WO2015121663A1 - Biomarqueurs destinés au cancer de la prostate - Google Patents
Biomarqueurs destinés au cancer de la prostate Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Definitions
- This invention relates to microRNA biomarkers useful in the diagnosis of prostate cancer.
- the biomarkers are also useful for the monitoring and/or treatment of prostate cancer.
- PC Prostate cancer
- PSA and PCA3 are currently the only two molecular markers approved for use in PC diagnosis. Reported specificities for the PSA test vary but in general are much less than 50% [e.g. see Reference 2; and Table 2 of Reference 3]. Although the reported specificity for PCA3 is higher than PSA (approximately 70-80% only), it has a much poorer sensitivity (approximately 60-70%) [3, 4, 5]. The metrics obtained from a combination of the two tests (PSA and PCA3) are modest: the Area Under the Curve (AUC) values for PSA alone was 0.63, and for PSA + PCA3 was 0.71 [4].
- AUC Area Under the Curve
- PC and other conditions of the prostate such as benign prostatic hypertrophy (BPH) and prostatitis
- BPH benign prostatic hypertrophy
- prostatitis these conditions can coexist and diagnosis of a benign condition may distract the clinician from a concomitant diagnosis of PC.
- BPH benign prostatic hypertrophy
- patients with BPH often present with urinary obstructive symptoms due to the enlarged prostate compressing the urethra.
- those with BPH also often have the symptoms of urinary frequency, urgency, hesitancy, incomplete bladder emptying and the need to strain.
- elevated PSA levels are seen in various prostate conditions, such as BPH and PC, thus reducing the specificity and diagnostic accuracy of using PSA as a population screening tool for PC.
- the invention is based on the identification of correlations between prostate cancer (PC) and the levels of small non-coding microRNAs (miRNAs).
- the inventors have identified miRNAs whose expression profiles can be used to indicate that a subject has PC or not. These miRNAs are present at significantly different levels in subjects with PC and without PC, so the miRNAs function as biomarkers of PC. Detection of the levels of any of the biomarkers in a subject sample would provide a diagnostic indicator of whether the subject has PC or not.
- the invention can be used to discriminate between PC and a confounding prostate condition, such as BPH and prostatitis, where inflammation and similar symptoms, such as raised PSA levels, are common.
- the invention can also be useful as a population screening tool for PC, particularly for discriminating between PC and a confounding prostate condition.
- the inventors have identified three such biomarkers and they are listed in Table 1.
- the invention uses at least one of them to assist in discriminating between PC and a confounding prostate condition, such as BPH or prostatitis, by measuring level(s) of the miRNAs.
- the invention provides a method for distinguishing between PC and non-PC in a subject having a prostate condition, comprising determining in a sample from the subject the level of a Table 1 biomarker, wherein the level of the biomarker provides a diagnostic indicator of whether the subject has PC or not.
- the invention also provides a method for analysing a sample from a subject having a prostate condition, comprising a step of determining the level of a Table 1 biomarker in the sample, wherein the level of the biomarker provides a diagnostic indicator of whether the subject has PC or not.
- the subject may have symptoms common to BPH and PC, in which case the method of the invention would provide a diagnostic indicator of whether the subject has PC or not.
- the subject may have BPH, in which case the method of the invention would provide a diagnostic indicator of whether the subject additionally has PC or not.
- the invention also provides a method for analysing a sample from a subject having a serum PSA level of 3ng/ml or more, and/or who is aged 50 or more, comprising a step of determining the level of a Table 1 biomarker in the sample, wherein the level of the biomarker provides a diagnostic indicator of whether the subject has PC or not.
- the invention also provides a method for diagnosing if a subject has PC or not, comprising determining in a sample from the subject the level of a Table 1 biomarker, wherein the level of the biomarker provides a diagnostic indicator of whether the subject has PC or not; wherein the subject: (i) has a prostate condition; (ii) has a serum PSA level of 3ng/ml or more; and/or (iii) is aged 50 or more.
- Analysis of a single Table 1 biomarker can be performed, and detection of the miRNA can provide a useful diagnostic indicator for PC even without considering any of the other Table 1 biomarkers.
- the sensitivity and specificity of diagnosis can be improved, however, by combining data for multiple biomarkers. It is thus preferred to analyse more than one Table 1 biomarkers.
- Analysis of two or more different biomarkers (a "panel") can enhance the sensitivity and/or specificity of diagnosis compared to analysis of a single biomarker. Each different biomarker in a panel is shown in a different row in Table 1.
- the invention also provides a method for analysing a sample from a subject, comprising a step of determining the levels of two or three different biomarkers of Table 1, wherein the levels of the biomarkers provide a diagnostic indicator of whether the subject has PC or not.
- the invention also provides a diagnostic device for use in diagnosis of PC, wherein the device permits determination of the levels of two or three Table 1 biomarkers.
- the invention also provides a diagnostic device for use in discriminating between PC and non-PC in a subject having a prostate condition, wherein the device permits determination of the levels of two or three Table 1 biomarkers.
- the Table 1 biomarkers ca n be also used in combination with one or more of: (a) known biomarkers for PC, which may or may not be miRNAs; and/or (b) other information about the subject from whom a sample was taken e.g. age, genotype, ethnicity), weight, other clinically-relevant data or phenotypic information; and/or (c) other diagnostic tests or clinical indicators for PC, which can include, but not limited to, Gleason score, PSA levels, tumour grading (TMN score) etc. Such combinations can enhance the sensitivity and/or specificity of diagnosis.
- the methods of the invention can comprise a step of determining:
- a sample from the subject contains a known biomarker selected from the group consisting of: the miRNAs listed in Table 2, PSA antigen, PCA3 antigen and/or mRNA, DD3 antigen and/or mRNA, AMACR antigen and/or mRNA, EPCA antigen and/or mRNA, EPCA-2 antigen and/or mRNA, TMPRSS2:ERG gene fusions and sarcosine (and optionally, any other known biomarkers e.g. see above); wherein detection of the known biomarker provides a second diagnostic indicator of whether the subject has PC; (c) the subject's age, and combining the different diagnostic indicators to provide an aggregate diagnostic indicator of whether the subject has PC or not.
- a known biomarker selected from the group consisting of: the miRNAs listed in Table 2, PSA antigen, PCA3 antigen and/or mRNA, DD3 antigen and/or mRNA, AMACR antigen and/or mRNA, EPCA antigen and
- samples used in (a) and (b) may be the same or different.
- the value of y is 1 or more e.g. 1, 2 or 3.
- the methods of the invention can comprise the steps of: (a) determining the levels of y biomarkers of Table 1 in a sample from the subject; and (b) comparing the determination from step (a) to data obtained from samples from subjects without PC and/or from subjects with PC, wherein the comparison provides a diagnostic indicator of whether the subject has PC not.
- the com parison in step (b) can use a classifier a lgorithm as discussed in more detail below.
- the invention also provides a method for analysing a sample from a subject, comprising a step of determining in a sample from the subject the levels of one or more biomarkers listed Table 1, also one or both of: (i) one or more biomarkers listed in Table 2, and (ii) one or more of the known PC biomarkers mentioned above e.g. PSA and/or PCA3, wherein the levels of the biomarkers provide a diagnostic indicator of whether the subject has PC or not.
- Preferred panels have up to 3 biomarkers, e.g. those listed in Tables 8, 10, 12 and 15.
- the invention also provides a method for diagnosing if a subject has PC or not, comprising a step of determining in a sample from the subject the levels of one or more biomarkers listed Ta ble 1, and also one or both of: (i) one or more biomarkers listed in Table 2, and (ii) one or more of the known PC biomarkers mentioned above e.g. PSA and/or PCA3, wherein the levels of the biomarkers provide a diagnostic indicator of whether the subject has PC or not.
- Preferred panels have up to 3 biomarkers, e.g. those listed in Tables 8, 10, 12 and 15.
- the invention also provides a diagnostic device for use in diagnosis of PC, wherein the device permits determination of the levels of one or more biomarkers listed Table 1, and also one or both of: (i) one or more biomarkers listed in Ta ble 2, and (ii) one or more of the known PC biomarkers mentioned above e.g. PSA and/or PCA3.
