WO2010018585A2 - Compositions et procédés de pronostic d'un mélanome - Google Patents

Compositions et procédés de pronostic d'un mélanome Download PDF

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WO2010018585A2
WO2010018585A2 PCT/IL2009/000803 IL2009000803W WO2010018585A2 WO 2010018585 A2 WO2010018585 A2 WO 2010018585A2 IL 2009000803 W IL2009000803 W IL 2009000803W WO 2010018585 A2 WO2010018585 A2 WO 2010018585A2
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nucleic acid
seq
mir
mirna
survival
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PCT/IL2009/000803
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WO2010018585A3 (fr
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Eva Hernando-Monge
Ilana Belitskaya-Levy
Moshe Hoshen
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New York University
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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
    • C12Q1/6809—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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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 to compositions and methods for the prognosis of melanoma patients after surgical operation. Specifically the invention relates to microRNA molecules associated with the prognosis of melanoma, as well as various nucleic acid molecules relating thereto or derived therefrom.
  • MM Malignant melanoma
  • MM Malignant melanoma
  • melanomas are characterized by frequent chromosomal aberrations associated with tumor progression.
  • melanomas display a characteristic pattern of genomic alterations involving microRNA genes.
  • miRNAs are endogenous non-coding small RNAs that interfere with the translation of coding messenger RNAs (mRNAs) in a sequence- specific manner, playing a critical role in the control of gene expression during development and tissue homeostasis (Yi et al, Nat Genet 2006;38:356-362).
  • miRNAs have been shown to be deregulated in human cancer, and their specific over- or under-expression has been shown to correlate with particular tumor types (Calin and Croce, Nat Rev Cancer 2006;6:857-866), as well as to predict patient outcome (Yu et al, Cancer Cell 2008; 13:48-57).
  • miRNA over-expression results in reduced expression of tumor suppressor genes, while loss of miRNA expression often leads to oncogene activation.
  • ignitican associations nave een emons ra e e ween expression or mi signatures and clinical outcome of lung adenocarcinoma, chronic lymphocytic leukemia, breast and pancreatic cancers.
  • the impact of miRNA expression signatures on clinical outcome in melanoma has not yet been reported. Prognosis and staging of melanoma is an important tool in weighing the efficacy and cost-effectiveness of alternative treatments.
  • altered expression levels of any of SEQ ID NO: 21, 32-40, 1-20, 22-31, 41-66 or variants thereof are indicative of cancer prognosis: life expectancy of the patient, expected recurrence-free survival, response to treatment and risk of recurrence of melanoma.
  • a method for determining a prognosis for melanoma in a subject comprising:
  • the nucleic acid sequence is selected from the group consisting of SEQ ID NOS: 21, 32-40, 1-19, 22, 24-31 and 41-66 and sequences at least about 80% identical thereto, and a decreased expression level of any of said nucleic acid sequence compare to the threshold express on eve s n cat ve o poor prognos s o said subject, hi other embodiments the nucleic acid sequence is selected from the group consisting of SEQ ID NOS: 20 and 23 and sequences at least about 80% identical thereto, and an increased expression level of any of said nucleic acid sequence compared to the threshold expression level is indicative of poor prognosis of said subject.
  • the nucleic acid sequence is selected from the group consisting of SEQ TD NOS: 21, 32-40, 2-3, 6, 8, 12-15, 18, 19, 22, 24, 49-57 and 66 and sequences at least about 80% identical thereto, and a decreased expression level of any of said nucleic acid sequence compared to the threshold expression level is indicative of poor prognosis of said subject.
  • the subject is a human.
  • the method is used to determine a course of treatment of the subject.
  • the biological sample obtained from the subject is selected from the group consisting of a bodily fluid, a cell line or a tissue sample, hi certain embodiments the tissue is a fresh, frozen, fixed, wax-embedded or formalin- fixed, paraffin-embedded (FFPE) tissue.
  • FFPE paraffin-embedded
  • said bodily fluid is blood, hi certain embodiments said tissue is a skin tissue.
  • the expression levels are determined by a method selected from the group consisting of nucleic acid hybridization, nucleic acid amplification, or a combination thereof.
  • the nucleic acid hybridization is performed using a solid-phase nucleic acid biochip array or in situ hybridization.
  • the nucleic acid amplification method is realtime PCR.
  • the PCR method comprises forward and reverse primers.
  • the real-time PCR method further comprises a probe.
  • the probe is complementary to SEQ TD NOS: 1-66, to a fragment thereof or to a sequence at least about 80% identical thereto.
  • the invention further provides a kit for prognosis of melanoma, said kit comprising a probe comprising a nucleic acid sequence that is complementary to a sequence selected from SEQ ID NO: 1-66, to a fragment thereof or to a sequence at least about 80% identical thereto.
  • the kit further comprises forward and reverse primers.
  • said kit comprises reagents for performing in situ hybridization analysis.
  • Figure 1 shows differential expression of miRs (in fluorescence units), comparing the median values of each miR in all "short-lived” patients (y-axis represents survival below 36 months) with the corresponding median for "long-lived” patients (x- axis represents survival above 36 months).
  • the parallel lines describe a fold change between groups of 1.5 in either direction.
  • miRs differentially expressed are MID-00466 (SEQ ID NO: 19), hsa-miR-199a-5p (SEQ ID NO: 2), hsa-miR-143 (SEQ ID NO: 3), hsa-miR-23b (SEQ ID NO: 4), hsa-miR-27a (SEQ ID NO: 49), hsa- miR-199a-3p (SEQ ID NO: 6), hsa-miR-497 (SEQ ID NO: 8) and hsa-miR-150 (SEQ ID NO: 21), which all have relatively high expression in the group of "long-lived” patients.
