsional freedom such as furan, benzene, or pyrrole rings.
  • Minor groove binders may be antibiotics such as netropsin, distamycin, berenil, pentamidine and other aromatic diamidines, Hoechst 33258, SN 6999, aureolic anti-tumor drugs such as chromomycin and mithramycin, CC- 1065, dihydrocyclopyrroloindole tripeptide (DPI 3 ), l,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI 3 ), and related compounds and analogues, including those described in Nucleic Acids in Chemistry and Biology, 2d ed., Blackburn and Gait, eds., Oxford University Press, 1996, and PCT
  • a minor groove binder may be a component of a primer, a probe, a hybridization tag complement, or combinations thereof. Minor groove binders may increase the T m of the primer or a probe to which they are attached, allowing such primers or probes to effectively hybridize at higher temperatures.
  • Host cell as used herein may be a naturally occurring cell or a transformed cell that may contain a vector and may support replication of the vector.
  • Host cells may be cultured cells, explants, cells in vivo, and the like.
  • Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells, such as CHO and
  • Identity or “identity” as used herein in the context of two or more nucleic acids or polypeptide sequences mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity.
  • In situ detection means the detection of expression or expression levels in the original site hereby meaning in a tissue sample such as biopsy.
  • Label as used herein means a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and other entities which can be made detectable.
  • a label may be incorporated into nucleic acids and proteins at any position. nucleic acid
  • Nucleic acid or “oligonucleotide” or “polynucleotide” as used herein mean at least two nucleotides covalently linked together.
  • the depiction of a single strand also defines the sequence of the complementary strand.
  • a nucleic acid also encompasses the complementary strand of a depicted single strand.
  • Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid.
  • a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.
  • a single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions.
  • a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.
  • Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine.
  • Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.
  • a nucleic acid will generally contain phosphodiester bonds, although nucleic acid analogs may be included that may have at least one different linkage, e.g., phosphoramidate, phospliorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages and peptide nucleic acid backbones and linkages.
  • Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, which are incorporated by reference.
  • Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included within one definition of nucleic acids.
  • the modified nucleotide analog may be located for example at the 5'-end and/or the 3'-end of the nucleic acid molecule.
  • Representative examples of nucleotide analogs may be selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase-modified ribonucleotides, i.e. ribonucleotides, containing a non-naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cytidines modified at the 5-position, e.g.
  • the 2'-OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or CN, wherein R is C 1 -C 6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.
  • Modified nucleotides also include nucleotides conjugated with cholesterol through, e.g., a hydroxyprolinol linkage as described in Krutzfeldt et al., Nature 438:685-689 (2005) and Soutschek et al., Nature 432:173-178 (2004), which are incorporated herein by reference.
  • Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments, to enhance diffusion across cell membranes, or as probes on a biochip.
  • the backbone modification may also enhance resistance to degradation, such as in the harsh endocytic environment of cells.
  • the backbone modification may also reduce nucleic acid clearance by hepatocytes, such as in the liver. Mixtures of naturally occurring nucleic acids and analogs may be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
  • Probe as used herein means an oligonucleotide capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. Probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. There may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids described herein. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence.
  • a probe may be single stranded or partially single and partially double stranded.
  • Probes may be directly labeled or indirectly labeled such as with biotin to which a streptavidin complex may later bind.
  • promoter as used herein means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell.
  • a promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same.
  • a promoter may also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • a promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
  • a promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.
  • Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or S V40 late promoter and the CMV IE promoter.
  • reference expression profile refers to a criterion expression value to which measured values are compared in order to determine the detection of a subject with colorectal cancer.
  • the reference may be based on a combine metric score. selectable marker
  • Selectable marker as used herein means any gene which confers a phenotype on a host cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct.
  • selectable markers include the ampicillin-resistance gene (Amp 1 ), tetracycline-resistance gene (Tc 1 ), bacterial kanamycin-resistance gene (Kan 1 ), zeocin resistance gene, the AURI-C gene which confers resistance to the antibiotic aureobasidin A, phosphinothricin-resistance gene, neomycin phosphotransferase gene (npt ⁇ ), hygromycin-resistance gene, beta- glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein (GFP)-encoding gene and luciferase gene.
