WO2008122447A2

WO2008122447A2 - Dna methylation assay for the diagnosis or prognosis of medical conditions

Info

Publication number: WO2008122447A2
Application number: PCT/EP2008/002850
Authority: WO
Inventors: Fabian Model; Andrew Sledziewski; Theo De Vos; Matthias Schuster; Jürgen Distler; Catherine Lofton-Day
Original assignee: Epigenomics Ag
Priority date: 2007-04-10
Filing date: 2008-04-10
Publication date: 2008-10-16
Also published as: WO2008122447A3

Abstract

The present invention relates to methods for performing DNA methylation assays of biological samples for features associated with disease conditions. The method is carried out on 2-8 aliquots of the ≥sample. The extent of methylation is used in the prediction of a medical condition. Among the conditions analysed are colorectal and breast cancer and among the genes analysed are SEPT9 and PITX2.

Description

DNA methylation assay for the diagnosis or prognosis of medical conditions

FIELD OF THE INVENTION

The present invention relates to methods for performing DNA methylation assays of biological samples for features associated with disease conditions.

BACKGROUND

Molecular disease markers. Molecular disease markers offer several advantages over other types of markers, one advantage being that even samples of very small sizes and/or samples whose tissue architecture has not been maintained can be analyzed quite efficiently. Within the last decade a large number of genes have been shown to be differentially expressed in disease conditions. However, the development of such molecular diagnostic or prognostic tests has been greatly limited by a lack of sensitivity or specificity.

Q?G island methylation.

The methylation of CpG islands has been shown to lead to the transcriptional silencing of certain genes that have been previously linked to the pathogenesis of various diseases, including cancers. CpG islands are short sequences which are rich in CpG dinucleotides and can usually be found in the 5' region of approximately 50% of all human genes. Methylation of the cytosines in these islands leads to the loss of gene expression and has been reported in the inactivation of the X chromosome and genomic imprinting.

Incidence and diagnosis of cancer.

Cancer is the second leading cause of death of the United States. Mortality rates could be significantly improved if current screening methods would be improved in terms of patient compliance, sensitivity and ease of screening. Current recommended methods for diagnosis of cancer are often invasive, expensive or are otherwise not suitable for application as population wide screening tests.

Development of medical tests. Two key evaluative measures of any medical screening or diagnostic test are its sensitivity and specificity, which measure how well the test performs in accurately detecting all affected individuals without exception, and without falsely including individuals who do not have the target disease (predicitive value). Historically, many diagnostic tests have been criticized due to poor sensitivity and specificity. A true positive (TP) result is where the test is positive and the condition is present. A false positive (FP) result is where the test is positive but the condition is not present. A true negative (TN) result is where the test is negative and the condition is not present. A false negative (FN) result is where the test is negative but the condition is not present. In this context: Sensitivity = TP/(TP+FN); Specificity = TN/(FP+TN); and Predictive value = TP/(TP+FP).

Sensitivity is a measure of a test's ability to correctly detect the target disease in an individual being tested. A test having poor sensitivity produces a high rate of false negatives, i.e., individuals who have the disease but are falsely identified as being free of that particular disease. The potential danger of a false negative is that the diseased individual will remain undiagnosed and untreated for some period of time, during which the disease may progress to a later stage wherein treatments, if any, may be less effective. An example of a test that has low sensitivity is a protein-based blood test for HIV. This type of test exhibits poor sensitivity because it fails to detect the presence of the virus until the disease is well established and the virus has invaded the bloodstream in substantial numbers. In contrast, an example of a test that has high sensitivity is viral-load detection using the polymerase chain reaction (PCR). High sensitivity is achieved because this type of test can detect very small quantities of the virus. High sensitivity is particularly important when the consequences of missing a diagnosis are high.

Specificity, on the other hand, is a measure of a test's ability to identify accurately patients who are free of the disease state. A test having poor specificity produces a high rate of false positives, i.e., individuals who are falsely identified as having the disease. A drawback of false positives is that they force patients to undergo unnecessary medical procedures treatments with their attendant risks, emotional and financial stresses, and which could have adverse effects on the patient's health. A feature of diseases which makes it difficult to develop diagnostic tests with high specificity is that disease mechanisms, particularly in cancer, often involve a plurality of genes and proteins. Additionally, certain proteins may be elevated for reasons unrelated to a disease state. Specificity is important when the cost or risk associated with further diagnostic procedures or further medical intervention are very high.

