WO2004087957A2 - Hypermethylated genes and cervical cancer - Google Patents

Abstract

The present invention provides improved methods and kits for detecting cervical cancer in a sample. This is done by detecting the hypermethylation status of a panel of genes.

Description

Hyperπiethylated Genes and cervical cancer

Field of the invention The invention relates to various methods and kits for use in the detection of cervical cancer.

Background to the invention 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).

Although it has been suggested that high risk HPV testing may well improve cervical cancer screening (7,8) the specificity for high grade cervical neoplasia of high risk HPV testing is relatively low (9) . Therefore new objective diagnostic methods based on molecular changes in cervical cancer are needed. Silencing of tumour suppressor- or other cancer- associated genes by hypermethylation of CpG islands, located in the promoter and/or 5' -regions of many genes, is a common feature of human cancer (10) . CpG island hypermethylation is often associated with a transcriptional block and loss of the relevant protein (10) . In addition to the functional implications of gene inactivation in tumour development, these aberrant methylation patterns represent excellent targets for novel diagnostic approaches based on methylation sensitive PCR techniques.

Recently Dong et al . showed that promoter hypermethylation of at least one of the genes pl6, DAPK, MGMT, APC, HIC-1, and E-cadherin occurred in 79% of cervical cancer tissues and in none of normal cervical tissues from 24 hysterectomy specimens. Virmani et al . detected aberrant methylation of at least one of the genes pl6, RAR/3. , FHIT, GSTP1, MGMT and hMLHl in 14 of 19 cervical cancer tissue samples. These experiments were carried out using conventional methylation specific PCR (MSP) . In this approach DNA can be amplified using primer pairs designed to distinguish methylated from unmethylated DNA by taking advantage of sequence differences as a result of sodium-bisulfite treatment (11) . After sodium-bisulfite treatment unmethylated Cytosine's are converted to Uracil, and methylated Cytosine's remain unconverted.

An advancement of this technique is called realtime quantitative MSP (QMSP) which permits reliable quantification of methylated DNA. 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). Moreover, 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 .

Description of the invention The present invention seeks to provide improved methods to detect cervical cancer in a sample.

Accordingly, in a first aspect of the invention there is provided 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 LUX™ fluorogenic primers from Invitrogen™ 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.

In one embodiment of this aspect of 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. Alternatively, the primers designed to amplify the genes whose hypermethylation status is linked to the incidence of cervical cancer, possibly using PCR, could amplify different sized products that could be identified by, for example, separation by size of amplicon using gel electrophoresis .

It may not, in any case, be necessary to identify exactly which of the aforementioned panel of genes according to the invention is hypermethylated in order to accurately diagnose cervical cancer. Because 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.

In an alternative embodiment 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.

Preferably, 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.

Advantageously, 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.

In a second aspect of the invention there is provided 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 (Methyl -Guanine-Methyl-transferase) is an enzyme that functions to repair DNA. 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.

It has been discovered that, the hypermethylation of at least one of the genes in the aforementioned panel is seen in 79% of SCC patients and in no control patients. Thus detection of the hypermethylation status of this panel of genes provides a sensitive and specific test for cervical cancer, that can be used to augment current morphological testing methods.

In a preferred embodiment of the first and second aspects of the invention detection of the hypermethylation status of the panel of genes relies upon quantitative methylation specific PCR (QMSP) .

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. By designing 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. In a preferred embodiment of the invention 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.

However, the skilled reader will appreciate that alternatives to such probes would work equally with the invention. For example molecular beacon technology could be employed. In this system the 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. When 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) . Moreover, 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.

However, it is clear that other methods that can detect the hypermethylation status of the panel of genes can be used in the method of the invention. For example standard methylation specific PCR (MSP) could be used to detect the hypermethylation status of the panel of genes according to the invention. The primers represented by

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) 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.

In a preferred embodiment of the invention, 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. In a preferred embodiment of the invention, the internal reference gene used is β-actin.

Accordingly, 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.

In a preferred embodiment the methods of the invention will be used to detect squamous cell carcinoma (SCC) , a specific type of cervical cancer.

According to a preferred embodiment of the invention the sample tested would be a cervical scraping. However the method of the invention could also be carried out on tissue samples for example.

The methods described can be implemented using various kits of the invention. Accordingly, in a still further aspect 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.

DNA isolation reagents would be needed in order to purify DNA from samples, which may be cervical scrapings or tissue samples for example. Such DNA isolation reagents are well known in the art, for example phenol- chloroform extraction is a commonly used technique. Kits may include phosphate buffered saline (PBS) for suspending cells and wash buffer (10 mM HEPES-KOH

(pH=7.5); 1.5 mM MgCl₂; 10 mM KC1; 1 mM dithiothreitol) . 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.

As the QMSP technique is well known in the art such buffers and enzymes will be familiar to a person of skill in the art. Primers 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.

Any suitable fluorophore is included within the scope of the invention. Fluorophores that could possibly be used in the method of the invention include, by way of example, FAM, HEX™, NED™, ROX™, Texas Red™ etc. Similarly 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. Thus, reagents would be required for the sodium bisulphite treatment of the extracted DNA. Also required would be PCR enzymes, such as Taq polymerase in order to amplify the DNA sequences. As 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.