  • sensitivity the ampicillin-resistance gene
  • sensitivity used herein may mean a statistical measure of how well a binary classification test correctly identifies a condition, for example how frequently it correctly classifies a cancer into the correct type.
  • the sensitivity for class A is the proportion of cases that are determined to belong to class "A” by the test out of the cases that are in class "A”, as determined by some absolute or gold standard.
  • Specificity used herein may mean a statistical measure of how well a binary classification test correctly identifies a condition, for example how frequently it correctly classifies a cancer into the correct type.
  • the specificity for class A is the proportion of cases that are determined to belong to class "not A" by the test out of the cases that are in class
  • Stringent hybridization conditions mean conditions under which a first nucleic acid sequence (e.g., probe) will hybridize to a second nucleic acid sequence (e.g., target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-1O 0 C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • the T m may be the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., about 10-50 nucleotides) and at least about 60°C for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times background hybridization.
  • Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65 0 C.
  • substantially complementary as used herein means that a first sequence is at least
  • substantially identical means that a first and a second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.
  • the term "subject” refers to a mammal, including both human and other mammals.
  • the methods of the present invention are preferably applied to human subjects. target nucleic acid
  • Target nucleic acid as used herein means a nucleic acid or variant thereof that may be bound by another nucleic acid.
  • a target nucleic acid may be a DNA sequence.
  • the target nucleic acid may be RNA.
  • the target nucleic acid may comprise a mRNA, tRNA, shRNA, siRNA or Piwi-interacting RNA, or a pri-miRNA, pre-miRNA, miRNA, or anti- miRNA.
  • the target nucleic acid may comprise a target miRNA binding site or a variant thereof.
  • One or more probes may bind the target nucleic acid.
  • the target binding site may comprise 5-100 or 10-60 nucleotides.
  • the target binding site may comprise a total of 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, 30-40, 40- 50, 50-60, 61, 62 or 63 nucleotides.
  • the target site sequence may comprise at least 5 nucleotides of the sequence of a target miRNA binding site disclosed in U.S. Patent Application Nos. 11/384,049, 11/418,870 or 11/429,720, the contents of which are incorporated herein. tissue sample
  • tissue sample is tissue obtained from a tissue biopsy using methods well known to those of ordinary skill in the related medical arts.
  • the phrase "suspected of being cancerous" as used herein means a cancer tissue sample believed by one of ordinary skill in the medical arts to contain cancerous cells. Methods for obtaining the sample from the biopsy include gross apportioning of a mass, microdissection, laser-based microdissection, or other art-known cell-separation methods. variant
  • nucleic acid means (i) a portion of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequence substantially identical thereto.
  • Vector as used herein means a nucleic acid sequence containing an origin of replication.
  • a vector may be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
  • a vector may be a DNA or RNA vector.
  • a vector may be either a self-replicating extrachromosomal vector or a vector which integrates into a host genome. wild type
  • wild type sequence refers to a coding, a non-coding or an interface sequence which is an allelic form of sequence that performs the natural or normal function for that sequence. Wild type sequences include multiple allelic forms of a cognate sequence, for example, multiple alleles of a wild type sequence may encode silent or conservative changes to the protein sequence that a coding sequence encodes.
  • the present invention employs miRNA for the identification, classification and diagnosis of colorectal cancer.
  • a gene coding for a microRNA may be transcribed leading to production of an miRNA precursor known as the pri-miRNA.
  • the pri-miRNA may be part of a polycistronic RNA comprising multiple pri-miRNAs.
  • the pri-miRNA may form a hairpin structure with a stem and loop.
  • the stem may comprise mismatched bases.
  • the hairpin structure of the pri-miRNA may be recognized by Drosha, which is an RNase III endonuclease. Drosha may recognize terminal loops in the pri-miRNA and cleave approximately two helical turns into the stem to produce a 60-70 nucleotide precursor known as the pre-miRNA.
  • Drosha may cleave the pri-miRNA with a staggered cut typical of RNase III endonucleases yielding a pre-miRNA stem loop with a 5' phosphate and ⁇ 2 nucleotide 3' overhang. Approximately one helical turn of the stem ( ⁇ 10 nucleotides) extending beyond the Drosha cleavage site may be essential for efficient processing.