  • the expression of hsa-miR-574-5p (SEQ ID NO: 20) is relatively high in the group of "short-lived" patients.
  • Figure 2 shows the inset p-values sorted and compared with a random distribution to correct for multiple testing.
  • the y-axis represents the sorted p-values, and the x-axis the sorted index. miRs with expression p-values 0.15 of that of their corresponding proportional ranking are deemed significant.
  • the dashed line represents the expected p-values, given a random distribution.
  • the solid line represents the expected p-values, allowing a false-detection rate of 0.15. The point at which the observed p-values cross this line determines the FDR threshold.
  • Figures 3A-F are boxplot presentations comparing the group of "long-lived" patients (survival above 36 months) (the right box) with the group of "short-lived” patients (survival below 36 months) (the left box), with regard to expression (in Iog 2 (fluorescence units)) of the following miRs:
  • Figure 3A lisa-miR-199a-3p (SEQ ID NO: 6)
  • Figure 3B hsa-miR-497 (SEQ ID NO: 8)
  • Figure 3C hsa-m - a- p S ID NO:
  • Figure 3D miR-23b (SEQ ID NO: 4)
  • Figure 3E miR-27b (SEQ ID NO: 7)
  • Figure 3F hsa-let-7a (SEQ ID NO: 1).
  • the "box” part contains 50% of the data, the line in the box indicates the median value, and the ends of the vertical lines indicate the minimum and maximum data values. Additional points represent
  • Figure 4 A shows a Kaplan-Meier plot, which corrects for patients who were censored (subjects that may have dropped out of the study and/or were lost to follow-up or deliberately withdrawn due to the culmination of the study).
  • the y-axis depicts the fraction of surviving patients, and the x-axis depicts months of survival, with regard to expression of hsa-miR-199a-5p (SEQ ID NO: 2) with the expression values Iog2 transformed.
  • Figure 4B shows multiple-hypothesis testing of hsa-miR-199a-5p (SEQ ID NO:
  • the miRs values were reallocated to patients at random to form a statistical ensemble of 100 possible miR-clinical outcome interactions.
  • a histogram shows the p-values for this ensemble.
  • Figure 5 shows a Kaplan-Meier plot in which data are presented for survival of subjects using scores from the combined expression levels of hsa-miR-497 (SEQ ID NO: 8), hsa-miR-199a-5p (SEQ ID NO: 2) and MID-00466 (SEQ ID NO: 19).
  • Figure 6 shows differential expression of miRs (in Iog 2 (fluorescence units)), comparing the median values of each miR in all "short-lived” patients (y-axis represents survival below 18 months) with the corresponding median for "long-lived” patients (x- axis represents survival above 18 months).
  • Median normalized fluorescence for each miRNA indicates expression levels as measured by microarray.
  • the parallel lines describe a fold change between groups of 1.5 in either direction.
  • miRs differentially expressed are hsa-miR-145 (SEQ ID NO: 32), hsa-miR-143 (SEQ ID NO: 3), hsa-miR-342-3p (SEQ ID NO: 33), hsa-miR-193a-3p (SEQ ID NO: 34), hsa-miR-193b (SEQ ID NO: 35), hsa-miR-155 (SEQ ID NO: 36), lisa-miR-497 (SEQ ID NO: 8), hsa- miR-455-3p (SEQ ID NO: 37), hsa-miR-28-3 ⁇ (SEQ ID NO: 38), hsa-miR-28-5p (SEQ ID NO: 39), hsa-miR-342-5p (SEQ ID NO: 40), and hsa-miR-150 (SEQ ID NO: 21), which all have relatively high expression in the group of "long-
  • Figure 7 shows validation of miRNA arrays by qRT-PCR. Fold change of miRNA expression measured by microRNA array (empty bars) and qRT-PCR (filled bars) in a subset of nine samples, including short-term (survival below 18 months) and long-term (survival above 18 months) survivors (hsa-miR-150 (SEQ ID NO: 21), hsa- miR-126 (SEQ ID NO: 47), hsa-miR-155 (SEQ ID NO: 36), hsa-miR-145 (SEQ ID NO: 32), hsa-miR-497 (SEQ ID NO: 8), hsa-miR-143 (SEQ ID NO: 3), hsa-miR-455-3p (SEQ ID NO: 37), hsa-miR-199a-5p (SEQ ID NO: 2), and hsa-miR-27a (SEQ ID NO: 49)) is depicted.
  • Figure 8 shows a Kaplan-Meier plot of survival, which corrects for patients who were censored and calculates the significance of separation using logranks based on the expression of the 'predictor miRNA', a subset of the predictive rm ' RNAs.
  • Figure 9 shows a Kaplan-Meier plot in which data are presented for survival of melanoma patients after surgical operation based on stage at recurrence combined with the expression of predictor miRNAs.
  • Figure 10 shows a Kaplan-Meier plot for survival of melanoma patients after surgical operation in which data are presented for survival of melanoma patients based on stage at recurrence.
  • Figure 11 shows a Kaplan-Meier plot for survival of melanoma patients after surgical operation in which data are presented for survival of melanoma patients based on site of metastasis.
  • Figure 12A shows a Kaplan-Meier plot for survival of melanoma patients after surgical operation.
  • data are presented for survival of melanoma patients based on melanoma stage.
  • Figures 13A-F are boxplot presentations comparing primary and metastatic tumor, with regard to expression of the following miRs:
  • Figure 13A hsa-miR-497 (SEQ ID NO: 8),
  • Figure 13B hsa-miR-145 (SEQ ID NO: 32),
  • Figure 13D hsa-miR-150 (SEQ ID NO: 21)
  • Figure 13E hsa-miR-155 (SEQ ID NO: 36)
  • Figure 13F hsa-miR-455-3p (SEQ ID NO: 37).