- the invention a lso provides a panel com prising one or more of the Table 1 biomarkers, also one or both of: (i) one or more of the biomarkers listed in Table 2; and (ii) PSA, PCA3, DD3, AMACR, EPCA, EPCA-2, TMPRSS2:ERG gene fusions, EGR/ETV1 gene rearrangements, PTEN gene loss, and/or sarcosine (and optionally, any other known biomarkers e.g. see above). Panels comprising these biomarkers are particularly useful for diagnosing PC, in particular discriminating between PC and a confounding prostate condition in a subject.
- the invention also provides a panel of biomarkers comprising one or more of the Table 1 biomarkers as biomarkers for PC diagnosis.
- the invention also provides a software product comprising (i) code that accesses data attributed to a sample, the data comprising measurement of y Table 1 biomarkers, and optionally other biomarkers (e.g. any of the Table 2 biomarkers), wherein y is 1, 2 or 3, (ii) code that executes an algorithm for assessing the data to represent a level of y of the biomarkers in the sample.
- the software product may also comprise (iii) code that executes an algorithm for assessing the result of step (ii) to provide a diagnostic indicator of whether the subject has PC.
- suitable algorithms for use in part (iii) include support vector machine algorithms, artificial neural networks, tree-based methods, genetic programming, etc.
- the algorithm can preferably classify the data of part (ii) to distinguish between PC subjects and non-PC subjects based on measured biomarker levels in samples taken from such subjects.
- the invention also provides methods for training such algorithms.
- the invention also provides a computer which is loaded with and/or is running a software product of the invention.
- the invention also extends to methods for communicating the results of a method of the invention.
- This method may involve communicating assay results and/or diagnostic results. Such communication may be to, for example, technicians, physicians or patients.
- detection methods of the invention will be performed in one country and the results will be communicated to a recipient in a different country.
- the invention also provides the use of a Table 1 biomarker to discriminate between PC and a confounding prostate condition in a subject.
- the invention also provides the use of a Table 1 biomarker to distinguish between PC and non-PC in a subject having a prostate condition.
- the invention also provides the use of a Table 1 biomarker as a diagnostic indicator of whether a subject has PC or not, wherein the subject: (i) has a prostate condition; (ii) has a serum PSA level of 3ng/ml or more; and/or (iii) is aged 50 or more.
- the invention also provides a method for analysing a sample from a subject, comprising a step of determining the level of a Table 1 biomarker, wherein the level of the biomarker provides a diagnostic indicator of whether the subject has PC or not, but does not provide a diagnostic indication of the aggressiveness or staging of the PC.
- the invention also provides the use of a Table 1 biomarker as a diagnostic marker for PC, but does not provide a diagnostic indication of the aggressiveness or staging of the PC.
- the invention uses hsa-miR-451a.
- the 23 miRNAs listed in Table 2 can be used in combination with the biomarkers of Table 1.
- the diagnostic performance can be increased when a combination of a Table 1 and a Table 2 marker is used.
- Further details of these 23 Table 2 miRNAs are given in Tables 13 and 14.
- Table 14 provides further details of the miRNA biomarkers, as provided by miRBase database (version 20, released, June 2013), such as the precursor hairpin pre-miRNA sequences and the genomic location of the miRNA gene. In some instances, multiple precursor pre-miRNAs (i.e. from different genomic locations) lead to the same mature miRNA sequence.
- a single pre-miRNA precursor may lead to one or more mature miRNA sequences, such as sequences excised from the 5' and 3' arms of the hairpin, as indicated in Table 14.
- the methods of the invention can involve detecting and determining the levels of the mature miRNA sequences that are excised from 5' and/or 3' arms of the pre-miRNA precursor, as indicated in Tables 1 and 2. These specific coding sequences are not limiting on the invention.
- the invention includes detecting and measuring the levels of polymorphic variants of these miRNAs.
- a database outlining in more detail the miRNAs listed in Tables 1 and 2 is available: MiRBase [6, 7, 8, 9] or, in relation to target prediction, the DIANA-microT [10, 11], microRNA.org [12], miRDB [13, 14], TargetScan [15] and PicTar [16] databases.
- detection of a single biomarker or panel of biomarkers comprised from Table 1 can provide useful diagnostic information, but each biomarker might not individually provide information which is useful i.e. a miRNA in Table 1 may be present in some, but not all, subjects with PC.
- a single biomarker might not provide universal diagnostic results, and to increase the overall confidence that an assay is giving sensitive and specific results across a disease population, it is advantageous to analyse a plurality of the Table 1 biomarkers, or a combination of Tables 1 and 2 biomarkers (i.e. a panel). For instance, a negative signal for a particular Table 1 miRNA is not necessarily indicative of the absence of PC (just as a low PSA concentration is not), but confidence that a subject does not have PC increases as the number of negative results increases. For example, in the diagnosis of PC if all 3 biomarkers are tested and are negative then the result provides a higher degree of confidence than if only one biomarker is tested and is negative.
- biomarker panels are most useful for enhancing the distinction seen between diseased and non-diseased samples.
- Preferred panels have up to 7 biomarkers, as the burden of measuring a higher number of markers is usually not rewarded by better sensitivity or specificity. Preferred panels are given below.
- a method for analysing a subject sample can function as a method for diagnosing if a subject has PC or not.
- a method may not always provide a definitive diagnosis and so a method for analysing a subject sample can sometimes function only as a method for aiding in the diagnosis of PC or non-PC, or as a method for contributing to a diagnosis of PC or non-PC, where the method's result may imply that the subject has PC (e.g. the disease is more likely than not).
- the invention is used for diagnosing PC in a subject, and the subject will be male.
- the invention is particularly useful for discriminating PC from confounding prostate conditions.
- the invention is particularly useful for diagnosing PC in subjects who present with symptoms similar to PC.
- the subject may present with a prostate condition.
- Conditions of the prostate include PC, Benign Prostatic Hyperplasia (BPH) (also known as Enlarged Prostate) and prostatitis. Diagnosis of these conditions and symptoms typically associated with these conditions are known in the art.
- BPH Benign Prostatic Hyperplasia
- the invention is useful for determining whether the subject has PC or other conditions of the prostate.
- the subject may have BPH. Similar to patients with PC, patients with BPH also often have the symptoms of urinary frequency, urgency, hesitancy, incomplete bladder emptying and the need to strain. Furthermore, elevated serum PSA levels (more than 3ng/ml) are often also seen in PC and BPH. For subjects who have BPH, the invention is useful for determining whether the subject has PC in addition to BPH.
- the subject is typically at least 50 years old. Subjects who are at least 50 years old are prone to developing prostate conditions, such as BPH. An estimated 50% of men have histologic evidence of BPH by age 50 years and 75% are thought to display such evidence by age 80 years [17]. Also, the risk of PC increases in these men, so for these subjects it may be appropriate to offer a screening service. Conditions of the prostate are usually associated with elevated levels of PSA in the serum. Typically, serum PSA levels of less than 3ng/ml are considered as normal, 3-10ng/ml may warrant further investigation, and >10ng/ml is high. The invention is particularly effective in diagnosing a subject who has an elevated serum PSA level, e.g. more than 3ng/mL, preferably more than 4ng/mL.
- the subject may present with a prostate condition, the subject may have a normal serum PSA level, i.e. less than 3ng/ml.
- the invention is also effective in diagnosing PC in such a subject.
- the subject may be pre-symptomatic for PC or may already be displaying clinical symptoms.
- the invention may be used to confirm or resolve another diagnosis.
- the subject may already have begun treatment for PC.
- the subject may already be known to be predisposed to development of PC e.g. due to family or genetic links.
- the subject may have no such predisposition, and may develop the disease as a result of environmental factors e.g. as a result of exposure to particular chemicals (such as toxins or pharmaceuticals), as a result of diet [18], as a result of infection, etc.
- the invention can be implemented relatively easily and/or cheaply in that the invention is not restricted to being used in patients who are already suspected of having PC and/or who already present with a prostate condition. Rather, it can be used to screen the general population or a high risk population e.g. men at least 20 years old (e.g. >25, >30, >35, >40, >45, >50, >55, >60, >65, >70).
- a high risk population e.g. men at least 20 years old (e.g. >25, >30, >35, >40, >45, >50, >55, >60, >65, >70).