  • the pre-miRNA may then be actively transported from the nucleus to the cytoplasm by Ran-GTP and the export receptor Ex-portin-5.
  • the pre-miRNA may be recognized by Dicer, which is also an RNase III endonuclease. Dicer may recognize the double-stranded stem of the pre-miRNA.
  • Dicer may also recognize the 5' phosphate and 3' overhang at the base of the stem loop. Dicer may cleave off the terminal loop two helical turns away from the base of the stem loop leaving an additional 5' phosphate and ⁇ 2 nucleotide 3' overhang.
  • the resulting siRNA-like duplex which may comprise mismatches, comprises the mature miRNA and a similar-sized fragment known as the miRNA*.
  • the miRNA and miRNA* may be derived from opposing arms of the pri-miRNA and pre-miRNA. MiRNA* sequences may be found in libraries of cloned miRNAs but typically at lower frequency than the miRNAs.
  • RISC RNA-induced silencing complex
  • RISC RNA-induced silencing complex
  • Various proteins can form the RISC, which can lead to variability in specificity for miRNA/miRNA* duplexes, binding site of the target gene, activity of miRNA (repression or activation), and which strand of the miRNA/miRNA* duplex is loaded in to the RISC.
  • the miRNA strand of the miRNA:miRNA* duplex is loaded into the RISC, the miRNA* may be removed and degraded.
  • the strand of the miRNA:miRNA* duplex that is loaded into the RISC may be the strand whose 5 1 end is less tightly paired. In cases where both ends of the miRNA:miRNA* have roughly equivalent 5' pairing, both miRNA and miRNA* may have gene silencing activity.
  • the RISC may identify target nucleic acids based on high levels of complementarity between the miRNA and the mRNA, especially by nucleotides 2-7 of the miRNA. Only one case has been reported in animals where the interaction between the miRNA and its target was along the entire length of the miRNA.
  • mir-196 mediates the cleavage of the Hox B8 mRNA (Yekta et al 2004, Science 304-594). Otherwise, such interactions are known only in plants (Bartel & Bartel 2003, Plant Physiol 132-709).
  • the target sites in the mRNA may be in the 5' UTR, the 3' UTR or in the coding region.
  • multiple miRNAs may regulate the same mRNA target by recognizing the same or multiple sites.
  • the presence of multiple miRNA binding sites in most genetically identified targets may indicate that the cooperative action of multiple RISCs provides the most efficient translational inhibition.
  • miRNAs may direct the RISC to downregulate gene expression by either of two mechanisms: mRNA cleavage or translational repression.
  • the miRNA may specify cleavage of the mRNA if the mRNA has a certain degree of complementarity to the miRNA. When a miRNA guides cleavage, the cut may be between the nucleotides pairing to residues 10 and 11 of the miRNA.
  • the miRNA may repress translation if the miRNA does not have the requisite degree of complementarity to the miRNA. Translational repression may be more prevalent in animals since animals may have a lower degree of complementarity between the miRNA and the binding site.
  • any pair of miRNA and miRNA* there may be variability in the 5' and 3' ends of any pair of miRNA and miRNA*. This variability may be due to variability in the enzymatic processing of Drosha and Dicer with respect to the site of cleavage. Variability at the 5' and 3' ends of miRNA and miRNA* may also be due to mismatches in the stem structures of the pri-miRNA and pre-miRNA. The mismatches of the stem strands may lead to a population of different hairpin structures. Variability in the stem structures may also lead to variability in the products of cleavage by Drosha and Dicer. Nucleic Acids
  • Nucleic acids are provided herein.
  • the nucleic acids comprise the sequence of SEQ TD NOS: 1-271 or variants thereof.
  • the variant may be a complement of the referenced nucleotide sequence.
  • the variant may also be a nucleotide sequence that is substantially identical to the referenced nucleotide sequence or the complement thereof.
  • the variant may also be a nucleotide sequence which hybridizes under stringent conditions to the referenced nucleotide sequence, complements thereof, or nucleotide sequences substantially identical thereto.
  • the nucleic acid may have a length of from 10 to 250 nucleotides.
  • the nucleic acid may have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or 250 nucleotides.
  • the nucleic acid may be synthesized or expressed in a cell (in vitro or in vivo) using a synthetic gene described herein.