Furthermore, a kit for use in the MSP method of detecting cervical cancer in a sample is provided, and 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.

Once again, as the technique is well known in the ^' art such buffers and enzymes will be familiar to a person of skill in the art.

In a further aspect of the invention there is provided 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.

The method relies upon use of QMSP to achieve real time quantification of hypermethylation levels of certain gene sequences as aforementioned. In a preferred embodiment of the invention 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.

However, the skilled reader will appreciate that alternatives to such probes would work equally with the invention. For example molecular beacon technology could be employed.

In a most preferred embodiment the cervical cancer is squamous cell carcinoma (SCC) . However, the technique may be applicable to other cervical cancers, such as adenocarcinoma for example.

In a most preferred embodiment the panel of genes will comprise at least the following genes: pl6, MGMT, GSTP1, DAP-kinase and APC.

These five 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.

Therefore, in a further preferred embodiment the panel of genes comprises at least the following genes: pl6, DAP-kinase and APC . Preferably, 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.

Advantageously, 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.

Furthermore, 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.

In order to detect the hypermethylation status of the genes of interest, as mentioned above, primers are required that allow amplification of the region affected by sodium bisulphite treatment . A forward and reverse primer will normally be provided. Additionally for the

QMSP technique, in order to allow real time detection, a fluorescent probe is required. A list of suitable forward and reverse primers and probes for the various genes of interest is given below. It should be noted that the invention is not limited to these specific sequences. Any primers and probes that allow suitable detection of the hypermethylation status of the relevant genes by QMSP will fall within the scope of the invention. Similarly, conservative substitutions in the sequences that do not significantly affect the specificity of binding to the target sequences are within the scope of the invention.

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.

(a) APC methylation specific, SEQ ID NO:l 5 ' -GAA CCA AAA CGC TCC CCA T-3 '

SEQ ID NO: 2 5 ' -TTA TAT GTC GGT TAC GTG CGT TTA TAT-3

SEQ ID NO: 3 6FAM5 ' -CCC GTC GAA TAC CCG CCCG ATT A- 3 ' TAMRA

(b) pl6 methylation specific,

SEQ ID NO: 4 5'- TTA TTA GAG GGT GGG GCG GAT CGC-3 ' SEQ ID NO: 5 5 ' -GAC CCC GAA CCG CGA CCG TAA-3 '

SEQ ID NO: 6 6FAM5 ' -AGT AGT ATG GAG TCG GCG GCG GG- 3 'TAMRA

(c) GSTP1 methylation specific

SEQ ID NO: 7 5 ' -AGT TGC GCG GCG ATT TC-3 '

SEQ ID NO: 8 5 ' -GCC CCA ATA CTA AAT CAC GAC G-3 '

SEQ ID NO: 9 6FAM5 ' -CGG TCG ACG TTC GGG GTG TAG CG- 3 'TAMRA

(d) MGMT methylation specific

SEQ ID NO: 10 5 ' -CGA ATA TAC TAA AAC AAC CCG CG-3 ' SEQ ID NO: 11 5 ' -GTA TTT TTT CGG GAG CGA GGC-3' SEQ ID NO: 12 6FAM5 ' -AAT CCT CGC GAT ACG CAC CGT TTA

CG-3 'TAMRA

(e) DAPK methylation specific

SEQ ID NO: 13 5 ' -GGA TAG TCG GAT CGA GTT AAC GTC- 3 ' SEQ ID NO:14 5 ' -CCC TCC CAA ACG CCG A-3 ' SEQ ID NO: 15 6FAM5 ' -TTC GGT AAT TCG TAG CGG TAG GGT TTG G-3 'TAMRA

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, HEX™, NED™, ROX™, Texas Red™ etc.

Similarly, 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.

In a further preferred embodiment, 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.

In a further aspect of the invention there is provided 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.

A suitable internal standard gene is used with the QMSP technique. In a preferred embodiment, the internal reference gene is β-actin. However, other internal references can be used, as long as the hypermethylation status of these genes is not linked to cervical cancer. In a most preferred embodiment a forward and reverse primer (SEQ ID NO: 16 and 17 respectively) and a fluorescent probe (SEQ ID NO: 18) are used to detect the hypermethylation status of the relevant region of the β- 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.

(f) β-actin

SEQ ID NO: 16 5 ' -TGG TGA TGG AGG AGG TTT AGT AAG T-3 ' SEQ ID NO: 17 5 ' -AAC CAA TAA AAC CTA CTC CTC CCT TAA-3 ' SEQ ID NO: 18 6FAM5 ' -ACC ACC ACC CAA CAC ACA ATA ACA AAC ACA-3 'TAMRA.

In a most preferred embodiment the sample tested will be a cervical scraping as this sample type appears to give the most reliable results. However, the invention is not limited to only this sample type. For example, paraffin embedded tissue samples are also within the scope of the invention. Preferably 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

This ratio may require multiplication by a certain number in order to achieve a result that is more easily presented. The threshold is set to the geometrical phase of the amplification above the background. However the invention is not limited to this specific threshold. Any threshold can be used providing it gives sensitive and specific results.

