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WO2010020787A1 - Diagnostic et traitement des tumeurs - Google Patents

Diagnostic et traitement des tumeurs Download PDF

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Publication number
WO2010020787A1
WO2010020787A1 PCT/GB2009/002040 GB2009002040W WO2010020787A1 WO 2010020787 A1 WO2010020787 A1 WO 2010020787A1 GB 2009002040 W GB2009002040 W GB 2009002040W WO 2010020787 A1 WO2010020787 A1 WO 2010020787A1
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Prior art keywords
microrna
gene
family gene
tumour
methylation
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Amaia Lujambio
Manel Esteller
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MDxHealth SA
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OncoMethylome Sciences SA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to diagnosis and treatment of disease. More specifically, the invention relates to methods and kits for diagnosing or identifying a tumour, which include determining the methylation status, or the expression levels, or a combination thereof, of one or more genes encoding microRNAs. In particular the invention relates to the detection of tumours with metastatic potential or metastases or secondary tumours. The invention also relates to pharmacogenetic methods for determining suitable treatment regimens for tumours and methods for treating tumours. Related kits are also described.
  • miRNAs are small, non-coding RNAs, about 22 nucleotides long, that repress gene expression in a variety of eukaryotic organisms (1 ,2). In animals, these single stranded RNAs interact with specific target mRNAs through an almost perfect complementarity with sequences located in the 3'-untranslated region (UTR), where they induce mRNA degradation or translational inhibition (1 ,2). miRNAs play important roles in several cellular processes, such as proliferation, differentiation, apoptosis and development, by simultaneously controlling the expression levels of hundreds of genes (1 ,2). In human cancer, recent studies have shown that miRNA expression profiles differ between normal tissues and derived tumours and between tumour types (3,4).
  • RNAs such as miR- 148a, miR-34b/c and miR-9, that contribute in vitro and in vivo to the formation of lymph node metastasis, one of the first steps in the dissemination of human tumours.
  • the tumour may be any type of tumour.
  • the tumour is a solid tumour.
  • the tumour is selected from a lung, breast, colon, or head and neck tumour or a melanoma.
  • a tumour with metastatic potential is one which has the capability to metastasize.
  • a metastasis comprises tumour cells from a primary tumour which have left the site of the primary tumour. The cells may form a metastasis or secondary tumour at a new site within the body. The metastasis or secondary tumour thus comprises cells from the primary tumour and can be identified as such. The spread of metastases may occur via the lymphatic system or the vasculature, or both.
  • microRNA 148 family genes Methylation of specific microRNA genes has been experimentally linked to specific tumour types.
  • the invention is concerned with microRNA 148 family genes, microRNA 34 family genes and microRNA 9 family genes.
  • MIRN148A is the gene symbol for microRNA 148a (located on chromosome 7, sequence accession ID: NT_007819).
  • MIRN34C is the gene symbol for microRNA 148c (located on chromosome 11, sequence accession ID: NT_033899).
  • MIRN9-1 is the gene symbol for microRNA 9-1 (located on chromosome 1 , sequence accession ID: NT_004487).
  • MIRN9-2 is the gene symbol for microRNA 9-2 (located on chromosome 5, sequence accession ID: NT_006713)
  • MIRN9-3 is the gene symbol for microRNA 9-3 (located on chromosome 15, sequence accession ID: NT_010274).
  • MIRN MIRN
  • microRNA microRNA
  • miR- miR-
  • the microRNA 148 family gene is microRNA 148a.
  • the tumour may be selected from a lung or breast tumour or melanoma.
  • the methylation status or expression level may be confirmed by determining expression levels of TGIF. It is shown herein that TGIF levels correlate with the methylation status and expression level of the microRNA 148a gene. A corresponding increase in expression levels of TGIF indicates methylation (or hypermethylation) and/or reduced levels of expression of microRNA 148a.
  • the microRNA 34 family gene is microRNA 34b or microRNA 34c.
  • the tumour may be selected from a lung or breast tumour or a melanoma.
  • the methylation status or expression level may be confirmed by determining expression levels of C-MYC and/or CDK6. It is shown herein that C-MYC and CDK6 levels correlate with the methylation status and expression level of the microRNA 34b gene. A corresponding increase in expression levels of C-MYC and/or CDK6 indicates methylation (or hypermethylation) and/or reduced levels of expression of microRNA 34b.
  • the methylation status or expression level may be confirmed by determining expression levels of E2F3. It is shown herein that E2F3 levels correlate with the methylation status and expression level of the microRNA 34c gene. A corresponding increase in expression levels of E2F3 indicates methylation (or hypermethylation) and/or reduced levels of expression of microRNA 34c.
  • the microRNA 9 family gene is selected from microRNA 9-1 or microRNA 9-2 or microRNA 9-3.
  • the tumour that is diagnosed or identified may be selected from a lung, breast or head and neck tumour.
  • the microRNA 9 family gene is microRNA 9-2
  • the tumour may be selected from a lung, breast or head and neck tumour or a melanoma.
  • the microRNA 9 family gene is microRNA 9-3
  • the tumour may be selected from a colon, lung or breast tumour or a melanoma.
  • Methylation is an example of an epigenetic change.
  • "Epigenetic change” is defined herein to include alterations resulting in diminished gene expression potential, mediated by mechanisms other than alterations in the primary nucleotide sequence of a gene.
  • the epigenetic change is generally epigenetic silencing in this invention.
  • Epigenetic silencing may be caused by DNA methylation in certain embodiments.
  • the epigenetic change in the genes of present invention is generally epigenetic silencing caused by (aberrant) DNA methylation.
  • Aberrant methylation may also be referred to as hypermethylation and relates to increased levels of methylation within the gene or genes of interest.
  • methylation status refers to the presence or absence of a methylated cytosine residue in one or more CpG dinucleotides within the nucleic acid or gene of interest.
  • the CpG islands are found in the promoter region and may begin (just) upstream of a promoter and extend downstream into the transcribed region. Methylation of a CpG island at a promoter often prevents expression of the gene.
  • the islands can also surround the 5' region of the coding region of the gene as well as the 3' region of the coding region.
  • CpG islands can be found in multiple regions of a nucleic acid sequence including upstream of coding sequences in a regulatory region including a promoter region, in the coding regions (e.g.
  • exons downstream of coding regions in, for example, enhancer regions, and in introns. All of these regions can be assessed to determine their methylation status, as appropriate.
  • the methylation status of the gene is assessed by determining levels of methylation in the promoter, intron, exoni and/or exon2 region of the gene.
  • a "promoter” is a region upstream from the transcription start site (TSS), extending between approximately 10 Kb, 4 Kb, 3Kb, 1 Kb, 500 bp or 150 to 300 bp from the TSS.
  • TSS transcription start site
  • levels of methylation may be assessed in the intron and/or exon regions.
  • the region for assessment may be a region that comprises both intron and exon sequences and thus overlaps both regions. CpG islands are readily identifiable through a range of techniques, including sequencing and in silico predictive methods.
  • the methods of the invention may investigate an epigenetic change, and in particular the methylation status, of the relevant gene or genes around the TSS, the genomic location of which is shown herein (in Fig.7 for example) for each of the relevant genes.
  • the methods of the invention may investigate an epigenetic change, and in particular the methylation status within, or between, and optionally including, the primer binding sites of the primers listed in the tables.
  • the methods may investigate an epigenetic change, and in particular the methylation status, within or between the genomic locations listed in Table 5 which are the binding sites of the respectve primers.