  • the nucleic acid may be synthesized as a single strand molecule and hybridized to a substantially complementary nucleic acid to form a duplex.
  • the nucleic acid may be introduced to a cell, tissue or organ in a single- or double-stranded form or capable of being expressed by a synthetic gene using methods well known to those skilled in the art, including as described in U.S. Patent No. 6,506,559 which is incorporated by reference.
  • nucleic acid complexes The nucleic acid may further comprise one or more of the following: a peptide, a protein, a RNA-DNA hybrid, an antibody, an antibody fragment, a Fab fragment, and an aptamer.
  • the nucleic acid may comprise a sequence of a pri-miRNA or a variant thereof.
  • the pri-miRNA sequence may comprise from 45-30,000, 50-25,000, 100-20,000, 1,000-1,500 or 80-100 nucleotides.
  • the sequence of the pri-miRNA may comprise a pre-miRNA, miRNA and miRNA*, as set forth herein, and variants thereof.
  • the sequence of the pri-miRNA may comprise the sequence of SEQ ID NOS: 1-24, 33-34, 81-84, 86-105, 107-129, 132-155, 157-177; or variants thereof.
  • the pri-miRNA may form a hairpin structure.
  • the hairpin may comprise a first and a second nucleic acid sequence that are substantially complimentary.
  • the first and second nucleic acid sequence may be from 37-50 nucleotides.
  • the first and second nucleic acid sequence may be separated by a third sequence of from 8-12 nucleotides.
  • the hairpin structure may have a free energy of less than -25 Kcal/mole, as calculated by the Vienna algorithm, with default parameters as described in Hofacker et al., Monatshefte f. Chemie 125: 167-188 (1994), the contents of which are incorporated herein.
  • the hairpin may comprise a terminal loop of 4-20, 8-12 or 10 nucleotides.
  • the pri-miRNA may comprise at least 19% adenosine nucleotides, at least 16% cytosine nucleotides, at least 23% thymine nucleotides and at least 19% guanine nucleotides.
  • the nucleic acid may also comprise a sequence of a pre-miRNA or a variant thereof.
  • the pre-miRNA sequence may comprise from 45-90, 60-80 or 60-70 nucleotides.
  • the sequence of the pre-miRNA may comprise a miRNA and a miRNA* as set forth herein.
  • the sequence of the pre-miRNA may also be that of a pri-miRNA excluding from 0-160 nucleotides from the 5' and 3' ends of the pri-miRNA.
  • the sequence of the pre-miRNA may comprise the sequence of SEQ ID NOS: 1-24, 33-34, 81-84, 86-105, 107-129, 132-155, 157-177; or variants thereof.
  • the nucleic acid may also comprise a sequence of a miRNA (including miRNA*) or a variant thereof.
  • the miRNA sequence may comprise from 13-33, 18-24 or 21-23 nucleotides.
  • the miRNA may also comprise a total of at least 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 nucleotides.
  • the sequence of the miRNA may be the first 13-33 nucleotides of the pre-miRNA.
  • the sequence of the miRNA may also be the last 13-33 nucleotides of the pre-miRNA.
  • the sequence of the miRNA may comprise the sequence of SEQ ID NOS: 1- 1-11, 33-34, 81-84, 86-104, 124; or variants thereof.
  • Anti-miRNA The nucleic acid may also comprise a sequence of an anti-miRNA capable of blocking the activity of a miRNA or miRNA*, such as by binding to the pri-miRNA, pre- miRNA, miRNA or miRNA* (e.g. antisense or RNA silencing), or by binding to the target binding site.
  • the anti-miRNA may comprise a total of 5-100 or 10-60 nucleotides.
  • the anti-miRNA may also comprise a total of at least 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 nucleotides.
  • the sequence of the anti-miRNA may comprise (a) at least 5 nucleotides that are substantially identical or complimentary to the 5' of a miRNA and at least 5-12 nucleotides that are substantially complimentary to the flanking regions of the target site from the 5' end of the miRNA, or (b) at least 5-12 nucleotides that are substantially identical or complimentary to the 3' of a miRNA and at least 5 nucleotide that are substantially complimentary to the flanking region of the target site from the 3' end of the miRNA.
