WO2010135692A2 - Mirna biomarkers of prostate disease - Google Patents

Mirna biomarkers of prostate disease Download PDF

Info

Publication number
WO2010135692A2
WO2010135692A2 PCT/US2010/035839 US2010035839W WO2010135692A2 WO 2010135692 A2 WO2010135692 A2 WO 2010135692A2 US 2010035839 W US2010035839 W US 2010035839W WO 2010135692 A2 WO2010135692 A2 WO 2010135692A2
Authority
WO
WIPO (PCT)
Prior art keywords
mir
mirna
mirnas
serum
patients
Prior art date
Application number
PCT/US2010/035839
Other languages
French (fr)
Other versions
WO2010135692A3 (en
Inventor
Jeffrey Shelton
Kevin Kelnar
Stephanie Volz
Alex Adai
David Brown
Original Assignee
Asuragen, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Asuragen, Inc. filed Critical Asuragen, Inc.
Priority to CA2762986A priority Critical patent/CA2762986C/en
Priority to EP10720115A priority patent/EP2432895A2/en
Priority to AU2010249421A priority patent/AU2010249421A1/en
Publication of WO2010135692A2 publication Critical patent/WO2010135692A2/en
Publication of WO2010135692A3 publication Critical patent/WO2010135692A3/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/112Disease subtyping, staging or classification
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/16Primer sets for multiplex assays
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/178Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • PrCa Prostate cancer
  • PSA prostate specific antigen
  • DRE digital rectal examination
  • a definitive diagnosis of PrCa requires the direct observation of cancerous cells in a prostate tissue biopsy.
  • a prostate biopsy is typically acquired with the aid of a transrectal ultrasound probe, in which six to twelve tissue fragments are collected. Once collected, the tissue is fixed in a formalin solution and embedded in paraffin for sectioning. Formalin-fixed, paraffin embedded (FFPE) tissue sections are stained and analyzed by a pathologist. If cancerous cells are observed, the pathologist will most commonly identify the two most common architectural patterns of differentiation within the lesion and assign a grade (1 through 5) to each of these two differentiation patterns.
  • a Gleason Score is calculated as the sum of the grade for the most common pattern (which must represent at least 50% of the tumor) and the grade for the second Attorney Docket No. 10256.32-304
  • PSA testing in addition to DRE, has dramatically increased the number of prostate cancers that are identified at a point early enough so that radical prostatectomy can be curative.
  • the PSA assay still fails to detect ⁇ 15% of patients with cancer (Daneshgari et al., Urology 45:604-609 (1995)).
  • PSA testing has a relatively high false positive rate that is caused by the normal fluctuation of PSA levels due to a patient's age or to the presence of conditions such as prostatitis or benign prostatic hyperplasia (BPH).
  • a nucleic acid-based diagnostic assay has been developed as an alternative to PSA testing. This assay measures levels of PCA3 mRNA using Attorney Docket No. 10256.32-304
  • PCA3 is over-expressed in prostate tumors (Bussemakers et al., Cancer Res 59:5975-5979 (1999)). In clinical trials, the PCA3 test achieved 66-82% sensitivity and 76-89% specificity for PrCa (Fradet et al., Urology 64:311 -316 (2004)). PCA3 diagnostic assays are available but have yet to be approved by the FDA. Unfortunately, as with PSA, PCA3 testing exhibits disappointingly high false-positive and false-negative rates.
  • PCA3 test The apparent best application of the PCA3 test is as a reflex for the PSA test. Also, a major impediment to the adoption of the PCA3 test is that it requires a urine sample from patients following a prostatic massage, which may be poorly tolerated. It also requires that an individual submit both blood and urine samples to accommodate both the PSA and PCA3 tests.
  • the invention relates to the characterization of prostate diseases such as cancer or BPH by detecting miRNAs from serum or plasma.
  • the methods of the invention include both diagnosing disease, and evaluating the prognosis or aggressiveness of a prostate disease. Further, the methods may be used to characterize the progression of a prostate disease.
  • the patients tested using the methods of the invention may also be tested using PSA or PCA3 assays.
  • the diagnosis or prognosis may be achieved by amplifying and detecting the amount of certain miRNAs that are present in elevated or reduced levels in the serum or plasma of a subject with prostate disease.
  • one serum or plasma miRNA may be amplified and measured to characterize prostate disease, while in other embodiments, two or more miRNAs are detected from serum or plasma.
  • Some embodiments include amplifying and measuring a pair of miRNAs. In some instances, one miRNA in the pair is elevated in serum or plasma of patients with prostate disease, and the other miRNA in the pair is reduced. In other circumstances, both miRNAs in the pair can be elevated or both reduced.
  • non-miRNA biomarkers such as PSA may also be measured.
  • Some embodiments of the invention relate to diagnosis or prognosis of prostate cancer. Other embodiments of the invention include diagnosing BPH in a patient.
  • Figure 1 shows human precursor miRNA (pre-miRNA) sequences, as provided by Release 13.0 of the miRBase::Sequences Database (http://microrna.sanger.ac.uk; Griffiths-Jones et al., Nucleic Acids Research, 2008, 36, Database Issue, D154-D158; Griffiths-Jones et al., Nucleic Acids Research, 2006, 34, Database Issue, D140-D144; Griffiths-Jones, Nucleic Acids Research, 2004, 32, Database Issue, D109-D111 ).
  • the names of mature miRNAs from Tables 1 and 20 are also provided for each precursor sequence.
  • the methods of the invention provide assays for amplifying and measuring the amount of a miRNA in a serum or plasma sample, thereby characterizing a prostate disease.
  • miRNA includes human miRNAs, mature single stranded miRNAs, precursor miRNAs (pre-miR), and variants thereof, which may be naturally occurring.
  • miRNA also includes primary miRNA transcripts and duplex miRNAs.
  • the name of a specific miRNA refers to the mature miRNA.
  • miR-122a refers to a mature miRNA sequence derived from pre-miR-122.
  • RNAs For certain miRNAs, a single precursor contains more than one mature miRNA sequence. In other instances, multiple precursor miRNAs contain the same mature sequence. In some instances, mature miRNAs have been re-named based on new scientific consensus.
  • miR-213 refers to a mature miRNA from pre-miR-181a- 1 , and is also called miR-181a*.
  • miRNAs that have been re-named include miR-189 (also called miR-24 * ), which comes from pre-miR-24-1 ; miR-368 (also called miR-376c); and miR-422b (also called miR-378 * ).
  • miRNAs detected by assays of this application include naturally occurring sequences for the miRNAs.
  • Serum is typically the fluid, non-cellular portion of coagulated blood.
  • Plasma is also a non-cellular blood sample, but unlike serum, plasma contains clotting factors.
  • serum or plasma samples may be obtained from a human patient previously screened for prostate disease using PSA or PCA3 assays, or other diagnostic methods.
  • the patient has undergone a physical exam or biopsy to detect prostate disease. Additional embodiments include measuring miRNA in samples from patients previously or currently undergoing treatment for a prostate disease. The volume of plasma or serum obtained and used for the assay may be varied depending upon clinical intent.
  • clotting activators such as silica particles are added to the blood collection tube.
  • the blood is not treated to facilitate clotting.
  • Blood collection tubes are commercially available from many sources and in a variety of formats (e.g., Becton Dickenson Vacutainer® tubes-SSTTM, glass serum tubes, or plastic serum tubes).
  • the blood is collected by venipuncture and processed within three hours after drawing to minimize hemolysis and minimize the release of miRNAs from intact cells in the blood.
  • blood is Attorney Docket No. 10256.32-304
  • the blood may be fractionated by centrifugation to remove cellular components.
  • centrifugation to prepare serum can be at a speed of at least 500, 1000, 2000, 3000, 4000, or 5000 X G.
  • the blood can be incubated for at least 10, 20, 30, 40, 50, 60, 90, 120, or 150 minutes to allow clotting. In other embodiments, the blood is incubated for at most 3 hours.
  • plasma the blood is not permitted to coagulate prior to separation of the cellular and acellular components. Serum or plasma can be frozen after separation from the cellular portion of blood until further assayed.
  • RNA may be extracted from serum or plasma and purified using methods known in the art. Many methods are known for isolating total RNA, or to specifically extract small RNAs, including miRNAs.
  • the RNA may be extracted using commercially-available kits (e.g., Perfect RNA Total RNA Isolation Kit, Five Prime-Three Prime, Inc.; mirVanaTM kits, Ambion, Inc.).
  • RNA extraction methods previously published for the extraction of mammalian intracellular RNA or viral RNA may be adapted, either as published or with modification, for extraction of RNA from plasma and serum.
  • RNA may be extracted from plasma or serum using silica particles, glass beads, or diatoms, as in the method or adaptations described in U.S. Publication No. 2008/0057502. II. miRNA MARKERS FOR PROSTATE DISEASE
  • Certain embodiments of the invention provide serum or plasma miRNAs as markers for prostate disease.
  • miRNAs that are present at elevated levels in the serum or plasma of patients with prostate disease are used as markers.
  • miRNAs that have reduced levels are used as markers.
  • more than one miRNA from Attorney Docket No. 10256.32-304 are used as markers.
  • the miRNAs may all have elevated levels, all have reduced levels, or a mixture of miRNAs with elevated and reduced levels may be used.
  • the terms “reduced levels” or “elevated levels” refer to the amount of a miRNA in a serum or plasma sample from a patient compared to the amount of the miRNA in serum or plasma from a cohort or cohorts that do not have the prostate disease that the patient is being tested for. For instance, a miRNA that has reduced levels in the sera of prostate cancer patients is present at lower amounts in prostate cancer patient sera than in serum from a donor who does not have prostate cancer. For certain miRNAs, elevated levels in a patient serum or plasma sample indicates presence or prognosis for a prostate disease. Other miRNAs are present in reduced levels in patients with prostate disease.
  • Prostate disease can be classified as BPH or prostate cancer.
  • prostate cancer can be further characterized based on histopathology, aggressiveness, Gleason score, PSA measurements, and other methods known in the art.
  • the methods described herein can be used to characterize a patient with at least 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sensitivity. The degree of sensitivity indicates the percentage of patients with a disease who are positively characterized as having the disease.
  • the methods have at least 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% specificity (e.g., the percentage of non-diseased patients who are correctly characterized).
  • the assay parameters can be adjusted to optimize for both sensitivity and specificity.
  • the level of the miRNA marker will be compared to a control to determine whether the level is reduced or elevated.
  • the control may be Attorney Docket No. 10256.32-304
  • an external control such as a miRNA in a serum or plasma sample from a patient known to be free of prostate disease.
  • the external control may be a miRNA from a non-serum sample like a tissue sample or a known amount of a synthetic RNA.
  • An internal control may be a miRNA from the same serum or plasma sample being tested. The identity of a miRNA control may be the same as or different from the patient serum or plasma miRNA being measured.
  • Table 1 lists miRNAs that have elevated or reduced levels in serum from patients with prostate disease. These miRNAs may be used in accordance with the invention. Some of the miRNAs are useful for characterizing prostate cancer, including distinguishing cancer from BPH. Other miRNAs are suitable markers for identifying patients with BPH. In addition, some miRNAs may be used to predict the aggressiveness or outcome of prostate cancer.
  • Table 1 miRNAs with elevated or reduced levels in serum from patients with prostate disease. Levels are of miRNA in prostate cancer patients compared to patients with BPH or normal prostate.
  • a single miRNA may be used to characterize prostate cancer.
  • any one of the following miRNAs may be used to characterize prostate cancer, either alone or in combination with other markers: let-7a, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, miR-100, miR-101 , miR-103, miR- 105, miR-106a, miR-106b, miR-107, miR-10a, miR-10b, miR-122a, miR-125a, miR-125b, miR-126, miR-126 * , miR-128a, miR-128b, miR-129, miR-130b, miR- 132, miR-133a, miR-133b, miR-136, miR-138, miR-139, miR-140, miR-141 , miR- 142-3p, miR-142-5p, miR-143, miR-146a, miR-
  • any one of the following miRNAs may be used to characterize prostate cancer, either alone or in combination with other markers: let-7c, let-7d, let-7f, let-7g, let-7i, m ⁇ R-101 , miR-106b, miR-10a, miR-10b, miR-122a, miR-125a, miR-125b, miR-126, miR-126 * , miR-128a, miR-130b, miR- Attorney Docket No. 10256.32-304
  • miR-181b miR-181d, miR-192, miR-193a, miR-195, miR-196a, miR198, miR- 199a * , miR-199b, miR-200a, miR-200b, miR-200c, miR-202, miR-204, miR-205, miR-206, miR-212, miR-219, miR-220, miR-223, miR-23b, miR-24, miR25, miR- 26a, miR-27a, miR-296, miR-29b, miR-302b * , miR-30-a-3p, miR-30b,
  • a miRNA for characterizing prostate cancer is chosen from let-7d, miR-IOb, miR-122a, miR-139, miR-24, miR-204, miR-205, miR-206, miR-375, and miR-99b, and may be used alone or in combination with other markers.
  • the miRNA used for characterizing prostate cancer may be chosen from let-7d, miR-122a, miR-139, miR-204, miR-205, miR- 206, and miR-375.
  • miRNAs that have reduced levels in serum from patients with prostate disease are used as biomarkers. Certain miRNAs at reduced levels in serum can be used to distinguish patients with prostate cancer from normal patients or patients with BPH. These miRNA markers include let-7d, let-7f, let-7g, miR-132, miR-135a, miR-135b, miR-140, miR-143, miR-153, miR- Attorney Docket No. 10256.32-304
  • miR-15b miR-16, miR-17-5p, miR-181c, miR-182, miR-188, miR-195, miR- 196a, miR-197, miR198, miR-200a*, miR-202, miR-202*, miR-203, miR-20a, miR- 212, miR-22, miR-220, miR-223, miR-24, miR-26b, miR-301 , miR-302a * , miR-30- a-3p, miR-30b, miR-30c, miR-30d, miR-30e-3p, miR-32, miR-323, miR-331 , miR- 335, miR-346, miR-365, miR-367, miR-372, miR-373 * , miR-380-3p, miR-412, miR-425, miR-432 * , miR-449, miR-452 * , miR-489, miR-490, miR
  • two or more miRNAs are used to characterize prostate disease. Any one of the following miRNAs may be used in combination with at least one other serum miRNA in the methods of the invention: let-7a, let-7c, let-7d, let-7f, let-7g, miR-1 , miR-103, miR-106a, miR-106b, miR-107, miR-10b, miR-122a, miR-125a, miR-125b, miR-126, miR-126 * , miR-130a, miR- 130b, miR-132, miR-133b, miR-135a, miR-139, miR-140, miR-142-3p, miR-142- Attorney Docket No. 10256.32-304
  • any miRNA chosen from the following list can be used in combination with another serum miRNA biomarker to characterize prostate cancer: let-7a, !et-7c, let-7d, let-7f, let-7g, miR-1 , miR-103, miR-106a, miR-107, miR-10b, miR-122a, miR-125a, miR-125b, miR-126, miR-126 * , miR- 130a, miR-130b, miR-132, miR-139, miR-140, miR-142-5p, miR-143, miR-145, miR-146a, miR-148a, miR-155, miR-15a, miR-15b, miR-16, miR-17-5p, miR-181c, miR-185, miR-188, miR-18a, miR-18a * , miR-191 , miR-192, miR-193a, miR-194, miR-195, miR-196b
  • Some embodiments of the invention relate to amplifying and measuring at least a pair of miRNAs from serum
  • Table 2 includes pairs that may be used to characterize prostate cancer.
  • pairs of miRNA markers from serum can be used to predict the aggressiveness or stage of prostate cancer.
  • one of the miRNA pairs in Table 3 may be used to determine cancer aggressiveness
  • a miRNA is amplified prior to measurement.
  • the level of miRNA is measured during the amplification process.
  • the miRNA is not amplified prior to measurement Attorney Docket No. 10256.32-304
  • nucleic acid polymerization and amplification techniques include reverse transcription (RT), polymerase chain reaction (PCR), real-time PCR (quantitative PCR (q-PCR)), nucleic acid sequence-base amplification (NASBA), ligase chain reaction, multiplex ligatable probe amplification, invader technology (Third Wave), rolling circle amplification, in vitro transcription (IVT), strand displacement amplification, transcription-mediated amplification (TMA), RNA (Eberwine) amplification, and other methods that are known to persons skilled in the art.
  • more than one amplification method is used, such as reverse transcription followed by real time quantitative PCR (qRT-PCR) (Chen et al., Nucleic Acids Research, 33(20):e179 (2005)).
  • a typical PCR reaction includes multiple amplification steps, or cycles that selectively amplify target nucleic acid species: a denaturing step in which a target nucleic acid is denatured; an annealing step in which a set of PCR primers (forward and reverse primers) anneal to complementary DNA strands; and an elongation step in which a thermostable DNA polymerase elongates the primers. By repeating these steps multiple times, a DNA fragment is amplified to produce an amplicon, corresponding to the target DNA sequence.
  • Typical PCR reactions include 20 or more cycles of denaturation, annealing, and elongation.
  • the annealing and elongation steps can be performed concurrently, in which case the cycle contains only two steps. Since mature miRNAs are single-stranded, a reverse transcription reaction (which produces a complementary cDNA sequence) may be performed prior to PCR reactions.
  • Reverse transcription reactions include the use of, e.g., a RNA-based DNA polymerase (reverse transcriptase) and a primer.
  • a RNA-based DNA polymerase reverse transcriptase
  • primer reverse transcriptase
  • a set of primers is used for each target sequence.
  • the lengths of the primers depends on many factors, including, but not limited to, the desired hybridization temperature between the primers, the target nucleic acid sequence, and the complexity of the different target nucleic acid sequences to be amplified.
  • a primer is about 15 to about 35 nucleotides in length. In other embodiments, a primer is equal to or fewer than 15, 20, 25, 30, or 35 nucleotides in length. In additional embodiments, a primer is at least 35 nucleotides in length.
  • a forward primer can comprise at least one sequence that anneals to a miRNA biomarker and alternatively can comprise an additional 5' non-complementary region.
  • a reverse primer can be designed to anneal to the complement of a reverse transcribed miRNA. The reverse primer may be independent of the miRNA biomarker sequence, and multiple miRNA biomarkers may be amplified using the same reverse primer. Alternatively, a reverse primer may be specific for a miRNA biomarker.
  • two or more miRNAs are amplified in a single reaction volume.
  • One aspect includes multiplex q-PCR, such as qRT-PCR, which enables simultaneous amplification and quantification of at least two miRNAs of interest in one reaction volume by using more than one pair of primers and/or more than one probe.
  • the primer pairs comprise at least one amplification primer that uniquely binds each miRNA, and the probes are labeled such that they are distinguishable from one another, thus allowing simultaneous quantification of Attorney Docket No. 10256.32-304
  • Multiplex qRT-PCR has research and diagnostic uses, including but not limited to detection of miRNAs for diagnostic, prognostic, and therapeutic applications.
  • the qRT-PCR reaction may further be combined with the reverse transcription reaction by including both a reverse transcriptase and a DNA-based thermostable DNA polymerase.
  • a "hot start” approach may be used to maximize assay performance (U.S. Patent Nos. 5,411 ,876 and 5,985,619).
  • the components for a reverse transcriptase reaction and a PCR reaction may be sequestered using one or more thermoactivation methods or chemical alteration to improve polymerization efficiency (U.S. Patent Nos. 5,550,044, 5,413,924, and 6,403,341).
  • labels, dyes, or labeled probes and/or primers are used to detect amplified or unamplified miRNAs.
  • detection methods are appropriate based on the sensitivity of the detection method and the abundance of the target.
  • amplification may or may not be required prior to detection.
  • miRNA amplification is preferred.
  • a probe or primer may include Watson-Crick bases or modified bases.
  • Modified bases include, but are not limited to, the AEGIS bases (from Eragen Biosciences), which have been described, e.g., in U.S. Patent Nos. 5,432,272, 5,965,364, and 6,001 ,983.
  • bases are joined by a natural phosphodiester bond or a different chemical linkage.
  • linkages include, but are not limited to, a peptide bond or a Locked Nucleic Acid (LNA) linkage, which is described, e.g., in U.S. Patent No. 7,060,809.
  • LNA Locked Nucleic Acid
  • oligonucleotide probes or primers present in an amplification reaction are suitable for monitoring the amount of amplification product produced as a function of time.
  • probes having different single stranded versus double stranded character are used to detect the nucleic acid.
  • Probes include, but are not limited to, the 5'-exonuclease assay (e.g., TaqManTM) probes (see U.S. Patent No. 5,538,848), stem-loop molecular beacons (see, e.g., U.S. Patent Nos.
  • stemless or linear beacons see, e.g., WO 9921881, U.S. Patent Nos. 6,485,901 and 6,649,349), peptide nucleic acid (PNA) Molecular Beacons (see, e.g., U.S. Patent Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, e.g. U.S. Patent No. 6,329,144), non-FRET probes (see, e.g., U.S. Patent No. 6,150,097), SunriseTM /AmplifluorBTMprobes (see, e.g., U.S. Patent No.