SUMMARY OF THE INVENTION

The present invention provides a method for analyzing patient samples for DNA methylation characteristically associated with disease conditions. Although such methods are known in the art, the present invention provides a method for analyzing a plurality of aliquots of said samples and determining the presence or absence of a disease condition based upon the proportion of aliquots presenting with methylation, preferably above a pre-defined threshold value. Analysis of patient samples according to the method of the present invention enables the diagnosis or prognosis of diseases with an improved accuracy.

DETAILED DESCRIPTION OF THE INVENTION Definitions:

The term "target nucleic acid" refers to either a DNA sequence comprising one or more CpG dinucleotides, or the sequence thereof subsequent to treatment with a bisulfite reagent. The term "CpG island" refers to a contiguous region of genomic DNA that satisfies the criteria of (1) having a frequency of CpG dinucleotides corresponding to an "Observed/Expected Ratio" >0.6, and (2) having a "GC Content" >0.5. The term "CpG rich region" or "CpG dense region" refers to a contiguous region of genomic DNA that satisfies the criteria of having a frequency of CpG dinucleotides corresponding to an "Observed/Expected Ratio" >0.5.

CpG islands, and CpG rich or dense regions are typically, but not always, between about 0.2 to about 1 KB, or to about 2kb in length.

The term "poor prognosis" shall be taken to mean aggressive disease, less than average or median disease free survival, less than average or median metastasis free survival or less than average or median survival.

The term "methylation state" or "methylation status" refers to the presence or absence of 5- methylcytosine ("5-mCyt") at one or a plurality of CpG dinucleotides within a DNA sequence. Methylation states at one or more particular CpG methylation sites (each having two CpG dinucleotide sequences) within a DNA sequence include "unmethylated," "fully- methylated" and "hemi-methylated."

The term "bisulfite assay" shall be taken to mean an assay for determining the methylation status of one or more CpG position comprising I) a methylation specific bisulfite reagent conversion of a genomic DNA to a "bisulfite converted DNA" and II) determining the methylation specific conversion or non-conversion of CpG positions of the genomic DNA. The term 'AUC as used herein is an abbreviation for the area under a curve. In particular it refers to the area under a Receiver Operating Characteristic (ROC) curve. The ROC curve is a plot of the true positive rate against the false positive rate for the different possible cut points of a diagnostic test. It shows the trade-off between sensitivity and specificity depending on the selected cut point (any increase in sensitivity will be accompanied by a decrease in specificity). The area under an ROC curve (AUC) is a measure for the accuracy of a diagnostic test (the larger the area the better, optimum is 1 , a random test would have a ROC curve lying on the diagonal with an area of 0.5; for reference: J.P. Egan. Signal Detection

Theory and ROC Analysis, Academic Press, New York, 1975).

The term "microarray" refers broadly to both "DNA microarrays," and 'DNA chip(s),' as recognized in the art, encompasses all art-recognized solid supports, and encompasses all methods for affixing nucleic acid molecules thereto or synthesis of nucleic acids thereon.

The term "bisulfite reagent" refers to a reagent comprising bisulfite, disulfite, hydrogen sulfite or combinations thereof, useful as disclosed herein to distinguish between methylated and unmethylated CpG dinucleotide sequences.

The term "Methylation assay" refers to any assay for determining the methylation state of one or more CpG dinucleotide sequences within a sequence of DNA.

The term "MS. AP-PCR" (Methylation-Sensitive Arbitrarily-Primed Polymerase Chain

Reaction) refers to the art-recognized technology that allows for a global scan of the genome using CG-rich primers to focus on the regions most likely to contain CpG dinucleotides, and described by Gonzalgo et al., Cancer Research 57:594-599, 1997.

The term "MethyLight™" refers to the art-recognized fluorescence-based real-time PCR technique described by Eads et al., Cancer Res. 59:2302-2306, 1999.

The term "HeavyMethyl™" assay, in the embodiment thereof implemented herein, refers to an assay, wherein methylation specific blocking probes (also referred to herein as blockers) covering CpG dinucleotides between, or covered by the amplification primers enable methylation-specific selective amplification of a nucleic acid sample.

The term "HeavyMethyl™ MethyLight™" assay, in the embodiment thereof implemented herein, refers to a HeavyMethyl™ MethyLight™ assay, which is a variation of the

MethyLight™ assay, wherein the MethyLight™ assay is combined with methylation specific blocking probes covering CpG dinucleotides between the amplification primers.

The term "Ms-SNuPE" (Methylation-sensitive Single Nucleotide Primer Extension) refers to the art-recognized assay described by Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531,

1997.