  • the "box" part contains 50% of the data, the line in the box indicates the median value (50 th percentile), and the ends of the vertical lines indicate the minimum and maximum data values.
  • Empty boxes normal tissue; hatched boxes: primary tumors in patients surviving more than 18 months; light gray boxes: primary tumors in patients surviving less than 18 months; cross-hatched boxes: metastatic tumors in patients surviving more than 18 months; dark gray boxes: metastatic tumors in patients surviving less than 18 months.
  • miRNA expression can serve as a novel tool for the prognosis of human melanoma. More particularly, it may serve for the prognosis of long survival versus short survival after surgical operation.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • “Attached” or “immobilized”, as used herein to refer to a probe and a solid support, 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 molecules.
  • 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 may mean a sample of biological tissue or fluid that comprises nucleic acids. Such samples include, but are not limited to, tissue isolated from animals. Biological samples may also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histological purposes, blood, plasma, serum, sputum, stool, tears, mucus, urine, effusions, amniotic fluid, ascitic fluid, hair, and skin. Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues.
  • 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 prognosis A forecast or prediction of the probable course or outcome of the cancer.
  • cancer prognosis includes the forecast or prediction of any one or more of the following: duration of survival of a patient susceptible to or diagnosed with a cancer, duration of recurrence-free survival, duration of progression-free survival of a patient susceptible to or diagnosed with a cancer, response to treatment in a group of patients susceptible to or diagnosed with a cancer, duration of response to treatment in a patient or a group of patients susceptible to or diagnosed with a cancer.
  • prognostic for cancer means providing a forecast or prediction of the probable course or outcome of the cancer.
  • prognostic for cancer comprises providing the forecast or prediction of (prognostic for) any one or more of the following: duration of survival of a patient susceptible to or diagnosed with a cancer, duration of recurrence-free survival, duration of progression-free survival of a patient susceptible to or diagnosed with a cancer, response to treatment in a group of patients susceptible to or diagnosed with a cancer, and duration of response to treatment in a patient or a group of patients susceptible to or diagnosed with a cancer.
  • prognosis may refer, in one embodiment, to "good prognosis", wherein “good prognosis”, in one embodiment, may refer to survival for more than 18 months and in another embodiment, may refer to survival for more than 36 months.
  • the term “good prognosis” is interchangeable with “better prognosis”. In another embodiment, the term “good prognosis” is interchangeable with "long-term survival”. In another embodiment, the term “good prognosis” is interchangeable with “long-lived”. In one embodiment, the term “prognosis may re er o poor prognosis , w erein poor prognosis , m one embodiment, may refer to survival for less than 18 months and in another embodiment, may refer to survival for less than 36 months. In one embodiment, the term “poor prognosis” is interchangeable with "worse prognosis", hi another embodiment, the term “poor prognosis” is interchangeable with "short-term survival”. In another embodiment, the term “poor prognosis” is interchangeable with "short-lived”. chemotherapeutic
  • a drug used to treat a disease especially cancer.
  • the drugs typically target rapidly dividing cells, such as cancer cells.
  • “Complement” or “complementary”, as used herein to refer to a nucleic acid may mean Watson-Crick (e.g., A-TfU 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. hi some embodiments the PCR C T signal is normalized such that the normalized
  • PCR C T remains inversed from the expression level.
  • 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 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” may mean qualitative or quantitative differences in the temporal and/or cellular gene expression patterns within and among cells and tissue.
  • a differentially expressed gene can 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 compa ⁇ son o wo or more s a es. qua i a ive y regu a e gene wi ex i i an expression pattern within a state or cell type that may be detectable by standard techniques. Some genes will 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, 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, and RNase protection. expression profile
  • “Expression profile”, as used herein, may mean 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, cRNA, etc., quantitative PCR, ELISA for quantification, 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, including all of the listed nucleic acid sequences.
  • the term "expression profile” may also mean 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.
  • FDR When performing multiple statistical tests, for example, in comparing the signal between two groups in multiple data features, there is an increasingly high probability of obtaining false positive results, by random differences between the groups that can reach levels that would otherwise be considered statistically significant. In order to limit the proportion of such false discoveries, statistical significance is defined only for data features in which the differences reach a p-value (such as by a two-sided t-test) below a threshold, which is ependent on the number o tests per orme an the distribution ot p- values obtained in these tests. FDR or false discovery rate is the probability that one of the "significant" results was actually false.
  • Gene 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 mRNA 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. identity
  • Identity as used herein in the context of two or more nucleic acids or polypeptide sequences, may 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.
  • Inhibit may mean prevent, suppress, repress, reduce or eliminate.
  • Label may mean 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. logistic regression
  • Logistic regression is part of a category of statistical models called generalized linear models. Logistic regression allows one to predict a discrete outcome, such as group membership, from a set of variables that may be continuous, discrete, dichotomous, or a mix of any of these. The dependent or response variable is dichotomous, for example, one of two possible types of cancer. Logistic regression models the natural log of the odds ratio, i.e., the ratio of the probability of belonging to the first group (P) over the probability of belonging to the second group (1-P), as a linear combination of the different expression levels (in log-space) and of other explaining variables.
  • the logistic regression output can be used as a classifier by prescribing that a case or sample will be classified into the first type if P is greater than 0.5 or 50%.
  • the calculated probability P can be used as a variable in other contexts such as a ID or 2D threshold classifier.
  • Logit score is a number between 0 and 1 that reflects the probability of a variable (or a combination of variables) to be in one of the two defined groups.