  • the methods may investigate the genomic region between (and including) the binding sites of the MSP primers (SEQ ID NOs 47 and 48 - miR-9-1 methylated, 49 and 50 - miR-9-1 unmethylated, 51 and 52 - miR-9-2 methylated, 53 and 54 - miR-9-2 unmethylated, 55 and 56 - miR-9-3 methylated, 57 and 58 - miR-9-3 unmethylated, 59 and 60 - miR- 34b/c methylated, 61 and 62 - miR-34b/c unmethylated, 63 and 64 - miR-148a methylated, 65 and 66 - miR-148a unmethylated respectively) or the bisulphite sequencing primers (SEQ ID Nos 13 and 14 - miR-9-1 , 15 and 16 - miR-9-2, 17 and 18 - miR-9-3, 19 and 20 - miR-34b/c, 21 and 22 - miR-148a respectively).
  • the MSP primers SEQ ID
  • Determination of the methylation status may be achieved through any suitable means. Suitable examples include bisulphite genomic sequencing and/or by methylation specific
  • Techniques for assessing methylation status are based on distinct approaches. Some include use of endonucleases. Such endonucleases may either preferentially cleave methylated recognition sites relative to non-methylated recognition sites or preferentially cleave non-methylated relative to methylated recognition sites. Some examples of the former are Ace III, Ban I, BstN I, Msp I, and Xma I. Examples of the latter are Ace II, Ava
  • cleavage pattern is indicative for the presence or absence of a methylated CpG dinucleotide.
  • Cleavage patterns can be detected directly, or after a further reaction which creates products which are easily distinguishable. Means which detect altered size and/or charge can be used to detect modified products, including but not limited to electrophoresis, chromatography, and mass spectrometry.
  • the identification of methylated CpG dinucleotides may utilize the ability of the methyl binding domain (MBD) of the MeCP2 protein to selectively bind to methylated DNA sequences (Cross et al, 1994; Shiraishi et al, 1999).
  • MBD methyl binding domain
  • the MBD may also be obtained from MBP, MBP2, MBP4, poly-MBD (Jorgensen et al., 2006) or from reagents such as antibodies binding to methylated nucleic acid.
  • the MBD may be immobilized to a solid matrix and used for preparative column chromatography to isolate highly methylated DNA sequences. Variant forms such as expressed His-tagged methyl-CpG Q _
  • binding domain may be used to selectively bind to methylated DNA sequences.
  • restriction endonuclease digested genomic DNA is contacted with expressed His-tagged methyl-CpG binding domain.
  • Other methods are well known in the art and include amongst others methylated-CpG island recovery assay (MIRA).
  • MIRA methylated-CpG island recovery assay
  • Another method, MB-PCR uses a recombinant, bivalent methyl-CpG-binding polypeptide immobilized on the walls of a PCR vessel to capture methylated DNA and the subsequent detection of bound methylated DNA by PCR.
  • Suitable chemical reagents include hydrazine and bisulphite ions.
  • the methods of the invention may use bisulphite ions, in certain embodiments.
  • the bisulphite conversion relies on treatment of DNA samples with sodium bisulphite which converts unmethylated cytosine to uracil, while methylated cytosines are maintained (Furuichi et al., 1970). This conversion finally results in a change in the sequence of the original DNA.
  • primers for assessing the methylation status at CpG dinucleotides.
  • Two approaches to primer design are possible. Firstly, primers may be designed that themselves do not cover any potential sites of DNA methylation. Sequence variations at sites of differential methylation are located between the two primers and visualisation of the sequence variation requires further assay steps. Such primers are used in bisulphite genomic sequencing, COBRA, Ms-SnuPE and several other techniques.
  • primers may be designed that hybridize specifically with either the methylated or unmethylated version of the initial treated sequence. After hybridization, an amplification reaction can be performed and amplification products assayed using any detection system known in the art. The presence of an amplification product indicates that a sample hybridized to the primer.
  • the specificity of the primer indicates whether the DNA had been modified or not, which in turn indicates whether the DNA had been methylated or not. If there is a sufficient region of complementarity, e.g., 12, 15, 18, or 20 nucleotides, to the target, then the primer may also contain additional nucleotide residues that do not interfere with hybridization but may be useful for other manipulations. Examples of such other residues may be sites for restriction endonuclease cleavage, for ligand binding or for factor binding or linkers or repeats.
  • the oligonucleotide primers may or may not be such that they are specific for modified methylated residues.
  • oligonucleotide probes may hybridize directly to modified nucleic acid or to further products of modified nucleic acid, such as products obtained by amplification.
  • Probe-based assays exploit oligonucleotide hybridisation to specific sequences and subsequent detection of the hybrid. There may also be further purification steps before the amplification product is detected e.g. a precipitation step.
  • Oligonucleotide probes may be labelled using any detection system known in the art. These include but are not limited to fluorescent moieties, radioisotope labelled moieties, bioluminescent moieties, luminescent moieties, chemiluminescent moieties, enzymes, substrates, receptors, or ligands.
  • DNA may be amplified using primer pairs designed to distinguish methylated from unmethylated DNA by taking advantage of sequence differences as a result of sodium-bisulphite treatment (Herman et al.,1996; and WO 97/46705).
  • bisulphite ions modify non-methylated cytosine bases, changing them to uracil bases.
  • Uracil bases hybridize to adenine bases under hybridization conditions.
  • oligonucleotide primer which comprises adenine bases in place of guanine bases would hybridize to the bisulphite-modified DNA, whereas an oligonucleotide primer containing the guanine bases would hybridize to the non-modified (methylated) cytosine residues in the DNA.
  • Amplification using a DNA polymerase and a second primer yield amplification products which can be readily observed, which in turn indicates whether the DNA had been methylated or not.
  • PCR is a preferred amplification method
  • variants on this basic technique such as nested PCR and multiplex PCR are also included within the scope of the invention.
  • a preferred embodiment for assessing the methylation status of the relevant gene requires amplification to yield amplification products.
  • the presence of amplification products may be assessed directly using methods well known in the art. They simply may be visualized on a suitable gel, such as an agarose or polyacrylamide gel. Detection may involve the binding of specific dyes, such as ethidium bromide, which intercalate into double-stranded DNA and visualisation of the DNA bands under a UV illuminator for example.
  • Another means for detecting amplification products comprises hybridization with oligonucleotide probes. Alternatively, fluorescence or energy transfer can be measured to determine the presence of the methylated DNA.
  • a specific example of the MSP technique is designated real-time quantitative MSP (QMSP), and permits reliable quantification of methylated DNA in real time or at end point.
  • Real-time methods are generally based on the continuous optical monitoring of an amplification procedure and utilise fluorescently labelled reagents whose incorporation in a product can be quantified and whose quantification is indicative of copy number of that sequence in the template.
  • fluorescently labelled reagents whose incorporation in a product can be quantified and whose quantification is indicative of copy number of that sequence in the template.
  • One such reagent is a fluorescent dye, called SYBR Green I that preferentially binds double-stranded DNA and whose fluorescence is greatly enhanced by binding of double-stranded DNA.
  • labelled primers and/or labelled probes can be used for quantification.
  • Real-Time PCR detects the accumulation of amplicon during the reaction. Real-time methods do not need to be utilised, however. Many applications do not require quantification and Real-Time PCR is used only as a tool to obtain convenient results presentation and storage, and at the same time to avoid post-PCR handling. Thus, analyses can be performed only to confirm whether the target DNA is present in the sample or not. Such end-point verification is carried out after the amplification reaction has finished. This knowledge can be used in a medical diagnostic laboratory to diagnose or identify a tumour in a patient. End-point PCR fluorescence detection techniques may employ the same approaches as widely used for Real Time PCR.
  • quantitation may be on an absolute basis, or may be relative to a constitutively methylated DNA standard, or may be relative to an unmethylated DNA standard.
  • Methylation status may be determined by using the ratio between the signal of the marker under investigation and the signal of a reference gene where the methylation status is known (such as ⁇ -actin for example), or by using the ratio between the methylated marker and the sum of the methylated and the non-methylated marker.
  • absolute copy number of the methylated marker gene can be determined.