  • the sequence of the anti-miRNA may comprise the compliment of SEQ ID NOS: 1-11, 33-34, 81-84, 86-104, 124; or variants thereof.
  • the nucleic acid may also comprise a sequence of a target microRNA binding site or a variant thereof.
  • the target site sequence may comprise a total of 5-100 or 10-60 nucleotides.
  • the target site sequence may also comprise a total of at least 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 or 63 nucleotides.
  • the target site sequence may comprise at least 5 nucleotides of the sequence of SEQ ID NOS: 1-11, 33-34, 81-84, 86-104, 124.
  • a synthetic gene comprising a nucleic acid described herein operably linked to a transcriptional and/or translational regulatory sequence.
  • the synthetic gene may be capable of modifying the expression of a target gene with a binding site for a nucleic acid described herein. Expression of the target gene may be modified in a cell, tissue or organ.
  • the synthetic gene may be synthesized or derived from naturally-occurring genes by standard recombinant techniques.
  • the synthetic gene may also comprise terminators at the 3 '-end of the transcriptional unit of the synthetic gene sequence.
  • the synthetic gene may also comprise a selectable marker.
  • a vector comprising a synthetic gene described herein.
  • the vector may be an expression vector.
  • An expression vector may comprise additional elements.
  • the expression vector may have two replication systems allowing it to be maintained in two organisms, e.g., in one host cell for expression and in a second host cell
  • the expression vector may contain at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct.
  • the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector.
  • the vector may also comprise a selectable marker gene to allow the selection of transformed host cells.
  • a host cell comprising a vector, synthetic gene or nucleic acid described herein.
  • the cell may be a bacterial, fungal, plant, insect or animal cell.
  • the host cell line may be DG44 and DUXBlI (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), Rl 610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/0 (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-IcIBPT (bovine endothelial cells), RAJI (human lymphocyte) and 293 (human kidney).
  • Host cell lines may be available from commercial services, the American Tissue Culture Collection or from published literature.
  • a probe is provided herein.
  • a probe may comprise a nucleic acid.
  • the probe may have a length of from 8 to 500, 10 to 100 or 20 to 60 nucleotides.
  • the probe may also have a length of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
  • the probe may comprise a nucleic acid of 18-25 nucleotides.
  • a probe may be capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. Probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
  • a probe may be single stranded or partially single and partially double stranded. The strandedness of the probe is dictated by the structure, composition, and properties of the target sequence. Probes may be directly labeled or indirectly labeled.
  • the probe may be a test probe.
  • the test probe may comprise a nucleic acid sequence that is complementary to a miRNA, a miRNA*, a pre-miRNA, or a pri-miRNA.
  • the sequence of the test probe may be selected from SEQ ID NOS: 47-57, 77-78, 178-195, 198- 203, 205-218, 220-224; or variants thereof.
  • the probe may further comprise a linker.
  • the linker may be 10-60 nucleotides in length.
  • the linker may be 20-27 nucleotides in length.
  • the linker may be of sufficient length to allow the probe to be a total length of 45-60 nucleotides.
  • the linker may not be capable of forming a stable secondary structure, or may not be capable of folding on itself, or may not be capable of folding on a non-linker portion of a nucleic acid contained in the probe.
  • the sequence of the linker may not appear in the genome of the animal from which the probe non-linker nucleic acid is derived.
  • Target sequences of a cDNA may be generated by reverse transcription of the target RNA.
  • Methods for generating cDNA may be reverse transcribing polyadenylated RNA or alternatively, RNA with a ligated adaptor sequence.
  • the RNA may be ligated to an adapter sequence prior to reverse transcription.
  • a ligation reaction may be performed by T4 RNA ligase to ligate an adaptor sequence at the 3' end of the RNA.
  • Reverse transcription (RT) reaction may then be performed using a primer comprising a sequence that is complementary to the 3' end of the adaptor sequence.
  • Polyadenylated RNA may be used in a reverse transcription (RT) reaction using a poly(T) primer comprising a 5' adaptor sequence.
  • the poly(T) sequence may comprise 8, 9, 10, 11, 12, 13, or 14 consecutive thymines.
  • the reverse transcription primer may comprise SEQ ID NO: 80 or variants thereof.