In a most preferred embodiment 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. However, 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.

Similarly 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.

In a further aspect the invention provides certain probes and primers that can be used in the methods of the invention. As stated above 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. Also provided are the primers and probes required for amplification of the internal reference gene -actin, as shown in SEQ ID NO's 15-18.

This aspect 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.

In a preferred embodiment 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.

In a further preferred embodiment 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.

A suitable internal standard gene is used with the QMSP technique. In a preferred embodiment the internal reference gene is β-actin. However, other internal references can be used, as long as the hypermethylation status of these genes is not linked to cervical cancer. In a most preferred embodiment 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 β-actin gene, as shown above. It will be appreciated, however, that any primers and probes that allow detection of the relevant region of the β-actin gene by QMSP will fall within the scope of the invention.

Once again the 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.

The 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 .

In further embodiments the kit may also contain 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.

In a still further embodiment the kits of the invention may further include components necessary foar the QMSP reaction. Thus, reagents would be required for the sodium bisulphite treatment of the extracted DNA. Also required would be PCR enzymes, such as Taq polymerase in order to amplify the DNA sequences. As 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.

It is known that hypermethylation of genes, particularly in the CpG island regions, can lead to cancer. 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.

Therefore, in a further aspect of the invention there is provided 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.

The decreased 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. For example, reverse-transriptase-PCR (RT-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) .

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. Examples of such 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 (β- actin, as internal reference) .

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.

In a further aspect of the invention 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.

Accordingly, included in the kits would be 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 (β-actin, as internal reference) .

However, 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.

In one embodiment the kits will further contain reagents neccessary for RT-PCR. Such components may include, by way of example, a reverse transcriptase such as AMV reverse transcriptase for first strand cDNA synthesis. For second strand cDNA synthesis and subsequent amplification 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 . A kit for use with a microarray method is also provided. Such a 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.

The invention will be further understood with reference to the following examples, together with the accompanying tables and figures in which:

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 β-actin and the gene of interest DAPK. ΔRn is defined as the cycle- to-cycle change in the reporter fluorescence signal normalized to a passive reference fluorescence signal (log scale). Calculation of the ratio's are based on the cycle number where fluorescence of each reaction passes the threshold, which is set to the geometrical phase of the amplification above the background. In this example: in the tissue sample cycle number for β-actin is 27 and for DAPK 32 which leads to a ratio of 27/32x10= 8.4. In the scraping the DAPK ratio is 27/29x10=9.3.

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.

Material and Methods To explore the diagnostic utility of QMSP for cervical cancer we examined the hypermethylation status of the tumour suppressor genes pi6, GSTPl, MGMT, DAP- kinase and APC in both cervical scrapings and cervical tissue samples obtained from cervical squamous cell cancer patients and controls.

Patients

Cervical cancer patients

In the period March 2001 - August 2002 all patients referred because of cervical cancer were asked to participate in our research program during their initial visit to the outpatient clinic of the University Hospital Groningen. For the present study only those patients were included in whom squamous cell cancer (SCC) was diagnosed and in whom cervical cancer had not been fully removed by exconisation or loop excision before referral. Bimanual examination under general anaesthesia was performed in all cervical cancer patients for staging in accordance with the FIGO criteria (0) . During this procedure lesion size (largest diameter) and tumour spread beyond the cervix was estimated routinely.

All specimens used for the study were collected during the initial visit or before bimanual examination under general anaesthesia.

Controls

As control patients 30 women without a history of abnormal Pap-smears who were planned to undergo hysterectomy because of non- (pre) malignant disease in the same period were asked to participate in our study. All specimens used for the study were collected during surgery .

The study was approved by the medical ethical committee of the University Hospital Groningen and all patients gave written informed consent.

Sample collection Cervical scrapings

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 Na₂HP0₄; 1.5 mM KH₂P0₄; 0.14 M NaC ■ 2.7 mM KC1 (pH 7.2)) and kept on ice until further processing. Of this cell suspension 1 mL was used four cytomorphological examination and 4 mL was centrifuged and washed with wash buffer (10 mM HEPES-KOH (pH=7.5); 1.5 mM MgCl₂; 10 mM KCl ; 1 mM dithiothreitol) (as described previously) (13) , after which half of the pellet was snap frozen in liquid nitrogen and then stored at -80° C until further use for DNA extraction. DNA was extracted using standard salt -chloroform extraction and ethanol precipitation for high molecular DNA and dissolved in 250 μL TE-4 buffer (10 mM Tris; 1 mM EDTA (pH 8.0) )

Tissue samples

Paraffin-embedded primary tumour tissue and control samples were prepared from unstained 10 μm 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 μL distilled water.

Real-time quantitative Methylation Specific PCR

Real-time quantitative MSP for APC, pl6, DAP- kinase, GSTPl and MGMT was performed after bisulfite treatment on denatured genomic DNA (11) as previously reported for APC and GSTPl (14,15) . As internal reference gene the /3-actin gene was chosen.

