WO2004087957A2 - Genes hypermethyles et cancer du col de l'uterus - Google Patents

Genes hypermethyles et cancer du col de l'uterus Download PDF

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WO2004087957A2
WO2004087957A2 PCT/IB2004/001516 IB2004001516W WO2004087957A2 WO 2004087957 A2 WO2004087957 A2 WO 2004087957A2 IB 2004001516 W IB2004001516 W IB 2004001516W WO 2004087957 A2 WO2004087957 A2 WO 2004087957A2
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genes
cervical cancer
qmsp
hypermethylation
primers
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WO2004087957A3 (fr
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N. Reesink-Peters
Ate G. J. Van Der Zee
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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
    • C12Q2600/112—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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention relates to various methods and kits for use in the detection of cervical cancer.
  • Cervical cancer is an important cause of death in women world wide, which is known to develop from cervical intraepithelial neoplasia (CIN) (1) There is a strong association between certain subtypes (high risk) of the Human Papillomavirus (HPV) and cervical cancer (2) . However, it is clear that other factors are also involved in cervical carcinogenesis because the majority of patients infected with HPV will not develop invasive cervical cancer (3) .
  • Cytomorphological examination of cervical smears is the most widely applied screening-method for cervical cancer and its precursors. Disadvantages include the high numbers of false-positive and false-negative cervical smears, leading to an overshoot of diagnostic procedures (4) or a delay in the diagnosis of cervical cancer. False-negative cytology may be found in about 50% of cases when previous negative smears are reviewed from the small proportion of screened women who develop invasive cancer (5,6).
  • QMSP realtime quantitative MSP
  • the method is based on the continuous optical monitoring of a fluorogenic PCR.
  • This PCR approach is more sensitive and more specific than conventional PCR and can therefore detect aberrant methylation patterns in human samples with substantial (1:10.000) contamination of normal DNA (12).
  • this PCR reaction is amenable to high-throughput techniques allowing the analysis of close to 400 samples in less then 2 hours without requirement for gel- electrophoresis .
  • the present invention seeks to provide improved methods to detect cervical cancer in a sample.
  • a method of detecting cervical cancer in a sample comprising detecting the hypermethylation status of a panel of genes whose hypermethylation status is linked to the incidence of cervical cancer in a multiplex experiment.
  • Hypermethylation is most commonly associated with promoter regions of genes. Therefore, in most cases methods of detection of hypermethylation will focus on this area of the gene. However, the invention is not limited to the promoter regions. If the gene is hypermethylated elsewhere leading to cervical cancer, it could be included in the methods of the invention by use of appropriate primers and probes .
  • a multiplex experiment is defined herein as one which allows detection of cervical cancer by analysis of the hypermethylation status of a number of genes whose hypermethylation status is linked to cervical cancer using a single sample.
  • Multiplexing provides technical advantages because cervical cancer can be accurately diagnosed from a single sample by identifying the hypermethylation status of the whole panel of genes. If many different samples are required for each gene of the panel to be analysed, this can lead to problems of variability between samples, possibly leading to less consistent and accurate detection of cervical cancer. Furthermore, it is preferable for patients if a minimum sample and minimum number of samples are required to achieve an accurate diagnosis.
  • Multiplexing can be performed by using labeled primers according to the LUXTM fluorogenic primers from InvitrogenTM or as described by Nazarenko et al . NAR 30:e37 (2002) and Nazarenko et al . NAR 30:2089-2095 (2002) .
  • This technology is based on labeling and designing at least one of the primers in the primer pair in such a way that it contains a hairpin structure.
  • a fluorescent label is attached to the same primer.
  • Said fluorophore can be FAM or JOE, for example.
  • the hairpin functions as a quencher.
  • the skilled reader would appreciate that alternatives to such probes would work equally well with the invention.
  • the hypermethylation status of each of the panel of genes can be detected in a single experiment. This could be the case, for example, if the hypermethylation status of each gene was detected using a probe with a different flurophore molecule attached that fluoresces at a different wavelength. Such fluorophores with different fluorescence emission spectra are well known in the art (also see below) . Thus, by way of example, the probes listed in SEQ ID NO's 3,6,9,12 and 15, with the internal reference probe represented by SEQ ID 18, could all have attached a different fluorophore molecule thus permitting their hypermethylation status to be detectable in the same single experiment.