  • the reverse transcript of the RNA may be amplified by real time PCR, using a specific forward primer comprising at least 15 nucleic acids complementary to the target nucleic acid and a 5' tail sequence; a reverse primer that is complementary to the 3' end of the adaptor sequence; and a probe comprising at least 8 nucleic acids complementary to the target nucleic acid.
  • the probe may be partially complementary to the 5' end of the adaptor sequence.
  • the amplification may be by a method comprising PCR.
  • the first cycles of the PCR reaction may have an annealing temp of 56°C, 57 0 C, 58 0 C, 59°C, or 60°C.
  • the first cycles may comprise 1-10 cycles.
  • the remaining cycles of the PCR reaction may be 60 0 C.
  • the remaining cycles may comprise 2-40 cycles.
  • the annealing temperature may cause the PCR to be more sensitive.
  • the PCR may generate longer products that can serve as higher stringency PCR templates.
  • the PCR reaction may comprise a forward primer.
  • the forward primer may comprise 15, 16, 17, 18, 19, 20, or 21 nucleotides identical to the target nucleic acid.
  • the 3' end of the forward primer may be sensitive to differences in sequence between a target nucleic acid and a sibling nucleic acid.
  • the forward primer may also comprise a 5' overhanging tail.
  • the 5' tail may increase the melting temperature of the forward primer.
  • the sequence of the 5' tail may comprise a sequence that is non-identical to the genome of the animal from which the target nucleic acid is isolated.
  • the sequence of the 5' tail may also be synthetic.
  • the 5' tail may comprise 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides.
  • the forward primer may comprise SEQ ID NOS: 36-46, 66-67, 225-250, 252-265, 267-271; or variants thereof.
  • the PCR reaction may comprise a reverse primer.
  • the reverse primer may be complementary to a target nucleic acid.
  • the reverse primer may also comprise a sequence complementary to an adaptor sequence.
  • the sequence complementary to an adaptor sequence may comprise SEQ ID NO: 80 or variants thereof.
  • Biochip A biochip is also provided.
  • the biochip may comprise a solid substrate comprising an attached probe or plurality of probes described herein.
  • the probes may be capable of hybridizing to a target sequence under stringent hybridization conditions.
  • the probes may be attached at spatially defined locations on the substrate. More than one probe per target sequence may be used, with either overlapping probes or probes to different sections of a particular target sequence.
  • the probes may be capable of hybridizing to target sequences associated with a single disorder appreciated by those in the art.
  • the probes may either be synthesized first, with subsequent attachment to the biochip, or may be directly synthesized on the biochip.
  • the solid substrate may be a material that may be modified to contain discrete individual sites appropriate for the attachment or association of the probes and is amenable to at least one detection method.
  • substrate materials include glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica- based materials including silicon and modified silicon, carbon, metals, inorganic glasses and plastics.
  • the substrates may allow optical detection without appreciably fluorescing.
  • the substrate may be planar, although other configurations of substrates may be used as well. For example, probes may be placed on the inside surface of a tube, for flow- through sample analysis to minimize sample volume.
  • the substrate may be flexible, such as flexible foam, including closed cell foams made of particular plastics.
  • the substrate of the biochip and the probe may be derivatized with chemical functional groups for subsequent attachment of the two.
  • the biochip may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol groups.
  • the probes may be attached using functional groups on the probes either directly or indirectly using a linker.
  • the probes may be attached to the solid support by either the 5' terminus, 3' terminus, or via an internal nucleotide.
  • the probe may also be attached to the solid support non-covalently.
  • biotinylated oligonucleotides can be made, which may bind to surfaces covalently coated with streptavidin, resulting in attachment.
  • probes may be synthesized on the surface using techniques such as photopolymerization and photolithography.
  • a method of diagnosis comprises detecting a differential expression level of colorectal cancer-associated nucleic acids in a biological sample.
  • the sample may be derived from a patient. Diagnosis of a cancer state, and its histological type, in a patient may allow for prognosis and selection of therapeutic strategy. Further, the developmental stage of cells may be classified by determining temporarily expressed cancer-associated nucleic acids.
  • In situ hybridization of labeled probes to tissue sections and smears may be performed.