  • Suitable controls may need to be incorporated in order to ensure the method chosen is working correctly and reliably.
  • Suitable controls may include assessing the methylation status of a gene known to be methylated. This experiment acts as a positive control to help to ensure that false negative results are not obtained.
  • the gene may be one which is known to be methylated in the sample under investigation or it may have been artificially methylated, for example by using a suitable methyltransferase enzyme, such as Sssl methyltransferase.
  • the at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene may be assessed in normal (i.e. non-tumour) cells, following treatment with Sssl methyltransferase, as a positive control.
  • suitable negative controls may be employed with the methods of the invention.
  • suitable controls may include assessing the methylation status of a gene known to be unmethylated or a gene that has been artificially demethylated. This experiment acts as a negative control to ensure that false positive results are not obtained.
  • the at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene may be assessed in normal (non-tumour) cells as a negative control, since it has been shown for the first time herein that these genes are unmethylated in normal tissues.
  • PCR is the preferred nucleic acid amplification technique, other amplification techniques may also be utilised to detect the methylation status of the concerned gene.
  • Such amplification techniques are well known in the art, and include methods such as NASBA (Compton, 1991 ), 3SR (Fahy et al., 1991 ) and Transcription Mediated Amplification (TMA).
  • Other suitable amplification methods include the ligase chain reaction (LCR) (Barringer et al, 1990), selective amplification of target polynucleotide sequences (US Patent No. 6,410,276), consensus sequence primed polymerase chain reaction (US Patent No 4,437,975), arbitrarily primed polymerase chain reaction (WO 90/06995), invader technology, strand displacement technology, and nick displacement amplification (WO 2004/067726).
  • LCR ligase chain reaction
  • SBR consensus sequence primed polymerase chain reaction
  • WO 90/06995 arbitrarily primed polymerase chain reaction
  • invader technology strand displacement technology
  • nick displacement amplification WO 2004/067726
  • MSP primers are utilised in the methods of the invention.
  • Primers useful in MSP to determine the methylation status of the genes of interest are set forth in table 5 below. These primers may comprise, consist essentially of or consist of (any of) the nucleotide sequences set forth in the table. Primers of the invention preferably are designed to bind to fully methylated genomic sequences in the regions under investigation, although primers are also provided which bind to unmethylated sequences.
  • the invention provides primers for methylation-specific PCR selected from primers comprising, consisting essentially of or consisting of the nucleotide sequences set forth in table 5 (SEQ ID NOs 47 to 66) and functional derivatives thereof, as defined herein.
  • the methods of the invention using methylation specific PCR may comprise use of primer pairs selected from the primers comprising the nucleotide sequences set forth as SEQ ID NOs 47 to 66 or functional derivatives thereof in order to determine if the (appropriate) at least one gene is methylated.
  • Suitable primer pairs can readily be identified in table 5 (SEQ ID NOs 47 to 50 are miR-9-1 primers, SEQ ID NOs 51 to 54 are miR-9-2 primers, SEQ ID NOs 55 to 58 are miR-9-3 primers, SEQ ID NOs 59 to 62 are miR-34b/c primers, and SEQ ID NOs 63 to 66 are miR-148a primers)
  • bisulphite sequencing primers are utilised in the methods of the invention. Primers useful in bisulphite sequencing to determine the methylation status of the genes of interest are set forth in table 5 below. These primers may comprise, consist essentially of or consist of (any of) the nucleotide sequences set forth in the table.
  • the invention also relates to primers for bisulfite sequencing selected from primers comprising consisting essentially of or consisting of the nucleotide sequences set forth in table 5 (SEQ ID NOs 13 to 22) and functional derivatives thereof, as defined herein.
  • the methods of the invention employing bisulphite sequencing may be carried out using sequencing primer pairs selected from the primers comprising the nucleotide sequences set forth as SEQ ID NOs 13 to 22 or functional derivatives thereof in order to determine if the (appropriate) at least one gene is methylated.
  • Suitable primer pairs can readily be identified in table 5 (SEQ ID NOs 13 and 14 are miR-9-1 primers, SEQ ID NOs 15 and 16 are miR-9-2 primers, SEQ ID NOs 17 and 18 are miR-9-3 primers, SEQ ID NOs 19 and 20 are miR-34b/c primers and SEQ ID NOs 21 and 22 are miR-148a primers).
  • primers of the invention are summarized in the experimental part. It is noted that variants of these sequences may be utilised in the present invention. In particular, additional flanking sequences may be added, for example to improve binding specificity, as required. Variant sequences preferably have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (overall) nucleotide sequence identity with the nucleotide sequences of the primers and/or probes set forth herein.
  • the primers and probes may incorporate synthetic nucleotide analogues as appropriate or may be DNA, RNA or PNA based for example, or mixtures thereof.
  • the primers and probes may include modified oligonucleotides and other appending groups and labels provided that the functionality as a primer and/or probe in the methods of the invention is not compromised.
  • the application of the methods of present invention on small amounts of abnormally- methylated DNA, that are found in the test samples may require the amplification of the DNA of interest before testing for methylation of any specific gene. Suitable methods on whole genome amplification and libraries generation for such amplification (e.g.
  • Methylplex and Enzyplex technology are described in US2003/0143599, WO2004/081225 and WO2004/081183.
  • WO2005/090507 describes library generation/amplification methods that require either bisulphite conversion or non-bisulphite based applications. Bisulphite treatment may occur before or after library construction and may require the use of adaptors resistant to bisulphite conversion.
  • Meth-DOP-PCR (Di Vinci et al, 2006), a modified degenerate oligonucleotide-primed PCR amplification (DOP-PCR) that is combined with MSP, provides another suitable method for specific detection of methylation in small amount of DNA. An initial amplification of the gene or genes of interest, which is non-methylation specific may be carried out prior to the methylation detection method itself. Improved management of patient care may advantageously import these existing methods and techniques to supplement the methods of the invention.
  • methylation status is determined according to the methods of the invention using a suitable technique such as a technique selected from methylation specific PCR, heavymethyl, bisulphite sequencing, microarray techniques, real-time or end point amplification techniques, multiplex ligation-dependent probe amplification (MLPA) and restriction enzyme based methods such as COBRA, either alone or in combination.
  • a suitable technique such as a technique selected from methylation specific PCR, heavymethyl, bisulphite sequencing, microarray techniques, real-time or end point amplification techniques, multiplex ligation-dependent probe amplification (MLPA) and restriction enzyme based methods such as COBRA, either alone or in combination.
  • the methylation specific PCR may comprise real-time or end point methylation specific PCR.
  • the methods of the invention may involve determining epigenetic silencing (methylation) of the at least one gene through measurement of expression levels of the gene and wherein reduced expression of the gene is indicative of a tumour, in specific embodiments a tumour with metastatic capability or a metastasis or a secondary tumour.
  • the methods of the invention may comprise, consist essentially of or consist of determining the effect of methylation on expression of the gene or genes of interest. Expression may be compared with gene expression in one or more control cells in which the methylation status and corresponding expression levels are known. Positive and negative controls may be employed as required. Expression is determined at the RNA level. Any suitable technique may be employed.
  • RNA level is determined by reverse transcriptase polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcriptase polymerase chain reaction
  • Methods employing nucleic acid probe hybridization to the relevant transcript(s) of an appropriate gene may be employed for measuring the presence and/or level of the RNA.
  • Such methods include use of nucleic acid probe arrays (microarray technology) and Northern blots. Advances in genomic technologies now permit the simultaneous analysis of thousands of genes, although many are based on the same concept of specific probe-target hybridization. Sequencing-based methods are an alternative. These methods started with the use of expressed sequence tags (ESTs), and now include methods based on short tags, such as serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS).