The term "MSP" (Methylation-specific PCR) refers to the art-recognized methylation assay described by Herman et al. Proc. Natl. Acad. ScL USA 93:9821-9826, 1996, and by US Patent

No. 5,786,146. The term "COBRA" (Combined Bisulfite Restriction Analysis) refers to the art-recognized methylation assay described by Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997. The term "MCA" (Methylated CpG Island Amplification) refers to the methylation assay described by Toyota et al., Cancer Res. 59:2307-12, 1999, and in WO 00/26401 Al. The term "aliquot" shall be taken to mean one of a plurality of portions of a biological sample wherein unless otherwise stated said plurality of aliquots are each of comparable size, weight and/or DNA concentration.

The term "clinical sample" shall be taken to mean a biological sample taken from an individual comprising DNA.

The term "hybridisation" is to be understood as a bond of an oligonucleotide to a complementary sequence along the lines of the Watson-Crick base pairings in the sample DNA, forming a duplex structure.

"Stringent hybridisation conditions," as defined herein, involve hybridising at 68°C in 5x SSC/5x Denhardt's solution/1.0% SDS, and washing in 0.2x SSC/0.1% SDS at room temperature, or involve the art-recognized equivalent thereof (e.g., conditions in which a hybridisation is carried out at 60⁰C in 2.5 x SSC buffer, followed by several washing steps at 37⁰C in a low buffer concentration, and remains stable). Moderately stringent conditions, as defined herein, involve including washing in 3x SSC at 42°C, or the art-recognized equivalent thereof. The parameters of salt concentration and temperature can be varied to achieve the optimal level of identity between the probe and the target nucleic acid. Guidance regarding such conditions is available in the art, for example, by Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N. Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N. Y.) at Unit 2.10. The terms "Methylation-specific restriction enzymes" or "methylation-sensitive restriction enzymes" shall be taken to mean an enzyme that selectively digests a nucleic acid dependant on the methylation state of its recognition site. In the case of such restriction enzymes which specifically cut if the recognition site is not methylated or hemimethylated, the cut will not take place, or with a significantly reduced efficiency, if the recognition site is methylated. In the case of such restriction enzymes which specifically cut if the recognition site is methylated, the cut will not take place, or with a significantly reduced efficiency if the recognition site is not methylated. Preferred are methylation-specific restriction enzymes, the recognition sequence of which contains a CG dinucleotide (for instance cgcg or cccggg). Further preferred for some embodiments are restriction enzymes that do not cut if the cytosine in this dinucleotide is methylated at the carbon atom C5. "Non-methylation-specific restriction enzymes" or "non-methylation-sensitive restriction enzymes" are restriction enzymes that cut a nucleic acid sequence irrespective of the methylation state with nearly identical efficiency. They are also called "methylation-unspecific restriction enzymes." In reference to composite array sequences, the phrase "contiguous nucleotides" refers to a contiguous sequence region of any individual contiguous sequence of the composite array, but does not include a region of the composite array sequence that includes a "node," as defined herein above.

Reference to a gene shall also be taken to include its entire sequence including promoter and other regulatory regions. Furthermore, reference to a gene shall be taken to include the CpG rich regions and islands comprised within or associated in a regulatory capacity therewith.

In one embodiment the present invention provides a method for determining the presence or absence of a medical condition or the prognosis of a patient by determining the presence or absence of methylation within at least one target nucleic acid in a biological sample by independently determining the methylation status of said target nucleic acid(s) within a plurality of aliquots of said sample, and determining the presence or absence of said medical condition according to the proportion of aliquots with target nucleic acid methylation. In a first step a plurality of aliquots of the biological sample are taken. Said plurality of aliquots are between two and eight, but more preferably between two and three. In the second step the presence of methylation within the target nucleic acid(s) is determined in each of the aliquots. In the third step of the method the presence or absence of said medical condition (or the prognosis thereof) is determined according to the number of aliquots comprising methylated target nucleic acids.

The method of the present invention is preferably utilised in determining the presence or absence of a medical condition selected from the group consisting of cancers, solid tumors, cell proliferative disorders. In an alternative embodiment the method of the invention may be used for determining the prognosis (i.e. disease progression) of a patient diagnosed with cancers, solid tumors or cell proliferative disorders

Preferred is a cancer selected from the group consisting of soft tissue, skin, leukemia, renal, prostate, brain, bone, blood, lymphoid, stomach, head and neck, colon or breast cancer. Further preferred is a cancer selected from the group consisting of colon, colorectal, breast, leukaemia, prostate and lung.