  • the primers designed to amplify the genes whose hypermethylation status is linked to the incidence of cervical cancer could amplify different sized products that could be identified by, for example, separation by size of amplicon using gel electrophoresis .
  • the panel of genes are selected in order to provide a test where hypermethylation of any one of the genes indicates cervical cancer in the sample, so long as one of the genes provides a positive signal for hypermethylation this may be sufficient to detect cervical cancer.
  • Methods that rely on an output of positive hypermethylation of at least one of the genes are included within the scope of the invention.
  • a single sample may be divided into several samples, each of which would be used in an individual experiment to detect the hypermethylation status of only one of the panel of genes whose hypermethylation status is linked to the incidence of cervical cancer. Therefore, a single sample could provide the hypermethylation status for each gene which would then be used to diagnose cervical cancer status. Each separate experiment would utilise the primers and/or probes required to detect the hypermethylation status of one of the panel of genes.
  • the genes will not previously have been characterised as genes whose hypermethylation status is linked to cervical cancer.
  • Candidate genes could be tested using the method of the invention to investigate whether their hypermethylation status is linked to, and therefore could improve, the sensitivity of detection of cervical cancer. Such identified genes could then be added to the panel in order to increase the sensitivity of the detection tests for cervical cancer. A potentially large number of genes could, therefore, be used in the test, to increase sensitivity of the test as long as specificity of the detection method is maintained.
  • These genes could possibly be candidate tumour suppressor genes or other cancer associated genes, where hypermethylation, particularly of CpG islands, can cause a transcriptional block leading to a loss of expression of the functional protein, which in turn can contribute to cervical cancer.
  • the method of the invention may decrease the number of false negative results when compared with morphological classification.
  • False negative results are an inherent problem of morphological classification due mainly to the inherent subjectivity of the test. Sampling errors and processing artefacts may also increase the likelihood of false negative results. Furthermore, due to the subjectivity of the test in many cases the significance of the results are not clear cut, and this can lead to a need for regular further testing, including invasive tests.
  • a method of detecting cervical cancer in a sample comprising detecting the hypermethylation status of a panel of genes whose hypermethylation status is linked to the incidence of cervical cancer, wherein the panel of genes comprises at least the following genes: pl6, DAP-kinase, APC, MGMT and GSTP1.
  • pl6 is a cell cycle control gene, involved in regulating progression of the cell cycle from Gl to S phase.
  • MGMT Metal -Guanine-Methyl-transferase
  • GSTP1 encodes a glutathione S-transferase, which are important for detoxifying chemicals that can damage cells.
  • Death- associated Protein (DAP) Kinase plays an important role in apoptotic cell death.
  • detection of the hypermethylation status of the panel of genes relies upon quantitative methylation specific PCR (QMSP) .
  • QMSP quantitative methylation specific PCR
  • the preferred method according to the first and second aspect of the invention therefore, relies upon use of QMSP to achieve real time quantification of hypermethylation levels of certain gene sequences.
  • QMSP is a technique well characterised in the art for real time detection of the hypermethylation status of genes.
  • the method relies on the fact that after sodium- bisulfite treatment unmethylated Cytosine's are converted to Uracil, and methylated Cytosine's remain unconverted.
  • suitable primers that take advantage of the sequence differences, methylated genes can be distinguished from those that are not methylated.
  • QMSP is based on the continuous optical monitoring of a fluorogenic PCR. Therefore, in addition to the primers a fluorescent probe is required.
  • a Taqman ® (Applied Biosystems) probe will be used. Such probes are widely commercially available, and the Taqman ® system (Applied Biosystems) is well known in the art.
  • Taqman ® probes anneal between the upstream and downstream primer in a PCR reaction. They contain a 5 ' -fluorophore and a 3 '-quencher. During amplification the 5 '-3' exonuclease activity of the Taq polymerase cleaves the fluorophore off the probe. Since the fluorophore is no longer in close proximity to the quencher, the fluorophore will be allowed to fluoresce. The resulting fluorescence can be measured, and is in direct proportion to the amount of target sequence that is being amplified.
  • beacons are hairpin- shaped probes with an internally quenched fluorophore whose fluorescence is restored when bound to its target.
  • the loop portion acts as the probe while the stem is formed by complimentary "arm" sequences at the ends of the beacon.
  • a fluorophore and quenching moiety are attached at opposite ends, the stem keeping each of the moieties in close proximity, causing the fluorophore to be quenched by energy transfer.