- the subject will typically be a male human being.
- the invention is useful in non-human organisms e.g. mouse, rat, rabbit, guinea pig, cat, dog, horse, pig, cow, or non-human primate (monkeys or apes, such as macaques or chimpanzees).
- non-human embodiments any method used for detection of miRNAs by the invention will typically be based on the relevant non-human ortholog of the human miRNA disclosed herein.
- animals can be used experimentally to monitor the impact of a therapeutic on a particular biomarker.
- the invention analyses samples from subjects.
- sample can include miRNAs suitable for detection by the invention, but the sample will typically be (homogenised) tissue and/or a body fluid.
- Suitable body fluids include, but are not limited to blood, tissue, saliva, prostate tissue, prostate fluid (i.e. fluid which immediately surrounds the prostate in vivo), prostatic secretions, lymphatic fluid, a wound secretion, urine, faeces, mucus, sweat, tears and/or cerebrospinal fluid.
- the sample is typically blood, tissue or urine.
- a method of the invention involves an initial step of obtaining the sample from the subject. I n other embodiments, however, the sample is obtained separately from and prior to performing a method of the invention. After a sample has been obtained then methods of the invention could be performed in vitro. I n other embodiments, however, a method of the invention involves detecting the presence and/or absence of the miRNA in vivo, for example, but not limited to, use of a detection probe (e.g. a radioactive probe) as a tracer for molecular imaging.
- a detection probe e.g. a radioactive probe
- Detection of biomarkers may be performed directly on a sample taken from a subject, or the sample may be treated between being taken from a subject and being analysed.
- a blood sample may be treated by adding anti-coagula nts (e.g. EDTA), followed by removing cells and cellular debris, leaving plasma containing free-circulating miRNA for analysis.
- a blood sample may be allowed to coagulate, followed by removing cells and various clotting factors, leaving serum containing free-circulating miRNA for analysis.
- the invention preferably involves determining the levels of the biomarkers in plasma obtained from blood samples from the subject. Without wishing to be bound by theory, it is hypothesised that during the coagulation process, blood cells are exposed to a stressful environment resulting in the stimulation of the release of miRNAs and other RNAs, thereby changing the true repertoire of circulating miRNA [19]. Therefore, plasma is considered to be most useful for studying circulating miRNAs.
- the invention preferably does not involve determining the levels of the biomarkers in serum obtained from blood samples from the subject.
- the invention also provides a method for analysing plasma obtained from a blood sample from a subject, comprising a step of determining the level of one or more Table 1 biomarker in the plasma, wherein the level of the biomarker provides a diagnostic indicator of whether the subject has PC or not.
- the invention provides a method for diagnosing PC a subject, comprising determining in plasma obtained from a blood sample from the subject the level of a Table 1 biomarker, wherein the level of the biomarker provides a diagnostic indicator of whether the subject has PC or not.
- the invention also provides the use of one or more Table 1 biomarker as a diagnostic marker for PC in plasma obtained from a blood sample from a subject.
- faeces samples can be used. Faeces samples usually require physical treatment prior to miRNA detection e.g. suspension, homogenisation and centrifugation. For some body fluids, though, such separation treatments are not usually required (e.g. urine, tears or saliva) but other treatments may be used. For example, various types of sample may be subjected to treatments such as dilution, aliquoting, sub-sampling, heating, freezing, irradiation, etc. between being taken from the body and being analysed e.g. plasma is usually stored, frozen prior to analysis. Also, addition of processing reagents is typical for various sample types e.g. addition of anticoagulants to blood samples.
- a tissue sample can be preserved with a fixative (e.g. formalin) before it is analysed.
- a preserved sample can also be embedded (e.g. formalin-fixed, paraffin-embedded (FFPE) samples).
- FFPE paraffin-embedded
- a fresh tissue sample can be used, and this sample is fresh frozen, without fixatives.
- biomarkers of the invention may show a 'field-effect' within the prostate gland, whereby differential relative expression profiles (e.g. PC sample compared to a control) can be observed in prostate tissue samples that are obtained from any part of a prostate lobe, wherein the prostate lobe contains cancerous cells or the cancer foci.
- prostate tissue samples are obtained from: (i) all major regions of the prostate so as to ensure complete "geographic" coverage, and/or (ii) any region of the prostate that may be suspected to be cancerous, e.g. suspicious on transrectal ultrasound or magnetic resonance imaging.
- TRUS transrectal ultrasound-guided prostate
- biomarkers of the invention that demonstrate a field-effect, e.g. hsa-miR-451a and the panels in Table 15, selecting sample regions that are suspected to be cancerous or the cancer foci is not essential for detecting PC. Hence, these biomarkers are able to detect or predict PC from a more generalised, less targeted, sampling of the prostate during a routine biopsy procedure.
- a method of the invention can include determining the expression level of a miRNA in a tissue sample from any region of the prostate, wherein the expression level of the miRNA indicates that the subject has PC.
- the method can further comprise determining the expression level of the miRNA in a control, and comparing the expression levels of the miRNA in the tissue sample and in the control, wherein a difference in the expression levels indicate that the subject has PC.
- the sample can be from a region in the prostate that has been suspected to be cancerous or a region in the prostate that has not been suspected to be cancerous.
- a method of the invention can include determining the expression level of a miRNA in a tissue sample from any part of a prostate lobe, wherein the expression level of the miRNA indicates that the subject has PC.
- the method can further comprise determining the expression level of the miRNA in a control, and comparing the expression levels of the miRNA in the tissue sample and in the control, wherein a difference in the expression levels indicate that the subject has PC.
- the sample can be from a part of a prostate lobe that has been suspected to be cancerous or a part of a prostate lobe that has not been suspected to be cancerous.
- a method of the invention uses a biomarker of the invention, e.g. hsa-miR-451a and the panels in Table 15, that demonstrates a field-effect
- the method does not include identifying a cancerous region or a cancer foci before obtaining a biopsy sample.
- Table 1 lists three human miRNA molecules and methods of the invention can involve detecting and determining the level of any of the three miRNA biomarkers in a sample.
- the invention can additionally involve detecting and determining the 23 human miRNA molecules listed in Table 2.
- Table 14 provides nucleotide sequences for these miRNA molecules, but polymorphisms of miRNA are known in the art and so the invention can also involve detecting and determining the level of a polymorphic miRNA variant of these listed miRNA sequences.
- RNA detection methods are well known in the art, e.g. microarray analysis and NanoString's nCounter technology, polymerase chain reaction (PCR)-based methods (e.g. reverse transcription PCR, RT-PCR), in-situ hybridisation (ISH)-based methods (e.g. fluorescent ISH, FISH), northern blotting, sequencing (e.g. next-generation sequencing), fluorescence-based detection methods, etc.
- PCR polymerase chain reaction
- ISH in-situ hybridisation
- ISH in-situ hybridisation
- sequencing e.g. next-generation sequencing
- fluorescence-based detection methods etc.
- RT-qPCR where the detection is fluorescence-based (e.g. TaqMan ® or SYBR ® Green).
- Detection of a miRNA typically involves contacting ("hybridising") a sample with a complementary detection probe (e.g. a synthetic oligonucleotide strand), wherein a specific (rather tha n non-specific) binding reaction between the sample and the complementary probe indicates the presence of the miRNA of interest.
- a complementary detection probe e.g. a synthetic oligonucleotide strand
- the miRNA in the sample is amplified prior to detection, e.g. by reverse transcription of the miRNA to produce a complementary DNA (cDNA) strand, and the derived cDNA can be used as a template in the subsequent PCR reaction.
- nucleic acids which ca n be used, for exam ple, as hybridisation probes for specific detection of miRNA in biological samples or as single-stranded primers to amplify the miRNA.
- nucleic acid in general means a polymeric form of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides, and/or their analogs. It includes DNA, RNA, DNA/RNA hybrids. It also includes DNA or RNA analogs, such as those containing modified backbones (e.g. peptide nucleic acids (PNAs) or phosphorothioates) or modified bases.
- Nucleic acid according to the invention ca n take various forms (e.g. single-stranded, primers, probes, labelled etc. ). Primers and probes are generally single-stranded.