Any clinical or biological sample comprising DNA of the subject may be used in the method of the present invention. Particularly preferred are cell lines, histological slides, biopsies, paraffin-embedded tissue, body fluids, ejaculate, urine, blood plasma, blood serum, whole blood, isolated blood cells, cells isolated from the blood, sputum, nipple aspirate fluid , lymphatic fluid, ductal lavage fluid, fine needle aspirate, biological matter derived from bronchoscopy, bronchial lavage, bronchial alveolar lavage, bronchial brushing, bronchial abrasion. The present invention is particularly suited to the diagnosis of cancer by means of methylation analysis of body fluid samples. Particularly preferred body fluid samples for the diagnosis of cancers include but are not limited to clinical samples selected from the group consisting of ejaculate, urine, blood plasma, blood serum, whole blood, isolated blood cells, cells isolated from the blood, sputum, nipple aspirate fluid, lymphatic fluid, ductal lavage fluid, fine needle aspirate, bronchial lavage and bronchial alveolar lavage. Particularly preferred for the diagnosis of prostate cancers are clinical samples selected from the group consisting of ejaculate, urine, blood plasma, blood serum, whole blood, isolated blood cells, cells isolated from the blood. Particularly preferred for the diagnosis of oral and esophagel cancers are clinical samples selected from the group consisting of sputum, oral swabs, cheek cell, blood plasma, blood serum, whole blood, isolated blood cells, cells isolated from the blood. Particularly preferred for the diagnosis of colon or colorectal cancers are clinical samples selected from the group consisting of blood plasma, blood serum, whole blood, isolated blood cells, cells isolated from the blood. Particularly preferred for the diagnosis of breast cancers are clinical samples selected from the group consisting of nipple aspirate fluid , lymphatic fluid, , ductal lavage fluid, fine needle aspirate, blood plasma, blood serum, whole blood, isolated blood cells, cells isolated from the blood. Particularly preferred for the diagnosis of lung cancers are clinical samples selected from the group consisting of biological matter derived from bronchoscopy, bronchial lavage, bronchial alveolar lavage, bronchial brushing, bronchial abrasion, blood plasma, blood serum, whole blood, isolated blood cells, cells isolated from the blood. Particularly preferred for the diagnosis of leukemias are clinical samples selected from the group consisting of blood plasma, blood serum, whole blood, isolated blood cells, cells isolated from the blood.

Analysis of the methylation status of each of the individual aliquots requires the isolation of DNA from the biological or clinical sample, preferably this is carried out prior to the isolation of aliquots from said sample. Methods for the isolation of DNA are known in the art. Genomic DNA may be isolated by any means standard in the art, including the use of commercially available kits. Briefly, wherein the DNA of interest is encapsulated in by a cellular membrane the biological sample must be disrupted and lysed by enzymatic, chemical or mechanical means. The DNA solution may then be cleared of proteins and other contaminants, e.g., by digestion with proteinase K. The genomic DNA is then recovered from the solution. This may be carried out by means of a variety of methods including salting out, organic extraction or binding of the DNA to a solid phase support. The choice of method will be affected by several factors including time, expense and required quantity of DNA. In the second step of the method the methylation status of at least one target nucleic acid is determined. It is preferred that the target nucleic acid comprises a sequence of a gene (including its promoter or regulatory regions) the methylation of which is associated with the presence of the disease or medical condition and which is not methylated (or less methylated) in healthy tissues. Preferably said target nucleic acid comprises 5, 10 , 15, 20, 50 or 100 CpG dinucleotides and has a "Observed/Expected Ratio" >0.3. It is also preferred that said target nucleic acid comprises a CpG island or CpG rich sequence sequence of said gene (including its promoter or regulatory regions). The methylation status of said target nucleic acid is preferably the methylation status of one or a plurality of CpG dinucleotides within the target nucleic acid. Preferably the methylation status of at least 1, 5, 10 , 15 or 20 CpG dinucleotides is determined. The methylation status may then be expressed as a composite value of the plurality of the analysed CpG dinucleotides (for example, as a sum or average).

Preferred target nucleic acids are nucleic acids comprising one or more CpG dinucleotides of genes (including promoter and regulatory regions) or sequences selected from the group consisting of Septin 9; Septin 9 CpG rich region; SNDl; PCDHGC3; EDNRB; STOM; GLI3; RXFP3; RASSF2; Q8N2B6; PCDHlO; LIMKl; TFAP2E; PTGER4; SHOX2; RASSF2A; TFAP2E; CCND2 ; RASSFlA ; MSF ; SEQ ID NO: 22; PRDM6; LMXlA ; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; NR2E1; SCGB3A1; SLIT2; SEQ ID NO: 31; DAPKl; PITX2. Particularly preferred are target nucleic acids comprising one or more CpG dinucleotides of the sequence of said gene(s) according to SEQ ID NO: 1 to SEQ ID NO: 33 (see Table 1). Also preferred are target nucleic acids wherein all CpG dinucleotides thereof are comprised within SEQ ID NO: 1 to SEQ ID NO: 33 (see Table 1).