The amplicon sizes for the QMSP were 74bp for APC

(position 761-834, deposited at GenBank as accession no. U02509) , 150 bp for pl6 (25-174, U12818) , 140 bp for GSTPl (1033-1172, M24485) , 122 bp for MGMT (1029-1150, X61657) , 98 bp for DAPK (5-102, X76104) and 133 bp for /3-actin (390-522, Y00474) . In all cases, the first primer is the forward primer, the second is the reverse primer, and the third is the TaqMan probe. The sequences were the following:

(a) APC methylation specific,

SEQ ID NO:l 5 ' -GAA CCA AAA CGC TCC CCA T-3 '

SEQ ID NO: 2 5 ' -TTA TAT GTC GGT TAC GTG CGT TTA TAT-3 '

SEQ ID NO: 3 6FAM5 ' -CCC GTC GAA TAC CCG CCCG ATT A- 3 ' TAMRA

(b) pl6 methylation specific,

SEQ ID NO: 4 5'- TTA TTA GAG GGT GGG GCG GAT CGC-3 ' SEQ ID NO: 5 5 ' -GAC CCC GAA CCG CGA CCG TAA-3 '

SEQ ID NO: 6 6FAM5 ' -AGT AGT ATG GAG TCG GCG GCG GG-

3 'TAMRA

(c) GSTPl methylation specific

SEQ ID NO: 7 5 ' -AGT TGC GCG GCG ATT TC-3 ' SEQ ID NO: 8 5 ' -GCC CCA ATA CTA AAT CAC GAC G-3' SEQ ID NO: 9 6FAM5 ' -CGG TCG ACG TTC GGG GTG TAG CG-

3 'TAMRA

(d) MGMT methylation specific

SEQ ID NO: 10 5 ' -CGA ATA TAC TAA AAC AAC CCG CG-3* SEQ ID NO: 11 5 ' -GTA TTT TTT CGG GAG CGA GGC-3 ' SEQ ID NO: 12 6FAM5 ' -AAT CCT CGC GAT ACG CAC CGT TTA

CG-3 'TAMRA

(e) DAPK methylation specific

SEQ ID NO: 13 5 ' -GGA TAG TCG GAT CGA GTT AAC GTC-3 ' SEQ ID NO: 14 5 ' -CCC TCC CAA ACG CCG A-3 ' SEQ ID NO: 15 6FAM5 ' -TTC GGT AAT TCG TAG CGG TAG GGT TTG G-3 'TAMRA

(f) β-actin

SEQ ID NO: 16 5 ' -TGG TGA TGG AGG AGG TTT AGT AAG T-3'

SEQ ID NO: 17 5 ' -AAC CAA TAA AAC CTA CTC CTC CCT TAA-3'

SEQ ID NO: 18 6FAM5 ' -ACC ACC ACC CAA CAC ACA ATA ACA AAC ACA-3 'TAMRA.

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 β-actin DNA. All data presented are within this linear range of amplification. All of the assays were performed at least twice.

Cytomorphological examination

After scraping of the cervix lmL of cell suspension was diluted with ethanol-carbowax (7% Polyethylenglycol , 43% distilled water, 50% ethanoll00%) and after resuspending centrifuged for 10 minutes at 1000 rpm. The cell -pellet was resuspended in ethanol -carbowax until appropriate cell numbers were achieved. Hettich cytospins (Hettich centrifuge, Depex b.v., Veenendaal, the Netherlands) were made on Poly L Lysin (Sigma chemical c.o., St. Louis, MO, USA) treated slides by centrifugation for 10 minutes at 1000 rpm. Cytospins were Papanicolaou stained and routinely classified according to a modified Papanicolaou system (16) without knowledge of clinical data.

Statistical Analysis

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 β-actin PCR crossed threshold before or at 40 cycles. When DNA input was not adequate, results for hypermethylation were not analyzed.

Differences in ratios between cancer patients and controls were tested with the Mann-Whitney U test and were considered to be significant when associated with p ≤ 0.05. Because of the exploratory character of this study it was decided to choose the cut-off values for tumour suppressor gene hypermethylation ratio's 0.01 higher than the highest control ratio observed. To be called positive SCC scrapings ratio's had to be higher than the cut-off values. After calculating these ratio's the frequency of positivity for tumour suppressor gene hypermethylation of cancer samples was analyzed. Analyses were carried out using the SPSS software package (Chicago, IL, USA) .

Results In the period March 2001 - August 2002 a total of 60 women were referred to our outpatient clinic because of cervical cancer. Of these women six did not want to, participate in our research program. Of the remaining 54 women 41 were diagnosed with squamous cell cancer but 11 had already been treated with exconisation or loop excision before referral. Therefore the cervical scrapings of 30 SCC women were eligible for analysis. Of the SCC patients four (13%) were diagnosed with FIGO stage IA, 14 (47%) with FIGO stage IB/IIA, and 12 (40%) with FIGO stage IIB-IV. In 10 SCC patients insufficient tissue samples were available for hypermethylation analyses. In all 30 control patients both scraping and tissue samples were available for analysis. The median age of the SCC patients was 51 (Interquartile-range 41 - 59) and of the control patients 48 (IQ-range 44 - 56) .