- the nucleic acid can be identical or complementary to the mature miRNA sequences of the invention that are listed in Table 14, i.e. to any one of SEQ I D NOs: 1-6 and 44-47.
- the nucleic acid may comprise sequences found in the miRBase database.
- the nucleic acid ca n comprise a nucleotide sequence that has >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >96%, >97%, >98%, >99% or more identity to any one of SEQ ID NOs: 1-6 and 44-47. Identity between sequences is preferably determined by the Smith-Waterman homology search algorithm as described below.
- the nucleic acid ca n comprise a nucleotide sequence that has >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95%, >96%, >97%, >98%, >99% or more complementarity to any one of SEQ I D NOs: 1-6 and 44-47.
- complementarity when used in relation to nucleic acids refers to Watson-Crick base pairing.
- the complement of C is G
- the complement of G is C
- the complement of A is T (or U)
- T or U
- T the complement of T (or U) is A.
- bases such as I (the purine inosine) e.g. to complement pyrimidines (C or T).
- nucleic acid is DNA
- U in a RNA sequence
- T in the DNA
- RNA RNA
- T in a DNA sequence
- the nucleic acid may be 12 or more, e.g. 12, 13, 14, 15, 16, 17 or 18, etc. (e.g. up to 50) nucleotides in length.
- the nucleic acid may be 15-30 nucleotides in length, 10-25 nucleotides in length, 15-25 nucleotides in length, or 20-25 nucleotides in length.
- the nucleic acid may include sequences that do not hybridise to the miRNA biomarkers, and/or amplified products thereof.
- the nucleic acid may contain additional sequences at the 5' end or at the 3' end.
- the additional sequences can be a linker, e.g. for cloning or PCR purposes.
- Nucleic acid of the invention may be attached to a solid support (e.g. a bead, plate, filter, film, slide, microarray support, resin, etc. ). Nucleic acid of the invention may be labelled e.g. with a radioactive or fluorescent label, or a biotin label. This is particularly useful where the nucleic acid is to be used in detection techniques e.g. where the nucleic acid is a primer or as a probe. Methods for preparing fluorescent labelled probes, e.g. for fluorescent in-situ hybridisation FISH analysis, are known in the art, and FISH probes can be obtained commercially, e.g. from Exiqon.
- a solid support e.g. a bead, plate, filter, film, slide, microarray support, resin, etc.
- Nucleic acid of the invention may be labelled e.g. with a radioactive or fluorescent label, or a biotin label. This is particularly useful where the nucleic acid is to
- the invention may use in-situ hybridisation (ISH)-based methods, e.g. fluorescent in-situ hybridisation (FISH).
- Hybridization reactions can be performed under conditions of different "stringency” followed by washing.
- the nucleic acid of the invention hybridise under high stringency conditions, such that the nucleic acid specifically hybridises to a miRNA in an amount that is detectably stronger than non-specific hybridisation.
- Relatively high stringency conditions include, for example, low salt and/or high temperature conditions, such as provided by about 0.02-0.1 M NaCI or the equivalent, at temperatures of about 50-70°C.
- a stringent wash removes non-specific probe binding and overloaded probes.
- Relatively stringent wash conditions include, for example, low salt and/or presence of detergent, e.g. 0.02 % SDS in IX Saline-Sodium Citrate (SSC) at about 50°C.
- detergent e.g. 0.02 % SDS in IX Saline-Sodium Citrate (SSC) at about 50°C.
- a sample that potentially contains the biomarkers are simultaneously contacted with multiple oligonucleotide complementary detection probes, or PCR primers/probes ("multiplexed") in a single reaction compartment, whereby a reaction compartment is defined as, but not limited to, a microtitre well, microfluidic chamber or detection pore.
- these multiple biomarkers could either be contacted with its complementary detection probe in separate, individual reaction compartments and/or; experiments could be separated over time and using different platform technologies in either multiplexed single reaction compartments or separate, individual reaction compartments.
- Microarray and PCR usage for the detection of miRNAs is well known in the art e.g. see references 20,21,22,23. Microarrays may be prepared by various techniques, such as those disclosed in references 24, 25, & 26. Methods based on nucleic acid amplification are also well known in the art. Methods and apparatus for detecting binding reactions on DNA microarrays are now standard in the art. Preferred detection methods are fluorescence-based detection methods.
- the invention includes detecting and determining a plurality of biomarkers, i.e. in a panel.
- the data derived from a panel can be combined in a multivariate analysis [27].
- the combination of biomarkers may increase the classification power relative to a single biomarker.
- a panel of biomarkers can also be evaluated simultaneously or in series. For evaluation in series, the data derived for each biomarker can be combined after analysing the biomarker, e.g.
- a sample could be split into sub-samples and the sub-samples could be assayed in series.
- the diagnostic indicators obtained on a subset of the panel may indicate that a patient has PC without requiring analysis of any further members of the panel.
- Such incomplete analysis of the panel is encompassed by the invention because of the intention or potential of the method to analyse the complete panel.
- some embodiments of the invention can include a contribution from known tests for PC, such as PSA and/or PCA3 tests. Any known tests can be used e.g. total PSA score, PSA velocity, the PROGENSATM assay for urinary PCA3 mRNA, etc. Data interpretation
- the invention involves a step of determining the level(s) of Table 1 biomarker(s).
- this determination for a particular marker can be a simple yes/no determination (qualitative), whereas other embodiments may require a quantitative or semi-quantitative determination, still other embodiments may involve a relative determination (e.g. a ratio relative to another marker, or a measurement relative to the same marker in a control sample), and other embodiments may involve a threshold determination (e.g. a yes/no determination whether a level is above or below a threshold).
- a skilled person can easily determine the relative change (e.g. up-regulation or down- regulation) for any given miRNA marker relative to any particular control of interest (e.g. a negative control or a positive control) in any given sample (e.g. a blood sample or a prostate sample).
- a control sample can be a positive control sample or a negative control sample. Typically the control sample is age-matched against the test subject.
- a positive control sample includes samples from confirmed cases of the presence of PC.
- pathological examination of prostate samples is currently the only accurate diagnostic test for PC.
- the grade of PC which is a measure of how aggressive the cancer cells are, is determined pathologically based on a clinically defined scale called the "Gleason scale" [28].
- the lowest grade of PC would have a Gleason score of 6 (3+3).
- a positive control sample for the methods of the invention would have a Gleason score of 6 or more.
- a negative control sample includes samples from confirmed cases of the absence of PC.
- a non-PC sample can be a subject with presentation of other conditions or diseases, e.g. a prostate condition such as BPH or prostatitis, but not PC. Pathological examination of a prostate sample of a non-PC subject would give a Gleason score of less than 6.
- a non-PC sample is preferably from a subject with BPH, but not PC.
- the absolute levels of a biomarker in a particular control sample may be different from that in a nother control sample (e.g. samples of a non-PC subject who has bladder cancer).
- a nother control sample e.g. samples of a non-PC subject who has bladder cancer.
- the relative expression profiles e.g. up- or down-regulation or fold-changes
- PC samples compared to non-PC samples i.e. a negative control sample
- biomarkers will be measured to provide quantitative or semi-quantitative results (whether as relative concentration, absolute concentration, fold-change, etc. ) as this gives more data for use with classifier algorithms.
- raw data obtained from an assay for determining the presence, absence, or level requires some sort of manipulation prior to their use. For instance, the nature of most detection techniques means that some signal will sometimes be seen even if no miRNA is actually present and so this noise may be removed before the results are interpreted. Similarly, there may be a background level of the miRNA in the general population which needs to be compensated for. Data may need scaling or standardising to facilitate inter-experiments comparisons.
- replicate measurements will usually be performed (e.g. using duplicate or triplicate reactions during RT-qPCR) to determine intra-assay variation and average values from the replicates can be compared (e.g. the median value of the PCR product).
- standard markers can be used to determine inter-assay variation and to permit calibration and/or normalisation e.g. a RT-qPCR reaction can include one or more 'standards', of known concentration, to determine the amplification efficiency of the PCR reaction, and to permit estimation of the total miRNA content of an unknown sample, relative to other unknown samples.
- an assay might include a step of analysing the level of one or more control marker(s) in a sample e.g. levels of a miRNA unrelated to PC.