It is further preferred that at least one or more of said CpG dinucleotides are located within CpG rich regions or CpG islands. Also preferred are CpG dinucleotides located within evolutionary conserved regions of the Homo Sapiens genome, as compared to other mammals. Particularly preferred are such evolutionary conserved CpG rich regions or CpG islands.

In one embodiment of the method said target nucleic acid is located within the genes or sequences selected from the group consisting of Septin 9; Septin 9 CpG rich region; SNDl; PCDHGC3; EDNRB; STOM; GLI3; RXFP3; RASSF2; Q8N2B6; PCDHlO; LIMKl; TFAP2E; PTGER4; SHOX2; RASSF2A; TFAP2E; CCND2 ; RASSFlA ; MSF ; SEQ ID NO: 22; PRDM6; LMXlA ; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; NR2E1; SCGB3A1 ; SLIT2; SEQ ID NO: 31; DAPKl; PITX2 and said corresponding medical condition(s) is/are selected from Table 1. In a further embodiment of the method said target nucleic acid comprises CpG comprised within SEQ ID NO: 1 to SEQ ID NO: 33 (see Table 1) and said corresponding medical condition(s) is/are selected from Table 1.

Determination of the methylation status may be carried out by any means known in the art. Various methylation assay procedures are known in the art, and can be used in conjunction with the present invention. These assays allow for determination of the methylation state of one or a plurality of CpG dinucleotides (e.g., CpG islands) within a DNA sequence. Preferably said means is a chemical assay, a bisulfite assay, an enzymatic assay or combinations thereof. It is particularly preferred that said assay is a quantitave assay that determines the level (e.g. percent, fraction, ratio, proportion or degree) of methylation at a particular CpG dinucleotide(s). In a preferred embodiment said assay is a bisulfite assay. It is particularly preferred that the treatment with a bisulfite reagent is carried out upon the biological or clinical sample prior to the aliquoting of the sample.

Methods of said treatment are known in the art (e.g. PCT/EP2004/011715, which is incorporated by reference in its entirety). It is preferred that the bisulfite treatment is conducted in the presence of denaturing solvents such as but not limited to n-alkylenglycol, particularly diethylene glycol dimethyl ether (DME), or in the presence of dioxane or dioxane derivatives. In a preferred embodiment the denaturing solvents are used in concentrations between 1% and 35% (v/v). It is also preferred that the bisulfite reaction is carried out in the presence of scavengers such as but not limited to chromane derivatives, e.g., 6-hydroxy-2, 5,7,8, -tetramethylchromane 2-carboxylic acid or trihydroxybenzoe acid and derivates thereof, e.g. Gallic acid (see: PCT/EP2004/011715 which is incorporated by reference in its entirety). The bisulfite conversion is preferably carried out at a reaction temperature between 30⁰C and 70⁰C, whereby the temperature is increased to over 85°C for short periods of times during the reaction (see: PCT/EP2004/011715 which is incorporated by reference in its entirety). The bisulfite treated DNA is preferably purified prior to the further method steps. This may be conducted by any means known in the art, such as but not limited to ultrafiltration, preferably carried out by means of Microcon^Λ(TM) columns (manufactured by Millipore^Λ(TM)). The purification is carried out according to a modified manufacturer's protocol (see: PCT/EP2004/011715 which is incorporated by reference in its entirety).

Subsequent to the aliquoting of the genomic DNA each aliquot is assayed for the presence of methylation. Preferably said assay is a PCR assay. Preferably said amplificates are 100 to 2,000 base pairs in length. The set of primer oligonucleotides preferably includes at least two oligonucleotides whose sequences are each reverse complementary, identical, or hybridise under stringent or highly stringent conditions to an at least 16-base-pair long segment of the base sequences of one of SEQ ID NO: 34 to SEQ ID NO: 165 (see Table 2) and sequences complementary thereto.