  • 0 refers to longer survival time and 1 refers to shorter survival time.
  • 1D/2D threshold classifier may mean an algorithm for classifying a case or sample such as a cancer sample into one of two possible types such as two types of cancer or two types of prognosis (e.g., good and bad).
  • ID threshold classifier the decision is based on one variable and one predetermined threshold value; the sample is assigned to one class if the variable exceeds the threshold and to the other class if the variable is less than the threshold.
  • a 2D threshold classifier is an algorithm for classifying into one of two types based on the values of two variables. A score may be calculated as a function (usually a continuous function) of the two variables; the decision is then reached by comparing the score to the predetermined threshold, similar to the ID threshold classifier. mismatch
  • “Mismatch” means a nucleobase of a first nucleic acid that is not capable of pairing with a nucleobase at a corresponding position of a second nucleic acid.
  • nucleic acid "Nucleic acid” or “oligonucleotide” or “polynucleotide”, as used herein, may 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.
  • 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 3 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, phosphorothioate, 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.
  • 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., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified at the 8-position, e.g., 8-bromo guanosme; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6- methyl adenosine are suitable.
  • uridines or cytidines modified at the 5-position e.g., 5-(2-amino)propyl uridine, 5-bromo uridine
  • 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 2005;438:685-689, Soutschek et al, Nature 2004;432:173-178, and U.S. Patent Publication No. 20050107325, 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 and kidney.
  • probes may mean 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.
  • 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 such as with biotin to which a streptavidin complex may later bind. reference value
  • the term "reference value” means a value that statistically correlates to a particular outcome when compared to an assay result. In some embodiments the reference value is determined from statistical analysis of studies that compare microRNA expression with known clinical outcomes.
  • the reference value may be a threshold score value or a cutoff score value. Typically, a reference value will be a threshold above which one outcome is more probable and below which an alternative outcome is more probable.
  • sensitivity "Sensitivity”, as 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 out of two possible types. 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
  • Specificity 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 out of two possible types.
  • 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 "not A”, as determined by some absolute or gold standard.
  • Stringent hybridization conditions may 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 5-10°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 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 6O 0 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.
  • 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°C.
  • substantially complementary as used herein, may mean that a first sequence is at least 60%-99% identical to the complement of a second sequence over a region of 8- 50 or more nucleotides, or that the two sequences hybridize under stringent hybridization conditions.
  • substantially identical may mean that a first and second sequence are at least 60%-99% identical over a region of 8-50 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.
  • subject refers to a mammal, including both human and other mammals. The methods of the present invention are preferably applied to human subjects. threshold expression level
  • threshold expression level refers to a criterion expression profile to which measured values are compared in order to determine the prognosis of a subject with melanoma.
  • the reference expression profile may be based on the expression of the nucleic acids, or may be based on a combined metric score thereof.
  • tissue sample is tissue obtained from a tissue biopsy using methods well known to those of ordinary skill in the related medical arts.
  • sample of being cancerous 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. treat
  • Tuat or “treating”, as used herein when referring to protection of a subject from a condition, may mean preventing, suppressing, repressing, or eliminating the condition.
  • Preventing the condition involves administering a composition described herein to a subject prior to onset of the condition.
  • Suppressing the condition involves administering the composition to a subject after induction of the condition but before its clinical appearance.
  • Repressing the condition involves administering the composition to a subject after clinical appearance of the condition such that the condition is reduced or prevented from worsening.
  • Elimination of the condition involves administering the composition to a subject after clinical appearance of the condition such that the subject no longer suffers from the condition.
  • tumor "Tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. variant
  • Vector as used herein to refer to a nucleic acid, may mean (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 sequences substantially identical thereto.
  • vector "Vector”, as used herein, may mean 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.
  • Malignant skin cancer or tumor relates to a malignant skin cancer or tumor of varying degree of severity and having the tendency to spread or "metastasize” in advanced stages of the disease.
  • cancer or “neoplasm” generally means a malignant disease and is characterized by an uncontrolled growth of tumor or cancer cells. Tumors may spread locally as a primary tumor mass or spread to distant parts of the body, i.e., metastasize.
  • Stage 0 Melanoma cells are found only in the outer layer of skin cells and have not invaded deeper tissues.
  • Stage I The tumor is no more than 1 millimeter thick, and there may be epidermal ulceration, or the tumor is between 1 and 2 millimeters thick and there is no ulceration.
  • the melanoma cells have not spread to nearby lymph nodes.
  • Stage II The tumor is at least 1 millimeter thick. The tumor is between 1 and 2 millimeters thick with ulceration, or the thickness of the tumor is more than 2 millimeters and there may be ulceration.
  • the melanoma cells have not spread to nearby lymph nodes.
  • Stage III This stage is divided into three groups, as follows: Stage IIIA: The melanoma has spread to 1 to 3 lymph nodes near the primary tumor, but the nodes are not enlarged and the cells can only be seen under a microscope. The melanoma is not ulcerated and has not spread to other areas of the body.
  • Stage IIIB (includes 3 different situations): •
  • the melanoma has spread to 1 to 3 lymph nodes near the primary tumor but the nodes are not enlarged and the cells can only be seen under a microscope.
  • the melanoma is ulcerated but has not spread to other areas of the body.
  • the melanoma is not ulcerated. It has spread to 1 to 3 lymph nodes near the primary tumor and the lymph nodes are enlarged. The melanoma has not spread to other areas of the body.
  • the melanoma may or may not be ulcerated. It has spread to small areas of skin or lymphatic channels close to the original melanoma but the lymph nodes do not contain melanoma cells. The melanoma has not spread to other areas of the body. Stage IIIC (includes 2 different situations): • The melanoma is ulcerated. It has spread to 1 to 3 lymph nodes near the primary tumor. The lymph nodes contain melanoma cells and are enlarged. The melanoma has not spread to other areas of the body.