- Signal may be adjusted according to distribution in a single experiment. For instance, signals in a single reaction experiment may be expressed as a percentage of interquartile differences e.g. as [observed signal - 25th percentile] / [75th percentile - 25th percentile]. This percentage may then be normalised e.g. using a standard quantile normalisation matrix, such as disclosed in reference 29, in which all percentage values on a single reaction are ranked and replaced by the average of percentages for miRNAs with the same rank across all reactions. Overall, this process gives data distributions with identical median and quartile values. Data transformations of this type are standard in the art for permitting valid inter-array comparisons despite variation between different experiments.
- the level of a biomarker relative to a single baseline level may be defined as a fold difference. Normally it is desirable to use techniques that can indicate a change of at least 1.5-fold e.g. >1.75-fold, >2-fold, >2.5-fold, >5-fold, etc.
- the measured level(s) of Table 1 biomarker(s), after any compensation/normalisation/efr., can be transformed into a diagnostic result respectively in various ways. This transformation may involve an algorithm which provides a diagnostic result as a function of the measured level(s). Where a panel is used then each individual biomarker may make a different contribution to the overall diagnostic result and so two biomarkers may be weighted differently.
- linear or non-linear classifier algorithms can be used. These algorithms can be trained using data from any particular technique for measuring the marker(s). Suitable training data will have been obtained by measuring the biomarkers in "case” and "control" samples i.e. samples from subjects known to suffer from PC and from subjects known not to suffer from PC. Most usefully the control samples will also include samples from subjects with a related disease which is to be distinguished from the disease of interest e.g. it is useful to train the algorithm with data from subjects with BPH and/or with data from subjects with cancer(s) other than PC. The classifier algorithm is modified until it can distinguish between the case and control samples e.g.
- a method of the invention may include a step of analysing biomarker levels in a subject's sample by using a classifier algorithm which distinguishes between PC subjects and non-PC subjects based on measured biomarker levels in samples taken from such subjects.
- classifier algorithms are available e.g. linear discriminant analysis, naive Bayes classifiers, regression modelling, perceptrons, support vector machines (SVM) [30] and genetic programming (GP) [31], as well as a series of statistical methods including, but not limited to, Principal Component Analysis (PCA), unsupervised hierarchical clustering and linear modelling.
- PCA Principal Component Analysis
- GP is particularly useful as it generally selects relatively small numbers of biomarkers and overcomes the problem of trapping in a local maximum which is inherent in many other classification methods.
- SVM-based approaches have previously been used for PC diagnosis by classifying images of prostate tissue [32,33], patient data [34], or gene expression levels [35].
- these approaches can potentially distinguish PC subjects from subjects with (i) other prostate conditions such as BPH and prostatitis and (ii) other forms of cancer.
- the biomarkers in Table 1 ca n be used to train such algorithms to reliably make such distinctions.
- the resulting data will be ana lysed for any potential signatures relating to differences between patient cohorts referring to levels of statistical significance (genera lly p ⁇ 0.05), multiple testing correction and fold changes within the expression data that could be indicative of biological effect (normally it is desirable to use techniques that can indicate a change of at least 1.5 fold e.g. >1.75 fold, >2-fold, >2.5-fold, >5-fold, etc.).
- references herein to detecting a biomarker may not be references to absolute detection but rather (as is standard in the art) to a level above the levels seen in an appropriate negative control sample.
- Such controls may be assayed in parallel to a test sample but it can be more convenient to use an absolute control level based on empirical data, or to analyse data using an algorithm which can (e.g. by previous training) use biomarker levels to distinguish samples from disease patients vs. non-disease patients.
- the level of a particular biomarker in a sample from a PC-diseased subject may be a bove or below the level seen in a negative control sample (i.e. from a non-PC subject).
- the expression of miRNAs can either be up-regulated or down-regulated depending on the state of the individual. I n a control population of healthy individuals there may thus be significa nt levels of miRNAs disclosed in Table 1 and these may occur at a significant frequency in the population.
- the level and frequency of these biomarkers may be a ltered in a disease cohort, compared with the control cohort. An analysis of the level and frequency of these biomarkers in the case and control populations may identify differences which provide diagnostic information.
- the level of the miRNAs of the invention may increase or decrease in a PC sample, compared with a non-PC sample.
- a method of the invention will involve determining whether a sa mple expresses a biomarker level which is associated with PC.
- a method of the invention ca n include a step of comparing biomarker levels in a subject's sample to levels in (i) a sample from a subject with PC (i.e. a positive control sample) and/or (ii) a samples form a subject without PC (i.e. a negative control sample). The comparison provides a diagnostic indicator of whether the subject has PC.
- An aberrant level of one or more biomarker(s), as compa red to known or standard expression levels of those biomarker(s) in a sample from a patient without PC indicates that the subject has PC.
- I n an embodiment where the method comprises comparing the level of the biomarker in the subject's sa mple and the same biomarker in a negative control sam ple, (i) if the level of the biomarker is different to that in the negative control sample, this indicates that the subject has PC; or (ii) if the level of the bioma rker is as expected in the negative control sa mple, this indicates that the subject does not have PC.
- I n an embodiment where the method comprises comparing the level of the biomarker in the subject's sample and the same biomarker in a positive control sample, (i) if the level of the biomarker is different to those in the positive control sam ple, this indicates that the subject does not have PC; or (ii) if the level of the biomarker is as expected in the positive control sample, this indicates that the subject has PC.
- I n an embodiment where a biomarker expresses at a lower level tha n a threshold value in a negative control sample, and expresses at a higher level than the threshold value in a positive control sample, when the level of the biomarker in the subject's sample is compared with the threshold value, (i) if the level of the biomarker is higher than the threshold value, this indicates that the subject has PC; and (ii) if the level of the biomarker is lower than the threshold value, this indicates that the subject does not have PC.
- I n an embodiment where a biomarker expresses at a higher level than a threshold value in a negative control sample, and expresses at a lower level than the threshold value in a positive control sample, when the level of the biomarker in the subject's sample is compared with the threshold value, (i) if the level of the biomarker is lower than the threshold value, this indicates that the subject has PC; and (ii) if the level of the biomarker is higher than the threshold value, this indicates that the subject does not have PC.
- the levels of the biomarkers of the invention in a PC sa mple should be different from those seen in a negative control sample.
- Advanced statistical tools can be used to determine whether two levels are the same or different. For example, an in vitro diagnosis will rarely be based on comparing a single determination. Rather, an appropriate number of determinations will be made with an appropriate level of accuracy to give a desired statistical certainty with an acceptable sensitivity and/or specificity.
- Levels of miRNAs can be measured quantitatively to permit proper comparison, and enough determinations will be made to ensure that any difference in levels can be assigned a statistical significance to a level of p ⁇ 0.05 or better.
- the number of determinations will vary according to various criteria (e.g. the degree of variation in the baseline, the degree of up-regulation in disease states, the degree of noise, etc. ) but, again, this falls within the normal design capabilities of a person of ordinary skill in this field.
- interquartile differences of normalised data can be assessed, and the threshold for a positive signal (i.e. indicating the presence or absence of a particular miRNA) can be defined as requiring that miRNAs in a sample hybridise with the complementary detection probe with at least a log change +/-0.585 th a n the interquartile difference above the 75th percentile.
- Other criteria are familiar to those skilled in the art and, depending on the assays being used, they may be more appropriate than quantile normalisation.
- Other methods to normalise data include data transformation strategies known in the art e.g. scaling, log normalisation, median normalisation, etc.
- Methods of the invention may have sensitivity of at least, but not limited to, 50% (e.g. >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >96%, >97%, >98%, >99%).
- Methods of the invention may have specificity of at least, but not limited to, 50% (e.g. >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >96%, >97%, >98%, >99%).
- Data obtained from methods of the invention, and/or diagnostic information based on those data may be stored in a computer medium (e.g. in RAM, in non-volatile computer memory, on CD-ROM, DVD) and/or may be transmitted between computers e.g. over the I nternet.
- a computer medium e.g. in RAM, in non-volatile computer memory, on CD-ROM, DVD
- a method of the invention indicates that a subject has PC
- further steps may then follow.