  • ESTs expressed sequence tags
  • SAGE serial analysis of gene expression
  • MPSS massively parallel signature sequencing
  • Differential display techniques provide yet another means of analyzing gene expression; this family of techniques is based on random amplification of cDNA fragments generated by
  • RNA encoded by the microRNA 148 family gene, microRNA 34 family gene or microRNA 9 family gene there will be reduced levels or none of the relevant RNA encoded by the microRNA 148 family gene, microRNA 34 family gene or microRNA 9 family gene. In certain embodiments this will present a negative result.
  • use of suitable controls ensures that false diagnoses will not be made, for example caused by degraded or non-specific reagents.
  • the same reagent can be tested on samples in which it is known that the at least one microRNA
  • microRNA 34 family gene is expressed.
  • microRNA 9 family gene is expressed. A positive result in this control sample, combined with a negative result in the test sample provides a confident diagnosis of a tumour and removes any doubt over the quality of the reagent.
  • the methods of the invention may further comprise, consist essentially of or consist of determining whether use of a demethylating agent can restore expression of the gene or genes of interest. If the result is positive, this indicates that the methylation is the cause of the loss of expression.
  • Any suitable demethylating agent may be employed, of which many are known.
  • the demethylating agent comprises, consists essentially of or consists of 5-aza-2-deoxycytidine.
  • the decreased level of expression may, as necessary, be measured in order to determine if it is statistically significant in the sample. This helps to provide a reliable test for the methods of the invention. Any method for determining whether the expression level of the microRNA 148 family gene, microRNA 34 family gene or microRNA 9 family gene is significantly reduced may be utilised. Such methods are well known in the art and routinely employed. For example, statistical analyses may be performed using an analysis of variance test. Typical P values for use in such a method would be P values of ⁇ 0.05 or 0.01 or 0.001 when determining whether the relative expression or activity is statistically significant. A change in expression may be deemed significant if there is at least a 10% decrease for example. The test may be made more selective by making the change at least 15%, 20%, 25%, 30%, 35%, 40% or 50%, for example, in order to be considered (statistically) significant.
  • the decreased level of expression of the microRNA 148 family gene, microRNA 34 family gene or microRNA 9 family gene is determined with reference to a control sample.
  • This control sample may be taken from normal (i.e. non tumourigenic) tissue in the subject, where expression of the microRNA 148 family gene, microRNA 34 family gene or microRNA 9 family gene is normal. Additionally or alternatively control samples may also be utilised in which there is known to be a lack of expression of the concerned gene. Suitable additional controls may also be included (to ensure that the test is working properly) such as measuring levels of expression or activity of a suitable reference gene in both test and control samples.
  • the expression level of the microRNA 148 family gene, microRNA 34 family gene or microRNA 9 family gene is determined using a suitable technique such as a technique selected from microarray techniques, real-time or end point amplification techniques, in particular RT-PCR, and northern blotting.
  • a suitable technique such as a technique selected from microarray techniques, real-time or end point amplification techniques, in particular RT-PCR, and northern blotting.
  • the methods of the invention may incorporate a suitable negative and/or positive control against which the (relative) levels of methylation or expression can be determined.
  • Such controls are routinely employed and readily performed by one skilled in the art. Testing can be performed diagnostically or in conjunction with a therapeutic regimen.
  • Epigenetic loss of function of at least one microRNA 148 family gene, microRNA 34 family gene or microRNA 9 family gene can potentially be rescued by the use of DNA demethylating agents and/or DNA methyltransferase inhibitors. Testing can be used to determine what therapeutic or preventive regimen to employ on a patient and be used to monitor efficacy of a therapeutic regimen.
  • the invention provides a method for predicting the likelihood of successful treatment of a tumour with a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor and/or a nucleic acid comprising, consisting essentially of or consisting of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene, or the corresponding RNA, the method comprising determining the methylation status or expression levels of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene in a test sample obtained from a subject, wherein the presence of methylation (or hypermethylation) or low or reduced levels of expression (caused by methylation (or hypermethylation) of the gene) is indicative of the likelihood of successful treatment being higher than if the at least one gene is not methylated (or is hypomethylated) or expression levels are higher.
  • the invention provides a method for predicting the likelihood of resistance to treatment of a tumour with a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor and/or a nucleic acid comprising, consisting essentially of or consisting of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene, or the corresponding RNA, the method comprising determining the methylation status or expression levels of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene in a test sample obtained from a subject, wherein the absence of methylation (or absence of hypermethylation) or high or increased levels of expression
  • the invention provides a method of selecting a suitable treatment regimen for a tumour comprising determining the methylation status or expression levels of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene in a test sample obtained from a subject, wherein the presence of methylation (or hypermethylation) or low or reduced levels of expression (caused by methylation (or hypermethylation) of the gene) results in selection of a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor and/or a nucleic acid comprising, consisting essentially of or consisting of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene, or the corresponding RNA, for treatment.
  • miRNAs from the 34 family have been shown to regulate directly (the oncogene) BCL2 (40) and high BCL-2 expression may be a useful prognostic factor for treatment of NSCLC patients treated with cisplatin-based concurrent chemoradiotherapy (41 ).
  • the treatment may comprise use of platinum-based concurrent chemoradiotherapy in certain embodiments.
  • the platinum-based chemotherapy may be cisplatin in certain embodiments.
  • methylation and reduced expression of the at least one gene selected from a microRNA 34 family gene, a microRNA 148 family gene and a microRNA 9 family gene is a reliable indicator of a metastatic tumour or a tumour with metastatic potential.
  • the treatment adopted may be more aggressive than if a primary tumour alone is detected.
  • the invention provides a method of determining the extent or aggressiveness of treatment for a tumour comprising determining the methylation status or expression levels of at least one gene selected from a microRNA
  • microRNA 34 family gene a microRNA 148 family gene and a microRNA 9 family gene in a test sample obtained from a subject, wherein the presence of methylation or low or reduced levels of expression, caused by methylation of the gene (which acts as an indicator of a tumour with metastatic potential or a metastasis or secondary tumour, such as a tumour with metastases in lymph nodes), results in a more extensive or aggressive treatment
  • Treatment of a primary tumour may begin with surgical resection.
  • the more extensive or aggressive treatment may involve specific targeting of the metastases.
  • the treatment employed may comprise use of additional techniques such as chemotherapy, radiation therapy, biological therapy, hormone therapy, cryosurgery, or a combination of these in addition to or as an alternative to surgical resection of the tumour.
  • the treatment may be according to one of the methods of the invention as described herein.
  • the more extensive or aggressive treatment may involve an increased frequency and/or dosage of the relevant treatment or treatments as appropriate, as determined by a range of factors such as the age and general health of the patient.
  • miRNAs from the 34 family have been shown to regulate directly (the oncogene) BCL2 (40) and high BCL- 2 expression may be a useful prognostic factor for treatment of NSCLC patients treated with cisplatin-based concurrent chemoradiotherapy (41).
  • the treatment may comprise use of platinum-based concurrent chemoradiotherapy in certain embodiments.
  • the platinum-based chemotherapy may be cisplatin in certain embodiments.
  • the diagnostic methods of the invention may also identify patients at particular risk for developing or suffering from metastases or secondary tumours. Such patients may then be tested at increased frequency to determine the progression or advancement of their condition. This may also assist the direction and monitoring the effect of treatment.