  • the beacon detects its target, it undergoes a conformational change forcing the stem apart, thus separating the fluorophore and quencher. This causes the energy transfer to be disrupted to restore fluorescence.
  • This QMSP approach is more sensitive and more specific than conventional PCR and can, therefore, detect aberrant methylation patterns in human samples with substantial (1:10.000) contamination of normal DMA (12) .
  • this PCR reaction is amenable to high- throughput techniques allowing the analysis of approximately 400 samples in less then 2 hours without requirement for gel-electrophoresis .
  • QMSP is a preferable technique to MSP in the performance of the methods of the invention that take place using a cervical scraping sample, because MSP may not be sufficiently sensitive to distinguish cancer from normal tissues and low grade dysplasias.
  • SEQ ID NO's 1 and 2, 4 and 5, 7 and 8, 10 and 11, 13 and 14, 16 and 17 could be used as primers in a suitable MSP reaction (see below) .
  • Alternative methods of detection of hypermethylation status of the panel of genes may not depend upon the PCR reaction. Examples of techniques that could be used include mass spectrometry, including MALDI mass spectrometry, and use of microarray technology (Motorola, Nanogen) . With respect to a microarray, multiple suitable CpG island tags could be arrayed as templates on a solid support.
  • the solid support may be a microchip for example.
  • Amplicons such as those amplified by the primers as set out in SEQ ID NO's 1 and 2, 4 and 5, 7 and 8, 10 and 11, 13 and 14, 16 and 17 (as internal reference) may be prepared from t ⁇ st samples and also control cells (positive and negative controls) . These amplicons may then be used to probe the arrays in order to detect the hypermethylation status of the panel of genes and therefore provide a cervical cancer diagnosis.
  • the read out from the methods will preferably be a fluorescent read out, but may comprise, for example, an electrical read out.
  • detection of the hypermethylation status of the genes by QMSP will depend upon the primer and probe sequences set out in SEQ ID NO's 1-15.
  • Furthemore, QMSP requires an internal standard gene against which hypermethylation of the panel of genes can be measured.
  • the internal reference gene used is ⁇ -actin.
  • detection of the hypermethylation status of the jS-actin gene by QMSP may be carried out using the primer and probe sequences set out in SEQ ID NO's 16-18.
  • the methods of the invention will be used to detect squamous cell carcinoma (SCC) , a specific type of cervical cancer.
  • SCC squamous cell carcinoma
  • the sample tested would be a cervical scraping.
  • the method of the invention could also be carried out on tissue samples for example.
  • kits of the invention provides a kit for use in the QMSP method of detecting cervical cancer in a sample including gene specific primers and probes and means for contacting said primers and probes with said sample.
  • the kits may further comprise DNA isolation reagents, polymerase enzymes for amplification, sodium bisulphite, QMSP specific buffers etc.
  • Kits may include phosphate buffered saline (PBS) for suspending cells and wash buffer (10 mM HEPES-KOH
  • DNA may extracted using standard salt-chloroform techniques and therefore such reagents would be included in the kits of the invention. Ethanol precipitation may be used to obtain high molecular weight DNA, and such reagents used in this technique could be included within the scope of the invention.
  • TE buffer (10 mM Tris; 1 mM EDTA (pH 8.0)) may also be included for dissolving DNA samples, alternatively, for example, distilled water could be used.
  • Probes are designed that amplify the region of the genes that will be affected by sodium bisulphite treatment depending upon the hypermethylation status of the genes.
  • Probes generally containing a fluorescer and a quencher at opposite ends (e.g. Taqman probes or molecular beacons) will also be designed such that they bind in between the primers that amplify the hypermethylated region.
  • Fluorophores that could possibly be used in the method of the invention include, by way of example, FAM, HEXTM, NEDTM, ROXTM, Texas RedTM etc.
  • the kits of the invention are not limited to a single quencher. Quenchers, for example Dabcyl and TAMRA are well known quencher molecules that could be used in the method of the invention.
  • Kits of the invention may further include components necessary for the QMSP reaction.
  • reagents would be required for the sodium bisulphite treatment of the extracted DNA.
  • PCR enzymes such as Taq polymerase in order to amplify the DNA sequences.
  • the QMSP technique is well known in the art the reagents neccessary for its implementation will also be well known to one of skill in the art . Any such reagents are included in the scope of the present invention.