- the subject may undergo confirmatory diagnostic procedures, such as those involving physical inspection of the subject, and/or may be treated with therapeutic agent(s) suitable for treating PC.
- Methods of the invention may involve testing samples from the same subject at two or more different points in time.
- the invention also includes an increasing or decreasing level of the biomarker(s) over time.
- Methods which determine changes in biomarker(s) over time can be used, for instance, to monitor the efficacy of a therapy being administered to the subject (e.g. in theranostics).
- the therapy may be administered before the first sample is taken, at the same time as the first sample is taken, or after the first sample is taken.
- the invention can be used to monitor a subject who is receiving PC therapy.
- Current therapies for PC include chemotherapy and/or hormone therapy.
- Hormone therapy seeks to block access of dihydrotestosterone (DHT) to prostate cells or to block the effects of DHT within prostate cells.
- Anti-androgens are medications such as flutamide, bicalutamide, nilutamide, and cyproterone acetate which directly block the actions of testosterone and DHT within PC cells. They may be given in combination with drugs such as ketoconazole and aminoglutethimide which block the production of adrenal androgens.
- the invention also provides a method for monitoring development of PC in a subject, comprising steps of: (i) determining the levels of zi biomarker(s) of Table 1 in a first sample from the subject taken at a first time; and (ii) determining the levels of z 2 biomarker(s) of Table 1 in a second sample from the subject taken at a second time, wherein: (a) the second time is later tha n the first time; (b) one or more of the z 2 biomarker(s) were present in the first sample; and (c) a change in the level(s) of the biomarker(s) in the second sample compared with the first sample indicates that PC is in remission or is progressing.
- the method monitors the biomarker(s) over time, with changing levels indicating whether the disease is getting better or worse.
- the disease development can be either an improvement or a worsening, and this method may be used in various ways e.g. to monitor the natural progress of a disease, or to monitor the efficacy of a therapy being administered to the subject.
- a subject may receive a therapeutic agent before the first time, at the first time, or between the first time and the second time.
- zi is 1 or more e.g. 1, 2 or 3.
- z 2 is 1 or more e.g. 1, 2 or 3.
- the values of zi and z 2 may be the same or different. If they a re different, it is usual that zi>z 2 as the later analysis (z 2 ) can focus on biomarkers which were already detected in the earlier analysis; in other embodiments, however, z 2 ca n be larger tha n zi e.g. if previous data have indicated that an expanded panel should be used; in other embodiments e.g. so that, for convenience, the same panel can be used for both analyses.
- z : >l or z 2 >l the biomarkers are different biomarkers.
- the invention also provides a method for monitoring development of PC in a subject, comprising steps of: (i) determining the level of at least wi Table 1 biomarkers in a first sample taken at a first time from the subject; and (ii) determining the level of at least w 2 Table 1 biomarkers in a second sample taken at a second time from the subject, wherein: (a) the second time is later than the first time; (b) at least one biomarker is common to both the Wi and w 2 biomarkers; (c) the level of at least one biomarker common to both the ⁇ : and w 2 biomarkers is different in the first and second samples, thereby indicating that the PC is progressing or regressing.
- the method monitors the range of biomarkers over time, with a broadening in the number of detected biomarkers indicating that the disease is getting worse.
- this method may be used to monitor disease development in various ways.
- the value of ⁇ : is 1 or more e.g. 1, 2 or 3.
- the value of w 2 is 2 or more e.g. 1, 2 or 3.
- the values of ⁇ : and w 2 may be the same or different. If they are different, it is usual that w 2 >w lt as the later analysis should focus on a biomarker panel that is at least as wide as the number already detected in the earlier analysis. There will usually be an overlap between the wi and w 2 biomarkers (including situations where they are the same, such that the same biomarkers are measured at two time points) but it is also possible for wi and w 2 to have no biomarkers in common.
- the methods involve a first time and a second time, these times may differ by at least 1 day, 1 week, 1 month or 1 year. Samples may be taken regularly. The methods may involve measuring biomarkers in more than 2 sam ples ta ken at more than 2 time points i.e. there may be a 3rd sample, a 4th sample, a 5th sample, etc. I n related embodiments of the invention, the results of monitoring a therapy are used for future thera py prediction.
- the miRNAs listed in Table 1 can be useful for imaging.
- a labelled, synthetic miRNA complementary to a miRNA(s) listed in Table 1 could be used for the identification, in ex vivo (e.g. tissue samples taken from biopsies), and in vivo (e.g. magnetic resonance imaging (MRI), positron emission tomography (PET) computed tomography (CT) scans of patients) samples of miRNAs associated with PC.
- MRI magnetic resonance imaging
- PET positron emission tomography
- CT computed tomography
- the miRNAs listed in Table 1 can be useful for analysing tissue samples by staining e.g. using standard FISH.
- a fluorescently labelled miRNA, complementary in sequence to the miRNAs outlined in Table 1 can be contacted with a tissue sample to visualise the location of the miRNA.
- a single sample could be stained against multiple miRNAs, and these different miRNAs may be differentially labelled to enable them to be distinguished.
- a plurality of different samples can each be stained with a single, labelled miRNA.
- the invention provides a labelled nucleic acid which can hybridise to miRNA(s) listed in Table 1.
- the miRNA may be, but not limited to, a human miRNA, as discussed above. Any suitable label can be used e.g.
- miRNAs can be used in methods of in vivo and/or in vitro imaging.
- microRNA-based therapy The miRNAs listed in Table 1 can be useful for miRNA-based therapy, e.g., antisense therapy.
- antisense therapy There is literature precedent outlining the use of antisense therapy to manage cancer [36].
- a synthetic miRNA complementary to a miRNA(s) listed in Ta ble 1 could be used to stimulate cell death of cancerous cells (associated with PC).
- in vivo antisense therapy could be used to introduce miRNA complementary to a miRNA(s) listed in Table 1 to specifically bind to, and abrogate, overexpression of specific miRNA(s) associated with PC.
- the invention provides a nucleic acid which hybridises to miRNA(s) listed in Table 1 and which is conjugated to a cytotoxic agent.
- the miRNA may be, but not limited to, a human miRNA, as discussed above. Any suitable cytotoxic agent can be used. These conjugates miRNAs can be used in methods of therapy.
- the invention provides a complementary miRNA which recognises a miRNA(s) listed in Table 1 for the purposes of miRNA-based therapies which include, but not limited to, antisense therapy.
- the invention also uses panels of biomarkers.
- Panels of particular interest for the diagnosis of PC consist of, or comprise, the combinations of biomarkers from Table 1, which are listed in Tables 4-5 and 7-8.
- Other panels of particular interest for the diagnosis of PC consist of, or comprise, one or more Table 1 biomarkers in combination with one or more Table 2 biomarkers, and these panels are listed in Tables 9-12 and 15 (which show panels of two or three biomarkers).
- Preferred panels comprise hsa-miR-451a.
- the different panels listed in each of Tables 4-5, 7-12 and 15 can be expanded by adding further biomarker(s) to create a larger panel.
- the further biomarkers can usefully be selected from known biomarkers (such as PSA, PCA3, DD3, AMACR, EPCA, EPCA-2, sarcosine, etc.; see above), or from Table 13, or from Table 1, or from Table 2 where appropriate. In general the addition does not decrease the sensitivity or specificity of the panel shown in the Tables.
- Such panels include, but are not limited to:
- a panel comprising or consisting of hsa-miR-451a and hsa-miR-16-5p.
- a panel comprising or consisting of hsa-miR-451a and hsa-miR-20a-5p.
- a panel comprising or consisting of the three biomarkers in Table 1, which are hsa- miR-451a, hsa-miR-16-5p and hsa-miR-20a-5p.
- a panel comprising or consisting of 2 different biomarkers, namely: (i) a biomarker selected from Table 1 and (ii) a further biomarker selected from Table 2. Examples of such a panel are provided in Tables 9 and 11. • A panel comprising or consisting of 3 different biomarkers, namely: (i) a biomarker selected from Table 1 and (ii) two further biomarkers selected from Table 2. Examples of such a panel are provided in Tables 8, 10, 12 and 15.
- a panel comprising or consisting of hsa-miR-375, hsa-miR-451a and hsa-miR-665.
- a panel comprising or consisting of hsa-miR-33b-3p, hsa-miR-451a and hsa-miR-99b- 3p.