  • one or more of the primers in an amplification reaction can include a label.
  • different probes or primers comprise detectable labels that are distinguishable from one Attorney Docket No. 10256.32-304
  • nucleic acid such as the probe or primer, may be labeled with two or more distinguishable labels.
  • a label is attached to one or more probes and has one or more of the following properties: (i) provides a detectable signal; (ii) interacts with a second label to modify the detectable signal provided by the second label, e.g., FRET (Fluorescent Resonance Energy Transfer); (iii) stabilizes hybridization, e.g., duplex formation; and (iv) provides a member of a binding complex or affinity set, e.g., affinity, antibody-antigen, ionic complexes, hapten- ligand (e.g., biotin-avidin).
  • use of labels can be accomplished using any one of a large number of known techniques employing known labels, linkages, linking groups, reagents, reaction conditions, and analysis and purification methods.
  • MiRNAs can be detected by direct or indirect methods.
  • a direct detection method one or more miRNAs are detected by a detectable label that is linked to a nucleic acid molecule.
  • the miRNAs may be labeled prior to binding to the probe. Therefore, binding is detected by screening for the labeled miRNA that is bound to the probe.
  • the probe is optionally linked to a bead in the reaction volume.
  • nucleic acids are detected by direct binding with a labeled probe, and the probe is subsequently detected.
  • the nucleic acids such as amplified miRNAs
  • FlexMAP Microspheres Luminex
  • Some methods may involve detection with polynucleotide probes modified with fluorescent labels or branched DNA (bDNA) detection, for example.
  • bDNA branched DNA
  • nucleic acids are detected by indirect detection methods.
  • a biotinylated probe may be combined with a stretavidin-conjugated dye to detect the bound nucleic acid.
  • the streptavidin molecule binds a biotin label on amplified miRNA, and the bound miRNA is detected by detecting the dye molecule attached to the streptavidin molecule.
  • the streptavidin-conjugated dye molecule comprises Phycolink® Streptavidin R-Phycoerythrin (PROzyme). Other conjugated dye molecules are known to persons skilled in the art.
  • Labels include, but are not limited to: light-emitting, light-scattering, and light-absorbing compounds which generate or quench a detectable fluorescent, chemiluminescent, or bioluminescent signal (see, e.g., Kricka, L., Nonisotopic DNA Probe Techniquies, Academic Press, San Diego (1992) and Garman A., Non-Radioactive Labeling, Academic Press (1997).).
  • Fluorescent reporter dyes useful as labels include, but are not limited to, fluoresceins (see, e.g., U.S. Patent Nos. 5,188,934, 6,008,379, and 6,020,481), rhodamines (see, e.g., U.S.
  • Tetramethylrhodamine, and/or Texas Red as well as any other fluorescent moiety capable of generating a detectable signal.
  • fluorescein dyes include, but are not limited to, 6-carboxyfluorescein; 2',4',1 ,4,-tetrachlorofluorescein; and 2 l ,4',5',7',1 ,4-hexachlorofluorescein.
  • the fluorescent label is selected from SYBR-Green, 6-carboxyfluorescein ("FAM”), TET, ROX, VICTM, and JOE.
  • labels are different fluorophores capable of emitting light at different, spectrally-resolvable wavelengths (e.g., 4- differently colored fluorophores); certain such labeled probes are known in the art and described above, and in U.S. Patent No. 6,140,054.
  • a dual labeled fluorescent probe that includes a reporter fluorophore and a quencher fluorophore is used in some embodiments. It will be appreciated that pairs of fluorophores are chosen that have distinct emission spectra so that they can be easily distinguished.
  • labels are hybridization-stabilizing moieties which serve to enhance, stabilize, or influence hybridization of duplexes, e.g., intercaiators and intercalating dyes (including, but not limited to, ethidium bromide and SYBR-Green), minor-groove binders, and cross-linking functional groups (see, e.g., Blackburn et al., eds. "DNA and RNA Structure” in Nucleic Acids in Chemistry and Biology (1996)).
  • intercaiators and intercalating dyes including, but not limited to, ethidium bromide and SYBR-Green
  • minor-groove binders include, but not limited to, ethidium bromide and SYBR-Green
  • cross-linking functional groups see, e.g., Blackburn et al., eds. "DNA and RNA Structure” in Nucleic Acids in Chemistry and Biology (1996)).
  • methods relying on hybridization and/or ligation to quantify miRNAs may be used, including oligonucleotide ligation (OLA) methods and methods that allow a distinguishable probe that hybridizes to the target nucleic acid sequence to be separated from an unbound probe.
  • OLA oligonucleotide ligation
  • HARP-like probes as disclosed in U.S. Publication No. 2006/0078894 may be used to measure the quantity of miRNAs.
  • a probe inactivation region comprises a subset of nucleotides within the target hybridization region of the probe.
  • a post-hybridization probe inactivation step is carried out using an agent which is able to distinguish between a HARP probe that is hybridized to its targeted nucleic acid sequence and the corresponding unhybridized HARP probe. The agent is able to inactivate or modify the unhybridized HARP probe such that it cannot be amplified.
  • a probe ligation reaction may be used to quantify miRNAs.
  • MLPA Multiplex Ligation-dependent Probe Amplification
  • pairs of probes which hybridize immediately adjacent to each other on the target nucleic acid are ligated to each other only in the presence of the target nucleic acid.
  • MLPA probes have flanking PCR primer binding sites. MLPA probes can only be amplified if they have been ligated, thus allowing for detection and quantification of miRNA biomarkers.
  • RNA isolation For serum preparation, ten (10) ml of whole blood was collected from patients and normal donors using a BD Vacutainer® glass serum tube (Becton, Dickinson and Company; Franklin Lakes, NJ, USA; cat. no. 366441 ). Following blood collection, tubes were incubated at room temperature for up to 2.5 hours to allow the blood to clot. Tubes were centrifuged for ten minutes at 2,000 x g. Serum was transferred to a new tube using a serological pipette and frozen at - 80 0 C until it was processed for RNA isolation.
  • BD Vacutainer® glass serum tube Becton, Dickinson and Company; Franklin Lakes, NJ, USA; cat. no. 366441 .
  • Serum RNA was purified using the organic extraction of the mirVana PARISTM Kit (Part No. AM1556; Applied Biosystems/Ambion; Austin, TX, USA), with the following modifications. Following the addition of acid phenoLchloroform and vortexing, samples were incubated on ice for 5 min then centrifuged at 13,000 x g for 10 min at 4 0 C. The aqueous layer was removed, extracted with Attorney Docket No. 10256.32-304
  • RNA template Serum RNA (0.25 ⁇ l per reaction) was added and mixed. RT reactions were incubated in a 384-well GeneAmp® PCR System 9700 (Applied Biosystems) at 16 0 C for 30 minutes, then at 42 0 C for 30 minutes, then at 85 0 C for 5 minutes. RT reactions were then frozen at -20 0 C. All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified. Table 5. Reverse transcription reaction components.
  • PCR components (Table 6) were assembled on ice prior to the addition of cDNA (4 ⁇ l) from the RT reaction. Reactions were incubated in an ABI PRISMTM 7900HT Fast Real-Time PCR system (Applied Biosystems) at 95 0 C for 1 minute, then for 50 cycles at 95 0 C for 5 seconds and 60 0 C for 30 seconds. Results were analyzed with the 7900HT Fast Real-Time PCR system SDS V2.3 software (Applied Biosystems). All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified. Table 6. PCR components.
  • the average dCt values for each miRNA in the normal donor, BPH, and PrCa patient samples were calculated.
  • the average dCt values for the PrCa patient samples were subtracted from the average dCt values for the normal donor or BPH samples, and the average dCt values for the BPH samples were subtracted from the average dCt values for normal donor samples to determine the variance in the miRNA levels between the three patient sets. Student's t-test was then used to determine the potential of various miRNAs to distinguish the sera of PrCa patients from sera of normal donors or patients with BPH.
  • Table 7 provides the average dCt values, the difference in miRNA levels between patient sets, and p-values for each miRNA tested. miRNAs that are present at reduced or elevated levels in PrCa patients compared to BPH or normal patients represent biomarkers of prostate cancer that can be used to diagnose prostate cancer using the serum from a patient.
  • Example 2 Combinations of miRNAs that distinguish sera of prostate cancer patients from sera of BPH patients and normal donors.
  • the inventors evaluated pairs of miRNA biomarkers for their abilities to distinguish sera of PrCa patients from sera of BPH patients and normal donors. Un-normalized qRT-PCR data generated using the samples described above in Example 1 were used to calculate dCt values for each pair of miRNAs that was evaluated. The dCt values of the various miRNA pairs in PrCa and norma! serum samples were analyzed using Receiver-Operator Characteristic (ROC) analysis to identify the miRNA pairs having the ability to distinguish sera of PrCa patients from sera of BPH patients and normal donors.
  • ROC Receiver-Operator Characteristic
  • a Receiver Operator Characteristic (ROC) curve is a graphical plot of the sensitivity vs. specificity for a binary classifier system as its discrimination threshold is varied.
  • ROC analysis provides a tool to select possibly optimal models and to discard suboptimal ones independently from (and prior to specifying) the class distribution
  • AUC area underthe ROC curve
  • Advantages of the ROC technique include (1) it does not assume a parametric form of the class probability as required in the logistic regression method, (2) it is adaptable to outcome-dependent samplings, e.g. the case-control design, which are widely used in medical studies, and (3) it is Attorney Docket No. 10256.32-304
  • ROC curves are used to evaluate the capacities of individual and combinations of biomarker candidates to distinguish patient classes.
  • the abundance(s) of biomarker candidates are measured.
  • the measurements are used to develop classifiers whose true positive and false positive rates are plotted in a ROC curve.
  • the AUC is then calculated for each classifier.
  • An ideal classifier has an ROC AUC value of 1 , and classifiers can be ranked based upon how close their ROC AUC is to 1.
  • the miRNA pairs in Table 8 and the individual miRNA biomarkers in Table 9 are all possible targets for diagnosing prostate cancer using serum.
  • the inventors selected fourteen miRNAs (miR-24, miR-125b, miR-340, miR-375, miR- 15b, miR-204, miR-205, miR-206, miR-122a, miR-483, let-7d, let-7f, miR-191 , and miR-26a) identified in Example 1 and quantified those miRNAs in the sera of 12 PrCa patients, 12 BPH patients, and 12 normal donors (Table 10) using qRT- PCR.
  • Patient and normal serum samples were purchased from ProteoGenex, Inc. (Culver City, cA, USA). Serum preparation and serum RNA isolation were performed as described in Example 1.
  • microRNA levels were determined by qRT-PCR using TaqMan® MicroRNA Assays (Applied Biosystems; Foster City, CA, USA) specific for each miRNA.
  • Reverse transcription (RT) reaction components were assembled on ice, as shown above in Example 1 (Table 5), prior to the addition of RNA template. Serum RNA (0.25 ⁇ l per reaction) was added and mixed. RT reactions were incubated in a 384-well GeneAmp® PCR System 9700 (Applied Biosystems) at Attorney Docket No. 10256.32-304
  • PCR components (Table 11) were assembled on ice prior to the addition of cDNA (4 ⁇ l) from the RT reaction. Reactions were incubated in an ABI PRISMTM 7900HT Fast Real-Time PCR system (Applied Biosystems) at 95 0 C for 1 minute, then for 50 cycles at 95 0 C for 5 seconds and 60 0 C for 30 seconds. Results were analyzed with the 7900HT Fast Real-Time PCR system SDS V2.3 software (Applied Biosystems). All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified. Table 11. PCR components.
  • qRT-PCR data were initially assessed for outliers. All miRNAs in a given sample with raw Ct readings of 50 were eliminated from further analysis. miR-103 served as an internal control, and its Ct was subtracted from the raw Ct readings for each miRNA in the corresponding sample to produce a dCt for each miRNA that was detected. Normalized values were used to estimate the relative abundance of each miRNA in the samples.
  • Average dCt values for each miRNA in the normal donor, BPH patient, and PrCa patient samples were calculated. Average dCt values for PrCa patient samples were subtracted from average dCt values for normal donor samples and BPH patient samples to determine the variance in the levels of the miRNAs between the patient sets Student's t-test was then used to determine the potential of various miRNAs to distinguish the sera of PrCa patients from sera of normal donors and BPH patients. Table 12 shows average dCt values for the three sample types, and the difference between PrCa samples and normal or BPH samples.
  • the inventors selected 11 miRNA biomarkers (miR- 24, miR-125b, miR-375, miR-204, miR-205, miR-206, miR-122a, let-7d, miR-10b, miR-99b, miR-22) and 1 1 internal control miRNAs (miR-191 , miR-16, miR-181a, miR-21 , miR-26a, miR-106a, m ⁇ R-155, m ⁇ R-30a-5p, let-7a, let-7c, m ⁇ R-222) and Attorney Docket No. 10256.32-304
  • PrCa patient and normal donor serum samples for this Example were purchased from a different vendor (ProMedDx, LLC; Norton, MA, USA), than those samples used for screening and validation in Examples 1 , 2, and 3 (ProteoGenex, Inc.).
  • the two vendors use different methods for serum preparation from patient samples.
  • ten (10) ml of whole blood was collected using a BD VacutainerTM SSTTM plastic serum tube (Becton, Dickinson and Company; Franklin Lakes, NJ, USA; cat. no. 367985). The tubes were incubated at room temperature for 10 to 60 minutes to allow the blood to clot. The tubes were centrifuged for 10 minutes at 3000 - 3500 RPM. Serum was transferred to a new tube using a serological pipette and frozen at -80 0 C until it was subjected to RNA isolation. Serum RNA was purified as described in Example 1.
  • MicroRNA levels were determined by qRT-PCR using TaqMan® MicroRNA Assays (Applied Biosystems; Foster City, CA, USA) specific for each miRNA. Reverse transcription (RT) reaction components were assembled on ice prior to the addition of RNA template (Table 14). Serum RNA (1 ⁇ l per reaction) was added and mixed. RT reactions were incubated in a 384-well GeneAmp® Attorney Docket No. 10256.32-304
  • PCR components (Table 15) were assembled on ice prior to the addition of cDNA (4 ⁇ l) from the RT reaction. Reactions were incubated in an ABI PRISMTM 7900HT Fast Real-Time PCR system (Applied Biosystems) at 95 0 C for 1 minute, then for 50 cycles at 95 0 C for 5 seconds and 60 X for 30 seconds. Results were analyzed with SDS V2.3 (Applied Biosystems). All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified.
  • qRT-PCR data for each miRNA in each sample was normalized by subtracting the Ct value for miR-103 from the Ct value for the miRNA from the same sample. The resulting values, called dCt, were used to calculate the average dCt values for each miRNA in the normal donor and PrCa patient samples. Average dCt values for PrCa patient samples were subtracted from average dCt values for normal donor samples to determine the variance in the levels of the miRNAs between the patient sets. Student's t-test was then used to determine the potential of various miRNAs to distinguish the sera of PrCa patients from sera of normal donors. Table 16 shows average dCt values for the three sample types, and the difference between PrCa samples and normal or BPH samples. Table 16. Prostate cancer biomarkers. SD, standard deviation.
  • RNA levels were determined by qRT-PCR using TaqMan® MicroRNA Assays (Applied Biosystems; Foster City, CA, USA) specific for each miRNA.
  • Reverse transcription (RT) reaction components (Table 18) were assembled on ice prior to the addition of RNA template. Plasma RNA (0.25 ⁇ l per reaction) was added and mixed. RT reactions were incubated in a 384-well GeneAmp® PCR System 9700 (Applied Biosystems) at 16 0 C for 30 minutes, then at 42 °C for 30 minutes, then at 85 0 C for 5 minutes. RT reactions were then frozen at -20 °C.
  • Table 21 miRNA biomarker pairs that can be used to identify plasma from prostate cancer patients.
  • a key issue associated with prostate cancer patients is determining whether treatment is necessary. Many patients are diagnosed at very early stages of disease Because most cases of prostate cancer are unlikely to progress to metastatic disease, it is often unnecessary to subject an individual, especially one of advanced age, to aggressive treatment Distinguishing patients with relatively benign prostate cancer from patients with an aggressive form of the disease is vital to determining the appropriate level of therapeutic intervention
  • miRNA pairs were capable of distinguishing those serum samples (Table 23; low, samples from PrCa patients with Gleason scores ⁇ 6; High, samples from PrCa patients with Gleason scores >6.). These miRNA pairs are useful for predicting aggressiveness of prostate cancer using patient serum samples.
  • miRNA levels were determined by qRT-PCR using TaqMan® MicroRNA Assays (Applied Biosystems; Foster City, CA, USA) specific for each miRNA.
  • Reverse transcription (RT) reaction components (Table 26) were assembled on ice prior to the addition of RNA template. Serum RNA (1.0 ⁇ l per reaction) was added and mixed. RT reactions were incubated in a 384-well GeneAmp® PCR System 9700 (Applied Biosystems) at 4 0 C for 30 minutes, Attorney Docket No. 10256.32-304
  • PCR components (Table 27) were assembled on ice prior to the addition of cDNA (4 ⁇ l) from the RT reaction. Reactions were incubated in an ABI PRISMTM 7900HT Fast Real-Time PCR system (Applied Biosystems) at 95 °C for 1 minute, then for 50 cycles at 95 0 C for 5 seconds, then at 60 0 C for 30 seconds. Data were extracted and exported with SDS V2.3 (Applied Biosystems). The data were analyzed with Microsoft Excel (Microsoft Corporation). All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified. Table 27. PCR components.
  • qRT-PCR data were initially assessed for outliers. All miRNAs in a given sample with raw Ct readings of >40 were eliminated from further analyses. qRT-PCR data for each miRNA in each sample were normalized by subtracting the Ct value for miR-103 from the Ct value for the miRNA in the same sample. The resulting dCt values, were used to calculate the average dCt values for each miRNA in the normal donor and PrCa patient samples. Average dCt values for PrCa patient samples were subtracted from average dCt values for normal donor samples to determine the variance in the levels of the miRNAs between the patient sets.
  • Table 28 lists the difference in average Ct between normal and prostate cancer samples, as well as p-values for PrCa samples compared to normal or BPH samples, and normal samples compared to BPH samples. These miRNAs are biomarkers of prostate cancer that can be used to diagnose prostate cancer using the serum from a patient. Table 28. Prostate cancer biomarkers.
  • the inventors used the data for the same miRNA biomarkers to identify pairs of miRNAs with the capacity to distinguish sera of PrCa patients from sera of BPH patients and normal donors. Un-normalized qRT-PCR data were used to calculate dCt values for each pair of miRNAs that was evaluated. The dCt values of the various miRNA pairs in PrCa and normal serum samples were analyzed using Receiver-Operator Characteristic (ROC) analysis to identify the miRNA pairs having the ability to distinguish sera of PrCa patients from sera of BPH patients and normal donors. Data from the best biomarker pairs are presented in Table 29. Numerous miRNAs were used in multiple biomarkers pairs, indicating the strength of their variable levels in sera of prostate cancer patients and normal donors. Attorney Docket No. 10256.32-304
  • miR-125b and miR-24 were quantified in the sera of 50 normal male donors, 40 patients with benign prostatic hyperplasia (BPH), 16 patients with prostate cancer who were undergoing hormone treatment and/or chemotherapy, and 33 untreated prostate cancer patients.
  • BPH benign prostatic hyperplasia
  • Serum RNA was purified using the organic extraction of the mirVana PARISTM Kit (Part No. AM1556; Applied Biosystems/Ambion; Austin, TX, USA), Attorney Docket No. 10256.32-304
  • RNA template (Table 30). Serum RNA (1 ⁇ l per reaction) was added and mixed. RT reactions were incubated in a 384-well GeneAmp® PCR System 9700 (Applied Biosystems) at 4 0 C for 30 minutes, then at 16 0 C for 30 minutes, then at 42 0 C for 30 minutes, then at 85 0 C for 5 minutes. RT reactions were then frozen at -20 0 C. All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified. Thus, the assays are useful to monitor patients' response to treatment. Attorney Docket No. 10256.32-304
  • PCR components (Table 31) were assembled on ice prior to the addition of cDNA (4 ⁇ l) from the RT reaction. Reactions were incubated in an ABI PRISMTM 7900HT Fast Real-Time PCR system (Applied Biosystems) at 95 C for 1 minute, then for 50 cycles at 95 0 C for 5 seconds and 60 0 C for 30 seconds. Results were analyzed with SDS V2.3 software (Applied Biosystems). All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified.
  • a diagnostic threshold of 6.00 dCt or 7.00 dCt was used to classify serum samples from donors.
  • a threshold of 6.00 dCt to identify serum from prostate cancer patients i.e., a serum sample with dCt ⁇ 6.00 is indicative of prostate cancer
  • Three (3) of 16 prostate cancer patients undergoing treatment were classified as positive using the 6 dCt cutoff.
  • Table 32 shows miR-125b/miR-24 dCt following qRT-PCR quantification of miR-125b and miR-24 in serum samples from normal male donors (Normal), benign prostatic hyperplasia patients (BPH), treated prostate cancer patients (Tr-PrCa), and untreated prostate cancer patients (PrCa). Values Attorney Docket No. 10256.32-304