  • kits for use in the MSP method of detecting cervical cancer in a sample which would include, for example, DNA isolation reagents and means for contacting said primers with said sample.
  • the kits may further comprise gene specific primers , enzymes for amplification, sodium bisulphite and MSP specific buffers.
  • a method for detecting the incidence of cervical cancer in a sample comprising use of quantitative methylation specific PCR (QMSP) in order to detect the hypermethylation status of a panel of genes whose hypermethylation status is linked to the incidence of cervical cancer.
  • QMSP quantitative methylation specific PCR
  • the method relies upon use of QMSP to achieve real time quantification of hypermethylation levels of certain gene sequences as aforementioned.
  • a Taqman ® (Applied Biosystems) probe will be used. Such probes are widely commercially available, and the Taqman ® system (Applied Biosystems) is well known in the art.
  • the cervical cancer is squamous cell carcinoma (SCC) .
  • SCC squamous cell carcinoma
  • the technique may be applicable to other cervical cancers, such as adenocarcinoma for example.
  • the panel of genes will comprise at least the following genes: pl6, MGMT, GSTP1, DAP-kinase and APC.
  • tumour suppressor genes are preferred because hypermethylation has been demonstrated for these genes in squamous cell head and neck and cervical cancer (17,18,19) and the assessment of hypermethylation status by QMSP has already been optimized for these genes. It has been discovered that with use of this panel of genes hypermethylation of at least one of the genes is seen in 79% of SCC patients and in no control patients. However, APC, pl6 and DAP-Kinase appear to be most sensitive for SCC and therefore a panel containing at least these genes should give a reliable detection. of the presence of cervical cancer.
  • the panel of genes comprises at least the following genes: pl6, DAP-kinase and APC .
  • additional genes added to the panel will not have been previously characterised as genes whose hypermethylation status is linked to cervical cancer.
  • Candidate genes could be tested using the method of the invention to investigate whether their hypermethylation status is linked to, and therefore may improve, the sensitivity of detection of cervical cancer.
  • An increased number of genes may, therefore, possibly be used in the test, to increase sensitivity of the test as long as specificity in the QMSP reaction is maintained.
  • These genes may be candidate tumour suppressor genes or other cancer associated genes, where hypermethylation, particularly of CpG islands can cause a transcriptional block leading to a loss of expression of the functional protein, which in turn can cause cervical cancer.
  • the methods of the invention may decrease the number of false negative results when compared with morphological classification.
  • False negative results are an inherent problem of morphological classification due mainly to the inherent subjectivity of the test. Sampling errors and processing artefacts may also increase the likelihood of false negative results. Furthermore, due to the subjectivity of the test in many cases the significance of the results are not clear cut, and this can lead to a need for regular further testing, including invasive tests.
  • the invention may also allow more sensitive detection of cervical cancer, requiring less cells in order to achieve an accurate diagnosis. This will have practical benefits for patients where cervical scraping can lead to physical discomfort.
  • primers are required that allow amplification of the region affected by sodium bisulphite treatment .
  • a forward and reverse primer will normally be provided.
  • the primers and probes may typically be synthesised nucleic acid sequences. However, the sequences may also incorporate non-natural or derivatised bases, provided specificity of binding to the target sequences is retained.
  • FAM is a well known and commercially available fluorophore.
  • the invention is not limited to using this specific fluorophore. Any suitable fluorophore is included within the scope of the invention.
  • Alternative fluorophores that could possibly be used in the method of the invention include, by way of example, HEXTM, NEDTM, ROXTM, Texas RedTM etc.
  • the invention is not limited to the commercially available quencher TAMRA.
  • Other quenchers are well known in the art, for example Dabcyl is a well known quencher molecule that could be used in the method of the invention.
  • detection of the hypermethylation status of the panel of genes by QMSP depends on at least the primer and probe sequences set out in SEQ ID NO's 1-6 and 13-15. Therefore, this method would allow detection of the hypermethylation status of a panel of 3 preferred genes, namely pl6, DAP-kinase and APC. These genes have been shown by the inventors to be most closely linked to the incidence of SCC. Again, the invention is not limited to only these specific primers and probes. Any such primers and probes that would amplify the panel of preferred genes will fall within the scope of the invention.