  • the skilled artisan can make a diagnosis, a prognosis, or a prediction based on the findings. It is further understood that the genes which indicate the diagnosis may differ from those which indicate the prognosis and molecular profiling of the condition of the cells may lead to distinctions between responsive or refractory conditions or may be predictive of outcomes. Kits
  • kits may comprise a nucleic acid described herein together with any or all of the following: assay reagents, buffers, probes and/or primers, and sterile saline or another pharmaceutically acceptable emulsion and suspension base.
  • the kits may include instructional materials containing directions (e.g., protocols) for the practice of the methods described herein.
  • the kit may be used for the amplification, detection, identification or quantification of a target nucleic acid sequence.
  • the kit may comprise a ⁇ oly(T) primer, a forward primer, a reverse primer, and a probe.
  • compositions described herein may be comprised in a kit.
  • reagents for isolating miRNA, labeling miRNA, and/or evaluating a miRNA population using an array are included in a kit.
  • the kit may further include reagents for creating or synthesizing miRNA probes.
  • the kits will thus comprise, in suitable container means, an enzyme for labeling the miRNA by incorporating labeled nucleotide or unlabeled nucleotides that are subsequently labeled. It may also include one or more buffers, such as reaction buffer, labeling buffer, washing buffer, or a hybridization buffer, compounds for preparing the miRNA probes, components for in situ hybridization and components for isolating miRNA.
  • kits of the invention may include components for making a nucleic acid array comprising miRNA, and thus, may include, for example, a solid support.
  • a nucleic acid array comprising miRNA may include, for example, a solid support.
  • CRC patients were defined as patients with histologically confirmed colorectal adenocarcinoma, regardless of stage. Patients should not have been operated nor received any anti-neoplastic therapy prior to study entry, to avoid any possible influence of such interventions on microRNA expression. Healthy controls were people in whom the presence of CRC was excluded by complete colonoscopy within six months prior to study entry. Colonoscopy was done as a screening test or as an investigational procedure for suspected cancer or benign conditions. All participants had to be 18 years or older at registration and to be able to sign and understand the informed consent. The study protocol was approved by the local institutional review board.
  • PAP poly (A) polymerase
  • MnC12 MnC12
  • ATP ATP
  • the cDNA was amplified by real time PCR; this reaction contained a microRNA-specific forward primer, a TaqMan probe complementary to the 3' of the specific microRNA sequence as well as to part of the polyA adaptor sequence, and a universal reverse primer complementary to the 3' sequence of the oligodT tail.
  • 11 microRNAs were measured for normalization purposes: hsa-miR-126 (SEQ ID NO: 25), hsa-miR-19a (SEQ ID NO: 27), hsa-miR-92a (SEQ ID NO: 26), hsa-miR-423-3p (SEQ ID NO: 28), hsa- miR-22 (SEQ ID NO: 29), hsa-miR-24 (SEQ ID NO: 30), hsa-let-7b (SEQ ID NO: 31), hsa- let-7d (SEQ ID NO: 32), hsa-miR-23a (SEQ ID NO: 33), hsa-miR-148a (SEQ ID NO: 34), and hsa-miR-27a (SEQ ID NO: 35).
  • Data analysis and statistics were measured for normalization purposes: hsa-miR-126 (SEQ ID NO: 25), hsa-miR-19a (SEQ ID
  • the data was rescaled by subtracting for each sample the mean C T of all 363 microRNAs, and adding back the mean CT across all measured samples.
  • Twenty-two microRNAs were chosen for the second stage using the following criteria: 11 differentially expressed microRNAs with the smallest p-values (two-sided unpaired t-test) among those with fold change (exponent base 2 of the difference in the median normalized C T of the two groups) above 1.5; and 11 additional microRNAs with smallest variation in levels were chosen for normalization purposes.
  • These 22 microRNAs were measured on the larger cohort, and the data was rescaled by subtracting for each sample the mean CT of the 11 normalization microRNAs, and adding back the mean CT (of the normalization microRNAs) across all measured samples.
  • Protocols for extracting and quantifying microRNA in serum and other body fluids were developed.
  • the sensitivity and specificity of this qRT-PCR method makes it possible to monitor the minute amount of microRNA present in cell-free body fluids (Fig. 1).
  • microRNA profile was consistent among the healthy controls, with a mean correlation coefficient (between all pairs of individuals) of 0.90 (Figs. 1 A-IB). The median standard deviation for these microRNAs was less than 1 C T (Fig. 1C).