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

This application describes miRNAs that may be used as serum or plasma biomarkers for characterizing prostate disease in a patient. These miRNA biomarkers may be used alone or in combination with other markers for the diagnosis, prognosis, or monitoring of diseases such as prostate cancer.

Description

Attorney Docket No. 10256.32-304
MIRNA BIOMARKERS OF PROSTATE DISEASE
[001] This application claims priority to U.S. Provisional Application No. 61/180,760, filed May 22, 2009, which is incorporated by reference in its entirety.
[002] Work described in this application was partially funded by the Federal government under National Cancer Institution/NIH Grant No. 1 R43CA135917-01. Accordingly, the Federal government may have certain rights in this invention.
[003] Prostate cancer (PrCa) is the most commonly diagnosed cancer and the second leading cause of cancer deaths in men, following only by lung cancer (Jemal et al., CA Cancer J Clin. 58(2):71-96 (2008)). The detection, diagnosis, and monitoring of PrCa involves a combination of assays (e.g., detection of prostate specific antigen (PSA) in blood), physical examination (e.g., digital rectal examination (DRE)), and observation of prostate tissue biopsy samples for cancerous cells.
[004] Currently, a definitive diagnosis of PrCa requires the direct observation of cancerous cells in a prostate tissue biopsy. A prostate biopsy is typically acquired with the aid of a transrectal ultrasound probe, in which six to twelve tissue fragments are collected. Once collected, the tissue is fixed in a formalin solution and embedded in paraffin for sectioning. Formalin-fixed, paraffin embedded (FFPE) tissue sections are stained and analyzed by a pathologist. If cancerous cells are observed, the pathologist will most commonly identify the two most common architectural patterns of differentiation within the lesion and assign a grade (1 through 5) to each of these two differentiation patterns. A Gleason Score (GS) is calculated as the sum of the grade for the most common pattern (which must represent at least 50% of the tumor) and the grade for the second Attorney Docket No. 10256.32-304
most common pattern (representing 5-50% of the tumor), thereby resulting in a GS ranging from 2 to 10 (Gleason, Cancer Chemother Rep 50:125-128 (1966), Gleason, Hum Pathol 23:273-279 (1992)). Higher Gleason Scores, particularly those > 5, are correlated with less favorable patient prognoses (Egevad et al., BJU lnt 89:538-542 (2002)). The GS is the most widespread method of PrCa tissue grading in use today, and the accurate assessment of Gleason grades and score is currently the most useful factor in predicting the course of the disease and the probable outcome.
[005] The adoption of PSA testing, in addition to DRE, has dramatically increased the number of prostate cancers that are identified at a point early enough so that radical prostatectomy can be curative. However, the PSA assay still fails to detect ~15% of patients with cancer (Daneshgari et al., Urology 45:604-609 (1995)). In addition, PSA testing has a relatively high false positive rate that is caused by the normal fluctuation of PSA levels due to a patient's age or to the presence of conditions such as prostatitis or benign prostatic hyperplasia (BPH). Early detection, due in part to PSA testing, has reduced detected tumor size to the point where PSA contribution from BPH is nearly indistinguishable from PrCa tumors (Stamey et al., J Urol 172:1297-1301 (2004)). Therefore, at the time of tumor detection, PSA measurements are not reliable for distinguishing between BPH and prostate cancer. Additionally, data suggests that the PSA-based PrCa screening may result in the diagnosis of PrCa in approximately 36.5% of men who are not destined to have clinical progression of the cancer (Etzioni et al., J Natl Cancer Inst 94:981-990 (2002)). In most of these patients, the disease is indolent.
[006] A nucleic acid-based diagnostic assay has been developed as an alternative to PSA testing. This assay measures levels of PCA3 mRNA using Attorney Docket No. 10256.32-304
qRT-PCR (de Kok et al., Cancer Res 62:2695-2698 (2002); Hessels et al., Eur Urol 44:8-15; discussion 15-16 (2003)). PCA3 is over-expressed in prostate tumors (Bussemakers et al., Cancer Res 59:5975-5979 (1999)). In clinical trials, the PCA3 test achieved 66-82% sensitivity and 76-89% specificity for PrCa (Fradet et al., Urology 64:311 -316 (2004)). PCA3 diagnostic assays are available but have yet to be approved by the FDA. Unfortunately, as with PSA, PCA3 testing exhibits disappointingly high false-positive and false-negative rates. The apparent best application of the PCA3 test is as a reflex for the PSA test. Also, a major impediment to the adoption of the PCA3 test is that it requires a urine sample from patients following a prostatic massage, which may be poorly tolerated. It also requires that an individual submit both blood and urine samples to accommodate both the PSA and PCA3 tests.
[007] Additional diagnostic assay alternatives are therefore needed. An assay that performs better than the PSA and PCA3 tests in one or more criteria would be a welcome addition to clinicians and patients who are seeking to diagnose and manage prostate cancer.
[008] We herein describe methods for diagnosing prostate disease by measuring miRNAs from serum or plasma.
[009] In some embodiments, the invention relates to the characterization of prostate diseases such as cancer or BPH by detecting miRNAs from serum or plasma. The methods of the invention include both diagnosing disease, and evaluating the prognosis or aggressiveness of a prostate disease. Further, the methods may be used to characterize the progression of a prostate disease. The patients tested using the methods of the invention may also be tested using PSA or PCA3 assays. Attorney Docket No. 10256.32-304
[010] In certain embodiments of the invention, the diagnosis or prognosis may be achieved by amplifying and detecting the amount of certain miRNAs that are present in elevated or reduced levels in the serum or plasma of a subject with prostate disease. In some instances, one serum or plasma miRNA may be amplified and measured to characterize prostate disease, while in other embodiments, two or more miRNAs are detected from serum or plasma. Some embodiments include amplifying and measuring a pair of miRNAs. In some instances, one miRNA in the pair is elevated in serum or plasma of patients with prostate disease, and the other miRNA in the pair is reduced. In other circumstances, both miRNAs in the pair can be elevated or both reduced. In certain embodiments, non-miRNA biomarkers such as PSA may also be measured. Some embodiments of the invention relate to diagnosis or prognosis of prostate cancer. Other embodiments of the invention include diagnosing BPH in a patient.
[011] Additional embodiments of the invention are discussed throughout this application. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. The embodiments in the Example section are understood to be embodiments of the invention that are applicable to all aspects of the invention.
[012] It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within Attorney Docket No. 10256.32-304
the spirit and scope of the invention will become apparent to those skilled in the art from this application.
BRIEF DESCRIPTION OF THE DRAWINGS
[013] Figure 1 shows human precursor miRNA (pre-miRNA) sequences, as provided by Release 13.0 of the miRBase::Sequences Database (http://microrna.sanger.ac.uk; Griffiths-Jones et al., Nucleic Acids Research, 2008, 36, Database Issue, D154-D158; Griffiths-Jones et al., Nucleic Acids Research, 2006, 34, Database Issue, D140-D144; Griffiths-Jones, Nucleic Acids Research, 2004, 32, Database Issue, D109-D111 ). The names of mature miRNAs from Tables 1 and 20 are also provided for each precursor sequence. EXEMPLARY EMBODIMENTS
[014] In certain aspects, the methods of the invention provide assays for amplifying and measuring the amount of a miRNA in a serum or plasma sample, thereby characterizing a prostate disease.
[015] To assist in understanding the present invention, certain terms are first defined. Additional definitions are provided throughout the application.
[016] As used herein, the term "microRNA" (miRNA or miR) includes human miRNAs, mature single stranded miRNAs, precursor miRNAs (pre-miR), and variants thereof, which may be naturally occurring. In some instances, the term "miRNA" also includes primary miRNA transcripts and duplex miRNAs. Unless otherwise noted, when used herein, the name of a specific miRNA refers to the mature miRNA. For example, miR-122a refers to a mature miRNA sequence derived from pre-miR-122. The sequences for particular miRNAs, including human mature and precursor sequences, are reported in the miRBase::Sequences Database (http://microrna.sanger.ac.uk (version 15 Attorney Docket No. 10256.32-304
released April 2010); Griffiths-Jones et al., Nucleic Acids Research, 2008, 36, Database Issue, D154-D158; Griffiths-Jones et al., Nucleic Acids Research, 2006, 34, Database Issue, D140-D144; Griffiths-Jones, Nucleic Acids Research, 2004, 32, Database Issue, D109-D111). For certain miRNAs, a single precursor contains more than one mature miRNA sequence. In other instances, multiple precursor miRNAs contain the same mature sequence. In some instances, mature miRNAs have been re-named based on new scientific consensus. For example, miR-213, as used herein, refers to a mature miRNA from pre-miR-181a- 1 , and is also called miR-181a*. Other miRNAs that have been re-named include miR-189 (also called miR-24*), which comes from pre-miR-24-1 ; miR-368 (also called miR-376c); and miR-422b (also called miR-378*). The skilled artisan will appreciate that scientific consensus regarding the precise nucleic acid sequence for a given miRNA, in particular for mature forms of the miRNAs, may change with time. MiRNAs detected by assays of this application include naturally occurring sequences for the miRNAs.
[017] The term "characterizing" includes making diagnostic or prognostic determinations or predictions of disease. In some instances, "characterizing" includes identifying whether a subject has a disease such as cancer or BPH. Additionally, "characterizing" includes distinguishing patients with prostate cancer from patients having other prostate diseases. In other circumstances, "characterizing" includes determining the stage or aggressiveness of a disease state such as prostate cancer, or determining an appropriate treatment method for prostate disease.
[018] The use of the word "a", "an" or "the" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it Attorney Docket No. 10256.32-304
is also consistent with the meaning of "one or more," "at least one," and "one or more than one." I. SAMPLES
[019] Serum is typically the fluid, non-cellular portion of coagulated blood. Plasma is also a non-cellular blood sample, but unlike serum, plasma contains clotting factors. In some embodiments, serum or plasma samples may be obtained from a human patient previously screened for prostate disease using PSA or PCA3 assays, or other diagnostic methods. In other embodiments, the patient has undergone a physical exam or biopsy to detect prostate disease. Additional embodiments include measuring miRNA in samples from patients previously or currently undergoing treatment for a prostate disease. The volume of plasma or serum obtained and used for the assay may be varied depending upon clinical intent.
[020] One of skill in the art will recognize that many methods exist for obtaining and preparing serum samples. Generally, blood is drawn into a collection tube using standard methods and allowed to clot. The serum is then separated from the cellular portion of the coagulated blood. In some methods, clotting activators such as silica particles are added to the blood collection tube. In other methods, the blood is not treated to facilitate clotting. Blood collection tubes are commercially available from many sources and in a variety of formats (e.g., Becton Dickenson Vacutainer® tubes-SST™, glass serum tubes, or plastic serum tubes).
[021] In some methods, the blood is collected by venipuncture and processed within three hours after drawing to minimize hemolysis and minimize the release of miRNAs from intact cells in the blood. In some methods, blood is Attorney Docket No. 10256.32-304
kept on ice until use. The blood may be fractionated by centrifugation to remove cellular components. In some embodiments, centrifugation to prepare serum can be at a speed of at least 500, 1000, 2000, 3000, 4000, or 5000 X G. In certain embodiments, the blood can be incubated for at least 10, 20, 30, 40, 50, 60, 90, 120, or 150 minutes to allow clotting. In other embodiments, the blood is incubated for at most 3 hours. When using plasma, the blood is not permitted to coagulate prior to separation of the cellular and acellular components. Serum or plasma can be frozen after separation from the cellular portion of blood until further assayed.
[022] Before analysis, RNA may be extracted from serum or plasma and purified using methods known in the art. Many methods are known for isolating total RNA, or to specifically extract small RNAs, including miRNAs. The RNA may be extracted using commercially-available kits (e.g., Perfect RNA Total RNA Isolation Kit, Five Prime-Three Prime, Inc.; mirVana™ kits, Ambion, Inc.). Alternatively, RNA extraction methods previously published for the extraction of mammalian intracellular RNA or viral RNA may be adapted, either as published or with modification, for extraction of RNA from plasma and serum. RNA may be extracted from plasma or serum using silica particles, glass beads, or diatoms, as in the method or adaptations described in U.S. Publication No. 2008/0057502. II. miRNA MARKERS FOR PROSTATE DISEASE
[023] Certain embodiments of the invention provide serum or plasma miRNAs as markers for prostate disease. In some embodiments, miRNAs that are present at elevated levels in the serum or plasma of patients with prostate disease are used as markers. In other embodiments, miRNAs that have reduced levels are used as markers. In some embodiments, more than one miRNA from Attorney Docket No. 10256.32-304
serum or plasma will be used as markers. When more than one miRNA biomarker is used, the miRNAs may all have elevated levels, all have reduced levels, or a mixture of miRNAs with elevated and reduced levels may be used.
[024] The terms "reduced levels" or "elevated levels" refer to the amount of a miRNA in a serum or plasma sample from a patient compared to the amount of the miRNA in serum or plasma from a cohort or cohorts that do not have the prostate disease that the patient is being tested for. For instance, a miRNA that has reduced levels in the sera of prostate cancer patients is present at lower amounts in prostate cancer patient sera than in serum from a donor who does not have prostate cancer. For certain miRNAs, elevated levels in a patient serum or plasma sample indicates presence or prognosis for a prostate disease. Other miRNAs are present in reduced levels in patients with prostate disease.
[025] Prostate disease can be classified as BPH or prostate cancer. In some embodiments, prostate cancer can be further characterized based on histopathology, aggressiveness, Gleason score, PSA measurements, and other methods known in the art. In some embodiments, the methods described herein can be used to characterize a patient with at least 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sensitivity. The degree of sensitivity indicates the percentage of patients with a disease who are positively characterized as having the disease. In additional embodiments, the methods have at least 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% specificity (e.g., the percentage of non-diseased patients who are correctly characterized). The assay parameters can be adjusted to optimize for both sensitivity and specificity.
[026] In some cases, the level of the miRNA marker will be compared to a control to determine whether the level is reduced or elevated. The control may be Attorney Docket No. 10256.32-304
an external control, such as a miRNA in a serum or plasma sample from a patient known to be free of prostate disease. In other circumstances, the external control may be a miRNA from a non-serum sample like a tissue sample or a known amount of a synthetic RNA. An internal control may be a miRNA from the same serum or plasma sample being tested. The identity of a miRNA control may be the same as or different from the patient serum or plasma miRNA being measured.
[027] Table 1 lists miRNAs that have elevated or reduced levels in serum from patients with prostate disease. These miRNAs may be used in accordance with the invention. Some of the miRNAs are useful for characterizing prostate cancer, including distinguishing cancer from BPH. Other miRNAs are suitable markers for identifying patients with BPH. In addition, some miRNAs may be used to predict the aggressiveness or outcome of prostate cancer.
Table 1. miRNAs with elevated or reduced levels in serum from patients with prostate disease. Levels are of miRNA in prostate cancer patients compared to patients with BPH or normal prostate.
Figure imgf000011_0001
Attorney Docket No 10256 32-304
Figure imgf000012_0001
Attorney Docket No. 10256.32-304
Figure imgf000013_0001
Attorney Docket No. 10256.32-304
Figure imgf000014_0001
Attorney Docket No. 10256 32-304
Figure imgf000015_0001
Attorney Docket No. 10256.32-304
Figure imgf000016_0001
[028] In some embodiments, a single miRNA may be used to characterize prostate cancer. For example, any one of the following miRNAs may be used to characterize prostate cancer, either alone or in combination with other markers: let-7a, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, miR-100, miR-101 , miR-103, miR- 105, miR-106a, miR-106b, miR-107, miR-10a, miR-10b, miR-122a, miR-125a, miR-125b, miR-126, miR-126*, miR-128a, miR-128b, miR-129, miR-130b, miR- 132, miR-133a, miR-133b, miR-136, miR-138, miR-139, miR-140, miR-141 , miR- 142-3p, miR-142-5p, miR-143, miR-146a, miR-146b, miR-147, miR-148a, miR- 148b, miR-151 , miR-152, miR-154, miR-154*, miR-15b, miR-16, miR-17-3p, miR- 17-5p, miR-181a, miR-181 b, miR-181d, miR-182, miR-184, miR-186, miR-189, Attorney Docket No. 10256.32-304