Real-time Quantitative Methylation Specific PCR Adequacy of QMSP

In two SCC and three control scrapings too little DNA was present to perform QMSP. In all others DNA input was adequate and β-actin PCR crossed threshold with a mean of 30.5 cycles (SD 3.0) in scrapings and 31.6 cycles (SD 1.1) in primary tissues. QMSP positive scrapings and tissue samples crossed the threshold at 36.4 (SD 4.5) and 38.0 cycles (SD 5.5), respectively. To analyze the reproducibility of QMSP differences between cycle numbers of duplicate measurements were calculated. For the 3-actin positive duplicate measurements the mean difference in cycle number was 0.66 (SD 1.45) in scrapings and 0.39 (SD 0.38) in tissue samples (p=0.98). The differences between two positive measurements of the five tumour suppressor genes were larger with a trend for higher differences in tissue samples than in scrapings. The mean difference in scrapings was 1.94 (SD 2.67) and in tissue samples 3.83 (SD 4.19) (p=0.07). When APC, pl6, GSTPl, MGMT and DAPK QMSP's were taken together 78 duplicate analyses gave positive results in at least one of two measurements in scrapings. Of these 78 analyses 19 (24%) gave a positive result in only one of two measurements and were therefore regarded negative. In tissue samples the same phenomenon was more frequently observed in 44 (65%) of 68 duplicate analyses (p < 0.001) . Because of these less reliable results in our paraffin embedded tissue samples only data from cervical scrapings were used for further analyses .

Clinical data and QMSP

For GSTPl no scraping tested positive and for MGMT the two scrapings that were positive were taken from control patients. For APC, pl6 and DAPK hypermethylation ratio's are presented in figure 2. In SCC patients DAPK- ratio's were significantly higher than DAPK-ratio's of controls (p<0.001). Cut-off for ratio's were defined to be 9.65 for APC, 6.95 for pl6, 7.80 for MGMT and 5.74 for DAPK. Using these ratio's hypermethylation of at least one of the five tested genes was seen in 22 of 28 (79%) scrapings of SCC patients and by definition in none of the controls (Table 1) . One SCC scraping tested positive for hypermethylation of both APC and DAPK.

Morphologic Pap-smear classification and real-time MSP

For two SCC patients and four controls no morphologic Pap-smear classification was obtained. In the seven cervical scrapings that had too little epithelial cells to be classified only one had too little DNA for QMSP while in none of the remaining six hypermethylation status was positive (Table 2) . In three scrapings DNA input was not adequate although the scraping could be morphologically classified. Although the cervical epithelium of all control patients was histologically diagnosed to be normal, six of the scrapings were cytologically classified in the category borderline dysplasia. In SCC patients no morphological classification could be made in 6 of 28 scrapings and one of 28 scrapings cervical cancer was under-diagnosed with only borderline dysplasia. In this borderline dysplastic scraping hypermethylation was detected and the sample scored positive.

Discussion

Cervical cancer deaths in countries with extensive screening programs partly reflect the drawbacks of current screening methods. The Pap-smear has false negatives rates of 2-40 % due to a combination of sampling error, processing artifacts and the nature of subjective interpretation (1,5,6). Moreover, as many as 20% of all Pap smears are interpreted as ASCUS (atypical squamous cells of undetermined significance) or Borderline dysplastic, leading to increased surveillance frequency and more invasive tests in many of these patients. These drawbacks of conventional screening have urged the search for an improvement of diagnostic adequacy of the Pap test . In this study we show that cervical scrapings can be used to detect hypermethylation of tumour suppressor genes in SCC and that QMSP is a promising new diagnostic tool in screening for cervical cancer.

The five tumour suppressor genes analyzed in the present study were chosen because for these genes hypermethylation was demonstrated in squamous cell head and neck and cervical cancer (17,18,19) and the assessment of hypermethylation status by QMSP had already been optimized for these genes. MGMT (20) is a DNA repair gene, GSTPl (21) is a detoxifying gene, DAE>- kinase is a proapoptotic gene and potentially inhibits metastasis (22) , and both pl6 and APC are important in cell cycle control (23,24).

Although none of these tumour suppressor genes were selected because of known high specificity for cervical SCC, 79% of cervical scrapings of SCC patients were scored as hypermethylation positive for at least one of the analyzed genes. The distributions of ratio's of pl6, APC and DAPK (figure 2) show that DAPK is most specific for SCC because hypermethylation for DAPK was either scored negative or ratio's were above control patients ratio's. Including more genes with these specific features may lead to the development of a specific squamous cervical cancer panel which should improve the sensitivity of the test without causing a decrease in the specificity for QMSP.