In one embodiment of the method, the methylation status of pre-selected CpG positions within the target nucleic acid, may be detected by use of methylation-specifϊc primer oligonucleotides. This technique (MSP) has been described in United States Patent No. 6,265,171 to Herman. The use of methylation status specific primers for the amplification of bisulfite treated DNA allows the differentiation between methylated and unmethylated nucleic acids. MSP primers pairs contain at least one primer which hybridises to a bisulfite treated CpG dinucleotide. Therefore, the sequence of said primers comprises at least one CpG dinucleotide. MSP primers specific for non-methylated DNA contain a "T' at the position of the C position in the CpG. Preferably, therefore, the base sequence of said primers is required to comprise a sequence having a length of at least 9 nucleotides which hybridises to a treated nucleic acid sequence according to one of SEQ ID NO: 34 to SEQ ID NO: 165 (see Table 2) and sequences complementary thereto, wherein the base sequence of said oligomers comprises at least one CpG dinucleotide. A further embodiment of the method comprises the use of blocker oligonucleotides (the HeavyMethyl™ assay). The use of such blocker oligonucleotides has been described by Yu et al., BioTechniques 23:714-720, 1997. Blocking probe oligonucleotides are hybridised to the bisulfite treated nucleic acid concurrently with the PCR primers. PCR amplification of the nucleic acid is terminated at the 5' position of the blocking probe, such that amplification of a nucleic acid is suppressed where the complementary sequence to the blocking probe is present. The probes may be designed to hybridize to the bisulfite treated nucleic acid in a methylation status specific manner. For example, for detection of methylated nucleic acids within a population of unmethylated nucleic acids, suppression of the amplification of nucleic acids which are unmethylated at the position in question would be carried out by the use of blocking probes comprising a 'CpA' or 'TpA' at the position in question, as opposed to a 'CpG' if the suppression of amplification of methylated nucleic acids is desired. For PCR methods using blocker oligonucleotides, efficient disruption of polymerase-mediated amplification requires that blocker oligonucleotides not be elongated by the polymerase. Preferably, this is achieved through the use of blockers that are 3'-deoxyoligonucleotides, or oligonucleotides derivitized at the 3' position with other than a "free" hydroxyl group. For example, 3'-O-acetyl oligonucleotides are representative of a preferred class of blocker molecule.

Additionally, polymerase-mediated decomposition of the blocker oligonucleotides should be precluded. Preferably, such preclusion comprises either use of a polymerase lacking 5 '-3' exonuclease activity, or use of modified blocker oligonucleotides having, for example, thioate bridges at the 5'-terminii thereof that render the blocker molecule nuclease-resistant. Particular applications may not require such 5' modifications of the blocker. For example, if the blocker- and primer-binding sites overlap, thereby precluding binding of the primer (e.g., with excess blocker), degradation of the blocker oligonucleotide will be substantially precluded. This is because the polymerase will not extend the primer toward, and through (in the 5 '-3' direction) the blocker — a process that normally results in degradation of the hybridized blocker oligonucleotide.

A particularly preferred blocker/PCR embodiment, for purposes of the present invention and as implemented herein, comprises the use of peptide nucleic acid (PNA) oligomers as blocking oligonucleotides. Such PNA blocker oligomers are ideally suited, because they are neither decomposed nor extended by the polymerase.

Preferably, therefore, the base sequence of said blocking oligonucleotides is required to comprise a sequence having a length of at least 9 nucleotides which hybridises to a treated nucleic acid sequence according to one of SEQ ID NO: 34 to SEQ ID NO: 165 (see Table 2) and sequences complementary thereto, wherein the base sequence of said oligonucleotides comprises at least one CpG, TpG or CpA dinucleotide.

The fragments obtained by means of the amplification can carry a directly or indirectly detectable label. In yet a further embodiment of the method, the genomic methylation status of the CpG positions may be ascertained by means of oligonucleotide probes (as detailed above) that are hybridised to the bisulfite treated DNA concurrently with the PCR amplification primers (wherein said primers may either be methylation specific or standard). A particularly preferred embodiment of this method is the use of fluorescence-based Real Time Quantitative PCR (Heid et al., Genome Res. 6:986-994, 1996; also see United States Patent No. 6,331,393) employing a dual-labelled fluorescent oligonucleotide probe (TaqMan™ PCR, using an ABI Prism 7700 Sequence Detection System, Perkin Elmer Applied Biosystems, Foster City, California). The TaqMan™ PCR reaction employs the use of a non-extendible interrogating oligonucleotide, called a TaqMan™ probe, which, in preferred embodiments, is designed to hybridise to a CpG-rich sequence located between the forward and reverse amplification primers. The TaqMan™ probe further comprises a fluorescent "reporter moiety" and a "quencher moiety" covalently bound to linker moieties (e.g., phosphoramidites) attached to the nucleotides of the TaqMan™ oligonucleotide. For analysis of methylation within nucleic acids subsequent to bisulfite treatment, it is required that the probe be methylation specific, as described in United States Patent No. 6,331,393, (hereby incorporated by reference in its entirety) also known as the MethyLightTM™ assay. Variations on the TaqMan™ detection methodology that are also suitable for use with the described invention include the use of dual-probe technology (Lightcycler™) or fluorescent amplification primers (Sunrise™ technology). Both these techniques may be adapted in a manner suitable for use with bisulfite treated DNA, and moreover for methylation analysis within CpG dinucleotides.