miR-190, miR-191 , miR-192, miR-193a, miR-193b, miR-194, miR-195, miR-196a, miR-196b, miR-197, miR198, miR-199a*, miR-199b, miR-19a, miR-19b, miR- 200a, miR-200a*, miR-200b, miR-200c, miR-202, miR-202*, miR-204, miR-205, miR-206, miR-208, miR-20a, miR-20b, miR-21 , miR-210, miR-212, miR-215, miR- 218, miR-219, miR-220, miR-222, miR-223, miR-224, miR-23a, miR-23b, miR-24, miR25, miR-26a, miR-26b, miR-27a, miR-27b, miR-296, miR-29a, miR-29b, miR- 29c, miR-302a, miR-302b*, miR-302d, miR-30-a-3p, miR-30a-5p, miR-30b, miR- 30c, miR-30d, miR-30θ-3p, miR-30θ-5p, miR-32, miR-320, miR-323, miR-324-3p, miR-326, miR-328, miR-33, miR-331 , miR-335, miR-338, miR-339, miR-340, miR- 342, miR-345, miR-346, miR-34a, miR-34b, miR-361 , miR-368, miR-369-5p, miR- 370, miR-372, miR-373*, miR-375, miR-376a, miR-376a*, miR-376b, miR-377, miR-379, miR-380-5p, miR-381 , miR-383, miR-409-5p, miR-412, miR-423, miR- 424, miR-429, miR-432*, miR-448, miR-451 , miR-452*, miR-455, miR-483, miR- 485-3p, miR-487b, miR-488, miR-489, miR-490, miR-491 , miR-492, miR-494, miR-496, miR-497, miR-498, miR-499, miR-501 , miR-502, miR-503, miR-504, miR-505, miR-507, miR-510, miR-512-5p, miR-515-3p, miR-515-5p, miR-518b, miR-518d, miR-518e, miR-518f, miR-519c, miR-519d, miR-519e, miR-520a*, miR- 520b, miR-520d, miR-520h, miR-521 , miR-523, miR-525*, miR-526a, miR-526b, miR-526b*, miR-527, miR-532, miR-542-5p, miR-552, miR-565, miR-566, miR- 575, miR-576, miR-584, miR-592, miR-638, mir660, miR-9, miR-9*, miR-92, miR- 93, miR-95, miR-98, miR-99a, and miR-99b.
[029] In other embodiments, any one of the following miRNAs may be used to characterize prostate cancer, either alone or in combination with other markers: let-7c, let-7d, let-7f, let-7g, let-7i, mϊR-101 , miR-106b, miR-10a, miR-10b, miR-122a, miR-125a, miR-125b, miR-126, miR-126*, miR-128a, miR-130b, miR- Attorney Docket No. 10256.32-304
133b, miR-136, miR-138, miR-139, miR-140, miR-141 , miR-142-5p, miR-143, miR-147, miR-148a, miR-151 , miR-152, miR-154, miR-15b, miR-17-3p, miR-17- 5p, miR-181b, miR-181d, miR-192, miR-193a, miR-195, miR-196a, miR198, miR- 199a*, miR-199b, miR-200a, miR-200b, miR-200c, miR-202, miR-204, miR-205, miR-206, miR-212, miR-219, miR-220, miR-223, miR-23b, miR-24, miR25, miR- 26a, miR-27a, miR-296, miR-29b, miR-302b*, miR-30-a-3p, miR-30b, miR-30c, miR-30e-3p, miR-32, miR-324-3p, miR-326, miR-33, miR-331 , miR-335, miR-340, miR-345, miR-34a, miR-361 , miR-368, miR-369-5p, miR-370, miR-375, miR-376a, miR-376a*, miR-376b, miR-379, miR-380-5p, miR-409-5p, miR-412, miR-423, miR-429, miR-432*, miR-448, miR-451 , miR-452*, miR-483, miR-489, miR-490, miR-494, miR-496, miR-497, miR-499, miR-502, miR-503, miR-505, miR-507, miR-515-3p, miR-515-5p, miR-518e, miR-519c, miR-519d, miR-519e, miR-520a*, miR-520h, miR-523, miR-526b*, miR-527, miR-532, miR-542-5p, miR-552, miR- 584, miR-9, miR-9*, miR-92, ιmiR-99a, and miR-99b.
[030] In some embodiments, a miRNA for characterizing prostate cancer is chosen from let-7d, miR-IOb, miR-122a, miR-139, miR-24, miR-204, miR-205, miR-206, miR-375, and miR-99b, and may be used alone or in combination with other markers. In other embodiments, the miRNA used for characterizing prostate cancer may be chosen from let-7d, miR-122a, miR-139, miR-204, miR-205, miR- 206, and miR-375.
[031] In certain methods, miRNAs that have reduced levels in serum from patients with prostate disease are used as biomarkers. Certain miRNAs at reduced levels in serum can be used to distinguish patients with prostate cancer from normal patients or patients with BPH. These miRNA markers include let-7d, let-7f, let-7g, miR-132, miR-135a, miR-135b, miR-140, miR-143, miR-153, miR- Attorney Docket No. 10256.32-304
154*, miR-15b, miR-16, miR-17-5p, miR-181c, miR-182, miR-188, miR-195, miR- 196a, miR-197, miR198, miR-200a*, miR-202, miR-202*, miR-203, miR-20a, miR- 212, miR-22, miR-220, miR-223, miR-24, miR-26b, miR-301 , miR-302a*, miR-30- a-3p, miR-30b, miR-30c, miR-30d, miR-30e-3p, miR-32, miR-323, miR-331 , miR- 335, miR-346, miR-365, miR-367, miR-372, miR-373*, miR-380-3p, miR-412, miR-425, miR-432*, miR-449, miR-452*, miR-489, miR-490, miR-492, miR-493*, miR-494, miR-503, miR-504, miR-505, miR-512-5p, miR-514, miR-515-5p, miR- 516-5p, miR517a, miR-518c, miR-518d, miR-518e, miR-518f, miR-519c, miR- 519d, miR-519e, miR-519e*, miR-520a*, miR-520b, miR-520d, miR-520d*, miR- 52Oe, miR-520f, miR-520h, miR-521 , miR-525*, miR-526b, miR-527, miR-542-3p, miR-542-5p, miR-552, miR-565, miR-566, miR-575, miR-584, miR-605, miR-638, miR-652, miR-9, miR-9*, miR-340, miR-26a, miR-191 , let-7a, miR-103, miR-519b, miR-93, miR-98, let-7b, let-7e, miR-1 , miR-107, miR-128b, miR-129, miR-134, miR-145, miR-183, miR-18a*, miR-196b, miR-20b, miR-211 , miR-224, miR-28, miR-299-3p, miR-299-5p, miR-302a, miR-302d, miR-31 , miR-328, miR-330, miR- 342, miR-369-3p, miR-369-5p, miR-379, miR-423, miR-432, miR-452, miR-485- 3p, miR-487b, miR-488, miR-493, miR-501 , miR-510, miR-515-3p, mir-518a, miR- 518b, miR-525, miR-526a, miR-539, miR-508, miR-155, let-7c, miR-106a, and miR-222.
[032] In certain embodiments, two or more miRNAs are used to characterize prostate disease. Any one of the following miRNAs may be used in combination with at least one other serum miRNA in the methods of the invention: let-7a, let-7c, let-7d, let-7f, let-7g, miR-1 , miR-103, miR-106a, miR-106b, miR-107, miR-10b, miR-122a, miR-125a, miR-125b, miR-126, miR-126*, miR-130a, miR- 130b, miR-132, miR-133b, miR-135a, miR-139, miR-140, miR-142-3p, miR-142- Attorney Docket No. 10256.32-304
5p, miR-143, miR-145, miR-146a, miR-146b, miR-148a, miR-148b, miR-152, miR- 154*, miR-155, miR-15a, mir-15b, miR-16, miR-17-5p, miR-181 , miR-181c, miR- 181d, miR-185, miR-187, miR-188, miR-18a, miR-18a*, miR-191 , miR-192, miR- 193a, miR-194, miR-195, miR-196b, miR-197, miR-199a*, miR-199b, miR-19a, miR-200a, miR-200c, miR-204, miR-206, miR-20a, miR-20b, miR-212, miR-214, miR-218, miR-22, miR-221 , miR-222, miR-223, miR-224, miR-23a, miR-23b, miR- 24, miR25, miR-26a, miR-26b, miR-27a, miR-27b, miR-28, miR-29a, miR-29c, miR-301 , miR-30a-5p, miR-30b, miR-30c, miR-30d, miR-30e-3p, miR-32, miR- 328, miR-331 , miR-335, miR-339, miR-340, miR-342, miR-345, miR-346, miR- 34a, miR-361 , miR-365, miR-374, miR-375, miR-378, miR-422b, miR-423, miR- 425, miR-432, miR-432*, miR-483, miR-495, miR-501 , miR-539, miR-565, miR- 566, miR-576, miR-584, miR-618, miR-638, mir-660, miR-92, miR-93, miR-98, and miR-99b.
[033] In other embodiments, any miRNA chosen from the following list can be used in combination with another serum miRNA biomarker to characterize prostate cancer: let-7a, !et-7c, let-7d, let-7f, let-7g, miR-1 , miR-103, miR-106a, miR-107, miR-10b, miR-122a, miR-125a, miR-125b, miR-126, miR-126*, miR- 130a, miR-130b, miR-132, miR-139, miR-140, miR-142-5p, miR-143, miR-145, miR-146a, miR-148a, miR-155, miR-15a, miR-15b, miR-16, miR-17-5p, miR-181c, miR-185, miR-188, miR-18a, miR-18a*, miR-191 , miR-192, miR-193a, miR-194, miR-195, miR-196b, miR-197, miR-199a*, miR-19a, miR-200a, miR-200c, miR- 204, miR-206, miR-20a, miR-20b, miR-212, miR-214, miR-22, miR-222, miR-223, miR-224, miR-23a, miR-23b, miR-24, miR25, miR-26a, miR-26b, miR-28, miR- 29a, miR-29c, miR-301 , miR-30a-5p, miR-30b, miR-30c, miR-30d, miR-30e-3p, miR-32, miR-328, miR-331 , miR-335, miR-339, miR-340, miR-342, miR-345, miR- Attorney Docket No. 10256.32-304
346, mιR-34a, miR-361 , miR-365, miR-374, miR-375, miR-422b, miR-423, miR- 425, mιR-432*, mιR-483, miR-501 , mιR-565, mιR-566, mιR-576, mιR-584, miR- 638, mιr-660, miR-92, miR-93, miR-98, and mιR-99b.
[034] Some embodiments of the invention relate to amplifying and measuring at least a pair of miRNAs from serum Table 2 includes pairs that may be used to characterize prostate cancer. Table 2. miRNA pairs for diagnosis of prostate cancer from serum samples
Figure imgf000021_0001
Attorney Docket No. 10256 32-304
Figure imgf000022_0001
Attorney Docket No. 10256.32-304
Figure imgf000023_0001
[035] In certain embodiments, pairs of miRNA markers from serum can be used to predict the aggressiveness or stage of prostate cancer. For example, one of the miRNA pairs in Table 3 may be used to determine cancer aggressiveness
Table 3. Biomarker pairs that can be used to identify serum from prostate cancer patients with high Gleason scores.
Figure imgf000023_0002
[036] Other miRNAs and groups of miRNAs that can be used in the methods of the invention will be apparent from the Examples described herein. III. METHODS TO MEASURE THE LEVEL OF A miRNA
[037] Many methods of measuring the levels or amounts of miRNAs are contemplated. Any reliable, sensitive, and specific method can be used. In some embodiments, a miRNA is amplified prior to measurement. In other embodiments, the level of miRNA is measured during the amplification process. In still other methods, the miRNA is not amplified prior to measurement Attorney Docket No. 10256.32-304
A. Amplification Reactions
[038] Many methods exist for amplifying miRNA nucleic acid sequences such as mature miRNAs, precursor miRNAs, and primary miRNAs. Suitable nucleic acid polymerization and amplification techniques include reverse transcription (RT), polymerase chain reaction (PCR), real-time PCR (quantitative PCR (q-PCR)), nucleic acid sequence-base amplification (NASBA), ligase chain reaction, multiplex ligatable probe amplification, invader technology (Third Wave), rolling circle amplification, in vitro transcription (IVT), strand displacement amplification, transcription-mediated amplification (TMA), RNA (Eberwine) amplification, and other methods that are known to persons skilled in the art. In certain embodiments, more than one amplification method is used, such as reverse transcription followed by real time quantitative PCR (qRT-PCR) (Chen et al., Nucleic Acids Research, 33(20):e179 (2005)).
[039] A typical PCR reaction includes multiple amplification steps, or cycles that selectively amplify target nucleic acid species: a denaturing step in which a target nucleic acid is denatured; an annealing step in which a set of PCR primers (forward and reverse primers) anneal to complementary DNA strands; and an elongation step in which a thermostable DNA polymerase elongates the primers. By repeating these steps multiple times, a DNA fragment is amplified to produce an amplicon, corresponding to the target DNA sequence. Typical PCR reactions include 20 or more cycles of denaturation, annealing, and elongation. In many cases, the annealing and elongation steps can be performed concurrently, in which case the cycle contains only two steps. Since mature miRNAs are single-stranded, a reverse transcription reaction (which produces a complementary cDNA sequence) may be performed prior to PCR reactions. Attorney Docket No. 10256.32-304
Reverse transcription reactions include the use of, e.g., a RNA-based DNA polymerase (reverse transcriptase) and a primer.
[040] In PCR and q-PCR methods, for example, a set of primers is used for each target sequence. In certain embodiments, the lengths of the primers depends on many factors, including, but not limited to, the desired hybridization temperature between the primers, the target nucleic acid sequence, and the complexity of the different target nucleic acid sequences to be amplified. In certain embodiments, a primer is about 15 to about 35 nucleotides in length. In other embodiments, a primer is equal to or fewer than 15, 20, 25, 30, or 35 nucleotides in length. In additional embodiments, a primer is at least 35 nucleotides in length.
[041] In a further aspect, a forward primer can comprise at least one sequence that anneals to a miRNA biomarker and alternatively can comprise an additional 5' non-complementary region. In another aspect, a reverse primer can be designed to anneal to the complement of a reverse transcribed miRNA. The reverse primer may be independent of the miRNA biomarker sequence, and multiple miRNA biomarkers may be amplified using the same reverse primer. Alternatively, a reverse primer may be specific for a miRNA biomarker.
[042] In some embodiments, two or more miRNAs are amplified in a single reaction volume. One aspect includes multiplex q-PCR, such as qRT-PCR, which enables simultaneous amplification and quantification of at least two miRNAs of interest in one reaction volume by using more than one pair of primers and/or more than one probe. The primer pairs comprise at least one amplification primer that uniquely binds each miRNA, and the probes are labeled such that they are distinguishable from one another, thus allowing simultaneous quantification of Attorney Docket No. 10256.32-304
multiple miRNAs. Multiplex qRT-PCR has research and diagnostic uses, including but not limited to detection of miRNAs for diagnostic, prognostic, and therapeutic applications.
[043] The qRT-PCR reaction may further be combined with the reverse transcription reaction by including both a reverse transcriptase and a DNA-based thermostable DNA polymerase. When two polymerases are used, a "hot start" approach may be used to maximize assay performance (U.S. Patent Nos. 5,411 ,876 and 5,985,619). For example, the components for a reverse transcriptase reaction and a PCR reaction may be sequestered using one or more thermoactivation methods or chemical alteration to improve polymerization efficiency (U.S. Patent Nos. 5,550,044, 5,413,924, and 6,403,341).
B. Detection of miRNAs
[044] In certain embodiments, labels, dyes, or labeled probes and/or primers are used to detect amplified or unamplified miRNAs. The skilled artisan will recognize which detection methods are appropriate based on the sensitivity of the detection method and the abundance of the target. Depending on the sensitivity of the detection method and the abundance of the target, amplification may or may not be required prior to detection. One skilled in the art will recognize the detection methods where miRNA amplification is preferred.
[045] A probe or primer may include Watson-Crick bases or modified bases. Modified bases include, but are not limited to, the AEGIS bases (from Eragen Biosciences), which have been described, e.g., in U.S. Patent Nos. 5,432,272, 5,965,364, and 6,001 ,983. In certain aspects, bases are joined by a natural phosphodiester bond or a different chemical linkage. Different chemical Attorney Docket No. 10256.32-304
linkages include, but are not limited to, a peptide bond or a Locked Nucleic Acid (LNA) linkage, which is described, e.g., in U.S. Patent No. 7,060,809.
[046] In a further aspect, oligonucleotide probes or primers present in an amplification reaction are suitable for monitoring the amount of amplification product produced as a function of time. In certain aspects, probes having different single stranded versus double stranded character are used to detect the nucleic acid. Probes include, but are not limited to, the 5'-exonuclease assay (e.g., TaqMan™) probes (see U.S. Patent No. 5,538,848), stem-loop molecular beacons (see, e.g., U.S. Patent Nos. 6,103,476 and 5,925,517), stemless or linear beacons (see, e.g., WO 9921881, U.S. Patent Nos. 6,485,901 and 6,649,349), peptide nucleic acid (PNA) Molecular Beacons (see, e.g., U.S. Patent Nos. 6,355,421 and 6,593,091), linear PNA beacons (see, e.g. U.S. Patent No. 6,329,144), non-FRET probes (see, e.g., U.S. Patent No. 6,150,097), Sunrise™ /AmplifluorB™probes (see, e.g., U.S. Patent No. 6,548,250), stem-loop and duplex Scorpion™ probes (see, e.g., U.S. Patent No. 6,589,743), bulge loop probes (see, e.g., U.S. Patent No. 6,590,091), pseudo knot probes (see, e.g., U.S. Patent No. 6,548,250), cyclicons (see, e.g., U.S. Patent No. 6,383,752), MGB Eclipse™ probe (Epoch Biosciences), hairpin probes (see, e.g., U.S. Patent No. 6,596,490), PNA light-up probes, antiprimer quench probes (Li et al., Clin. Chem. 53:624-633 (2006)), self-assembled nanoparticle probes, and ferrocene-modified probes described, for example, in U.S. Patent No. 6,485,901.
[047] In certain embodiments, one or more of the primers in an amplification reaction can include a label. In yet further embodiments, different probes or primers comprise detectable labels that are distinguishable from one Attorney Docket No. 10256.32-304
another. In some embodiments a nucleic acid, such as the probe or primer, may be labeled with two or more distinguishable labels.
[048] In some aspects, a label is attached to one or more probes and has one or more of the following properties: (i) provides a detectable signal; (ii) interacts with a second label to modify the detectable signal provided by the second label, e.g., FRET (Fluorescent Resonance Energy Transfer); (iii) stabilizes hybridization, e.g., duplex formation; and (iv) provides a member of a binding complex or affinity set, e.g., affinity, antibody-antigen, ionic complexes, hapten- ligand (e.g., biotin-avidin). In still other aspects, use of labels can be accomplished using any one of a large number of known techniques employing known labels, linkages, linking groups, reagents, reaction conditions, and analysis and purification methods.
[049] MiRNAs can be detected by direct or indirect methods. In a direct detection method, one or more miRNAs are detected by a detectable label that is linked to a nucleic acid molecule. In such methods, the miRNAs may be labeled prior to binding to the probe. Therefore, binding is detected by screening for the labeled miRNA that is bound to the probe. The probe is optionally linked to a bead in the reaction volume.
[050] In certain embodiments, nucleic acids are detected by direct binding with a labeled probe, and the probe is subsequently detected. In one embodiment of the invention, the nucleic acids, such as amplified miRNAs, are detected using FlexMAP Microspheres (Luminex) conjugated with probes to capture the desired nucleic acids. Some methods may involve detection with polynucleotide probes modified with fluorescent labels or branched DNA (bDNA) detection, for example. Attorney Docket No. 10256.32-304
[051] In other embodiments, nucleic acids are detected by indirect detection methods. For example, a biotinylated probe may be combined with a stretavidin-conjugated dye to detect the bound nucleic acid. The streptavidin molecule binds a biotin label on amplified miRNA, and the bound miRNA is detected by detecting the dye molecule attached to the streptavidin molecule. In one embodiment, the streptavidin-conjugated dye molecule comprises Phycolink® Streptavidin R-Phycoerythrin (PROzyme). Other conjugated dye molecules are known to persons skilled in the art.
[052] Labels include, but are not limited to: light-emitting, light-scattering, and light-absorbing compounds which generate or quench a detectable fluorescent, chemiluminescent, or bioluminescent signal (see, e.g., Kricka, L., Nonisotopic DNA Probe Techniquies, Academic Press, San Diego (1992) and Garman A., Non-Radioactive Labeling, Academic Press (1997).). Fluorescent reporter dyes useful as labels include, but are not limited to, fluoresceins (see, e.g., U.S. Patent Nos. 5,188,934, 6,008,379, and 6,020,481), rhodamines (see, e.g., U.S. Patent Nos. 5,366,860, 5,847,162, 5,936,087, 6,051 ,719, and 6,191 ,278), benzophenoxazines (see, e.g., U.S. Patent No. 6,140,500), energy- transfer fluorescent dyes, comprising pairs of donors and acceptors (see, e.g., U.S. Patent Nos. 5,863,727; 5,800,996; and 5,945,526), and cyanines (see, e.g., WO 9745539), lissamine, phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham), Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, Attorney Docket No. 10256.32-304