Conventional MSP for the detection of tumour suppressor gene promoter hypermethylation in cervical scrapings is severely limited as a technique to detect cancer because hypermethylation also occurs in normal tissue and low grade dysplasia 's (18) . When scrapings contain cervical cancer cells they are present in a background of normal epithelial cells. With conventional MSP, scrapings from patients with no, low and high grade dysplastic lesions but free of cervical cancer could all score positive. By using a robust quantitative assay, a clear difference in DAPK methylation ratio's between scrapings of controls and of SCC patients was demonstrated. In this pilot study in a series of SCC patients who were already referred because of cervical cancer we chose to define cut-off values for the five tumour suppressor genes tested to be above the highest control patients ratio's because this would represent the optimal balance between sensitivity and specificity of the test. If the same values were used in a screening population false positives would most probably be found because the cut-off was chosen very close to the highest control patients ratio. To obtain more definitive cutoff levels for real screening situations sensitivity and specificity analyses need to be performed in a much larger suitable study population. Furthermore, the sensitivity of the QMSP to detect cervical SCC using only five genes might be lower in a pre-clinical population than the in the present study reported 79%. Still our data suggest that QMSP is a promising new tool for the detection of cervical cancer even in screening populations.

In the present study we show that QMSP results in cervical scrapings are robust . The use of cervical scrapings for conventional MSP was previously shown in patients with low grade and high grade intraepithelial lesions (18) by detecting hypermethylation in 30% of nondysplasia/low-grade CIN in 71% of high grade CIN patients. Dong et al . showed with conventional MSP that hypermethylation of DAPK was present in 61%, of APC in 13%, of pl6 in 39% and of MGMT in 10% of 31 paraffin embedded SCC tissue samples from Korea, which are percentages comparable to what was found in scrapings in the present study (17) . It is possible that for some methylated genes, the number of fully methylated alleles detected by the very stringent Taqman assay may be so low that there is stochastic (random) 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. In our paraffin embedded tissues QMSP results for /3-actin were highly reproducible but for hypermethylated genes results were less reliable. A study of QMSP for GSTPl in adenocarcinoma of the prostate however, showed tumour-specific results in both fresh frozen and paraffin embedded tissue samples (15) . In cervical scrapings none of the samples analyzed tested positive for GSTPl hypermethylation. Possibly differences in paraffin-embedding-protocols can lead to differences in hypermethylation quality by further DNA degradation. Analysis of fresh frozen, paraffin embedded tissue samples and cervical scrapings of one patient will give more insight in the best way to store patient samples in order to obtain reliable QMSP results.

Cytomorphologic assessment did not recognize one case of stage IB disease. This is not surprising because false-negative rates of the conventional Pap smear are reported to be between 2-40% (1,5,6) . In this morphologically false negative smear QMSP correctly identified a hypermethylation value consistent with cervical cancer. In seven of fifty four morphologically classified scrapings too little epithelial cells were present for diagnosis. This corresponds with the 12% of inadequate smears in a screening program that was reported previously (6) . Due to the study protocol only one-fifth of a scrapings cell suspension was available for morphological analysis. The same amount of cell suspension was used for QMSP and therefore the presented results give a fair comparison, yet the results of both morphological classification and QMSP might improve when the entire cell suspension was available. In the four SCC patients with cervical scrapings containing too little epithelial cells for classification no hypermethylation was found while in three DNA input was sufficient for QMSP. Possibly the scrapings were made from negative cervical cancers. QMSP in our paraffin embedded tissue showed that two tissue samples were hypermethylation negative and one had a ratio for APC of

8.7, while in the paired scraping the ratio for APC was

6.8, which was below the cut-off value. A future comparison between morphological assessment and QMSP in adequately stored tissue samples will clarify whether QMSP is suitable as a diagnostic tool in scrapings with very little epithelial cells.

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.

Table 1: Hypermethylation of multiple genes in cervical scrapings according to the defined cut-off ratio's

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.

^a N, number of cases analyzed ^b adequate DNA input, defined as β-actin real-time PCR positive before or at 40 cycles. ^c hypermethylation, positive hypermethylation status defined as hypermethylation found for ≥ 1 of the analyzed tumor suppressor genes

Table 2 B: Morphological Pap-smear classification related to FIGO stage and hypermethylation results in cervical scrapings of SCC patients.

^a N, number of cases analyzed ^b adequate DNA input, defined as β-actin real-time PCR positive before or at 40 cycles. ^c hypermethylation, positive hypermethylation status defined as hypermethylation found for ≥ 1 of the analyzed tumor suppressor genes

Reference List

(0) Petterson, F. (Ed.) Annual report on the results of treatment in gynaecological cancer, FIGO, Stockholm, Vol 20 (1988) .

(1) Cervical cancer. NIH Consens Statement 1996; 14 (1) :l-38.

(2) Zur Hausen H. Papillomavirus infections--a major cause of human cancers. Biochimica et Biophysica Acta 1996; 1288 (2) :F55-F78.

(3) Woodman CB, Collins S, Winter H, Bailey A, Ellis J, Prior P et al . Natural history of cervical human papillomavirus infection in young women: a longitudinal cohort study. Lancet 2001; 357 (9271) : 1831-1836.