  • a method for detecting the incidence of cervical cancer in a sample comprising use of quantitative methylation specific PCR (QMSP) in order to detect the hypermethylation status of a panel of genes whose hypermethylation status is linked to the incidence of cervical cancer, wherein detection of the hypermethylation status of the genes by QMSP depends on the primer and probe sequences set out in SEQ ID NO's 1- 15. Therefore, this method would allow detection of the hypermethylation status of a panel of 5 preferred genes, namely pl6, MGMT, GSTP1, DAP-kinase and APC. Any suitable primers and probes that would amplify the panel of preferred genes will fall within the scope of the invention.
  • QMSP quantitative methylation specific PCR
  • a suitable internal standard gene is used with the QMSP technique.
  • the internal reference gene is ⁇ -actin.
  • other internal references can be used, as long as the hypermethylation status of these genes is not linked to cervical cancer.
  • a forward and reverse primer SEQ ID NO: 16 and 17 respectively
  • a fluorescent probe SEQ ID NO: 18
  • SEQ ID NO: 18 is used to detect the hypermethylation status of the relevant region of the ⁇ - actin gene, as shown below. It will be appreciated, however, that any primers and probes that allow detection of the relevant region of the 3-actin gene by QMSP will fall within the scope of the invention.
  • the sample tested will be a cervical scraping as this sample type appears to give the most reliable results.
  • the invention is not limited to only this sample type.
  • paraffin embedded tissue samples are also within the scope of the invention.
  • the results of QMSP analysis are expressed as ratio's between two absolute measurements: 1) cycle number of crossing threshold for internal reference 2) cycle number of gene of interest
  • the threshold is set to the geometrical phase of the amplification above the background.
  • the invention is not limited to this specific threshold. Any threshold can be used providing it gives sensitive and specific results.
  • the ratio's obtained will be compared against a control ratio using the well known Mann Whitney U test in order to achieve a result as either positive or negative for cervical cancer.
  • Mann Whitney U test the well known Mann Whitney U test
  • the skilled person will realise that other forms of statistical analysis can be used in order to achieve a diagnostic result, and the invention is not limited to only this test.
  • An association at a level of p ⁇ 0.05 is preferred, but other significance levels are within the scope of the invention.
  • the level that the sample has to reach above control in order to be classified positive for cervical cancer can be balanced in order to achieve maximal sensitivity for the test, whilst retaining selectivity.
  • each primer and probe set comprising a forward and reverse primer and a fluorescent probe, allows specific amplification of a particular gene whose hypermethylation status is linked to cervical cancer.
  • the specific primers and probes are shown in SEQ ID NO's 1-15.
  • the primers and probes required for amplification of the internal reference gene -actin as shown in SEQ ID NO's 15-18.
  • kits containing components suitable for carrying out the method of the invention may be further embodied by kits containing components suitable for carrying out the method of the invention. Therefore, in a further aspect the invention provides a kit for use in detecting cervical cancer comprising a suitable set of primers and fluorescent probes that can be used to detect the hypermethylation status of genes linked to cervical cancer.
  • the primers and probes included in the kit comprise at least those represented as SEQ ID NO's 1-15. Therefore, such a kit would allow detection of the hypermethylation status of a panel of 5 preferred genes, namely pl6, MGMT, GSTPl, DAP-kinase and APC. Again, the invention is not limited to only these specific primers and probes. Any such primers and probes that would amplify the panel of preferred genes will fall within the scope of the invention.
  • the primers and probes included in the kit comprise at least those represented as SEQ ID NO's 1-6 and 12-15. Therefore, such a kit would allow detection of the hypermethylation status of a panel of 3 preferred genes, namely pl6, DAP- kinase and APC. These genes have been shown by the inventors to be most closely linked to the incidence of SCC. Again, the invention is not limited to only these specific primers and probes. Any such primers and probes that would amplify the panel of preferred genes will fall within the scope of the invention.
  • the internal reference gene is ⁇ -actin.
  • the reagent kit will contain a forward and reverse primer (SEQ ID NO: 16 and 17 respectively) and a fluorescent probe (SEQ ID NO: 18) suitable for detection of the hypermethylation status of the relevant region of the ⁇ -actin gene, as shown above. It will be appreciated, however, that any primers and probes that allow detection of the relevant region of the ⁇ -actin gene by QMSP will fall within the scope of the invention.
  • kits of the invention are not limited to using FAM as the only fluorophore, any suitable fluorophore is included within the scope of the invention. Similarly the kits of the invention are not limited to the commercially available quencher TAMRA.
  • kits of the invention may further comprise DNA isolation reagents in order to purify DNA from samples, which may be cervical scrapings or tissue samples for example .