Tetramethylrhodamine, and/or Texas Red, as well as any other fluorescent moiety capable of generating a detectable signal. Examples of fluorescein dyes include, but are not limited to, 6-carboxyfluorescein; 2',4',1 ,4,-tetrachlorofluorescein; and 2l,4',5',7',1 ,4-hexachlorofluorescein. In certain aspects, the fluorescent label is selected from SYBR-Green, 6-carboxyfluorescein ("FAM"), TET, ROX, VICTM, and JOE. For example, in certain embodiments, labels are different fluorophores capable of emitting light at different, spectrally-resolvable wavelengths (e.g., 4- differently colored fluorophores); certain such labeled probes are known in the art and described above, and in U.S. Patent No. 6,140,054. A dual labeled fluorescent probe that includes a reporter fluorophore and a quencher fluorophore is used in some embodiments. It will be appreciated that pairs of fluorophores are chosen that have distinct emission spectra so that they can be easily distinguished.
[053] In still a further aspect, labels are hybridization-stabilizing moieties which serve to enhance, stabilize, or influence hybridization of duplexes, e.g., intercaiators and intercalating dyes (including, but not limited to, ethidium bromide and SYBR-Green), minor-groove binders, and cross-linking functional groups (see, e.g., Blackburn et al., eds. "DNA and RNA Structure" in Nucleic Acids in Chemistry and Biology (1996)).
[054] In further aspects, methods relying on hybridization and/or ligation to quantify miRNAs may be used, including oligonucleotide ligation (OLA) methods and methods that allow a distinguishable probe that hybridizes to the target nucleic acid sequence to be separated from an unbound probe. As an example, HARP-like probes, as disclosed in U.S. Publication No. 2006/0078894 may be used to measure the quantity of miRNAs. In such methods, after hybridization Attorney Docket No. 10256.32-304
between a probe and the targeted nucleic acid, the probe is modified to distinguish the hybridized probe from the unhybridized probe. Thereafter, the probe may be amplified and/or detected. In general, a probe inactivation region comprises a subset of nucleotides within the target hybridization region of the probe. To reduce or prevent amplification or detection of a HARP probe that is not hybridized to its target nucleic acid, and thus allow detection of the target nucleic acid, a post-hybridization probe inactivation step is carried out using an agent which is able to distinguish between a HARP probe that is hybridized to its targeted nucleic acid sequence and the corresponding unhybridized HARP probe. The agent is able to inactivate or modify the unhybridized HARP probe such that it cannot be amplified.
[055] In an additional embodiment of the method, a probe ligation reaction may be used to quantify miRNAs. In a Multiplex Ligation-dependent Probe Amplification (MLPA) technique (Schouten et al., Nucleic Acids Research 30:e57 (2002)), pairs of probes which hybridize immediately adjacent to each other on the target nucleic acid are ligated to each other only in the presence of the target nucleic acid. In some aspects, MLPA probes have flanking PCR primer binding sites. MLPA probes can only be amplified if they have been ligated, thus allowing for detection and quantification of miRNA biomarkers. IV. EXAMPLES
[056] The following examples illustrate various embodiments of the invention and are not intended to limit the scope of the invention.
[057] The examples described herein include the use of qRT-PCR, which includes real-time monitoring of PCR products during the exponential phase instead of by an end-point measurement. The threshold cycle (Ct) measurements Attorney Docket No. 10256.32-304
in the examples refer to the number of cycles it takes to reach a pre-defined point in the fluorescent signal.
Example 1. MicroRNA serum biomarkers of prostate cancer and BPH
[058] miRNAs potentially relevant to carcinogenesis frequently exhibit differential expression in cancerous versus normal samples. In certain instances, differential expression in a disease sample leads to reduced or elevated levels of the miRNA in serum or plasma isolated from the individual. In addition, miRNAs with reduced or elevated levels in normal and cancerous samples may be used in the diagnosis of cancerous lesions and in patient prognosis. To identify miRNAs present in serum, that may be useful markers for diagnosis of prostate cancer and for establishing patient prognosis, the inventors evaluated miRNA levels in serum samples from twelve normal male donors, twelve PrCa patients, and twelve patients with BPH (Table 4). Patient and normal serum samples were purchased from ProteoGenex, Inc. (Culver City, Ca, USA).
Table 4. Histopathological data and patient information. PIN, prostate intraepithelial neoplasia. NA, not available.
Figure imgf000032_0001
Attorney Docket No. 10256.32-304
Figure imgf000033_0001
[059] For serum preparation, ten (10) ml of whole blood was collected from patients and normal donors using a BD Vacutainer® glass serum tube (Becton, Dickinson and Company; Franklin Lakes, NJ, USA; cat. no. 366441 ). Following blood collection, tubes were incubated at room temperature for up to 2.5 hours to allow the blood to clot. Tubes were centrifuged for ten minutes at 2,000 x g. Serum was transferred to a new tube using a serological pipette and frozen at - 80 0C until it was processed for RNA isolation.
[060] Serum RNA was purified using the organic extraction of the mirVana PARIS™ Kit (Part No. AM1556; Applied Biosystems/Ambion; Austin, TX, USA), with the following modifications. Following the addition of acid phenoLchloroform and vortexing, samples were incubated on ice for 5 min then centrifuged at 13,000 x g for 10 min at 4 0C. The aqueous layer was removed, extracted with Attorney Docket No. 10256.32-304
chloroform, and centrifuged again. The aqueous layer was removed from the second extraction, and 3M NaOAc (1/10 volume), glycogen (5mg/ml), and 100% ethanol (1.5 volume) were added to the samples. Lysate/ethanol mixtures were passed through a mirVana PARIS filter cartridge, and filters were washed once with 650 μl of Wash 1 buffer and twice with 650 μl of Wash 2/3 buffer, RNA was eluted with two aliquots of nuclease free water (50 μl) and stored at -80 0C.
[061] Levels of 329 miRNAs in serum samples were determined by qRT- PCR using TaqMan® MicroRNA Assays (Applied Biosystems; Foster City, CA, USA) specific for each miRNA. Reverse transcription (RT) reaction components (Table 5) were assembled on ice prior to the addition of RNA template. Serum RNA (0.25 μl per reaction) was added and mixed. RT reactions were incubated in a 384-well GeneAmp® PCR System 9700 (Applied Biosystems) at 16 0C for 30 minutes, then at 42 0C for 30 minutes, then at 85 0C for 5 minutes. RT reactions were then frozen at -20 0C. All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified. Table 5. Reverse transcription reaction components.
Figure imgf000034_0001
Attorney Docket No. 10256.32-304
[062] PCR components (Table 6) were assembled on ice prior to the addition of cDNA (4 μl) from the RT reaction. Reactions were incubated in an ABI PRISM™ 7900HT Fast Real-Time PCR system (Applied Biosystems) at 95 0C for 1 minute, then for 50 cycles at 95 0C for 5 seconds and 60 0C for 30 seconds. Results were analyzed with the 7900HT Fast Real-Time PCR system SDS V2.3 software (Applied Biosystems). All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified. Table 6. PCR components.
Figure imgf000035_0001
[063] The qRT-PCR data were initially assessed for outliers. All miRNAs in a given sample with raw Ct readings of 50 were eliminated from further analyses. All data from samples having fewer than 150 miRNAs with raw Ct values <50 were eliminated. The average raw Ct for 50 miRNAs that were detected in each sample analyzed was calculated for each of the individual samples. The average Ct for a given sample was subtracted from the raw Ct readings for each miRNA in the corresponding sample to produce a dCt for each miRNA that was detected. Attorney Docket No. 10256.32-304
[064] The average dCt values for each miRNA in the normal donor, BPH, and PrCa patient samples were calculated. The average dCt values for the PrCa patient samples were subtracted from the average dCt values for the normal donor or BPH samples, and the average dCt values for the BPH samples were subtracted from the average dCt values for normal donor samples to determine the variance in the miRNA levels between the three patient sets. Student's t-test was then used to determine the potential of various miRNAs to distinguish the sera of PrCa patients from sera of normal donors or patients with BPH. Table 7 provides the average dCt values, the difference in miRNA levels between patient sets, and p-values for each miRNA tested. miRNAs that are present at reduced or elevated levels in PrCa patients compared to BPH or normal patients represent biomarkers of prostate cancer that can be used to diagnose prostate cancer using the serum from a patient.
Table 7. Normalized qRT-PCR data for quantification of 329 miRNAs in serum from PrCa patients, BPH patients, and normal donors (norm).
Figure imgf000036_0001
Attorney Docket No. 10256.32-304
Figure imgf000037_0001
Attorney Docket No. 10256.32-304
Figure imgf000038_0001
Attorney Docket No. 10256 32-304
Figure imgf000039_0001
Attorney Docket No. 10256.32-304
Attorney Docket No. 10256 32-304
Figure imgf000041_0001
Attorney Docket No. 10256.32-304
Figure imgf000042_0001
Attorney Docket No. 10256.32-304
Figure imgf000043_0001
Example 2. Combinations of miRNAs that distinguish sera of prostate cancer patients from sera of BPH patients and normal donors.
[065] The inventors evaluated pairs of miRNA biomarkers for their abilities to distinguish sera of PrCa patients from sera of BPH patients and normal donors. Un-normalized qRT-PCR data generated using the samples described above in Example 1 were used to calculate dCt values for each pair of miRNAs that was evaluated. The dCt values of the various miRNA pairs in PrCa and norma! serum samples were analyzed using Receiver-Operator Characteristic (ROC) analysis to identify the miRNA pairs having the ability to distinguish sera of PrCa patients from sera of BPH patients and normal donors.
[066] A Receiver Operator Characteristic (ROC) curve is a graphical plot of the sensitivity vs. specificity for a binary classifier system as its discrimination threshold is varied. ROC analysis provides a tool to select possibly optimal models and to discard suboptimal ones independently from (and prior to specifying) the class distribution Recently, ROC analysis has been used in selecting and applying biomarkers for the diagnosis of disease (Pepe, 2003; Pepe et al., 2004) since the classification performance can be measured by the area underthe ROC curve (AUC). Advantages of the ROC technique include (1) it does not assume a parametric form of the class probability as required in the logistic regression method, (2) it is adaptable to outcome-dependent samplings, e.g. the case-control design, which are widely used in medical studies, and (3) it is Attorney Docket No. 10256.32-304
relatively straightforward to assign different 'costs' to false positives and false negatives (Pepe, 2003; Pepe et a/., 2005).
[067] For the purposes of this invention, ROC curves are used to evaluate the capacities of individual and combinations of biomarker candidates to distinguish patient classes. The abundance(s) of biomarker candidates are measured. The measurements are used to develop classifiers whose true positive and false positive rates are plotted in a ROC curve. The AUC is then calculated for each classifier. An ideal classifier has an ROC AUC value of 1 , and classifiers can be ranked based upon how close their ROC AUC is to 1.
[068] Three miRNA pairs (miR-125b + miR-24; miR-125b + miR-26a; miR- 432* + miR-483) accurately separated the twelve PrCa patient serum samples from the twelve normal donor serum samples and the twelve BPH patient serum samples (Table 8). Five additional miRNA pairs (miR-125b + miR-335; miR-125b + miR-342; let-7d + miR-125b; miR-125b + miR-197; miR-125b + miR-30c) accurately classified all but one of the 36 samples (ROC AUC >0.98) (Table 8), and 129 miRNA pairs had a ROC AUC score of at least 0.90. Data from 166 independent miRNAs was included at least once in the 221 total miRNA pairs (Table 8). Numerous miRNAs were used in multiple biomarkers pairs, indicating the strength of their variable levels in serums of prostate cancer patients and donors who did not have prostate cancer. Table 8. MicroRNA biomarker pairs for classifying prostate cancer serum.
Figure imgf000044_0001
Attorney Docket No. 10256.32-304
Figure imgf000045_0001
Attorney Docket No. 10256.32-304
Figure imgf000046_0001
Attorney Docket No. 10256.32-304
Figure imgf000047_0001
[069] The five miRNAs appearing most commonly in biomarker pairs were miR-125b, miR-375, miR-24, miR-340, and miR-483 (Table 9). Table 9. Prevalence of serum biomarkers in paired analysis.
Figure imgf000047_0002
Attorney Docket No. 10256.32-304
Figure imgf000048_0001
[070] The miRNA pairs in Table 8 and the individual miRNA biomarkers in Table 9 are all possible targets for diagnosing prostate cancer using serum.
Example 3. Validation of prostate cancer serum biomarkers.
[071] To assess the performances of the prostate cancer biomarkers, the inventors selected fourteen miRNAs (miR-24, miR-125b, miR-340, miR-375, miR- 15b, miR-204, miR-205, miR-206, miR-122a, miR-483, let-7d, let-7f, miR-191 , and miR-26a) identified in Example 1 and quantified those miRNAs in the sera of 12 PrCa patients, 12 BPH patients, and 12 normal donors (Table 10) using qRT- PCR. Patient and normal serum samples were purchased from ProteoGenex, Inc. (Culver City, cA, USA). Serum preparation and serum RNA isolation were performed as described in Example 1.
Table 10. Histopathological data and patient information. NA, not available; ND, not determined.
Figure imgf000048_0002
Attorney Docket No. 10256.32-304
Figure imgf000049_0001
[072] microRNA levels were determined by qRT-PCR using TaqMan® MicroRNA Assays (Applied Biosystems; Foster City, CA, USA) specific for each miRNA. Reverse transcription (RT) reaction components were assembled on ice, as shown above in Example 1 (Table 5), prior to the addition of RNA template. Serum RNA (0.25 μl per reaction) was added and mixed. RT reactions were incubated in a 384-well GeneAmp® PCR System 9700 (Applied Biosystems) at Attorney Docket No. 10256.32-304
4 0C for 30 minutes, then at 16 0C for 30 minutes, then at 42 0C for 30 minutes, then at 85 0C for 5 minutes. RT reactions were then frozen at -20 0C.
[073] PCR components (Table 11) were assembled on ice prior to the addition of cDNA (4 μl) from the RT reaction. Reactions were incubated in an ABI PRISM™ 7900HT Fast Real-Time PCR system (Applied Biosystems) at 95 0C for 1 minute, then for 50 cycles at 95 0C for 5 seconds and 60 0C for 30 seconds. Results were analyzed with the 7900HT Fast Real-Time PCR system SDS V2.3 software (Applied Biosystems). All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified. Table 11. PCR components.
Figure imgf000050_0001
[074] qRT-PCR data were initially assessed for outliers. All miRNAs in a given sample with raw Ct readings of 50 were eliminated from further analysis. miR-103 served as an internal control, and its Ct was subtracted from the raw Ct readings for each miRNA in the corresponding sample to produce a dCt for each miRNA that was detected. Normalized values were used to estimate the relative abundance of each miRNA in the samples. Attorney Docket No. 10256.32-304
[075] Average dCt values for each miRNA in the normal donor, BPH patient, and PrCa patient samples were calculated. Average dCt values for PrCa patient samples were subtracted from average dCt values for normal donor samples and BPH patient samples to determine the variance in the levels of the miRNAs between the patient sets Student's t-test was then used to determine the potential of various miRNAs to distinguish the sera of PrCa patients from sera of normal donors and BPH patients. Table 12 shows average dCt values for the three sample types, and the difference between PrCa samples and normal or BPH samples.
Table 12. miRNA serum biomarkers for prostate cancer. SD, standard deviation.
Figure imgf000051_0001
Example 4. Validation of prostate cancer serum biomarkers
[076] To assess the robustness of the prostate cancer biomarkers in diagnosing prostate cancer, the inventors selected 11 miRNA biomarkers (miR- 24, miR-125b, miR-375, miR-204, miR-205, miR-206, miR-122a, let-7d, miR-10b, miR-99b, miR-22) and 1 1 internal control miRNAs (miR-191 , miR-16, miR-181a, miR-21 , miR-26a, miR-106a, mιR-155, mιR-30a-5p, let-7a, let-7c, mιR-222) and Attorney Docket No. 10256.32-304
quantified those miRNAs, using qRT-PCR, in the sera of 25 PrCa patients and 25 normal donors (Table 13), which were independent from those serum samples previously described.
Table 13. Histopathological data and patient information. ND, not determined; NA, not available.
Figure imgf000052_0001
Attorney Docket No. 10256.32-304
Figure imgf000053_0001
[077] PrCa patient and normal donor serum samples for this Example were purchased from a different vendor (ProMedDx, LLC; Norton, MA, USA), than those samples used for screening and validation in Examples 1 , 2, and 3 (ProteoGenex, Inc.). In addition, the two vendors use different methods for serum preparation from patient samples. For these samples purchased from ProMedDx, ten (10) ml of whole blood was collected using a BD Vacutainer™ SST™ plastic serum tube (Becton, Dickinson and Company; Franklin Lakes, NJ, USA; cat. no. 367985). The tubes were incubated at room temperature for 10 to 60 minutes to allow the blood to clot. The tubes were centrifuged for 10 minutes at 3000 - 3500 RPM. Serum was transferred to a new tube using a serological pipette and frozen at -80 0C until it was subjected to RNA isolation. Serum RNA was purified as described in Example 1.
[078] MicroRNA levels were determined by qRT-PCR using TaqMan® MicroRNA Assays (Applied Biosystems; Foster City, CA, USA) specific for each miRNA. Reverse transcription (RT) reaction components were assembled on ice prior to the addition of RNA template (Table 14). Serum RNA (1 μl per reaction) was added and mixed. RT reactions were incubated in a 384-well GeneAmp® Attorney Docket No. 10256.32-304
PCR System 9700 (Applied Biosystems) at 4 0C for 30 minutes, then at 16 0C for
30 minutes, then at 42 0C for 30 minutes, then at 85 0C for 5 minutes. RT reactions were then frozen at -20 °C. All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified.
Table 14. Reverse transcription reaction components.
Figure imgf000054_0001
[079] PCR components (Table 15) were assembled on ice prior to the addition of cDNA (4 μl) from the RT reaction. Reactions were incubated in an ABI PRISM™ 7900HT Fast Real-Time PCR system (Applied Biosystems) at 95 0C for 1 minute, then for 50 cycles at 95 0C for 5 seconds and 60 X for 30 seconds. Results were analyzed with SDS V2.3 (Applied Biosystems). All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified.
Table 15. PCR components.
Figure imgf000054_0002
Attorney Docket No. 10256.32-304
Figure imgf000055_0001