(4) Herbert A. Is cervical screening working? A cytopathologist ' s view from the United Kingdom. Hum Pathol 1997; 28 (2) : 120-126.

(5) Robertson JH, Woodend B. Negative cytology preceding cervical cancer: causes and prevention. J Clin Pathol 1993; 46 (8) : 700-702.

(6) Koss LG. Cervical (Pap) smear. New directions. Cancer 1993; 71(4 Suppl) : 1406-1412.

(7) Bovicelli A, Bristow RE, Montz FJ. HPV testing: where are we now? Anticancer Res 2000; 20 (6C) :4673-4680.

(8) Sherman ME, Schiffman M, Cox JT. Effects of age and human papilloma viral load on colposcopy triage: data from the randomized Atypical Squamous Cells of Undetermined Significance/Low-Grade Squamous Intraepithelial Lesion Triage Study (ALTS) . J Nati Cancer Inst 2002; 94 (2) : 102-107.

(9) Kulasingam SL, Hughes JP, Kiviat NB, Mao C, Weiss

NS, Kuypers JM et al . Evaluation of human papillomavirus testing in primary screening for cervical abnormalities: comparison of sensitivity, specificity, and frequency of referral. JAMA 2002; 288 (14) : 1749-1757.

(10) Baylin SB, Esteller M, Rountree MR, Bachman KE, Schuebel K, Herman JG. Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. Hum Mol Genet 2001; 10 (7) : 687-692.

(11) Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Nati Acad Sci U S A 1996; 93 (18) : 9821-9826.

(12) Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D et al . MethyLight : a high-throughput assay to measure DNA methylation. Nucleic Acids Res 2000;

28 (8) :E32.

(13) Wisman GB, Hollema H, de Jong S, Ter Schegget J, Tjong AHS, Ruiters MH et al . Telomerase activity as a biomarker for (pre) neoplastic cervical disease in scrapings and frozen sections from patients with abnormal cervical smear. J Clin Oncol 1998; 16(6):2238- 2245.

(14) Usadel H, Brabender J, Danenberg KD, Jeronimo C, Harden S, Engles J et al . Quantitative adenomatous polyposis coli promoter methylation analysis in tumor tissue, serum, and plasma DNA of patients with lung cancer. Cancer Res 2002; 62 (2) : 371-375.

(15) Jeronimo C, Usadel H, Henrique R, Oliveira J, Lopes C, Nelson WG et al . Quantitation of GSTPl methylation in non-neoplastic prostatic tissue and organ-confined prostate adenocarcinoma. J Nati Cancer Inst 2001; 93 (22) :1747-1752.

(16) Hanselaar AG. kwaliteit van cytopathologisch onderzoek in het herziene bevolkingsonderzoek naar baarmoederhalskanker . Nederlands Tijdschrift voor Obstetrie en Gynaecologie 1996; 109:207-210.

(17) Dong SM, Kim HS, Rha SH, Sidransky D. Promoter hypermethylation of multiple genes in carcinoma of the uterine cervix. Clin Cancer Res 2001; 7 (7) : 1982-1986.

(18) Virmani AK, Muller C, Rathi A, Zoechbauer-Mueller S, Mathis M, Gazdar AF . Aberrant methylation during cervical carcinogenesis . Clin Cancer Res 2001; 7(3):584- 589.

(19) Sanchez-Cespedes M, Esteller M, Wu L, Nawroz-Danish H, Yoo GH, Koch WM et al . Gene promoter hypermethylation in tumors and serum of head and neck cancer patients. Cancer Res 2000; 60 (4) : 892-895.

(20) Gerson SL. Clinical relevance of MGMT in the treatment of cancer. J Clin Oncol 2002; 20 (9) : 2388-2399.

(21) McCarver DG, Hines RN. The ontogeny of human drug- metabolizing enzymes: phase II conjugation enzymes and regulatory mechanisms. J Pharmacol Exp Ther 2002; 300 (2) :361-366. (22) Inbal B, Bialik S, Sabanay I, Shani G, Kimchi A. DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. J Cell Biol 2002; 157 (3 ): 455-468.

(23) Lundberg AS, Hahn WC, Gupta P, Weinberg RA. Genes involved in senescence and immortalization. Curr Opin Cell Biol 2000; 12 (6) : 705-709.

(24) Sieber OM, Tomlinson IP, Lamlum H. The adenomatous polyposis coli (APC) tumour suppressor- -genetics, function and disease. Mol Med Today 2000; 6(12) :462-469

Claims

1. 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.

2. A method according to claim 1 wherein the hypermethylation status of the panel of genes is detected using quantitative methylation specific PCR (QMSP) .

3. A method according to claim 1 wherein the hypermethylation status of the panel of genes is detected using methylation specific PCR (MSP) .

4. A method according to any one of claims 1 to 3 wherein the hypermethylation status of the entire panel of genes is assessed in a single experiment.

5. A method according to any one of claims 1 to 3 wherein the hypermethylation status of each of the panel of genes is assessed in a separate reaction.