  • the melanoma may or may not be ulcerated. It has spread to 4 or more nearby lymph nodes, or to nearby lymph nodes that are clumped together, or it has spread to near y s n or ymp at c c anne s aroun e origina me anoma an near y lymph nodes. The lymph nodes are enlarged because of the melanoma. The melanoma has not spread to other areas of the body.
  • Stage IV The melanoma cells have spread to other organs, to lymph nodes, or to skin areas far away from the original tumor.
  • Surgery to remove the tumor is the primary treatment of all stages of melanoma. (The surgery may be local excision or lymphadenectomy, in which the lymph nodes are also removed.) Chemotherapy (regional or systemic), radiation therapy (external or internal) and immunotherapy may further be provided.
  • a gene coding for a miRNA 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 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 30-200 nt 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 stem ( ⁇ 10 nucleotides) extending beyond the Drosha cleavage site may be essential for efficient processing.
  • Drosha 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 30-200 nt precursor known as the pre-
  • 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. .
  • e miRNA may eventually become incorporated as a single-stranded RNA into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC).
  • 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 (repress or activate), and which strand of the miRNA/miRNA* duplex is loaded in to the RISC.
  • the miRNA* When 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' 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 2-8 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. This was shown for miR-196 and Hox B8 and it was further shown that miR-196 mediates the cleavage of the Hox B8 mRNA (Yekta et al, Science 2004; 304:594-596). Otherwise, such interactions are known only in plants (B artel & B artel, Plant Physiol 2003; 132:709-717).
  • miRNAs may direct the RISC to down-regulate 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. Alternatively, 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 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 are provided herein.
  • the nucleic acid may comprise the sequence of SEQ ID NOS: 1-66 presented in Table 1 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 may be 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.
  • MID-00466, MID-00405 and MID-00713 were cloned at Rosetta Genomics; the microRNA name is the miRBase registry name (release 10).
  • nucleic acid complex 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 also comprise a protamine-antibody fusion protein as described in Song et al. (Nature Biotechnology 2005;23:709-17) and Rossi (Nature Biotechnology 2005;23:682-4), the contents of which are incorporated herein by reference.
  • the protamine-fusion protein may comprise the abundant and highly basic cellular protein protamine. The protamine may readily interact with the nucleic acid.
  • the protamine may comprise the entire 51 -amino acid protamine peptide or a fragment thereof.
  • the protamine may be covalently attached to another protein, which may be a Fab.
  • the Fab may bind to a receptor expressed on a cell surface.
  • 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: 1-66 or variants thereof.
  • the pri-miRNA may form a hairpin structure.
  • the hairpin may comprise first and second nucleic acid sequence that are substantially complementary.
  • 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 less than -25 Kcal/mole, as calculated by the Vienna algorithm, with default parameters, as described in Hofacker et al. (Monatshefte f.
  • 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. c. Pre-miRNA
  • the nucleic acid may also comprise a sequence of a pre-miRNA or a variant thereof.
  • the pre-miRNA sequence may comprise from 45-200, 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: 1-66 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 maybe 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: 1-8, 19-21 and 25-30, and 32-49 or variants thereof. e. Anti-miRNA
  • the nucleic acid may also comprise a sequence of an anti-miRNA that is 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 nucleoti es.
  • e sequence o t e ant -m may com . nucleotides that are substantially identical or complementary to the 5' of a miRNA and at least 5-12 nucleotides that are substantially complementary 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 complementary to the 3' of a miRNA and at least 5 nucleotides that are substantially complementary to the flanking region of the target site from the 3' end of the miRNA.
  • the sequence of the anti-miRNA may comprise the complement of SEQ ID NOS: 1-8, 19-21, 25-30, and 32-49 or variants thereof.
  • a probe comprising a nucleic acid described herein is also provided. Probes may be used for screening and diagnostic methods. The probe may be attached or immobilized to a solid substrate, such as a biochip.
  • 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,
  • the probe may further comprise a linker sequence of from 10-60 nucleotides.
  • 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 a spatially defined address 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, as appreciated by those in the art.
  • the probes may either be synthesized first, with subsequent attachment to the biochip, or maybe 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.
  • substrates 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.
  • 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 a flexible foam, including closed-cell foams made of particular plastics.
  • 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 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 melanoma-associated nucleic acid in a biological sample.
  • the sample may be derived from a subject. Diagnosis of a disease state in a patient may allow for prognosis and selection of therapeutic strategy. Further, the developmental stage of cells may be determined by determining temporarily expressed melanoma-associated nucleic acids.
  • In situ hybridization of labeled probes to tissue sections 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 nucleic acids which indicate the diagnosis may differ from those which indicate the prognosis and molecular profiling of the condition of the cells may lead to dist nctions between responsive or refractory con t ons or may be predictive o outcomes.
  • kits A kit is also provided and 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 a kit for the amplification, detection, identification or quantification of a target nucleic acid sequence.
  • the kit may comprise a poly(T) primer, a forward primer, a reverse primer, and a probe.
  • Example 1 Correlation between differential miRNA expression and survival rates in melanoma patients
  • Example 1.1 Materials and methods Biological samples: Forty-eight metastatic melanoma specimens (formalin-fixed, paraffin-embedded, FFPE) provided by the NYU Interdisciplinary Melanoma Cooperative Group (IMCG; PI: I. Osman, MD) were used for this research. The 48 samples comprised lymph node and metastatic melanomas that were grouped together on the basis of similar biological/clinical behavior. Total RNA enriched in microRNA was isolated from the melanoma FFPE specimens, and all RNAs extracted were hybridized onto microarrays according to the RNA extraction and microRNA array platform protocols described below.