  • Figs. 1 A-IB The median standard deviation for these microRNAs was less than 1 C T (Fig. 1C).
  • Fig. 1C The median standard deviation for these microRNAs.
  • microRNAs are generally found in serum within a limited range. Importantly for their potential use as diagnostic tools, it was found that microRNA levels in unfrozen serum did not change substantially when samples were left at room temperatures for up to 4 hours, or by twice freezing and re-thawing of samples. This further indicates that microRNA levels in serum samples are sufficiently robust to serve as potential clinical biomarkers. In order to test whether circulating microRNAs can be used to identify CRC patients.
  • the serum levels of 363 microRNAs were measured in the sera of 10 metastatic CRC patients as compared with microRNA levels in the sera of 10 healthy individuals. From this small dataset a subset of 11 microRNAs were identified (Table 3) (Fig. ID) which showed the strongest differences between the two groups, and a further set of 11 microRNAs for normalization. The levels of these 22 microRNAs were measured in serum samples from the cohort of 44 CRC patients and 74 controls. This cohort included 5 of each group from the first set of samples. AU of the microRNAs showed the same direction of change in the large sample set as in the initial screen (Fig. 2A-2B).
  • hsa-miR-16 SEQ ID NO: 2
  • hsa-miR-125b SEQ ID NO: 3
  • These microRNAs have lower levels (higher C T ) in the sera of CRC patients (Fig.2).
  • a simple sum of the abundance signals (Cx' s) of these microRNAs can be used to identify CRC patients with 91% sensitivity and 72% specificity (Fig. 2C, solid line).
  • An alternative cutoff on the levels can reach specificity of 93% at a sensitivity of 55% (Fig. 2C, dashed line).
  • P-values are calculated on normalized C T - Fold-changes are calculated by the exponent (base 2) of the difference in the median normalized Cx of the two groups. "+” marks higher expression (lower Cx) and “— “ marks lower expression (higher Cx) in colorectal cancer patients.
  • Table 3 Comparison between microRNA levels in serum samples obtained from healthy controls and patients with colorectal cancer, based on qRT-PCR measurements
  • microRNAs up-regulated in serum samples obtained from CRC patients as compared to healthy individuals are presented in Figure 3.
  • Statistically significant microRNAs up- regulated in serum samples obtained from CRC patients as compared to healthy individuals are presented in Table 6.
  • Statistically significant microRNAs down regulated in serum samples obtained from CRC patients as compared to healthy individuals are presented in Table 7. Boxplots presentations comparing distributions of the presence of exemplified statistically significant microRNAs are shown in Figures 4A-4H.
  • Table 6 microRNAs up-regulated in serum samples obtained from CRC patients as compared to healthy individuals
  • Table 7 microRNAs down regulated in serum samples obtained from CRC patients as compared to healthy individuals

Abstract

La présente invention concerne un procédé de réalisation d’une détection précoce minimalement invasive d’un cancer colorectal et/ou de cellules précurseurs du cancer colorectal. Cette détection s’effectue au moyen de molécules de microARN associées au cancer colorectal, ainsi que de diverses molécules d’acide nucléique liées à celles-ci ou dérivées de celles-ci.
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US8735074B2 (en) 2009-08-28 2014-05-27 Asuragen, Inc. MiRNA biomarkers of lung disease
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EP2619587A1 (fr) * 2011-01-13 2013-07-31 Industrial Technology Research Institute Biomarqueurs pour la prédiction de récurrence du cancer colorectal
CN103384829A (zh) * 2011-01-13 2013-11-06 财团法人工业技术研究院 预测大肠直肠癌复发的生物标记
EP2619587A4 (fr) * 2011-01-13 2013-11-13 Ind Tech Res Inst Biomarqueurs pour la prédiction de récurrence du cancer colorectal
WO2013093635A2 (fr) 2011-10-21 2013-06-27 Hospital Clinic De Barcelona Microarn du plasma pour la détection du cancer colorectal précoce
EP2944700A1 (fr) 2011-10-21 2015-11-18 Hospital Clínic de Barcelona Micro-ARN à plasma permettant la détection du cancer colorectal au premier stade
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