  • the kit may also contain a means for removing cervical cells from a patient for analysis by QMSP.
  • a means for removing cervical cells from a patient for analysis by QMSP For example an Ayre's spatula and an endocervical brush may be used in order to obtain a cervical sample.
  • kits of the invention may further include components necessary foar the QMSP reaction.
  • reagents would be required for the sodium bisulphite treatment of the extracted DNA.
  • PCR enzymes such as Taq polymerase in order to amplify the DNA sequences.
  • the QMSP technique is well known in the art the reagents neccessary for its implementation will also be well known to one of skill in the art. Any such reagents are included in the scope of the present invention.
  • CpG island hypermethylation is often associated with a transcriptional block and subsequent loss of translation of the relevant protein. In many cases an important tumour suppressor protein is lost.
  • a method of detecting cervical cancer in a sample by detecting the RNA expression levels of a panel of genes whose hypermethylation status is linked to the incidence of cervical cancer.
  • the level of RNA will be decreased in a cancer sample, relative to a control, because hypermethylation of the gene whose hypermethylation status is linked to cancer leads to a decrease in transcription.
  • RNA levels may be detected using a number of techniques, all of which are well known in the art and rely on suitable isolation of the RNA.
  • reverse-transriptase-PCR is a well known technique.
  • RT-PCR relies upon an enzyme called reverse transcriptase which can use single stranded RNA as a template for production of double stranded cDNA (complementary DNA) which can subsequently be amplified using the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • the subsequent PCR step, following reverse transcription could be real-time quantitative PCR.
  • This would require, for example, fluorescent probes (such as Taqman probes or molecular beacons) in addition to suitable gene specific PCR primers, which would be specific for the genes whose hypermethylation status is linked to cervical cancer.
  • gene specific probes are represented in SEQ ID NO's 3 (APC methylation specific) , 6 (pl6 methylation specific) , 9 (GSTPl methylation specific) , 12 (MGMT methylation specific) , 15 (DAPK methylation specific) and 18 ( ⁇ - actin, as internal reference) .
  • RNA detection Another technique that may be used is microarray technology. If suitable tags, representing the genes whose hypermethylation status is linked to the incidence of cervical cancer were attached to a solid support, probes that detect expression of the panel of genes whose hypermethylation is linked to cervical cancer could be used in order to compare expression of the various RNA molecules in the test sample as compared to control samples.
  • the solid support may be in the form of a microchip (Motorola, Nanogen) .
  • Other techniques for RNA detection that could be used in this aspect of the invention include mass spectrometry, including MALDI mass spectrometry.
  • the read out from the various techniques may be a fluorescent read out, or alternatively through radiolabelling or electrical read-out.
  • kits allowing methods of detecting cervical cancer in a sample by detecting the RNA expression levels of a panel of genes whose hypermethylation status is linked to the incidence of cervical cancer to be carried out are provided.
  • sequence specific primers that would prime reverse transcription for the genes whose methylation status is linked to cervical cancer. Additionally sequence specific primers would also be required for the amplification stages. Primers for the amplification stages could comprise primers such as those shown in SEQ ID NO's 1 and 2 (APC methylation specific) , 4 and 5 (pl6 methylation specific) , 7 and 8 (GSTPl methylation specific) , 10 and 11 (MGMT methylation specific) , 13 and 14 (DAPK methylation specific) , 16 and 17 ( ⁇ -actin, as internal reference) .
  • the invention is not limited to these specific genes for detection of cervical cancer and so many other primers can be used to detect reduced expression of other genes whose hypermethylation status is linked to cervical cancer.
  • kits will further contain reagents neccessary for RT-PCR.
  • reagents neccessary for RT-PCR may include, by way of example, a reverse transcriptase such as AMV reverse transcriptase for first strand cDNA synthesis.
  • a DNA polymerase such as Tfl DNA polymerase would be required. It is important in RT- PCR reactions that conditions are kept ribonuclease (RNase) free. Therefore an RNase inhibitor would be included in the kit to prevent degradation of the RNA samples .
  • RNase ribonuclease
  • kit may include, by way of example, primers used to amplify the genes for which hypermethylation affects their expression levels to produce probes and means for contacting said primers with said sample.
  • the kit may optionally further comprise RNA isolation reagents, appropriate buffers and an array containing suitable tags attached to a support .