  • RNA extraction For FFPE samples, total RNA was isolated from seven to ten 10- ⁇ m- thick tissue sections using the microRNA extraction protocol developed at Rosetta Genomics. Briefly, the sample is incubated repeatedly in Xylene at 570 to remove paraffin excess, followed by repeated ethaiiol washes. Proteins are degraded by proteinase K solution at 45 0 C for a few hours. The RNA is extracted with acid phenolxhloroform, followed by ethanol precipitation and DNAse digestion. Total RNA quantity and quality is checked by spectrophotometer (Nanodrop ND-1000). microRNA array platform: Custom microarrays were produced by printing DNA oligonucleotide probes representing 911 human microRNAs.
  • Each probe printed in triplicate, carries a linker up to 22 nt long at the 3' end of the complement sequence of the microRNA, in addition to an amine group used to couple the probes to coated glass slides.
  • 20 ⁇ M of each probe were dissolved in 2X SSC + 0.0035% SDS and spotted in triplicate on Schott Nexterion® Slide E-coated microarray slides using a Genomic Solutions® BioRobotics MicroGrid II, according the MicroGrid manufacturer's directions.
  • Fifty-four negative control probes were designed using the sense sequences of different microRNAs.
  • Two groups of positive control probes were designed to hybridize to a miRNAarray: (i) synthetic small RNA were spiked to the RNA before labeling to verify the labeling efficiency and (ii) probes for abundant small RNA (e.g., small nuclear RNAs (U43, U49, U24, Z30, U6, U48, U44), 5.8s and 5s ribosomal RNA) were spotted on the array to verify RNA quality. The slides were blocked in a solution containing 50 mM ethanolamine, 1 M Tris (pH 9.0) and 0.1% SDS for 20 min at 50 ° C, then thoroughly rinsed with water and spun dry.
  • small RNA e.g., small nuclear RNAs (U43, U49, U24, Z30, U6, U48, U44), 5.8s and 5s ribosomal RNA
  • Cy-dye labeling of microRNA for microRNA array Five ⁇ g of total RNA were labeled by ligation (Thomson et al, Nature Methods 2004; 1:47-53) of an RNA-linker, p-rCrU- Cy/dye (Dharmacon), to the 3' end with Cy3 or Cy5.
  • the labeling reaction contained total RNA, spikes (0.1-20 fmoles), 300 ng RNA-linker-dye, 15% DMSO, Ix ligase buffer and 20 units of T4 RNA ligase (NEB) and proceeded at 4° C for 1 h, followed by 1 h at 37 0 C.
  • Arrays were scanned using an Agilent Microarray Scanner Bundle G2565BA (resolution of 10 ⁇ m at 100% power). Array images were analyzed using SpotReader software (Niles Scientific).
  • Statistical analyses Differential expression was performed in log-space, comparing logs of readings of the microarray data. Differentially expressed miRNAs were en e y us ng e - es ase proce ure ca e signi cance ana ysis o microarrays ("SAM". Tibshirani, R., Hastie, T., Narasimhan, B. & Chu, G., journal 2002). SAM produces a score for each gene on the basis of the expression change relative to the standard deviation of all expression levels. This procedure allows the control of false discovery rate (FDR). FDR analysis was performed using the Benjamini & Hochberg process, using an FDR rate of 0.15.
  • Univariate survival analysis was performed using Kaplan-Meier, and statistical significance was calculated using logrank between upper and lower groups, as appropriate. The threshold for further consideration was p ⁇ 0.05.
  • Multivariate survival analysis was performed using Cox regression. Scores based on the Cox regression coefficients were calculated by linear combination. The scores were then again used for univariate survival analysis.
  • Example 1.2 Differential expression in long-lived and short-lived melanoma patients
  • Figure 4B shows a histogram of p-values from multiple-hypothesis testing of hsa-miR-199a-5p (SEQ ID NO: 2). The adjusted p-value for hsa-miR-199a-5p is 0.03. Accordingly, high expression of this miR is a predictor of >3 -year survival.
  • Example 1.5 Combination of expression of specific miRs is indicative of prognosis of melanoma subjects
  • MiRs were sorted according to their individual p-values for 36-month survival.
  • each miR in the patients was divided into tertiles.
  • the odds ratios for each tertile, taking the lowest as reference were associated with the patients.
  • Each patient received a score which was the sum of his/her odds ratios. These scores were then used for univariate analysis, using Kaplan-Meier. As indicated in Figure 5, the fraction of surviving subjects with lower scores from the combined expression levels of hsa-miR-
  • the values of miR expression levels were determined by nucleic acid hybridization performed using a solid-phase nucleic acid biochip array, according to
  • Example 1.1 The median expression levels in short-lived and long-lived patients were compared in order to assess threshold expression levels.
  • Example 2 miRNAs as prognostic biomarkers for melanoma patients
  • Example 2.1 Materials and methods
  • FFPE formalin-fixed paraffin-embedded
  • Quantitative real-time PCR was performed by using miRNA-specific TaqMan MicroRNA Assay Kit (Applied Biosystems); 12.5 ng of total RNA was reversed transcribed using the corresponding RT Primer and the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). PCR was performed on 1.33 uL of RT products by adding the TaqMan PCR primers and the TaqMan Universal PCR Master Mix (Applied Biosystems). U6 and RNU44 small RNAs were used for normalization of input RNA/cDNA levels. The primers used in the PCR are part of the miRNA- amplification kit provided by Applied Biosystems. The whole sequence is unknown but must contain the perfect complementary sequence to the mature miRNA.