  • the invention further provides a method of determining the frequency of testing for a tumour (in particular a tumour with metastatic potential or a metastasis or secondary tumour, such as a tumour with metastases in lymph nodes) comprising determining the methylation status or expression levels of at least one gene selected from a microRNA 34 family gene, a microRNA 148 family gene and a microRNA 9 family gene in a test sample obtained from a subject, wherein the presence of methylation or low or reduced levels of expression, caused by methylation of the gene (which acts as an indicator of a tumour with metastatic potential or a metastasis or secondary tumour, such as a tumour with metastases in lymph nodes), results in more frequent testing (than if methylation is at lower levels or absent or there are higher or increased
  • the more frequent testing may involve performing the diagnostic methods of the invention at increased frequency in order to monitor the patient. Further increases in the level of methylation, or corresponding decreases in the level of expression of the relevant miRNA gene or genes provides an indication of the progression of the tumour. The monitoring may also be employed in conjunction with appropriate therapies to determine whether the treatment has been effective. A decrease in methylation or a corresponding increase in expression of the at least one gene selected from a microRNA 34 family gene, a microRNA 148 family gene and a microRNA 9 family gene provides an indication that the treatment is having the desired effect.
  • the invention also provides a method of determining the effect of treatment of a tumour (in particular a tumour with metastatic potential or a metastasis or secondary tumour, such as a tumour with metastases in lymph nodes) comprising determining the methylation status or expression levels of at least one gene selected from a microRNA 34 family gene, a microRNA 148 family gene and a microRNA 9 family gene in a test sample obtained from a subject both prior to and following the treatment, wherein the presence of decreased methylation or higher or increased levels of expression following the treatment (compared to the methylation status or expression level measured prior to treatment) provides an indication that the treatment has been effective.
  • a consistent level of methylation or expression following treatment suggests the treatment is having a maintenance effect and may be preventing further progression of the tumour.
  • An increased level of methylation or decreased expression following treatment may provide an indication of ineffective treatment. In these circumstances the methods may be repeated following further treatment, perhaps at an increased dosage or frequency, or alternative treatments may be explored.
  • the embodiments and optional features of the methods of the invention apply mutatis mutandis and are not repeated for reasons of conciseness.
  • all (variants of the) methods of determining the methylation status or expression level in the at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene may be employed appropriately.
  • the tumour is one with metastatic potential or a metastasis or secondary tumour.
  • the tumour may be a tumour with metastases in lymph nodes.
  • the methods of the invention are preferably in vitro methods carried out on a test sample.
  • the methods are non-invasive.
  • the methods may be used to identify any tumour.
  • the "test sample" in which the methylation status or expression levels of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene is determined is a sample comprising nucleic acid molecules from cells, and in particular cells suspected of being tumourigenic.
  • the sample may thus comprise, consist essentially of or consist of a tissue sample, body fluid, body fluid precipitate or lavage specimen in certain embodiments. Any suitable test sample representative of the tumour of interest may be utilised.
  • the test sample is obtained from a human subject.
  • Test samples for diagnostic, prognostic, or personalised medicinal uses can be obtained from surgical samples, such as biopsies or fine needle aspirates, from paraffin embedded tissues, from frozen tumour tissue samples, from fresh tumour tissue samples, from a fresh or frozen body fluid, for example.
  • surgical samples such as biopsies or fine needle aspirates
  • paraffin embedded tissues from frozen tumour tissue samples, from fresh tumour tissue samples, from a fresh or frozen body fluid, for example.
  • Non-limiting examples include whole blood, bone marrow, cerebral spinal fluid, peritoneal fluid, pleural fluid, lymph fluid, serum, plasma, urine, chyle, stool, ejaculate, sputum, nipple aspirate, saliva, swabs specimen, wash or lavage fluid and/or brush specimens.
  • an appropriate sample may be selected on the basis of the nature of the primary tumour and the location or possible location of a metastasis or secondary tumour.
  • the sample may comprise a lymph node or portion thereof or a related representative sample type, such as lymph fluid.
  • the tumour is selected from a lung, breast, colon, or head and neck tumour or a melanoma.
  • the sample may be selected appropriately based upon cells from the appropriate organ or related cells or body fluids.
  • the tissues and body fluids may be collected using any suitable methods, many of which are well known in the art.
  • the "nucleic acid” in the methods according to the invention is preferably deoxyribonucleic acid (DNA), in particular genomic DNA.
  • the methods of the invention may also include the step of obtaining the test sample in some embodiments.
  • the tissue sample or liquid sample comprising the cells may be lysed or need to be concentrated to create a mixture of biological compounds comprising nucleic acids and other components.
  • the nucleic acid may need to be cleared of proteins or other contaminants, e.g. by treatment with proteinase K.
  • Procedures for lysing or concentrating biological samples are known by the person skilled in the art and can be chemical, enzymatic or physical in nature. A combination of these procedures may be applicable in some embodiments. For instance, lysis may be performed using ultrasound, high pressure, shear forces, alkali, detergents or chaotropic saline solutions, or proteases or lipases.
  • nucleic acids are extracted from the test sample using a commercially available purification kit, such as the PUREGENE® DNA purification kit.
  • the sample may be centrifuged and nucleic acid purified from the sediment or pellet fraction, in particular using such a purification kit. Suitable purification kits are commercially available and would be well known to one skilled in the art.
  • test sample is generally obtained from a (human) subject suspected of being tumourigenic.
  • test sample is obtained from a subject undergoing routine examination and not necessarily being suspected of having a disease. Thus patients at risk can be identified before the disease has a chance to manifest itself in terms of symptoms identifiable in the patient.
  • sample is obtained from a subject undergoing treatment, or from patients being checked for recurrence of disease. The nature of the sample may depend upon the particular method of the invention that is being performed.
  • the invention is also concerned with methods of treatment that result from the discovery of the new miRNA family gene markers, whose methylation status is linked to the incidence of tumours and metastasis and is functionally linked to expression of tumour- associated genes.
  • the invention provides a method of treating a tumour in a subject comprising administering a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or a HDAC inhibitor to the subject in order to reduce the methylation (and thus increase the expression) of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene.
  • the subject is selected for treatment on the basis of one of the methods of the invention.
  • the invention also provides a method of treating a tumour comprising administering a nucleic acid comprising, consisting essentially of or consisting of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene, or the corresponding RNA, to the subject in order to complement microRNA function suppressed by methylation of the corresponding endogenous gene.
  • a nucleic acid comprising, consisting essentially of or consisting of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene, or the corresponding RNA
  • the embodiments and optional features of the methods of the invention apply mutatis mutandis and are not repeated for reasons of conciseness.
  • all (variants of the) methods of determining the methylation status or expression level in the at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene may be employed appropriately.
  • the tumour is one with metastatic potential or a metastasis or secondary tumour.
  • the tumour may be a tumour with metastases in lymph nodes.
  • the DNA demethylating agent may be any agent capable of up regulating transcription of at least one of the novel tumour suppressor genes (i.e. the relevant at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene).
  • the DNA methyltransferase inhibitor may be any suitable inhibitor of DNA methyltransferase activity or expression which is suitable for treating cancer in the presence of methylation of the at least one gene.
  • the DNA methyltransferase inhibitor may be one which reduces expression of DNMT genes, such as suitable antisense molecules, or siRNA molecules which mediate RNA interference (RNAi) for example.
  • RNAi RNA interference
  • the design of a suitable siRNA molecule is within the capability of the skilled person and suitable molecules can be made to order by commercial entities (such as Ambion).
  • the DNA methyltransferase gene is (human) DNMT1.
  • the agent may be a direct inhibitor of DNMTs.
  • modified nucleotides such as phosphorothioate modified oligonucleotides (fig 6 of Villar-Garea, A. And Esteller, M. DNA demethylating agents and chromatin-remodelling drugs: which, how and why? Current Drug Metabolism, 2003, 4, 11-31 ) and nucleosides and nucleotides such as cytidine analogues.
  • cytidine analogues include 5-azacytidine, 5-aza-2'-deoxycytidine, 5-fluouro-2'-deoxycytidine, pseudoisocytidine, 5,6-dihydro-5-azacytidine, 1 - ⁇ -D-arabinofuranosyl-5-azacytosine (known as cambarine) (see figure 4 of Villar-Garea, A. And Esteller, M. Current Drug Metabolism, 2003, 4, 11-31 ).