6. A kit for use in the method of any one of claims 1, 2, 4 or 5 comprising: a) gene specific primers and probes for the genes whose hypermethylation status is linked to the incidence of cervical cancer; b) means for contacting said primers and probes with said sample.

7. A kit for use in the method of any one of claims 1 or 3 to 5 comprising: a) gene specific primers for the genes whose hypermethylation status is linked to the incidence of cervical cancer; and b) means for contacting said primers with said sample.

8. A kit according to claim 6 further comprising QMSP specific buffers.

9. A kit according to claim 7 further comprising MSP specific buffers.

10. A kit according to any one of claims 6 to 9 further comprising DNA isolation reagents.

11. A kit according to any one of claims 6 to 10 further comprising enzymes for amplification of DNA.

12. A kit according to any one of claims 6 to 11 further comprising sodium bisulphite.

13. 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: pi6, DAP-kinase, APC, MGMT and GSTPl.

14. A method according to claim 13 wherein detection of the hypermethylation status of the panel of genes relies upon quantitative methylation specific PCR (QMSP) .

15. A method according to claim 14 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.

16. A method according to any one of claims 14 or 15 wherein the internal reference gene used in QMSP is β-actin.

17. A method according to claim 16 wherein the detection of the hypermethylation status of the β-actin gene by QMSP depends on the primer and probe sequences set out in SEQ ID NO's 16-18.

18. A method according to any one of claims 13 to

17 wherein the cervical cancer comprises squamous cell carcinoma (SCC) .

19. A method according to any one of claims 13 to

18 wherein the sample is a cervical scraping.

20. A method of detecting 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.

21. A method according to claim 20 wherein the cervical cancer comprises squamous cell carcinoma (SCC) .

22. A method according to claim 20 or 21 wherein the panel of genes comprises at least the following genes: pl6, DAP-kinase and APC.

23. A method according to claim 22 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 1-6 and 13-15.

24. A method according to any one of claims 20 or 21 wherein the panel of genes comprises at least the following genes: pl6, MGMT, GSTPl, DAP-kinase and APC.

25. A method according to claim 24 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.

26. A method according to any one of claims 20 to 25 wherein the internal reference gene used in QMSP is β-actin.

27. A method according to claim 26 wherein the detection of the hypermethylation status of the β-actin gene by QMSP depends on the primer and probe sequences set out in SEQ ID NO's 16-18.

28. A method according to any one of claims 20 to 27 wherein the sample is a cervical scraping.

29. A kit comprising a set of primers and probes for detecting the hypermethylation status of a panel of genes using QMSP in order to detect cervical cancer in a sample.

30. A kit according to claim 29, wherein the primers and probes comprise at least those set out in SEQ ID NO's 1-15.

31. A kit according to any one of claims 29 or 30, wherein the primers and probes comprise at least those set out in SEQ ID NO's 1-6 and 13-15.

32. A kit according to any one of claims 29 to 31 further comprising primers and probes suitable for detecting the hypermethylation status of an internal reference gene using QMSP.

33. A kit according to claim 32 wherein the internal reference gene is β-actin.

34. A kit according to claim 33 wherein the primers and probes used to detect the hypermethylation status of the internal reference gene are those set out in SEQ ID NO's 15-18

35. A kit according to any one of claims 29 to 34 further comprising suitable QMSP reagents.

36. A kit according to claim 35 wherein the QMSP reagents include sodium bisulphite.

37. A kit according to claim 35 or 36 further comprising PCR enzymes.

38. A method of detecting cervical cancer in a sample comprising detecting the RNA expression levels of a panel of genes whose hypermethylation status is linked to the incidence of cervical cancer.

39. A method according to claim 38 wherein RNA expression levels are detected by a reverse transcription step followed by a PCR step.

40. A kit for use in a method according to claim 39 comprising: a) 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 b) means for contacting said primers with said sample .

41. A kit according to claim 40 further comprising RNA isolation reagents.

42. A kit according to claim 40 or 41 further comprising suitable reverse transcriptase and polymerase enzymes .

43. A kit according to any one of claims 40 to 42 further comprising a suitable RNase inhibitor.

44. A kit according to claim 40 further comprising appropriate buffers.

45. A kit according to any one of claims 40 to 44 further comprising probes that allow real-time quantitative PCR to be carried out.

46. A method according to claim 38 wherein RNA expression levels are detected using microarray technology.

47. A kit for use in a method according to claim 46 comprising: a) an array containing tags representing the genes whose hypermethylation status is linked to the incidence of cervical cancer attached to a support, b) primers used to amplify the genes for which hypermethylation affects their expression levels to produce probes.

48. A kit according to claim 47 further comprising RNA isolation reagents.

49. A kit according to claim 47 or 48 further comprising appropriate buffers.

50. A method according to claim 38 wherein RNA expression levels are detected by mass spectrometry.

51. A kit for use in a method according to claim 50 comprising: a) 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, b) means for contacting said primers with said sample.

52. A kit according to claim 51 further comprising RNA isolation reagents.

53. A kit according to claim 51 or 52 further comprising appropriate buffers.

537833; MPS; MPS