[080] qRT-PCR data for each miRNA in each sample was normalized by subtracting the Ct value for miR-103 from the Ct value for the miRNA from the same sample. The resulting values, called dCt, were used to calculate the average dCt values for each miRNA in the normal donor and PrCa patient samples. Average dCt values for PrCa patient samples were subtracted from average dCt values for normal donor samples to determine the variance in the levels of the miRNAs between the patient sets. Student's t-test was then used to determine the potential of various miRNAs to distinguish the sera of PrCa patients from sera of normal donors. Table 16 shows average dCt values for the three sample types, and the difference between PrCa samples and normal or BPH samples. Table 16. Prostate cancer biomarkers. SD, standard deviation.
Figure imgf000055_0002
Attorney Docket No. 10256.32-304
Figure imgf000056_0001
EXAMPLE 5. qRT-PCR for evaluation of microRNA levels in plasma samples from prostate cancer patients
[081] To identify miRNAs present in plasma that may be useful markers for diagnosis of prostate cancer and for establishing patient prognosis, the inventors evaluated miRNA levels in plasma samples from seven PrCa patients and nineteen normal donors (Table 17). Plasma samples were purchased from ProteoGenex Inc. (Culver City, CA, USA). Table 17. Histopathological data and patient information. NA, not available.
Figure imgf000056_0002
Attorney Docket No. 10256.32-304
Figure imgf000057_0001
[082] For plasma preparation, 10 ml whole blood was collected into a lavender top veinous blood collection tube (Becton, Dickinson and Company; Franklin Lakes, NJ, USA). The sample was allowed to incubate at room temperature for up to two hours, then centrifuged at 2,000 x g for 15 minutes. Following centrifugation, plasma was removed to a fresh tube. Plasma RNA was purified using the organic extraction of the mirVana PARIS™ Kit (Part No. AM1556; Applied Biosystems/Ambion; Austin, TX, USA), with the following modifications. Following the addition of acid phenokchloroform and vortexing, samples were incubated on ice for five minutes then centrifuged at 13,000 x g for 15 minutes at 4 0C. The aqueous layer was removed, 3M NaOAc (1/10 volume), glycogen (5mg/ml), and 100% ethanol (1.5 volume) were added to the samples, and samples were mixed by inversion. Lysate/ethanol mixtures were passed through a mirVana PARIS™ filter cartridge, and filters were washed once with 650 μl of Wash 1 buffer and twice with 650 μl of Wash 2/3 buffer. RNA was eluted with nuclease free water (50 μl) and stored at -80 0C.
[083] Levels of 319 miRNAs were determined by qRT-PCR using TaqMan® MicroRNA Assays (Applied Biosystems; Foster City, CA, USA) specific for each miRNA. Reverse transcription (RT) reaction components (Table 18) were assembled on ice prior to the addition of RNA template. Plasma RNA (0.25 μl per reaction) was added and mixed. RT reactions were incubated in a 384-well GeneAmp® PCR System 9700 (Applied Biosystems) at 16 0C for 30 minutes, then at 42 °C for 30 minutes, then at 85 0C for 5 minutes. RT reactions were then frozen at -20 °C. Attorney Docket No. 10256.32-304
Table 18. Reverse transcription reaction components. All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified.
Figure imgf000058_0001
[084] PCR components (Table 19) were assembled on ice prior to the
addition of cDNA (2 μl) from the RT reaction. Reactions were incubated in an ABI PRISM™ 7900HT Fast Real-Time PCR system (Applied Biosystems) at 95 0C for 1 minute, then for 50 cycles at 95 0C for 5 seconds and 60 °C for 30 seconds. Results were analyzed with SDS V2.3 (Applied Biosystems).
Table 19. PCR components. All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified.
Figure imgf000058_0002
[085] The qRT-PCR data were initially assessed for outliers. All miRNAs in a given sample with raw Ct readings of 50 were eliminated from further Attorney Docket No. 10256,32-304
analysis. All data from samples with fewer than 150 miRNAs having raw Ct values < 50 were eliminated. The average raw Ct values for 50 miRNAs detected in each sample were calculated for each individual sample. The average Ct for a given sample was subtracted from the raw Ct values for each miRNA in the corresponding sample to produce a dCt for each miRNA that was detected. Table 20 provides the normalized values for samples from normal donors and prostate cancer patients, and shows the difference in level of miRNAs between the sample types. miRNAs that have elevated or reduced levels in plasma samples from prostate disease patients are listed in Table 20.
Table 20. Normalized qRT-PCR data for quantification of 278 miRNAs in plasma from PrCa patients and normal donors (Norm).
Figure imgf000059_0001
Attorney Docket No 10256 32-304
Figure imgf000060_0001
Attorney Docket No 10256 32-304
Figure imgf000061_0001
Attorney Docket No. 10256.32-304
Figure imgf000062_0001
Attorney Docket No 10256 32-304
Figure imgf000063_0001
Attorney Docket No. 10256.32-304
Figure imgf000064_0001
Attorney Docket No. 10256.32-304
Figure imgf000065_0001
Example 6. miRNA combinations that distinguish plasma of prostate cancer patients from plasma of normal donors
[086] The un-normalized qRT-PCR data generated as described in Example 5 was used to calculate dCts for each pair of miRNAs that was quantified The dCt values for the miRNA pairs from plasma samples of PrCa patients and normal donors were analyzed using Receiver-Operator Characteristic (ROC) analysis to determine which miRNA pairs have the potential to distinguish plasma samples from those two groups. One miRNA pair (let-7c:tnιR-326) (Table 21 ) accurately classified the 14 PrCa patient samples and 12 normal donor samples that were analyzed. Twenty additional miRNA pairs accurately classified all but one of the 26 samples (ROC AUC >0 98) (Table 21) and 221 miRNA pairs had an ROC AUC score of at least 0.93 Data from 166 independent miRNAs was included at least once in the 221 total miRNA pairs (Table 22). Several miRNAs were used in multiple biomarkers pairs, indicating that they are present at significantly different levels in the plasmas of prostate cancer patients and normal donors. The six miRNAs appearing most commonly in biomarker pairs were miR- 10b, mιR-192, mιR-206, miR-101 , miR-205, and mιR-16 (Table 22). The miRNA pairs in Table 21 and the individual miRNA biomarkers in Table 22 are all possible targets for diagnosing prostate cancer using plasma. Attorney Docket No 10256 32-304
Table 21. miRNA biomarker pairs that can be used to identify plasma from prostate cancer patients.
Figure imgf000066_0001
Table 22. miRNA biomarkers that can be used in combination to identify plasma from prostate cancer patients
Figure imgf000066_0002
Attorney Docket No 10256 32-304
Figure imgf000067_0001
Example 7. Serum biomarkers of prostate cancer aggressiveness
[087] A key issue associated with prostate cancer patients is determining whether treatment is necessary. Many patients are diagnosed at very early stages of disease Because most cases of prostate cancer are unlikely to progress to metastatic disease, it is often unnecessary to subject an individual, especially one of advanced age, to aggressive treatment Distinguishing patients with relatively benign prostate cancer from patients with an aggressive form of the disease is vital to determining the appropriate level of therapeutic intervention
[088] Histopathological methods have been developed that are reasonably accurate in estimating the aggressiveness of a tumor. The Gleason score corresponds with tumor aggressiveness; higher Gleason scores correspond with more aggressive tumors. To determine if individual miRNAs in serum can Attorney Docket No. 10256.32-304
distinguish prostate cancer patients with high Gleason scores (>6) from those with low Gleason scores (<6) or from patients with BPH, the inventors further analyzed the qRT-PCR data from Example 1.
[089] All miRNAs in a given sample with raw Ct values of 50 were eliminated from further analysis. Data from serum samples with fewer than 150 miRNAs that had raw Ct values <50 were also eliminated. Un-normalized qRT- PCR data were used to calculate dCts for each pair of miRNAs that were evaluated. The dCt values for miRNA pairs measured in serum samples from PrCa and BPH patients were used to identify those pairs that distinguished serum samples from PrCa patients with high Gleason scores from those with low Gleason scores and from serum samples of BPH patients. Thirty-five (35) miRNA pairs were capable of distinguishing those serum samples (Table 23; low, samples from PrCa patients with Gleason scores ≤6; High, samples from PrCa patients with Gleason scores >6.). These miRNA pairs are useful for predicting aggressiveness of prostate cancer using patient serum samples.
Table 23. Biomarker pairs that can be used to identify serum from prostate cancer patients with high Gleason scores.
Figure imgf000068_0001
Attorney Docket No. 10256.32-304
Figure imgf000069_0001
Example 8. Validation of prostate cancer serum biomarkers.
[090] The inventors assessed the performance of 29 miRNAs (Table 24) that were identified as prostate cancer biomarkers in Example 1 by quantifying those miRNAs in the sera of 12 PrCa patients and 12 normal donors (purchased from ProteoGenex, Inc.) and in the sera of 8 PrCa patients, 8 BPH patients, and 8 normal donors (purchased from ProMedDx, LLC) (Table 25) using qRT-PCR. Serum RNA was purified as described in Example 1. Table 24. miRNAs assayed. rnJRNA Assayed hsa-miR-125b hsa-miR-99b hsa-miR-10b hsa-miR-205 hsa-miR-206 hsa-miR-29a hsa-miR-29c hsa-miR-122a hsa-miR-16 hsa-miR-138 hsa-miR-34a hsa-miR-200a Attorney Docket No. 10256.32-304
Figure imgf000070_0001
Table 25. Histopathological data and patient information. ND, not determined Source 1 , ProteoGenex, INc; Source 2, ProMedDx, LLC.
Figure imgf000070_0002
Attorney Docket No. 10256.32-304
Figure imgf000071_0001
[091] miRNA levels were determined by qRT-PCR using TaqMan® MicroRNA Assays (Applied Biosystems; Foster City, CA, USA) specific for each miRNA. Reverse transcription (RT) reaction components (Table 26) were assembled on ice prior to the addition of RNA template. Serum RNA (1.0 μl per reaction) was added and mixed. RT reactions were incubated in a 384-well GeneAmp® PCR System 9700 (Applied Biosystems) at 4 0C for 30 minutes, Attorney Docket No. 10256.32-304
16 0C for 30 minutes, 42 0C for 30 minutes, and 85 0C for 5 minutes. RT reactions were then frozen and stored at -20 °C. All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified.
Table 26. Reverse transcription reaction components.
Figure imgf000072_0001
[092] PCR components (Table 27) were assembled on ice prior to the addition of cDNA (4 μl) from the RT reaction. Reactions were incubated in an ABI PRISM™ 7900HT Fast Real-Time PCR system (Applied Biosystems) at 95 °C for 1 minute, then for 50 cycles at 95 0C for 5 seconds, then at 60 0C for 30 seconds. Data were extracted and exported with SDS V2.3 (Applied Biosystems). The data were analyzed with Microsoft Excel (Microsoft Corporation). All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified. Table 27. PCR components.
Figure imgf000072_0002
Attorney Docket No. 10256.32-304
Figure imgf000073_0001
[093] The qRT-PCR data were initially assessed for outliers. All miRNAs in a given sample with raw Ct readings of >40 were eliminated from further analyses. qRT-PCR data for each miRNA in each sample were normalized by subtracting the Ct value for miR-103 from the Ct value for the miRNA in the same sample. The resulting dCt values, were used to calculate the average dCt values for each miRNA in the normal donor and PrCa patient samples. Average dCt values for PrCa patient samples were subtracted from average dCt values for normal donor samples to determine the variance in the levels of the miRNAs between the patient sets. Student's t-test was then used to determine the potential of various miRNAs to distinguish the sera of PrCa patients from sera of normal donors. Table 28 lists the difference in average Ct between normal and prostate cancer samples, as well as p-values for PrCa samples compared to normal or BPH samples, and normal samples compared to BPH samples. These miRNAs are biomarkers of prostate cancer that can be used to diagnose prostate cancer using the serum from a patient. Table 28. Prostate cancer biomarkers.
Figure imgf000073_0002
Attorney Docket No. 10256.32-304
Figure imgf000074_0001
[094] The inventors used the data for the same miRNA biomarkers to identify pairs of miRNAs with the capacity to distinguish sera of PrCa patients from sera of BPH patients and normal donors. Un-normalized qRT-PCR data were used to calculate dCt values for each pair of miRNAs that was evaluated. The dCt values of the various miRNA pairs in PrCa and normal serum samples were analyzed using Receiver-Operator Characteristic (ROC) analysis to identify the miRNA pairs having the ability to distinguish sera of PrCa patients from sera of BPH patients and normal donors. Data from the best biomarker pairs are presented in Table 29. Numerous miRNAs were used in multiple biomarkers pairs, indicating the strength of their variable levels in sera of prostate cancer patients and normal donors. Attorney Docket No. 10256.32-304
Table 29. MicroRNA biomarker pairs for classifying prostate cancer serum.
Figure imgf000075_0001
Attorney Docket No. 10256.32-304
Figure imgf000076_0001
Attorney Docket No. 10256.32-304
Figure imgf000077_0001
Example 9. Validation of prostate cancer serum biomarker pair.
[095] To further assess the specificity and sensitivity of the combination of miR-125b and miR-24 in diagnosing prostate cancer patients using serum samples, miR-125b and miR-24 were quantified in the sera of 50 normal male donors, 40 patients with benign prostatic hyperplasia (BPH), 16 patients with prostate cancer who were undergoing hormone treatment and/or chemotherapy, and 33 untreated prostate cancer patients.
[096] For serum preparation, 10 ml of whole blood were collected from patients and normal donors using a BD Vacutainer® glass serum tube (Becton, Dickinson and Company; Franklin Lakes, NJ, USA; cat. no. 366441). Following blood collection, tubes were incubated at room temperature for up to 2.5 hours to allow the blood to clot, then centrifuged for 10 minutes at 2,000 x g. Serum was transferred to a new tube using a serological pipette and frozen at -80 0C until it was processed for RNA isolation.
[097] Serum RNA was purified using the organic extraction of the mirVana PARIS™ Kit (Part No. AM1556; Applied Biosystems/Ambion; Austin, TX, USA), Attorney Docket No. 10256.32-304
with the following modifications. Following the addition of acid phenol:chloroform and vortexing, samples were incubated on ice for 5 min then centrifuged at 13,000 x g for 10 min at 4 0C. The aqueous layer was removed, extracted with chloroform, and centrifuged again. The aqueous layer was removed from the second extraction, and 3M NaOAc (1/10 volume), glycogen (5 mg/ml), and 100% ethanol (1.5 volume) were added to the samples. Lysate/ethanol mixtures were passed through a mirVana PARIS filter cartridge, and filters were washed once with 650 μl of Wash 1 buffer and twice with 650 μl of Wash 2/3 buffer. RNA was eluted with two aliquots of nuclease free water (50 μl) and stored at -80 0C.
[098] Levels of miR-125b and miR-24 were determined by qRT-PCR using TaqMan® MicroRNA Assays (Applied Biosystems; Foster City, CA, USA) specific for each miRNA. Reverse transcription (RT) reaction components were assembled on ice prior to the addition of RNA template (Table 30). Serum RNA (1 μl per reaction) was added and mixed. RT reactions were incubated in a 384-well GeneAmp® PCR System 9700 (Applied Biosystems) at 4 0C for 30 minutes, then at 16 0C for 30 minutes, then at 42 0C for 30 minutes, then at 85 0C for 5 minutes. RT reactions were then frozen at -20 0C. All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified. Thus, the assays are useful to monitor patients' response to treatment. Attorney Docket No. 10256.32-304
Table 30. Reverse transcription reaction components.
Figure imgf000079_0001
[099] PCR components (Table 31) were assembled on ice prior to the addition of cDNA (4 μl) from the RT reaction. Reactions were incubated in an ABI PRISM™ 7900HT Fast Real-Time PCR system (Applied Biosystems) at 95 C for 1 minute, then for 50 cycles at 95 0C for 5 seconds and 60 0C for 30 seconds. Results were analyzed with SDS V2.3 software (Applied Biosystems). All reaction components were as provided by the manufacturer (Applied Biosystems; Foster City, CA, USA) unless otherwise specified.
Table 31. PCR components.
Figure imgf000079_0002
Attorney Docket No. 10256.32-304
[0100] The Ct values for miR-24 in each sample were subtracted from those for miR-125b in the corresponding sample to produce a dCt value for each sample (Table 32). Smaller dCt values correspond to serum samples with relatively higher miR-125b levels and/or lower miR-24 levels.
[0101 ] A diagnostic threshold of 6.00 dCt or 7.00 dCt was used to classify serum samples from donors. Using a threshold of 6.00 dCt to identify serum from prostate cancer patients (i.e., a serum sample with dCt <6.00 is indicative of prostate cancer), the miR-125b/miR-24 dCt value correctly identified 25 out of 33 untreated prostate cancer patients (76% sensitivity) and mis-identified 2 out of 50 normal male donors (96% specificity) and 0 out of 40 benign prostatic hyperplasia patients (100% specificity). Three (3) of 16 prostate cancer patients undergoing treatment were classified as positive using the 6 dCt cutoff.
[0102] Using a threshold of 7.00 dCt to identify serum from prostate cancer patients (i.e., a serum sample with dCt <7.00 is indicative of prostate cancer), the miR-125b/miR-24 dCt value correctly identified 32 out of 33 prostate cancer patients (97% sensitivity) and mis-identified 5 out of 50 normal male donors (90% specificity) and 4 out of 40 benign prostatic hyperplasia patients (90% specificity). Nine (9) of the 16 prostate cancer patients undergoing treatment had dCt values ranging from 6.00 to 6.99 and three other treated patients had dCt values ranging from 7.00 to 7.10, indicating that treatment was changing the serum levels of one or both miRNA biomarkers.
[0103] Table 32 shows miR-125b/miR-24 dCt following qRT-PCR quantification of miR-125b and miR-24 in serum samples from normal male donors (Normal), benign prostatic hyperplasia patients (BPH), treated prostate cancer patients (Tr-PrCa), and untreated prostate cancer patients (PrCa). Values Attorney Docket No. 10256.32-304
marked with * denote serum samples with dCt values less than 6.00. Values marked with # denote serum samples with dCt values ranging from 6.00 to 6.99. Unmarked values denote samples with dCt values > 7.00.
Table 32. miR-125b/miR-24 dCt
Figure imgf000081_0001
Attorney Docket No. 10256.32-304
Figure imgf000082_0001
[0104] All references cited herein are incorporated herein by reference in their entirety. To the extent publications and patents or patent applications incorporated by reference contradict the invention contained in the specification, the specification will supersede any contradictory material.