  • the DNA methyltransferase inhibitor may comprise Decitabine.
  • Additional DNMT inhibitors include S-Adenosyl-Methionine (SAM) related compounds like ethyl group donors such as L-ethionine and non-alkylating agents such as S- adenosyl-homocysteine (SAH), sinefungin, (S)-6-methyl-6-deaminosine fungin, 6- deaminosinefungin, N4-adenosyl-N4-methyl-2,4-diaminobutanoic acid, 5'-methylthio-5'- deoxyadenosine (MTA)and 5'-amino-5'-deoxyadenosine (Villar-Garea, A. And Esteller, M. Current Drug Metabolism, 2003, 4, 11-31 ).
  • Useful DNMT inhibitors in the present invention comprise, consists essentially of or consists of 5-azacytidine and/or zebulaine.
  • DNA demethylating agents include organohalogenated compounds such as chloroform etc, procianamide, intercalating agents such as mitomycin C, 4-aminobiphenyl etc, inorganic salts of arsenic and selenium and antibiotics such as kanamycin, hygromycin and cefotaxim (Villar-Garea, A. And Esteller, M. DNA demethylating agents and chromatin-remodelling drugs: which, how and why? Current Drug Metabolism, 2003, 4, 11-31).
  • organohalogenated compounds such as chloroform etc, procianamide
  • intercalating agents such as mitomycin C, 4-aminobiphenyl etc
  • inorganic salts of arsenic and selenium such as kanamycin, hygromycin and cefotaxim (Villar-Garea, A. And Esteller, M. DNA demethylating agents and chromatin-remodelling drugs: which, how and why? Current Drug Metabolism, 2003, 4, 11-31).
  • HDAC inhibitors are similarly known in the art. Examples include trichostatin A (TSA), suberoyl hydroxamic acid (SBHA), 6-(3-chlorophenylureido)caproic hydroxamic acid (3-CI-UCHA), m-carboxycinnamic acid bishydroxylamide (CBHA), suberoylanilide hydroxamic acid (SAHA), azelaic bishydroxamic acid (ABHA), pyroxamide, scriptaid, aromatic sulfonamides bearing a hydroxamic acid group, oxamflatin, trapoxin, cyclic- hydroxamic-acid containing peptides, FR901228, MS-275, MGCD0103 (see www.methylgene.com), short-chain fatty acids and N-acetyldinaline.
  • TSA trichostatin A
  • SBHA suberoyl hydroxamic acid
  • the invention also provides a(n isolated) nucleic acid comprising, consisting essentially of or consisting of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene, or the corresponding RNA, for use in therapy.
  • the invention provides a(n isolated) nucleic acid comprising, consisting essentially of or consisting of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene, or the corresponding RNA, for use in treating a tumour .
  • a(n isolated) nucleic acid comprising, consisting essentially of or consisting of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene, or the corresponding RNA, in the manufacture of a medicament for treating a tumour .
  • the tumour is one with metastatic potential or a metastasis or secondary tumour.
  • the tumour is one with metastases in lymph nodes.
  • a pharmaceutical composition comprising a(n isolated) nucleic acid comprising, consisting essentially of or consisting of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene and/or the corresponding RNA.
  • an altered version at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene and/or the corresponding RNA may be utilised provided it retains substantially wild type, or improved, (tumour suppressor) activity.
  • adenoviruses preferred types include adenoviruses, retroviruses, in particular Moloney murine leukaemia virus (Mo-MLV), adeno-related viruses and herpes simplex virus type I.
  • Mo-MLV Moloney murine leukaemia virus
  • the gene of interest in this case at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene and/or the corresponding RNA, will be included in the viral genome, preferably in the "non-essential" region of the viral genome.
  • the virus may be made replication incompetent to prevent unwanted replication once the virus has been targeted.
  • the env gene (which encodes the viral vector's envelope) may be engineered or replaced with the env gene from a different virus to alter the range of cells the viral vector will "infect".
  • alteration of the viral tropism may be achieved by using suitable antibodies raised against antigenic determinants on the cell surface of the desired target cells.
  • the antibodies which include all derivatives thereof, such as scFV, nanobodies, VH domains, Fab fragements etc., may be genetically incorporated into the viral vectors to provide targeted gene delivery of the HDAC2 gene. Most preferred is use of scFV (Hedley et al., Gene Therapy (2006) 13, 88-94).
  • the viral vectors may have many genes removed, such as packaging genes, in order to reduce immunogenicity and/or infectivity. These functions may thus be supplied by a helper virus.
  • adenoviruses are a preferred vector according to the methods of the invention.
  • viral vectors include direct gene delivery, use of other delivery agents and use of molecular conjugates.
  • Tissue specific promoters may be employed as appropriate.
  • Direct gene delivery may be achieved for example by microinjection of a suitable vector, such as a plasmid carrying the at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene and/or the corresponding RNA, directly into the tissue of interest.
  • a suitable vector such as a plasmid carrying the at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene and/or the corresponding RNA
  • Alternatives include use of ballistic transformation, for example using vector coated onto suitable particles (e.g. gold particles).
  • Additional delivery agents include liposomes and derivatives thereof.
  • targeting proteins such as antibodies and derivatives thereof may be utilised in order to ensure delivery to the cells of interest.
  • Molecular conjugates may include suitable proteins conjugated to the DNA of interest using
  • the gene therapy aspects of the invention may incorporate any and all of the preferred aspects described in respect of the diagnostic and pharmacogenetic methods and also methods of treating a tumour as described above.
  • the diagnostic methods and/or the pharmacogenetic methods of the invention are carried out as a prelude to, or as an integral part of the methods of treating cancer according to the gene therapy aspects of the invention.
  • compositions include pharmaceutically acceptable carriers including, for example, non-toxic salts, sterile water or the like.
  • a suitable buffer may also be present allowing the compositions to be lyophilized and stored in sterile conditions prior to reconstitution by the addition of sterile water for subsequent administration.
  • the carrier may also contain other pharmaceutically acceptable excipients for modifying other conditions such as pH, osmolarity, viscosity, sterility, lipophilicity, somobility or the like.
  • Pharmaceutical compositions which permit sustained or delayed release following administration may also be used.
  • Suitable pharmaceutical compositions for use in the treatment methods or medical uses of the invention may be used together with other standard chemotherapeutic treatments which target tumour cells directly.
  • Non limiting examples include paclitaxel, cyclaphosphomide and 5-tumour-uracil (5-FU) and pharmaceutically acceptable derivatives thereof including salts, etc.
  • the therapeutic agent may, for example, be encapsulated and/or combined with suitable carriers in solid dosage forms for oral administration which would be well known to those of skill in the art or alternatively with suitable carriers for administration in an aerosol spray.
  • suitable carriers include tablets, capsules and liquids.
  • the therapeutic agent may be administered parenterally.
  • specific examples include intradermal injection, subcutaneous injection (which may advantageously give slower absorption of the therapeutic agent), intramuscular injection (which can provide more rapid absorption), intravenous delivery (meaning the drug does not need to be absorbed into the blood stream from elsewhere), sublingual delivery (for example by dissolving of a tablet under the tongue or by a sublingual spray), rectal delivery, vaginal delivery, topical delivery, transdermal delivery and inhalation.
  • the specific dosage regime may be calculated according to the body surface area of the patient or the volume of body space to be occupied, dependent on the particular route of administration to be used.
  • the amount of the composition actually administered will, however, be determined by a medical practitioner based on the circumstances pertaining to the disorder to be treated, such as the severity of the symptoms, the age, weight and response of the individual.
  • kits which may be used in order to carry out the methods of the invention.
  • the kits may incorporate any of the preferred features mentioned in connection with the various methods (and uses) of the invention herein.