The present invention provides bisulfite converted variants of the preferred target nucleic acids in Table 1. For each genomic target nucleic acid sequence four equivalent bisulfite converted sequence variants are provided. The sequences of SEQ ID NO: 34 TO SEQ ID NO: 165 provide non-naturally occurring modified versions of the nucleic acid according to SEQ ID NO: 1 TO SEQ ID NO: 33, wherein the modification of each genomic sequence results in the synthesis of a nucleic acid having a sequence that is unique and distinct from said genomic sequence as follows. For each sense strand genomic DNA, e.g., SEQ ID NO: 1, four converted versions are disclosed. A first version wherein "C" is converted to "T," but "CpG" remains "CpG" (i.e., corresponds to case where, for the genomic sequence, all "C" residues of CpG dinucleotide sequences are methylated and are thus not converted); a second version discloses the complement of the disclosed genomic DNA sequence (i.e. antisense strand), wherein "C" is converted to "T," but "CpG" remains "CpG" (i.e., corresponds to case where, for all "C" residues of CpG dinucleotide sequences are methylated and are thus not converted). The 'upmethylated' converted sequences of SEQ ID NO: 1 TO SEQ ID NO: 14 correspond to SEQ ID NO: 34 TO SEQ ID NO: 99. A third chemically converted version of each genomic sequences is provided, wherein "C" is converted to "T" for all "C" residues, including those of "CpG" dinucleotide sequences (i.e., corresponds to case where, for the genomic sequences, all "C" residues of CpG dinucleotide sequences are wwmethylated); a final chemically converted version of each sequence, discloses the complement of the disclosed genomic DNA sequence (i.e. α«//sense strand), wherein "C" is converted to "T" for all "C" residues, including those of "CpG" dinucleotide sequences (i.e., corresponds to case where, for the complement (antisense strand) of each genomic sequence, all "C" residues of CpG dinucleotide sequences are uranethylated). The 'downmethylated' converted sequences of SEQ ID NO: 1 TO SEQ ID NO: 33 correspond to SEQ ID NO: 100 TO SEQ ID NO: 165. See Table 2 for further details.

It is particularly preferred that the methylation assay is quantified by reference to a calibration assay, or standard curve. Such methods are known in the art.

In the final step of the method the presence or absence of the medical condition (preferably cancer) is determined wherein the presence thereof is determined if at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the aliquots contain methylated DNA. Particularly preferred is a method wherein the presence of a cancer is determined if at least 20%, 30% or 40% of the aliquots are methylated in the target nucleic acid. However due to so-called "background methylation" and the heterogenous nature of clinical samples (i.e. they may contain a small number of disease cells in a background of normal cells) it is particularly preferred that a threshold value is set and an aliquot is not determined as methylated unless it is methylated above this level. It is particularly preferred that the presence of methylation is determined when the said methylation is at least 1%, 2%, 5%, 10%, 20%, 30% or 50%. In this embodiment of the method it is particularly preferred if the methylation state of a target nucleic acid of a gene selected form the group consisting of Septin 9; Septin 9 CpG rich region; SNDl; PCDHGC3; EDNRB; STOM; GLI3; RXFP3; RASSF2; Q8N2B6; PCDHlO; LIMKl; TFAP2E; PTGER4; SHOX2; RASSF2A; TFAP2E; CCND2 ; RASSFlA ; MSF ; SEQ ID NO: 22; PRDM6; LMXlA ; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; NR2E1; SCGB3A1; SLIT2; SEQ ID NO: 31; DAPKl is determined, and that the disease condition is selected from Table 1.

In an alternative embodiment of the method, in the final step of the method the prognosis of the medical condition (preferably cancer) is determined wherein the a poor prognosis thereof is determined if at least 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the aliquots contain methylated DNA. Particularly preferred is a method wherein the a poor prognosis is determined if at least 20%, 30% or 40% of the aliquots are methylated in the target nucleic acid. However due to so-called "background methylation" and the heterogenous nature of clinical samples (i.e. they may contain a small number of disease cells in a background of normal cells) it is particularly preferred that a threshold value is set and an aliquot is not determined as methylated unless it is methylated above this level. It is particularly preferred that the presence of methylation is determined when the said methylation is at least 1%, 2%, 5%, 10%, 20%, 30% or 50%.