Claims

Attorney Docket No. 10256.32-304What is claimed is:
1. A method for characterizing prostate disease in a patient comprising the steps of: a. measuring the level of a miRNA in a serum sample, wherein the miRNA is chosen from let-7d, miR-10b, miR-122a, miR-139, miR-24, miR- 204, miR-205, miR-206, miR-375, and miR-99b; and b. determining whether the level of let-7d or miR-24 is reduced or whether the level of miR-10b, miR-122a, miR-139, miR-204, miR-205, miR- 206, miR-375, or miR-99b is elevated in the sample, thereby characterizing prostate disease in the patient.
2. The method of claim 1 , wherein the prostate disease is cancer.
3. The method of claim 2, wherein the stage of the cancer is characterized.
4. The method of claim 1 , wherein the progression of prostate disease is characterized.
5. The method of claim 1 , wherein the prostate disease is benign prostatic hyperplasia.
6. The method of claim 1 , further comprising amplifying the miRNA.
7. The method of claim 6, wherein the amplification is by quantitative reverse transcriptase polymerase chain reaction.
8. The method of claim 1 , wherein the miRNA is miR-24.
9. The method of claim 1 , further comprising amplifying, measuring, and determining reduced or elevated levels of a second miRNA.
10. The method of claim 9, wherein the first miRNA is miR-24 and the second miRNA is miR-125b. Attorney Docket No. 10256.32-304
11. A method for characterizing prostate disease in a patient comprising the steps of: a. measuring the level of a first miRNA and a second miRNA in a serum sample, wherein the miRNAs are chosen from the pairs listed in Table 4; and b. detecting reduced or elevated levels of the miRNAs in the sample, thereby characterizing prostate disease in the patient.
12. A method for characterizing prostate disease in a patient comprising the steps of: a. measuring the level of a first miRNA and a second miRNA in a serum sample, wherein the first miRNA is chosen from miR-125b, miR-122a, miR- 139, miR-375, miR-206, miR-205, miR-204, miR-16, miR-99b, let-7d, miR-24, miR-340, and miR-483; and b. detecting (1) whether the level of miR-16, let-7d, miR-24, or miR- 340 is reduced in the sample; or (2) whether the level of miR-125b, miR-122a, miR-139, miR-375, miR-206, miR-205, miR-204, miR-99b, or miR-483 is elevated in the sample; and c. further detecting whether the level of the second miRNA is elevated or reduced, thereby characterizing prostate disease in the patient.
13. The method of claim 12, wherein the first and second miRNAs are a pair chosen from miR-125b and miR-24; miR-125b and miR-26a; miR-432* and miR-483; miR-125b and miR-335; miR-125b and miR-342; let-7d and miR-125b; miR-125b and miR-197; miR-125b and miR-30c; miR-139 and let-7d; miR-125b and miR-16; miR-125b and miR-195; miR-16 and miR-139; miR-195 and miR- 139; miR-17-5p and miR-139; and miR-125b and mir-15b. Attorney Docket No. 10256.32-304
14. The method of claim 12, wherein the prostate disease is cancer.
15. The method of claim 14, wherein the stage of the cancer is characterized.
16. The method of claim 12, wherein the disease is benign prostatic hyperplasia.
17. The method of claim 12, further comprising amplifying the first miRNA and the second miRNA.
18. The method of claim 17, wherein the amplification is by quantitative reverse transcriptase polymerase chain reaction.
19. The method of claim 12, wherein the first miRNA is expressed at elevated levels in patients with prostate disease, and the second miRNA is expressed at reduced levels in patients with prostate disease.
20. The method of claim 12, wherein the first and the second miRNAs are expressed at elevated levels in patients with prostate disease.
21. The method of claim 12, wherein the first and the second miRNAs are expressed at reduced levels in patients with prostate disease.
22. The method of claim 12, wherein the progression of prostate disease is characterized.
PCT/US2010/035839 2009-05-22 2010-05-21 Mirna biomarkers of prostate disease WO2010135692A2 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA2762986A CA2762986C (en) 2009-05-22 2010-05-21 Mirna biomarkers of prostate disease
EP10720115A EP2432895A2 (en) 2009-05-22 2010-05-21 Mirna biomarkers of prostate disease
AU2010249421A AU2010249421A1 (en) 2009-05-22 2010-05-21 miRNA biomarkers of prostate disease

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US18076009P 2009-05-22 2009-05-22
US61/180,760 2009-05-22

Publications (2)

Publication Number Publication Date
WO2010135692A2 true WO2010135692A2 (en) 2010-11-25
WO2010135692A3 WO2010135692A3 (en) 2011-03-10

Family

ID=42751608

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2010/035839 WO2010135692A2 (en) 2009-05-22 2010-05-21 Mirna biomarkers of prostate disease