  • the array may be in the form of a microchip.
  • a kit for use in a method of detecting cervical cancer wherein RNA expression levels are detected by mass spectrometry may include components such as gene specific primers for the genes whose hypermethylation status is linked to the incidence of cervical cancer for reverse transcription and for second strand synthesis and further amplification and means for contacting said primers with said sample.
  • the kit may further include RNA isolation reagents and appropriate buffers. Such buffers are well known in the art.
  • Table 1 shows the number of positive hypermethylation results for the panel of 5 genes in cervical scrapings.
  • Table 2A compares the Pap-smear classification results with hypermethylation results in cervical scrapings from control patients.
  • Table 2B compares the Pap-smear classification results with hypermethylation results in cervical scrapings from SCC patients, related to FIGO stage (0) .
  • Figure 1 shows amplification plots for QMSP from a paired cervical scraping and tissue sample for a single SCC patient. Scraping and tissue show strong amplification of the internal reference gene ⁇ -actin and the gene of interest DAPK.
  • Figure 2 shows the distribution of (A) pl6, (B) APC and (C) DAP-kinase hypermethylation levels in cervical scrapings of SCC patients and controls. Each circle or triangle represents a different sample. The solid horizontal bars represent the cut-off values for the presented gene.
  • the cervix of both SCC and control patients was scraped with the blunt or pointed end of an Ayre's spatula and with an endocervical brush.
  • the scraped cells were suspended in 5 mL ice-cold phosphate buffered saline (PBS: 6.4 mM Na 2 HP0 4 ; 1.5 mM KH 2 P0 4 ; 0.14 M NaC ⁇ 2.7 mM KC1 (pH 7.2)) and kept on ice until further processing.
  • PBS ice-cold phosphate buffered saline
  • Paraffin-embedded primary tumour tissue and control samples were prepared from unstained 10 ⁇ m sections. If possible, tumour tissue was selected from an area with > 75% malignant cells. DNA was purified by phenol - chloroform extraction and ethanol precipitation and dissolved in 50 ⁇ L distilled water.
  • the amplicon sizes for the QMSP were 74bp for APC
  • the first primer is the forward primer
  • the second is the reverse primer
  • the third is the TaqMan probe. The sequences were the following:
  • Amplifications were carried out in 384-well plates. Each plate consisted of patient samples and multiple water blanks, as well as a positive and negative control. Serial dilutions of in vitro CpG methylated DNA with Sss I (New England Biolabs . Inc., Beverly, MA) were used for constructing the calibration curve on each plate. Dilution experiments showed linearity of amplification down to a dilution of 1:10,000 for methylated promoter DNA, as well as for unmethylated ⁇ -actin DNA. All data presented are within this linear range of amplification. All of the assays were performed at least twice.
  • QMSP analyses yielded values that were expressed as ratios between two absolute measurements ( (cycle number of crossing threshold for internal reference : cycle number of gene of interest) x 10) (figure 1) .
  • the presented positive ratios for APC, pl6, DAPK, GSTPl and MGMT per sample are the means of positive ratio's of in duplicate performed measurements for these genes.
  • a sample was called negative for the gene of interest when QMSP did not cross the threshold in one of the two, or in both measurements.
  • DNA input was called adequate when real-time ⁇ -actin PCR crossed threshold before or at 40 cycles. When DNA input was not adequate, results for hypermethylation were not analyzed.
  • MGMT is a DNA repair gene
  • GSTPl (21) is a detoxifying gene
  • DAE>- kinase is a proapoptotic gene and potentially inhibits metastasis (22)
  • pl6 and APC are important in cell cycle control (23,24).
  • the number of fully methylated alleles detected by the very stringent Taqman assay may be so low that there is stochastic (random) amplification.
  • stochastic amplification In conventional MSP blotting of the amplificated product assures that only a product of the right size is regarded positive and products obtained by random amplification are therefore excluded.
  • a solution for dealing with stochastic amplification could be to better select the relevant methylation sites tested by the primers and probes and to perform in triplicate analysis.
  • QMSP results for /3-actin were highly reproducible but for hypermethylated genes results were less reliable.
  • the QMSP technique on cervical scrapings is a promising new diagnostic tool for the detection of cervical cancer.
  • DAPK hypermethylation levels separated cancer cases and controls. By adding more tumour suppressor genes to the assay a higher detection rate may be obtained. Further exploratory studies and larger trials are necessary to better understand the use of this assay in the clinical setting.