In this embodiment of the method it is particularly preferred if the methylation state of a target nucleic acid of the gene PITX2 is determined, and that the disease condition is either of breast or prostate cancer.

In a further embodiment the method comprises the steps of the embodiments as disclosed above, briefly:

i) dividing the sample into 2-8 aliquots ii) determining the presence or absence of methylation within at least one target nucleic acid in each of said aliquots iii) determining the presence or absence of a medical condition according to the number of aliquots containing methylated DNA.

Furthermore in said embodiment the sample is divided into 3-5 aliquots, wherein at least one thereof is diluted to at least 1/5 of the original concentration and the presence of a medical condition is determined when at least two thirds of the non-diluted aliquots are positive for methylation OR at least one of the diluted aliquots is positive for methylation. It is preferred that the diluted aliquot(s) is/are diluted to at least 1/10 , 1/20 or 1/30 of the original concentration. In one embodiment said assay is performed upon 3 non-diluted replicates and 1 diluted replicate.

Table 1

Table 2

Claims

1. Method for determining the presence or absence of a medical condition or the prognosis thereof in a subject by determining the presence or absence of methylation within at least one target nucleic acid in a biological sample comprising i) dividing said sample into 2-8 aliquots ii) determining the presence or absence of methylation within at least one target nucleic acid in each of said aliquots iii) determining the presence or absence of a medical condition or the prognosis thereof according to the number of aliquots containing methylated DNA.

2. The method according to Claim 1 wherein in iii) the presence of a medical condition is determined if 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more of the samples contain methylated DNA.

3. The method according to any of claims 1 to 2 wherein in ii) the presence of methylation is determined when the said methylation is at least 1%, 2%, 5%, 10%, 20%, 30% or 50%.

4. The method according to any of claims 1 to 3 wherein said biological sample is selected from the group consisting of cell lines, histological slides, biopsies, paraffin- embedded tissue, body fluids, ejaculate, urine, blood plasma, blood serum, whole blood, isolated blood cells, cells isolated from the blood, sputum, nipple aspirate fluid , lymphatic fluid, ductal lavage fluid, fine needle aspirate, biological matter derived from bronchoscopy, bronchial lavage, bronchial alveolar lavage, bronchial brushing, bronchial abrasion and combinations thereof.

5. The method according to any of claims 1 to 4 wherein determining the presence or absence of methylation is determined by means of an enzymatic and/or bisulfite assay.

6. The method according to claim 5 wherein determining the presence or absence of methylation is determined by means of a bisulfite assay, and wherein treatment of the genomic DNA with a bisulfite reagent is carried out prior to i).

7. The method according to either of claims 5 or 6 wherein said assay is a PCR assay.

8. The method according to claim 7 wherein said assay is selected from the group consisting of realtime assay, MSP, MethyLight, HeavyMethyl and combinations thereof.

9. The method according to any of claims 1 to 8 wherein the target nucleic acid comprises one or more CpG dinucleotides of a sequence, gene or its promoter or regulatory region selected from the group consisting of Septin 9; Septin 9 CpG rich region; SNDl; PCDHGC3; EDNRB; STOM; GLD; RXFP3; RASSF2; Q8N2B6; PCDHlO; LIMKl; TFAP2E; PTGER4; SHOX2; RASSF2A; TFAP2E; CCND2 ; RASSFlA ; MSF ; SEQ ID NO: 22; PRDM6; LMXlA ; SEQ ID NO: 25; SEQ ID NO: 26; SEQ ID NO: 27; NR2E1; SCGB3A1; SLIT2; SEQ ID NO: 31; DAPKl ; PITX2.

10. The method according to Claim 9 wherein the target nucleic acid comprises one or more CpG dinucleotides of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 33.

11. The method according to any of claims 1 to 11 wherein said medical condition is selected from the group consisting of cancers, solid tumors, cell proliferative disorders.

12. A method according to any of claims 1 to 11 further comprising, i) dividing said sample into 3-5 aliquots, wherein at least one thereof is diluted to at least 1/5 of the original concentration; ii) determining the presence or absence of methylation within at least one target nucleic acid in each of said aliquots iii) determining the presence of a medical condition when at least two thirds of the non- diluted aliquots are positive for methylation OR at least one of the diluted aliquots is positive for methylation.

13. A method according to claim 12 wherein the diluted sample is diluted to at least 1/10 , 1/20 or 1/30 of the original concentration.

14. A method according to any of claims 12 to 13 wherein in step i. said assay is performed upon 3 non-diluted replicates and 1 diluted replicate.