  • the invention also relates to kits for use in, or for carrying out, any one of the methods of the invention comprising carrier means containing at least one primer pair for determining the methylation status or expression level of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene.
  • the primer pair may be comprised of any two primers of the invention that can be used in combination to determine the methylation status or expression level of at least one gene selected from a microRNA 148 family gene, a microRNA 34 family gene and a microRNA 9 family gene.
  • Suitable primers are described herein.
  • the primer pair investigates the methylation status of the at least one gene directly.
  • the primer may bind to sites of potential methylation in the gene. Binding of the primer may be dependent upon whether the genomic DNA in the test sample has been modified or not following treatment with a suitable reagent that modifies unmethylated cytosine residues but not methylated cytosine residues.
  • the primer can be designed to bind to either the sequence as modified or unmodified following the treatment.
  • the pimer pair is an MSP primer pair.
  • primers that do not themselves bind to potential sites of methylation may be employed in conjunction with blocking probes.
  • the blocking probes investigate methylation status and permit or prevent primer extension depending upon the methylation status.
  • the kits of the invention may incorporate such blocking probes, optionally together with the associated primers, which directly determine methylation status of the genomic DNA sample. Such kits permit the "heavymethyl" technique to be performed.
  • the microRNA 148 family gene is microRNA 148a.
  • the microRNA 34 family gene may be microRNA 34b or 34c.
  • the microRNA 9 family gene may be selected from microRNA 9-1 , 9-2 or 9-3.
  • the kits may incorporate at least one primer pair selected from the primer pairs of the invention. Suitable MSP and Bisulphite sequencing primer pairs are described herein with reference to table 5 (MSP primers are set forth as SEQ ID NOs 47 to 66 and bisulphite sequencing primers as SEQ ID NOs 13 to 22).
  • the kit of the invention further comprises a reagent which modifies unmethylated cytosine residues but does not methylated cytosine residues (or vice versa), in detectable fashion. This allows methylated residues to be distinguished from non-methylated residues.
  • the reagent converts unmethylated cytosine residues to a different nucleotide (uracil) but methylated residues are not converted.
  • the reagent comprises bisulphite, preferably sodium bisulphite but may comprise hydrazine for example.
  • kits of the invention may further comprise at least one labelled primer or probe to permit real-time or end point detection of methylation status or expression levels. As discussed herein, such real-time or end point detection reactions are often utilised in order to perform the methods of the invention.
  • FIG. 1a Schematic strategy used to identify DNA methylation-associated repression of miRNAs in metastatic cancer cell lines.
  • FIG. 2a Wound-healing assay. The relative invasion of c-shRNA cells compared with miRNA 34b/c transfected cells after 16 h is represented.
  • FIG. 2b Wound-healing assay. The relative invasion of c-shRNA cells compared with miRNA 148a transfected cells after 16 h is represented.
  • FIG. 2c Illustrative examples of the wound-healing assay in the c-shRNA and miRNA transfected metastatic cell lines at 0 h and 16 h.
  • Dot/dash (-•- • -) and thicker dash (- - - ) lines represent the initial and final leading edges of the invasion front, respectively.
  • miR-148a and miR-34b/c transfected cells show minor invasion capability.
  • FIG. 2f Effects of 34b/c miRNA transfection on in vivo growth of metastatic SIHN-011 B cells xenografted into nude mice. Tumour weight was measured at the end of the experiment.
  • FIG. 2h Representative large tumours on the right flank (c-shRNA cells), and the small tumour on the opposite flank, corresponding to miR-34b/c or miR-148a transfected cells respectively.
  • FIG. 2i - Example of the resected tumours are also shown.
  • FIG. 2k Illustrative examples of tumours formed from transplantation of metastatic SIHN-011 B cancer cells with stable transfection of miR-148a, miR-34b/c or a control vector (C-ShRNA) into athymic nude mice by tail-vein injection are shown.
  • C-ShRNA control vector
  • FIG. 3a Protein expression analysis by western blot for TGIF2, C-MYC, CDK6 and E2F3 in untreated and 5-aza-2'-deoxycytidine treated SIHN-011B cells.
  • the DNA demethylating agent induces downregulation of the oncoproteins.
  • FIG. 3b Transfection of metastatic SIHN-011B cells with the miR-148a, miR-34b and miR-34c precursor molecules also causes a reduction in the protein levels measured.
  • FIG. 4a Methylation-specific PCR analyses for miR-148a, miR-34b/c, miR-9-1 , miR-9-2 and miR-9-3 in primary human tumours derived from different tissues. The presence of a band under the U or M lanes indicates unmethylated or methylated sequences, respectively. Normal lymphocytes (NL) and in vitro methylated DNA (IVD) are shown as positive controls for the unmethylated and methylated sequences, respectively.
  • NL normal lymphocytes
  • IVD in vitro methylated DNA
  • FIG. 4b Association between miRNA methylation and upregulation of their corresponding oncoprotein targets in primary tumours.
  • Illustrative immunohistochemical examples demonstrate that C-MYC and CDK6 overexpression is associated with miR- 34b/c hypermethylation in lung tumours and melanomas, respectively.
  • FIG. 5a, 5b, 5c and 5d Bisulfite genomic sequencing analyses of illustrative miRNAs that show a methylated CpG island in normal and cancer cells. Eight single clones are represented for each sample. The CpG island is depicted and each vertical bar illustrates a single CpG. Black and white squares represent methylated and unmethylated CpG, respectively. NL, normal lymphocytes; NC, normal colon; NS, normal skin.
  • FIG. 6a and 6b Bisulfite genomic sequencing analyses of miR-9-1- and miR-9-2 that show cancer-specific CpG island hypermethylation. Eight single clones are represented for each sample. The CpG island is depicted and each vertical bar illustrates a single CpG. Black and white squares represent methylated and unmethylated CpG, respectively. NL, normal lymphocytes; NC, normal colon; NS, normal skin.
  • FIG. 7 - 5'RACE analyses of the five miRNAs in 5-aza-2 ' -deoxycytidine treated cells Black arrows represent the putative transcriptional start site. White portions indicate the position of the mature miRNAs while the grey lines represent the location of the bisulfite sequencing primers. Hatched lines depict the CpG islands.
  • FIG. 8a and 8b Bisulfite genomic sequencing analyses of CR612213 and BC021736 5'- ends. Eight single clones are represented for each sample. Each vertical bar illustrates a single CpG. The black arrow represents the transcriptional start site. Black and white squares represent methylated and unmethylated CpGs, respectively. NL, normal lymphocytes.
  • FIG. 9 Chromatin immunoprecipitation assay for histone modification marks in the miRNA associated CpG islands (examples in miR-9-1, miR-9-3 and miR-34b/c) in untreated (C) and 5-aza-2 ' -deoxycytidine (A) treated cells.
  • the presence of miRNA methylation is associated with the lack of histone modifications linked to transcriptional activity, such as acetylation of histones H4 (AcH4) and trimethylation of Lys4 of histone H3 (3mK4H3), whereas the opposite scenario is observed when DNA demethylation events are present by pharmacologic treatment with a DNA-demethylating agent. No AB, no antibody.
  • TSA trichostatin A
  • FIG. 11 Complementary sites between miR-34b and CDK6 and C-MYC; miR-34c and E2F3, and miR-148 and TGIF2.
  • the capital and bold letters identify perfect base matches according to the TARGETSCAN 4.1 software.
  • the base pairing between the miRNAs and the mutant target site is also represented.
  • FIG. 12 - Table representing the distribution of cases related to methylation and positive immunostaining (+). P value was calculated using Pearson ' s chi-square test.
  • FIG. 13a, 13b and 13c Frequency of CpG island hypermethylation of the metastasis- associated miRNAs according to the presence or absence of lymph node metastasis in different tumour types. P value calculated using Pearson ' s chi-square test is represented.