  • N number of cases analyzed b hypermethylation, positive hypermethylation status defined as hypermethylation found for ⁇ 1 of the analyzed tumour suppressor genes ,
  • Table 2A Morphological Pap-smear classification related to hypermethylation results in cervical scrapings of control patients.
  • Table 2 B Morphological Pap-smear classification related to FIGO stage and hypermethylation results in cervical scrapings of SCC patients.
  • McCarver DG Hines RN.

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Abstract

La prEsente invention concerne des mEthodes et des trousses perfectionnEes pour dEtecter le cancer du col de l'utErus dans un Echantillon. Ces procEdEs consistent A dEtecter l'Etat d'hypermEthylation d'un panel de gEnes.
PCT/IB2004/001516 2003-04-03 2004-04-05 Genes hypermethyles et cancer du col de l'uterus Ceased WO2004087957A2 (fr)

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WO2006007980A3 (fr) * 2004-07-16 2006-04-06 Oncomethylome Sciences S A Esr1 et cancer cervical
EP1764419A2 (fr) 2005-09-15 2007-03-21 Veridex, LLC Procédé pour détecter la méthylation des gènes pour diagnostiquer une maladie proliferative
WO2009037441A1 (fr) * 2007-09-17 2009-03-26 Oncomethylome Sciences Sa Détection de méthylation améliorée
WO2009115615A3 (fr) * 2008-03-21 2009-11-12 Oncomethylome Sciences S.A. Détection et pronostic du cancer du col de l’utérus
EP1869224A4 (fr) * 2005-04-15 2009-11-18 Oncomethylome Sciences Inc Marqueurs de méthylation pour le diagnostic et le traitement de cancers
WO2011036173A1 (fr) 2009-09-24 2011-03-31 Oncomethylome Sciences S.A. Détection et pronostic du cancer du col de l'utérus
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WO2000052204A2 (fr) * 1999-02-22 2000-09-08 Orntoft Torben F Expression genique dans les tumeurs de la vessie
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WO2005049861A3 (fr) * 2003-11-10 2005-11-24 Epigenomics Ag Procede d'analyse de troubles cellulaires proliferatifs gynecologiques
WO2006007980A3 (fr) * 2004-07-16 2006-04-06 Oncomethylome Sciences S A Esr1 et cancer cervical
US20140141998A1 (en) * 2005-02-14 2014-05-22 The Johns Hopkins University Neoplasia screening compositions and methods of use
EP1869224A4 (fr) * 2005-04-15 2009-11-18 Oncomethylome Sciences Inc Marqueurs de méthylation pour le diagnostic et le traitement de cancers
EP1869222A4 (fr) * 2005-04-15 2010-01-20 Oncomethylome Sciences S A Marqueur de méthylation pour le diagnostic et le traitement des cancers
EP1764419A2 (fr) 2005-09-15 2007-03-21 Veridex, LLC Procédé pour détecter la méthylation des gènes pour diagnostiquer une maladie proliferative
JP2007075114A (ja) * 2005-09-15 2007-03-29 Veridex Llc 遺伝子メチル化の検出
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US9050280B2 (en) 2007-09-17 2015-06-09 Mdxhealth Sa Methylation detection of MGMT
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US10041128B2 (en) 2008-03-21 2018-08-07 MD×Health SA Methylation of the EPB41L3 gene or the promoter of the EPB41L3 gene in a test sample comprising cervical cells
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WO2011036173A1 (fr) 2009-09-24 2011-03-31 Oncomethylome Sciences S.A. Détection et pronostic du cancer du col de l'utérus
US20130224738A1 (en) * 2012-02-23 2013-08-29 Medical Diagnostic Laboratories, Llc Detecting dna methylation of bcl2, cdkn2a and nid2 genes to predict bladder cancer in humans
US9096905B2 (en) * 2012-02-23 2015-08-04 Medical Diagnostic Laboratories, Llc Detecting DNA methylation of BCL2, CDKN2A and NID2 genes to predict bladder cancer in humans
WO2016048138A1 (fr) 2014-09-22 2016-03-31 Rijksuniversiteit Groningen Biomarqueurs destinés au cancer du col de l'utérus
CN107287294A (zh) * 2017-06-14 2017-10-24 广州中心法则生物科技有限公司 一种宫颈癌相关基因甲基化程度的检测引物、探针、试剂盒及其应用
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