Country Status (5)

Country Link
US (2) US8597892B2 (en)
EP (2) EP2432895A2 (en)
AU (1) AU2010249421A1 (en)
CA (1) CA2762986C (en)
WO (1) WO2010135692A2 (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011080315A1 (en) * 2009-12-30 2011-07-07 Febit Holding Gmbh Mirna fingerprint in the diagnosis of prostate cancer
WO2012089772A3 (en) * 2010-12-30 2013-08-22 Febit Holding Gmbh Complex set of mirnas as non-invasive biomarkers for prostate diseases
US20130331278A1 (en) * 2010-12-30 2013-12-12 Febit Holding Gmbh Complex set of mirnas as non-invasive biomarkers for prostate diseases
WO2014057279A1 (en) * 2012-10-10 2014-04-17 Institute Of Cancer Research: Royal Cancer Hospital Micro-rna biomarkers for prostate cancer
US9089589B2 (en) 2007-05-23 2015-07-28 University Of South Florida Micro-RNAs modulating immunity and inflammation
KR101559131B1 (en) 2013-11-18 2015-10-07 한국원자력의학원 Composition comprising miRNA for enhancing radiation sensitivity
CN107488735A (en) * 2017-10-10 2017-12-19 广州医科大学附属第二医院 Applications of the 5p of miR 339 in prostate cancer with osseous metastasis and TGF signal beta paths is suppressed

Families Citing this family (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130040303A1 (en) * 2011-08-08 2013-02-14 Eugenia Wang Biomarker for Alzheimer's Disease and/or Mild Cognitive Impairment, and Use Thereof
US9745578B2 (en) * 2011-11-30 2017-08-29 Cedars-Sinai Medical Center Targeting microRNA miR-409-3P to treat prostate cancer
EP3106168A3 (en) * 2011-11-30 2017-02-22 Cedars-Sinai Medical Center Targeting micrornas mir-409-5p, mir-379 and mir-154* to treat prostate cancer bone metastasis and drug resistant lung cancer
WO2014071226A1 (en) * 2012-11-02 2014-05-08 The Regents Of The University Of California Methods and systems for determining a likelihood of adverse prostate cancer pathology
WO2015042708A1 (en) 2013-09-25 2015-04-02 Bio-Id Diagnostic Inc. Methods for detecting nucleic acid fragments
US20160237505A1 (en) * 2013-10-15 2016-08-18 The Board Of Trustees Of The University Of Illinois Serum mirnas for the prognosis of prostate cancer
KR102557689B1 (en) * 2014-06-12 2023-07-20 국립연구개발법인 고쿠리츠간켄큐센터 Prostate cancer detection kit or device, and detection method
EP3256602B1 (en) * 2015-02-11 2019-09-04 QIAGEN GmbH A microrna-based method for early detection of prostate cancer in urine samples
WO2016134727A1 (en) * 2015-02-27 2016-09-01 Exiqon A/S A microrna-based method for assessing the prognosis of a prostate cancer patient
US20180238889A1 (en) * 2015-08-14 2018-08-23 Northwestern University THE SCANO-miR PLATFORM IDENTIFIES A DISTINCT CIRCULATING MICRORNA SIGNATURE FOR THE DIAGNOSIS OF DISEASE
KR102601499B1 (en) * 2015-11-06 2023-11-13 연세대학교 산학협력단 Method for diagnosing uqcrb related desease by measuring micro rna expression level
EP3390659A1 (en) * 2015-12-15 2018-10-24 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for diagnosing or prognosing prostate cancer
US20180371554A1 (en) * 2015-12-15 2018-12-27 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for diagnosing or prognosing prostate cancer
KR102052398B1 (en) 2018-04-18 2019-12-05 (주)유로테크 Biomarkers for diagnosis of prostate cancer and uses thereof
JP7378739B2 (en) * 2018-08-10 2023-11-14 東レ株式会社 Kits, devices and methods for the detection of prostate cancer
EP3699300A1 (en) 2019-02-22 2020-08-26 Institut d'Investigació Biomèdica de Bellvitge (IDIBELL) Seminal mirnas as non-invasive biomarkers for the diagnosis and/or prognosis of prostate cancer
WO2020190819A1 (en) * 2019-03-15 2020-09-24 Mir Scientific, Llc Methods for predicting prostate cancer and uses thereof
CN109880900B (en) * 2019-04-23 2022-04-01 中国人民解放军陆军军医大学 Application of hsa-miR-134-3p as molecular marker and kit
CN109880899B (en) * 2019-04-23 2022-04-01 中国人民解放军陆军军医大学 Application of hsa-miR-134-3p as acute altitude reaction susceptible molecular marker and kit
CN110354137B (en) * 2019-08-20 2023-08-04 中山大学附属第六医院 Application of miRNA-197-3p in preparation of anti-prostate cancer drugs
CN110643711B (en) * 2019-11-20 2023-03-10 广州医科大学附属第二医院 Biomarkers for bone metastasis of prostate cancer
US11211147B2 (en) 2020-02-18 2021-12-28 Tempus Labs, Inc. Estimation of circulating tumor fraction using off-target reads of targeted-panel sequencing
US11475981B2 (en) 2020-02-18 2022-10-18 Tempus Labs, Inc. Methods and systems for dynamic variant thresholding in a liquid biopsy assay
US11211144B2 (en) 2020-02-18 2021-12-28 Tempus Labs, Inc. Methods and systems for refining copy number variation in a liquid biopsy assay
CN113388674B (en) * 2021-04-30 2022-03-08 江汉大学 Application of miRNA as drug target for detecting or treating benign prostatic hyperplasia

Family Cites Families (44)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6713619B1 (en) 1980-08-29 2004-03-30 Massachusetts Institute Of Technology Oncogenes and methods for their detection
US5366860A (en) 1989-09-29 1994-11-22 Applied Biosystems, Inc. Spectrally resolvable rhodamine dyes for nucleic acid sequence determination
US5188934A (en) 1989-11-14 1993-02-23 Applied Biosystems, Inc. 4,7-dichlorofluorescein dyes as molecular probes
EP0515506B1 (en) 1990-02-16 2000-01-05 F. Hoffmann-La Roche Ag Improvements in the specificity and convenience of the polymerase chain reaction
US5432272A (en) 1990-10-09 1995-07-11 Benner; Steven A. Method for incorporating into a DNA or RNA oligonucleotide using nucleotides bearing heterocyclic bases
US5965364A (en) 1990-10-09 1999-10-12 Benner; Steven Albert Method for selecting functional deoxyribonucleotide derivatives
US5413924A (en) 1992-02-13 1995-05-09 Kosak; Kenneth M. Preparation of wax beads containing a reagent for release by heating
US5767259A (en) 1994-12-27 1998-06-16 Naxcor Oligonucleotides containing base-free linking groups with photoactivatable side chains
US5925517A (en) 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
US5538848A (en) 1994-11-16 1996-07-23 Applied Biosystems Division, Perkin-Elmer Corp. Method for detecting nucleic acid amplification using self-quenching fluorescence probe
CA2168712A1 (en) 1995-02-07 1996-08-08 John William Henderson Sutherland Use of exonuclease and/or glycosylase as supplements to anti-polymerase antibody to increase specificity in polymerase chain reaction
WO1997035589A1 (en) 1996-03-26 1997-10-02 Kopreski Michael S Method enabling use of extracellular rna extracted from plasma or serum to detect, monitor or evaluate cancer
US6020481A (en) 1996-04-01 2000-02-01 The Perkin-Elmer Corporation Asymmetric benzoxanthene dyes
AU713667B2 (en) 1996-04-12 1999-12-09 Phri Properties, Inc. Detection probes, kits and assays
US5863727A (en) 1996-05-03 1999-01-26 The Perkin-Elmer Corporation Energy transfer dyes with enhanced fluorescence
US5945526A (en) 1996-05-03 1999-08-31 Perkin-Elmer Corporation Energy transfer dyes with enhanced fluorescence
US5800996A (en) 1996-05-03 1998-09-01 The Perkin Elmer Corporation Energy transfer dyes with enchanced fluorescence
US5847162A (en) 1996-06-27 1998-12-08 The Perkin Elmer Corporation 4, 7-Dichlororhodamine dyes
SE506700C2 (en) 1996-05-31 1998-02-02 Mikael Kubista Probe and Methods for Analysis of Nucleic Acid
US6008379A (en) 1997-10-01 1999-12-28 The Perkin-Elmer Corporation Aromatic-substituted xanthene dyes
US6485901B1 (en) 1997-10-27 2002-11-26 Boston Probes, Inc. Methods, kits and compositions pertaining to linear beacons
EP1025120B1 (en) 1997-10-27 2010-08-18 Boston Probes, Inc. Methods, kits and compositions pertaining to pna molecular beacons
US5936087A (en) 1997-11-25 1999-08-10 The Perkin-Elmer Corporation Dibenzorhodamine dyes
US20060204989A1 (en) 1998-09-22 2006-09-14 Kopreski Michael S Comparative analysis of extracellular RNA species
US6140054A (en) 1998-09-30 2000-10-31 University Of Utah Research Foundation Multiplex genotyping using fluorescent hybridization probes
US6383752B1 (en) 1999-03-31 2002-05-07 Hybridon, Inc. Pseudo-cyclic oligonucleobases
US6140500A (en) 1999-09-03 2000-10-31 Pe Corporation Red-emitting [8,9]benzophenoxazine nucleic acid dyes and methods for their use
US6528254B1 (en) 1999-10-29 2003-03-04 Stratagene Methods for detection of a target nucleic acid sequence
US6191278B1 (en) 1999-11-03 2001-02-20 Pe Corporation Water-soluble rhodamine dyes and conjugates thereof
US6596490B2 (en) 2000-07-14 2003-07-22 Applied Gene Technologies, Inc. Nucleic acid hairpin probes and uses thereof
US6350580B1 (en) 2000-10-11 2002-02-26 Stratagene Methods for detection of a target nucleic acid using a probe comprising secondary structure
US6403341B1 (en) 2001-08-02 2002-06-11 Wayne M. Barnes Magnesium precipitate hot start method for PCR
CA2459347C (en) 2001-09-04 2012-10-09 Exiqon A/S Locked nucleic acid (lna) compositions and uses thereof
US6593091B2 (en) 2001-09-24 2003-07-15 Beckman Coulter, Inc. Oligonucleotide probes for detecting nucleic acids through changes in flourescence resonance energy transfer
EP1771563A2 (en) 2004-05-28 2007-04-11 Ambion, Inc. METHODS AND COMPOSITIONS INVOLVING MicroRNA
US20060078894A1 (en) 2004-10-12 2006-04-13 Winkler Matthew M Methods and compositions for analyzing nucleic acids
EP2479285B1 (en) * 2006-01-05 2014-05-14 The Ohio State University Research Foundation MicroRNA-based methods and compositions for the diagnosis and treatment of solid cancers
EP2487240B1 (en) * 2006-09-19 2016-11-16 Interpace Diagnostics, LLC Micrornas differentially expressed in pancreatic diseases and uses thereof
WO2008112283A2 (en) * 2007-03-12 2008-09-18 Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Microrna profiling of androgen responsiveness for predicting the appropriate prostate cancer treatment
US7993831B2 (en) 2007-09-14 2011-08-09 Asuragen, Inc. Methods of normalization in microRNA detection assays
CN101424640B (en) 2007-11-02 2012-07-25 江苏命码生物科技有限公司 Method for detecting miRNA in blood serum, detection kit, biochip, making method thereof and application method
JP2011517283A (en) 2008-02-28 2011-06-02 ジ・オハイオ・ステイト・ユニバーシティ・リサーチ・ファウンデイション MicroRNA-based methods and compositions for prognosis and treatment of prostate related disorders
US20110160290A1 (en) 2008-05-21 2011-06-30 Muneesh Tewari Use of extracellular rna to measure disease
WO2010004562A2 (en) 2008-07-09 2010-01-14 Rosetta Genomics Ltd. Methods and compositions for detecting colorectal cancer

Non-Patent Citations (17)

* Cited by examiner, † Cited by third party
Title
BUSSEMAKERS ET AL., CANCER RES, vol. 59, 1999, pages 5975 - 5979
CHEN ET AL., NUCLEIC ACIDS RESEARCH, vol. 33, no. 20, 2005, pages E179
DANESHGARI ET AL., UROLOGY, vol. 45, 1995, pages 604 - 609
DE KOK ET AL., CANCER RES, vol. 62, 2002, pages 2695 - 2698
EGEVAD ET AL., BJU INT, vol. 89, 2002, pages 538 - 542
ETZIONI ET AL., J NATL CANCER INST, vol. 94, 2002, pages 981 - 990
FRADET ET AL., UROLOGY, vol. 64, 2004, pages 311 - 316
GLEASON, CANCER CHEMOTHER REP, vol. 50, 1966, pages 125 - 128
GLEASON, HUM PATHOL, vol. 23, 1992, pages 273 - 279
GRIFFITHS-JONES ET AL., NUCLEIC ACIDS RESEARCH, vol. 34, 2006, pages D140 - D144
GRIFFITHS-JONES ET AL., NUCLEIC ACIDS RESEARCH, vol. 36, 2008, pages D154 - D158
GRIFFITHS-JONES, NUCLEIC ACIDS RESEARCH, vol. 32, 2004, pages D109 - D111
HESSELS ET AL., EUR UROI, vol. 44, 2003, pages 8 - 15
JEMAL, CA CANCERJ CLIN., vol. 58, no. 2, 2008, pages 71 - 96
LI ET AL., CLIN. CHEM., vol. 53, 2006, pages 624 - 633
SCHOUTEN ET AL., NUCLEIC ACIDS RESEARCH, vol. 30, 2002, pages E57
STAMEY ET AL., J UROL, vol. 172, 2004, pages 1297 - 1301

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9089589B2 (en) 2007-05-23 2015-07-28 University Of South Florida Micro-RNAs modulating immunity and inflammation
WO2011080315A1 (en) * 2009-12-30 2011-07-07 Febit Holding Gmbh Mirna fingerprint in the diagnosis of prostate cancer
US9745630B2 (en) 2009-12-30 2017-08-29 Hummingbird Diagnostics Gmbh MiRNA fingerprint in the diagnosis of prostate cancer
WO2012089772A3 (en) * 2010-12-30 2013-08-22 Febit Holding Gmbh Complex set of mirnas as non-invasive biomarkers for prostate diseases
US20130331278A1 (en) * 2010-12-30 2013-12-12 Febit Holding Gmbh Complex set of mirnas as non-invasive biomarkers for prostate diseases
WO2014057279A1 (en) * 2012-10-10 2014-04-17 Institute Of Cancer Research: Royal Cancer Hospital Micro-rna biomarkers for prostate cancer
KR101559131B1 (en) 2013-11-18 2015-10-07 한국원자력의학원 Composition comprising miRNA for enhancing radiation sensitivity
CN107488735A (en) * 2017-10-10 2017-12-19 广州医科大学附属第二医院 Applications of the 5p of miR 339 in prostate cancer with osseous metastasis and TGF signal beta paths is suppressed
CN107488735B (en) * 2017-10-10 2019-11-05 广州医科大学附属第二医院 MiR-339-5p is inhibiting the application in prostate cancer with osseous metastasis and TGF-β signal path

Also Published As

Publication number Publication date
EP2634266B1 (en) 2015-10-14
WO2010135692A3 (en) 2011-03-10
US8597892B2 (en) 2013-12-03
US20140235469A1 (en) 2014-08-21
EP2634266A1 (en) 2013-09-04
CA2762986C (en) 2018-03-06
AU2010249421A1 (en) 2011-12-08
EP2432895A2 (en) 2012-03-28
CA2762986A1 (en) 2010-11-25
US20100297652A1 (en) 2010-11-25

Similar Documents

Publication Publication Date Title
EP2634266B1 (en) miRNA biomarkers of prostate disease
US8735074B2 (en) MiRNA biomarkers of lung disease
US7993831B2 (en) Methods of normalization in microRNA detection assays
Lan et al. MicroRNAs as potential biomarkers in cancer: opportunities and challenges
EP2358912B1 (en) Methods for assessing rna patterns
Shen et al. MicroRNAs as potential biomarkers in human solid tumors
CN101921759B (en) Serum/plasma miRNA serum marker related to cervical carcinoma and precancerous lesions thereof and application thereof
US20160319364A1 (en) Reagents and Methods for miRNA Expression Analysis and Identification of Cancer Biomarkers
US20170002428A1 (en) Detection and quantification of microRNAs in the circulation and the use of circulating microRNAs as biomarkers in cancer
US20130157886A1 (en) Methods of detecting lung cancer
JP2013502931A5 (en)
US20130184169A1 (en) Methods for assessing rna patterns
Giannopoulou et al. Liquid biopsy in ovarian cancer
KR101693649B1 (en) Methods of predicting the tissue origin for adenocarcinomas in the liver using microRNA profiles
GB2465088A (en) Methods for characterising prostate cancer
Ghai et al. Circulating miRNAs as tumor biomarkers

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10720115

Country of ref document: EP

Kind code of ref document: A2

WWE Wipo information: entry into national phase

Ref document number: 2762986

Country of ref document: CA

Ref document number: 2010249421

Country of ref document: AU

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2010249421

Country of ref document: AU

Date of ref document: 20100521

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2010720115

Country of ref document: EP