  • miRNAs that were reactivated upon drug treatment miR-148a, miR-34b/c and miR-9 were found to undergo specific hypermethylation-associated silencing in cancer cells compared with normal tissues.
  • the reintroduction of miR-148a and miR-34b/c in cancer cells with epigenetic inactivation inhibited their motility, reduced tumour growth and inhibited metastasis formation in xenograft models, with an associated downregulation of the miRNA oncogenic target genes, such as C-MYC, E2F3, CDK6 and TGIF2.
  • miRNAs The expression of many miRNAs is tightly regulated according to cell-type (1 ,2), so it was not surprising to observe that 11 (65%) of the miRNAs were also densely methylated in normal tissues (Fig. 1a, Table 2 and Fig. 5). The previously mentioned miR-126 was one of these. However, and most importantly, miR148a, miR-34b/c, miR-9-1 , miR-9-2 and miR-9-3 were always unmethylated in all normal tissues studied (Fig. 1a-d, Table 2 and Fig. 6). The DNA methylation results were also confirmed using methylation-specific PCR (MSP) (Fig. 1 e). Thus, the CpG island hypermethylation of these miRNAs was cancer-specific.
  • MSP methylation-specific PCR
  • Hypermethylated miRNAs show metastasis tumour suppressor features in vitro and in vivo.
  • miR-148a, miR-34b/c, miR-9-1 , miR-9-2 and miR-9-3 in metastatic cancer cells we sought to demonstrate that the epigenetic inactivation of these miRNAs contributed to metastasis formation.
  • miR-148a and miR-34b/c epigenetic silencing mediates the activation of oncogenic and metastasis target genes.
  • epigenetic silencing of miR-148a and miR-34b/c had functional cancer relevance to overcome their putative metastasis tumour suppressor function.
  • Using computational prediction for miR-34b/c target genes we observed that C-MYC, CDK6 and E2F3 were excellent potential targets, whilst for miR-148a the TGIF2 gene was one of the best candidates.
  • metastasis-associated protein 1 (MTA1 ) is an essential downstream effector of the C- MYC oncoprotein (31), CDK6 is involved in cell-cycle progression and differentiation (32), and TGIF2 is overexpressed and amplified in aggressive ovarian tumours (33).
  • MTA1 metastasis-associated protein 1
  • TGIF2 is overexpressed and amplified in aggressive ovarian tumours
  • SIHN-011 B cells were transfected with miR-148a and miR-34b/c precursor molecules, which are designed to mimic endogenous miRs.
  • miR-148a overexpression of miR-148a induced a reduction of TGIF2 protein levels (Fig. 3b).
  • miR-34b/c miR-34b transfection decreased CDK6 and C-MYC protein levels, whilst miR-34c transfection induced the downregulation of E2F3 protein levels (Fig. 3b).
  • methylation status was analyzed by bisulfite genomic sequencing of both strands of the corresponding CpG islands. Eight independent clones were analyzed. The second analysis used methylation-specific PCR with primers specific for either the methylated or modified unmethylated DNA. The primers used are described in the SI Table S5.
  • Cells were transfected with pSILENCER/TM 4.1 -CMV Expression Vector (Ambion) containing the flanking regions of the mature microRNAs (miR-148a and miR-34b/c). The primers used are described in Table 5. Cells were selected with puromycin 48 h after transfection and then diluted to perform clonal selection. The mature microRNA expression in the selected clones was assessed by q RT-PCR, as described above.
  • the mean volume or tumour mass ⁇ SEM were calculated.
  • miRNA precursor molecules and negative control miRNA were purchased from Ambion. Experiments involving transient transfections of miRNAs were done using oligofectamine (Invitrogen) and 100 nmol/L RNA duplexes. The cells were collected 48 h and 72 h after transfection, and the expression of c-MYC, E2F3, CDK6, and TGIF2 was analyzed by western blot.
  • Luciferase constructs were made by ligating oligonucleotides containing the wild-type or mutant putative target site of the C-MYC, E2F3, CDK6 and TGIF2 3 ' -UTR into the multi- cloning site of the p-MIR Reporter Luciferase vector (Ambion).
  • Firefly luciferase reporter vector containing the wild-type or mutant oligonucleotides
  • 0.04 ⁇ g of control vector pRL-TK vector, Promega
  • pRL-TK vector Promega
  • miR-34b, miR-34c or miR-148a precursor ambion.
  • Firefly and Renilla luciferase activities were measured consecutively 48 h after transfection using Renilla for normalization in dual- luciferase assays (Promega). The experiments were performed in quadruplicate in three independent experiments. The mean luciferase levels ⁇ SEM were calculated for each group.
  • BS-9-1-as TAACTTTATAAAAACTCCACACCA
  • BS-9-2-S YGGAATAAATTTTGAAGGTAAT
  • 35 BS-126-s TAAATAGTTTTGGTTGTGTTTGG
  • pLUC-CDK6-mut-as AGCTTTGTCCCCGATTTCCAGGATCCACACTGCTTCTTA 83 pLUC-TGIF2-WT-s: CGCGTGGGATTTTCCCTCCCCACAGTGCACTGAGCAATGGA
  • GSP Gene-Specific Primers
  • miR-9-2-GSP1 CTCTTGCCAGACTCCAGGTC
  • miR-9-2-GSP2 TACTTGCCGCGCTTAAGATT
  • miR-34b/c-GSP1 CAGGCAATTCATTGGTTGAG 100 miR-34b/c-GSP2: CAGGCATCTTCTCTCGAAGG

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Abstract

La présente invention concerne une méthode de diagnostic ou d’identification d’une tumeur utilisée sur un échantillon de test prélevé chez un sujet. La méthode comprend la détermination de l’état de méthylation ou le niveau d’expression d’au moins un gène choisi parmi un gène de la famille des microARN 148, un gène de la famille des microARN 34 et un gène de la famille des microARN 9, la présence de méthylation ou des niveaux faibles ou réduits d’expression indiquant la présence d’une tumeur. Les méthodes peuvent être utilisées pour diagnostiquer ou identifier une tumeur potentiellement métastatique, une métastase ou une tumeur secondaire. Des méthodes apparentées sont utilisées pour prédire la probabilité de réussite d’un traitement d’une tumeur par un agent de déméthylation de l’ADN et/ou un inhibiteur des méthyltransférases de l’ADN et/ou un inhibiteur d’HDAC et/ou un acide nucléique comprenant, composé essentiellement de, ou composé d’au moins un gène choisi parmi un gène de la famille des microARN 148, un gène de la famille des microARN 34 et un gène de la famille des microARN 9, ou l’ARN correspondant, et pour choisir un schéma thérapeutique adapté à une tumeur. La présente invention concerne également des méthodes de traitement, des utilisations médicales, des compositions, des compositions, des amorces et des kits.
PCT/GB2009/002040 2008-08-22 2009-08-20 Diagnostic et traitement des tumeurs Ceased WO2010020787A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8491927B2 (en) 2009-12-02 2013-07-23 Nimble Epitech, Llc Pharmaceutical composition containing a hypomethylating agent and a histone deacetylase inhibitor
US10434090B2 (en) 2009-12-02 2019-10-08 Nimble Epitech, Llc Pharmaceutical composition containing a hypomethylating agent and a histone deacetylase inhibitor
CN102031309A (zh) * 2010-11-30 2011-04-27 华东师范大学 miRNA-34c化合物作为脑胶质瘤标志物的应用
RU2507268C1 (ru) * 2012-10-04 2014-02-20 Федеральное государственное унитарное предприятие "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов" (ФГУП "ГосНИИгенетика") Система маркеров на основе группы генов микрорнк для диагностики немелкоклеточного рака легкого, включая плоскоклеточный рак и аденокарциному

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