  • SAM Significance Analysis of Microarrays
  • FDR False Discovery Rate
  • Example 2.2 Longer survival time is associated with up-regulation of miRNA signature
  • Example 3 miRNAs as prognostic biomarkers for melanoma patients
  • Example 3.1 Materials and methods
  • AJCC American Joint Committee on Cancer
  • om rep es e hematoxylin and eosin (H&E), and evaluated for tumor content.
  • Congenital nevi and melanoma tissue found to have significant contamination with normal tissue (>20%) were macrodissected or sectioned on Leica MembraneSlides PEN [(polyethylene naphthalate)-Membrane 2.0 mm] and laser-capture microdissected, using a LMD6000 Leica Laser Micro-Dissection System.
  • RNA extraction Total RNA was extracted as follows. Briefly, ten sections of 10 ⁇ m of FFPE tissues were deparaffinized with xylene, washed in ethanol, and digested with proteinase K. The RNA was extracted with acid phenol: chloroform followed by ethanol precipitation and DNAse digestion, or using the Qiagen miRNeasy FFPE kit. Total RNA quantity and quality were evaluated using the spectrophotometer Nanodrop ND- 1000 (Thermo Scientific, Wilmington, DE) with an inclusion criterion of A260/A280 >1.8. miRNA microarray expression profiling and data pre-processing: miRNA microarrays were prepared as follows.
  • RNA samples 3.5 ⁇ g
  • RNA-linker p-rCrU-Cy/dye
  • Hybridization and washing of the microarray slides were performed.
  • Arrays were scanned using an Agilent Microarray Scanner Bundle G2565BA (resolution of 10 mm at 100% and 10% power).
  • Array images were analyzed using SpotReader software (Niles Scientific) to generate raw intensity data. Microarray spots were combined and signals normalized. Triplicate spots were combined into one signal by taking the logarithmic mean of the reliable spots.
  • Array data were log-transformed and quantile normalization was applied to make arrays comparable to one another. miRNAs with low variance across samples (i.e., coefficient of variation ⁇ 1% on the log-scale) were filtered out, leaving 610 miRNAs for analysis.
  • the expression level or signal of a microRNA refers to the normalized value.
  • SAM Significance Analysis of Microarrays
  • FDR False Discovery Rate
  • SAM computed the Cox regression coefficient for each miRNA.
  • SAM computed a Wilcoxon rank-sum statistic for each miRNA.
  • One thousand permutations of the data were used to select differentially expressed miRNAs. The significance cut-off was adjusted so as to set the median number of falsely discovered miRNAs to 0.
  • the method of pre-validation was used to compare the prediction accuracy of the miRNA signature to that of TNM stage, site of metastasis, age at recurrence and other clinical and demographic variables.
  • the PV miRNA predictor was compared to other predictors of survival in a multivariate Cox regression analysis of post-recurrence survival.
  • the Kaplan-Meier method was used to estimate the post- recurrence survival function.
  • the log-rank test was used to compare the survival distribution between groups. All analyses were performed using the R language for statistical computing and the Bioconductor software. Heatmap and hierarchical clustering analyses were done using Prism 4 software v4.0 (GraphPad Software, Inc.
  • Bioinformatics analysis DAVID bioinformatics resource (http://david.abcc.ncifcrf.gov) was used to conduct KEGG pathway analysis on predicted targets (according to TargetScan) of the six miRNAs most commonly associated with melanoma prognosis. Most frequently represented pathways are assigned a p-value calculated with a modified version of a Fisher-exact test (p-value cutoff of less than 0.1) showing significance of the association as compared to a random list using the human genome as a background.
  • qRT-PCR Quantitative real-time PCR analysis of miR- 126, miR-145, miR-143, miR-497, miR-150, miR-155, miR-342-3p and miR-455-3 ⁇ was performed using the miRNA-specific TaqMan MicroRNA Assay Kit (Applied Biosystems Inc, Foster City, CA). Total RNA template (12.5 ng) was reversed transcribed using the corresponding RT primer and the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) in a 15- ⁇ l reaction volume, using the following program: 30 min at 16 0 C, 30 min at 42°C, 5 min at 85 0 C and then holding at 4 0 C.
  • RT product (1.33 ⁇ l) was used in a total reaction volume of 20 ⁇ l for quantitative analysis by real-time PCR adding the TaqMan PCR primers and the TaqMan Universal PCR Master Mix (Applied Biosystems), using an Applied Biosystems 7500 Sequence detection system.
  • Thermal cycling program used for quantification was as follows: 5O 0 C for 2 min and 95 0 C for 10 min, followed by 50 cycles at 95°C for 15 s and 6O 0 C for 1 min.
  • RNU44 small nuclear RNA was used for normalization of input RNA/cDNA levels. Each measurement was performed in triplicate and no-template controls were included for each assay.
  • Example 3.2 A miRNA signature associates with post-recurrence survival (1.5-year cut-off)
  • microRNA expression profile of 59 human metastatic melanoma tumors was investigated using oligonucleotide microarrays containing approximately 900 miRNAs in triplicate.
  • miRNAs that could discriminate between "better prognosis” (patients who survived more than 18 months from the date of resection of the metastatic tumor) and "worse prognosis” (patients who survived less than 18 months) were analyzed.
  • Significance analysis of microarrays (SAM) was used to identify differentially expressed miRNAs with a false discovery rate set to 5%. As indicated in Figure 6, the data show that differences in miRNA expression levels associate with the post-recurrence survival of metastatic melanoma patients.
  • MID-00405 and MID-00713 were cloned at Rosetta Genomics; the microRNA name is the miRBase registry name (release 10).