- a panel comprising or consisting of hsa-miR-33b-3p, hsa-miR-451a and hsa-miR- 133a-3p.
- Preferred panels have three biomarkers in total.
- biomarkers can have different relative differential expression profiles in a PC sample compared to a control sample. Pairs of these biomarkers (i.e. where one is up- regulated and the other is down-regulated relative to the same control) may provide a useful way of diagnosing PC. For example, the inventors found that hsa-miR-451a is up- regulated in PC samples vs. BPH samples and hsa-miR-20a-5p is down-regulated in PC samples vs. BPH samples, so this pair would be useful. This divergent behaviour can enhance diagnosis of PC when a pair of the biomarkers is assessed in the same sample.
- a method of the invention can include a step of determining the expression levels of a first and a second biomarker of the invention in a subject's sample, wherein the first biomarker is up-regulated in a PC sample compared to a non-PC sample and the second biomarker is down-regulated in a PC sample compared to the same non-PC sample.
- a method of the invention can include: (i) determining the expression level of a first biomarker of the invention in a subject's sample, (ii) determining the expression level of a second biomarker of the invention in the subject's sample, wherein the first biomarker is up-regulated in a PC sample compared to a non-PC sample and the second biomarker is down-regulated in a PC sample compared to the same non-PC sample, and (iii) comparing the determinations of (i) and (ii) with a non-PC sample, a PC sample and/or an absolute value, wherein the comparison provides a diagnostic indicator of whether the subject has PC.
- Aberrant levels of the first and the second biomarkers, as compared to the known or standard expression levels of them in the non-PC sample or PC sample, and/or the absolute value indicate that the subject has PC.
- the invention provides diagnostic devices and kits for detecting the biomarkers of the invention.
- the invention also provides a diagnostic device for use in diagnosis of PC, wherein the device permits determination of the levels of one or more biomarkers listed Table 1, and either (i) one or more biomarkers listed in Table 2, or (ii) one or more of the known PC biomarkers mentioned above e.g. PSA and/or PCA3.
- the invention also provides a diagnostic device for use in discriminating between PC and a confounding prostate condition in a subject, wherein the device permits determination of the levels of one or more biomarkers listed Table 1, and either (i) one or more biomarkers listed in Ta ble 2, or (ii) one or more of the known PC biomarkers mentioned above e.g. PSA and/or PCA3.
- the invention also provides a kit comprising (i) a diagnostic device of the invention and (ii) instructions for using the device to detect any of the of the Table 1 biomarkers.
- the kit is useful in the diagnosis of PC.
- the kit is particularly useful in discriminating between PC and a confounding prostate condition in a subject.
- the invention also provides a kit comprising reagents for measuring the levels of any of the Table 1 biomarkers.
- the kit may also include reagents for determining whether a sample contains one or more of the known PC biomarkers mentioned above e.g. PSA and/or PCA3, and/or any of the biomarkers listed in Ta ble 2.
- the kit is useful in the diagnosis of PC.
- the kit is particularly useful in discriminating between PC and a confounding prostate condition in a subject.
- the invention also provides a kit comprising components for preparing a diagnostic device of the invention.
- the kit may comprise individua l detection reagents for a ny of the Table 1 biomarkers, such that a selection of those Table 1 biomarkers can be prepared.
- the invention also provides a product comprising (i) one or more detection reagents which permit measurement of any of the Table 1 biomarkers, and (ii) a sample from a subject.
- the invention has been described above by reference to miRNA bioma rkers. I n addition to these biomarkers, however, the invention ca n be used with other biological manifestations of the Table 1 miRNAs.
- the expression level of mRNA transcripts which are a target of a Table 1 miRNA can be measured, particularly in blood where changes in transcription level can easily be determined (such as in the potential disease blood samples).
- the copy number variation of a chromosomal location of a Table 1 miRNA can be measured e.g. to check for a chromosomal deletion or duplication events.
- the level of a regulator of transcription for a Table 1 miRNA can be measured e.g. the methylation status of the miRNA chromosomal region.
- a single pre-miRNA precursor may lead to one or more mature miRNA sequences, such as sequences excised from the 5' and 3' arms of the hairpin, as shown in Table 14.
- the invention can be used to look for other mature miRNA sequences from the same pre-miRNA precursor.
- other mature miRNA sequences from the same precursor in Table 14 may be appropriate biomarkers as well.
- composition comprising X may consist exclusively of X or may include something additional e.g. X + Y.
- references to a miRNA's ability to "hybridise" to a complementary oligonucleotide probe means that the miRNA and the complementary oligonucleotide probe interact that nonspecific binding will be minimised or eliminated.
- references to a "level" of a biomarker mean the amount of an analyte (e.g. a miRNA) measured in a sample and this encompasses relative and absolute concentrations of the analyte, analyte titres, relationships to a threshold, rankings, percentiles, etc.
- an analyte e.g. a miRNA
- An assay's "sensitivity" is the proportion of true positives which are correctly identified i.e. the proportion of PC subjects who test positive by a method of the invention. This can apply to individual biomarkers, panels of biomarkers, single assays or assays which combine data integrated from multiple sources e.g. PSA score and DRE. It can relate to the ability of a method to identify samples containing a specific analyte (e.g. miRNAs) or to the ability of a method to correctly identify samples from subjects with PC.
- a specific analyte e.g. miRNAs
- An assay's "specificity" is the proportion of true negatives which are correctly identified i.e. the proportion of subjects without PC who test negative by a method of the invention. This can apply to individual biomarkers, panels of biomarkers, single assays or assays which combine data integrated from multiple sources e.g. PSA score and DRE. It can relate to the ability of a method to identify samples containing a specific analyte (e.g. miRNAs) or to the ability of a method to correctly identify samples from subjects with PC.
- a specific analyte e.g. miRNAs
- a method comprising a step of mixing two or more components does not require any specific order of mixing.
- components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.
- references to a percentage sequence identity between two miRNA sequences means that, when aligned, that percentage of nucleotides are the same in comparing the two sequences.
- This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref.37.
- a preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62.
- the Smith-Waterman homology search algorithm is disclosed in ref.38. BRIEF DESCRIPTION OF DRAWINGS
- FIG. 1 shows receiver operating characteristic (ROC) curves for hsa-miR-375, hsa-miR- 451a, hsa-miR-665 and the combination of these three miRNAs.
- ROC receiver operating characteristic
- Figure 2 demonstrates the diagnostic performance ("call rate") of a panel of the invention (hsa-miR-375 + hsa-miR-451a + hsa-miR-665) compared to those of the individual miRNAs.
- the samples are diagnosed using the current 'gold standard' of histopathological examination ("Clinical diagnosis” column).
- EDTA anti-coagulant
- RNA total RNA, including miRNA
- the samples were run using the LifeTech ViiATM7 qPCR machine, capable of exciting the reporter dye (e.g. FAMTM dye) and capturing the emission via specific emission filters.
- the qPCR runs produce amplification traces, for each individual sample, with the intensity of fluorescence emission being directly proportional to the amount of amplification.
- the procedure of qPCR is well known in the art.
- a threshold value is applied to the raw data (using LifeTech's proprietary software analysis program), which generates a Ct value for each sample.
- the samples may be processed in triplicate and a mean and/or median value for this triplicate calculated.
- Analyses of qPCR data is known in the art.
- a logistic regression model was developed for each marker or combination of markers (up to 3 in total) and 95% confidence intervals for the corresponding ROC curve were generated for "Study 1"
- the "Study 1" data was used to train the statistical algorithm to select marker(s), based on their performance, to differentiate cancer samples from non-cancer samples.
- the performance of the derived panels was then ranked by combined S+S.
- Table 1 and Table 2 miRNAs Panels of combined Table 1 and Table 2 miRNAs were generated and the performance of these panels (sensitivity, specificity and S+S values) is provided in Tables 11-12. This highlights that the Table 2 miRNAs augment the performance of the Table 1 miRNAs in diagnosis of PC. It is noted that the performance of the "Study 2" panels is slightly reduced against that of "Study 1", but this is to be expected on a blinded sample set. Despite this, the performance of the panels of the invention, in terms of S+S, is still better than the performance of PSA.
- Figure 1 shows a ROC curve generated using the data set from this study.
- the ROC curve plots the performance of the 3-mer panel (hsa-miR-375 + hsa-miR-451a + hsa-miR-665) against the three individual miRNAs that make up this panel.
- the Area Under the Curve (AUC) values for hsa-miR-451a is 0.9 and for the 3-mer panel is 0.95. This demonstrates that the performance of the 3-mer panel augments the performance of the individual miRNAs.
- this 3-mer panel provides a much better AUC value than those of PSA and PSA+PCA3, which were determined to be 0.63 and 0.71, respectively [4].
- a comparison of the "call rate” is made between the 3-miRNA panel (hsa-miR-375 + hsa- miR-451a + hsa-miR-665) and the individual miRNAs.
- the comparison is made to the current 'gold standard' of histopathological examination ("Clinical diagnosis” column). Any variation from the "Clinical diagnosis” may be due to subtle biochemical changes, which would be indicative of cancer, and may yet demonstrate a true-positive result.
- Figure 2 demonstrates that the 3-miRNA panel (hsa-miR-375 + hsa-miR-451a + hsa-miR-665) produces a more accurate "call rate" when compared to the individual miRNAs.
- hsa-miR-665 provides 14 false-positives and 7 false-negatives of PC;
- hsa-miR-375 provides 10 false- positives and 8 false-negatives of PC;
- hsa-miR-451a provides 5 false-positives and 3 false- negatives of PC;
- the 3-mer panel provides only 3 false-positives and only 3 false- negatives of PC.
- Biomarkers of the invention demonstrate "field-effect"
- the expression levels of the miRNA biomarkers of the invention were analysed in prostate tissue samples.
- the inventors also tested whether the miRNA biomarkers of the invention show a 'field-effect' within prostate tissue.
- the concept of 'field-effect' within cancer dates back to the early 1950s when Slaughter et al. [43] described the phenomenon of abnormal tissue surrounding the primary site of oral squamous cell carcinoma. Since then, various researchers have demonstrated cancer field-effect within a variety of different tissues and organs, and that this field-effect has been attributed, in part, to aberrant DNA methylation in various genes (e.g. 44, 45, 46).
- hsa-miR-451a is a field-effect marker for PC (see Table 15).
- Using macro-dissected prostate tissue a selection of samples originally confirmed as histopathologically negative, dissected from the same lobe as the primary cancer foci, were found to be biochemically positive for PC using hsa-miR-451a as a biomarker.
- hsa-miR-451a can show a 'field-effect' within the prostate gland, whereby differential relative expression profiles (e.g. PC sample compared to a control) of these miRNAs can be observed in samples from any part of the same prostate lobe as where the cancer foci is situated.
- hsa-miR-451a and panels including hsa-miR-451a
- PC differential relative expression profiles
- the measured biomarker(s) can be (i) up-regulated (an increase in fold-change, when compared to negative control samples) or (ii) down-regulated (a decrease in fold-change, when compared to negative control samples).
- miRNA name gives the name of the human miRNA as provided by the specialist database, miRBase. The name is correct to miRBase version 20 (released, June 2013).
- the HGNC aims to give unique and meaningful names to every miRNA (and human gene).
- the HGNC number thus identifies a unique human gene.
- Inclusion on to HUGO is for human genes only.
- the measured biomarker(s) can be (i) up-regulated (an increase in fold-change, when compared to negative control samples) or (ii) down-regulated (a decrease in fold-change, when compared to negative control samples).
- miRNA name gives the name of the human miRNA as provided by the specialist database, miRBase. The name is correct to miRBase version 20 (released, June 2013).
- the HGNC aims to give unique and meaningful names to every miRNA (and human gene).
- the HGNC number thus identifies a unique human gene.
- Inclusion on to HUGO is for human genes only.
- Tables 3-5 list biomarkers and panels of biomarkers useful with the invention, for the diagnosis of PC, generated from the 'training' set of samples in Study 1.
- the measured biomarker(s) can be (i) up-regulated (an increase in fold-change, when compared to control samples) or (ii) down-regulated (a decrease in fold-change, when compared to control samples).
- Tables 6-8 list biomarkers and panels of biomarkers that are useful with the invention, for the diagnosis of PC, when the statistical algorithm generated in Study 2.
- the measured biomarker(s) can be (i) up-regulated (an increase in fold-change, when compared to negative control samples) or (ii) down-regulated (a decrease in fold-change, when compared to negative control samples).
- S+S is the sum of the sensitivity and specificity columns. These final two columns show the sensitivity and specificity of a test based solely on the relevant biomarker (or, for Tables 7-8, panel) shown in the left-hand column when applied to the samples used in the examples.
- Tables 9 and 10 list panels of biomarkers useful with the invention for the diagnosis of PC. Each panel includes biomarkers from any of the biomarkers listed in Table 1 or Table 2.
- the diagnostic performance (sensitivity + specificity) is generated in Study 1.
- the measured biomarker(s) can be (i) up-regulated (an increase in fold-change, when compared to negative control samples) or (ii) down-regulated (a decrease in fold-change, when compared to negative control samples).
- S+S is the sum of the sensitivity and specificity columns. These final two columns show the sensitivity and specificity of a test based solely on the relevant panel shown in the left-hand column when applied to the samples used in the examples.
- Tables 11 and 12 list the panels of biomarkers that are useful with the invention for the diagnosis of PC. Each panel includes biomarkers from any of the biomarkers listed in Table 1 and Table 2.
- the diagnostic performance (sensitivity + specificity) is generated from the statistical algorithm in Study 2.
- the measured biomarker(s) can be (i) up-regulated (an increase in fold-change, when compared to negative control samples) or (ii) down-regulated (a decrease in fold-change, when compared to negative control samples).
- Table 13 lists all the biomarkers listed in Table 1 and Table 2.
- Table 13 states the official name of the miRNA biomarkers (according to NCBI), as well as their unique Entrez GenelD number (“ID”) and Reference Sequence ID number (“RefSeq ID”).
- the "ID” column shows the Entrez Genel D number for the miRNA. An Entrez GenelD value is unique across all taxa.
- the "RefSeq I D" column shows the Reference Sequence I D number for the miRNA, processed by NCBI.
- a Reference Sequence I D provides a stable reference for genome annotation, gene identification and characterisation.
- a Reference Sequence ID number is unique across all taxa.
- Table 14 lists all the biomarkers listed in Table 1 and Table 2.
- Table 14 provides the accession number and sequence (according to miRBase) fo the precursor hairpin, as well as the mature, processed miRNAs (for both the 5' and 3' arm of the hairpin, where applicable). Additionally, th genomic location of the hairpin is also provided.
- the "Hairpin sequence” column gives the sequence information of the precursor hairpin, which is processed biologically, to yield the mature human miRNA, a provided by the specialist database, miRBase.
- the name is correct to miRBase version 20 (released, June 2013).
- the "Mature sequence (-3p)" column gives the sequence information of the mature, processed, miRNA located on the 3' arm, as provided by the specialist database miRBase. The name is correct to miRBase version 20 (released, June 2013).
- Table 15 Panels of biomarkers useful with the invention of field-effect detection
- Table 15 lists panels of biomarkers that are useful for detecting PC in prostate tissue sa mples, where the samples are obtained from the sam prostate lobe as the cancer foci (i.e. these biomarkers show a "field-effect").
- the measured biomarker(s) can be (i) up-regulated (an increase i fold-change, when compared to negative control) or (ii) down-regulated (a decrease in fold-change, when compared to negative control).
- S+S is the sum of the sensitivity and specificity columns. These final two columns show the sensitivity and specificity of a test based solely on the relevant biomarkers i the panel shown in the left-hand column.
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Abstract
La présente invention concerne des biomarqueurs de micro-ARN utiles dans le diagnostic du cancer de la prostate. Les biomarqueurs sont également utiles pour la surveillance et/ou le traitement du cancer de la prostate.
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GB201407003A GB201407003D0 (en) | 2014-04-17 | 2014-04-17 | Biomarkers for prostate cancer |
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CN116465920A (zh) * | 2023-04-03 | 2023-07-21 | 汕头大学医学院 | 用于诊断食管癌的代谢标志物 |
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