  • Example 3.3 Validation of miRNA arrays by qRT-PCR m order to validate the data obtained from the microarray platform, the expression of nine miRNAs was quantified using real time RT-PCR analysis. Specifically, the expression of miR-150, miR-126, miR-155, miR-145, miR-497, miR- 143, miR-455-3p, miR-199a-5p and miR-27a in ten tissue samples included in the microarray expression profiling was measured. The data were normalized to the endogenous control small nuclear RNA, RNU44. Real-time PCR showed that expression of all nine selected miRNAs was up-regulated in five cases with longer survival in comparison with five tumor samples with poor prognosis (Figure 7).
  • Example 3.4 Training error rates and cross-validation estimates of true error rates of classifiers based on 1000 randomizations
  • the median number of miRNAs used by the nearest shrunken centroids method was 2 (1-4 interquartile range).
  • the number of miRNAs used by the classification trees was 1 and 2.
  • a small number of miRNAs can therefore be used to predict post- recurrence survival.
  • the miRNA signatures obtained by the method of nearest shrunken centroids most often comprised miR-150, miR-455-3 ⁇ , miR-145, miR-155, miR-497 and miR-342-3 ⁇ .
  • the prediction accuracy using this combined miRNA set was estimated to be 81.4% (training error 84.7%).
  • Example 3.5 microRNA signature as an indicator o pat ent survival
  • FIG. 8 shows a Kaplan-Meier plot in which data are presented showing the probability of survival of melanoma patients relative to a miRNA predictor.
  • Figure 9 shows a Kaplan-Meier plot in which data are presented showing the probability of survival of melanoma patients relative to the same miRNA predictor described for Figure 8. Combining the miRNA predictor expression and stage at recurrence further subclassified patients into low, medium and high risk, and provided an optimal model of prediction of post-recurrence survival (p ⁇ 0.0001). The capacity of specific miRNAs to implement a molecular classification of melanoma is clearly shown by these findings.
  • Example 3.6 Classification of patients based on disease staging or site of metastasis
  • Figure 10 shows a Kaplan-Meier plot in which data are presented showing the probability of survival of melanoma patients based on melanoma staging.
  • patients with more advanced stage melanoma have a higher probability of shorter survival times.
  • Figure 11 shows a Kaplan-Meier plot in which data are presented showing the probability of survival of melanoma patients based on site of metastasis (brain, lymph node, visceral, or soft-tissue). Overall, the site of metastasis correlates with post- recurrence survival (p ⁇ 0.0026).
  • Example 3.7 Associations between predictive miRNAs and conventional prognostic factors
  • a model to predict post-recurrence survival based on clinico-pathological and miRNA expression predictors was designed.
  • Baseline stage IIIB ( 2) Baseline: brain mets ( 3) Baseline: stage IIIB
  • PV miRNA refers to the predictor value of a subgroup of six miRNAs with maximum predictive potential and minimum redundancy. This group includes miR-342- 3p (SEQ ID NO: 33), miR-150 (SEQ ID NO: 21), miR 455-3p (SEQ ID NO: 37), miR- 145 (SEQ ID NO: 32), miR-497 (SEQ ID NO: 8) and miR-155 (SEQ ID NO: 36).
  • the microRNA predictor associates with longer survival of melanoma patients, with a hazard ratio of 3.42 (95% confidence interval: [1.49, 7.86]) and p value of 0.0038.
  • stage and the miRNA predictor were removed.
  • An optimal Cox hazard regression showed that the miRNA predictor was able to increase the prognostic value of stage IV melanoma (Table 8).
  • Example 3.9 Predictive capacity of miRNAs in primary matched pairs
  • the clmicopathological features of these primary specimens are summarized in Table 9.
  • MiR-155 was up-regulated in both primary and metastatic tumors compared to nevi, although the differential expression between long- and short-term survival was appreciable only in the metastatic tumors, with high expression associated with longer survival.

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Abstract

L'invention porte sur des compositions et des procédés pour le pronostic de patients atteints de mélanome après une opération chirurgicale. Plus particulièrement, l'invention porte sur des molécules de micro-ARN associées au pronostic d'un mélanome, ainsi que diverses molécules d'acide nucléique liées à celles-ci ou issues de celles-ci.
PCT/IL2009/000803 2008-08-14 2009-08-13 Compositions et procédés de pronostic d'un mélanome WO2010018585A2 (fr)

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WO2011026909A1 (fr) * 2009-09-02 2011-03-10 L'oreal SIGNATURE microARN DE LA DIFFÉRENCIATION ÉPIDERMIQUE ET UTILISATIONS

Citations (2)

* Cited by examiner, † Cited by third party
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US20070065844A1 (en) * 2005-06-08 2007-03-22 Massachusetts Institute Of Technology Solution-based methods for RNA expression profiling
US20080076674A1 (en) * 2006-07-06 2008-03-27 Thomas Litman Novel oligonucleotide compositions and probe sequences useful for detection and analysis of non coding RNAs associated with cancer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070065844A1 (en) * 2005-06-08 2007-03-22 Massachusetts Institute Of Technology Solution-based methods for RNA expression profiling
US20080076674A1 (en) * 2006-07-06 2008-03-27 Thomas Litman Novel oligonucleotide compositions and probe sequences useful for detection and analysis of non coding RNAs associated with cancer

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011026909A1 (fr) * 2009-09-02 2011-03-10 L'oreal SIGNATURE microARN DE LA DIFFÉRENCIATION ÉPIDERMIQUE ET UTILISATIONS
US9090937B2 (en) 2009-09-02 2015-07-28 L'oreal Epidermal differentiation microrna signature and uses thereof

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