WO2008155549A2

WO2008155549A2 - Improved urine sample collecting and processing

Info

Publication number: WO2008155549A2
Application number: PCT/GB2008/002093
Authority: WO
Inventors: Katja Bierau; Madeleine Grooteclaes; Gaetan Otto; Joost Louwagie; Isabelle Renard
Original assignee: Oncomethylome Sciences Sa; Spencer, Matthew, Peter
Priority date: 2007-06-19
Filing date: 2008-06-19
Publication date: 2008-12-24
Also published as: WO2008155549A3; EP2173897A2

Abstract

A method for identifying, diagnosing and/or staging or otherwise characterising a urologic cancer or neoplasia in a subject comprises isolating cell-free DNA from a urine sample taken from the subject and analysing the DNA to determine whether the DNA contains an epigenetic modification. The epigenetic modification is indicative of the presence or stage or other characteristic of the urologic cancer or neoplasia and thus facilitates diagnosis, staging or otherwise characterising the urologic cancer or neoplasia. Stabilising buffers may be added to collected urine samples to enhance storage stability, even without the need for centrifugation, at various temperatures. Cell-free DNA may be isolated through appropriate filtration or capture.

Description

_ i _

IMPROVED URINE SAlNdPLE COLLECTING AND PROCESSING

FIELD OF THE INVENTION

The present invention is concerned with improved methods of collecting and processing samples, in particular urine samples. More particularly, the invention relates to methods for identifying, diagnosing, staging or otherwise characterising cancers, in particular urologic cancers. Certain methods involve isolating and analysing the cell- free DNA component of urine.

BACKGROUND OF THE INVENTION

In their earliest stages most cancers are clinically silent. Patient diagnosis typically involves invasive procedures that frequently lack sensitivity and accuracy. Highly reliable, non-invasive screening methods would permit easier patient screening, diagnosis and prognostic evaluation.

Tumor markers are biological substances that are usually produced by malignant tumors. Ideally a tumor marker should be tumor-specific, provide an indication of tumor burden and should be produced in sufficient amounts to allow the detection of minimal disease. Clinical usefulness of tumor markers is dependent on two variables: sensitivity and specificity. Typically, sensitivity and specificity are used as quantitative statistical parameters to describe the performance of diagnostic tests. The sensitivity expresses the percentage of true positives obtained using a diagnostic test, whilst specificity expresses the percentage of true negatives obtained through use of a particular diagnostic test. Thus, maximal levels of sensitivity and specificity are desirable. Most tumor markers used in clinical practice are tumor antigens, enzymes, hormones, receptors and growth factors that are detected by biochemical assays. The detection of DNA alterations such as mutations, deletions and epigenetic modifications provide another means for identifying tumors.

Epigenetic changes, particularly methylation of specific genes, are found to be involved in a variety of cancers including colon, lung, breast, ovarian and prostate cancer. Methylation is the main epigenetic modification in humans (Das et al. , 2004) .

DNA methylation markers are evaluated as potential genetic markers for detection of cancer because they offer certain advantages when compared to mutation markers. One of the most important advantages is that changes in methylation status occur at the early stages of cancer development and in many cases are tissue- and tumor-type specific (Esteller et al., 2001). A further advantage is that a methylation profile is preserved in purified isolated DNA. Also, methylation changes appear to precede apparent malignancy in many cases.

Bodily fluids provide a cost-effective and early non- invasive procedure for cancer detection. Various bodily fluids have been used for the molecular detection of cancer and the potential of urine-derived, cell-associated DNA in cancer detection has been described (Cairns et al., 2001). Human total urine has been shown to possess two size categories of DNA: a larger species, heterogeneous in size, but (typically) greater than lkb and a smaller species, the majority of which is between 150bp and 250bp. The large size class appears to be mainly derived from cells shed into the urine from the urinary tract. The kidney barrier prevents cells "upstream" of this point entering the urine. The small size class of DNA may be recovered from the supernatant following low-speed centrifugation of the urine. It comprises, inter alia, cell-free circulating DNA from the blood circulation that passed the kidney barrier into urine.

The potential usefulness of both classes of urine DNA in diagnosis of specific cancer types has been investigated. As the kidney does not allow passage of cancer cells into urine, the potential of small, cell-free urine DNA for detection of colorectal cancers and other cancers distant from the urinary tract has been investigated (Ying-Hsiu Su et al., 2004; Botezatu, I. et al. 2000; Bryzgunova et al., 2006) . The large size and cell-associated DNA on the other hand has been investigated for detecting prostate cancers that originate in the peripheral zone of the prostate gland for which the secretary ducts empty cells and cellular components directly into the urethra (Cairns et al., 2001) . Utting et al (2002) carried out microsatellite analysis of free tumour DNA in urine, serum and plasma of bladder cancer patients. In urine only 27% of tumor-specific alterations were detected. Serum and plasma performed better in each case. No relation between detected DNA alterations and tumor staging/grading nor tumor progression could be found.

Techniques such as polymerase chain reaction allow the detection of small quantities of tumor DNA in the context of a high level of background DNA derived from normal cells.

The main advantage of working with the cellular fraction is the reduced risk of contamination with tumor DNA derived from cancers distant from the urinary tract, since such tumor DNA will reside only in the small size fraction because of the kidney barrier.

Although tumor specificity of both DNA classes in urine has proven excellent, sensitivity is significantly hampered by the extremely low concentration of free nucleic acid in urine and because of impurities present in the large size population DNA. In the case of prostate cancer, improved sensitivity can be obtained using void urine after prostate massage which results in a higher shedding of cancer cells into the urinary tract (Goessl et al., 2000). However, current tools for cancer detection in the urine are still unsatisfactory and methods allowing improved sensitivity are needed.

DESCRIPTION OF THE INVENTION - SUMMARY

The present invention is based around the finding that cell- free DNA in urine is suitable for the analysis of epigenetic alteration-associated, in particular methylation-associated, gene silencing in human cancer cells, in particular in urologic, such as prostate, cancer cells. Unexpectedly, the sensitivity obtained with the cell-free DNA fraction from urine is higher than the sensitivity obtained with the traditionally used sediment fraction. The use of the cell- free DNA fraction thus leads to an improvement in the sensitivity and specificity of tumor marker detection in urine for cancers, in particular those cancers that release their cells and cellular components directly in the urethra. Consequently cell-free DNA may be useful in diagnosis, prognosis and successful treatment of such cancers. Accordingly, in a first aspect the invention provides a method for identifying, diagnosing and/or staging or otherwise characterising a urologic cancer or neoplasia in a subject, the method comprising the step of isolating cell- free DNA from a urine sample taken from the subject and analysing the isolated cell-free DNA to identify, diagnose, stage or otherwise characterise the urologic cancer or neoplasia.

By "urologic cancer or neoplasia" is meant a cancer or neoplasia of the urinary tract or the urogenital system. Thus, the cancer or neoplasia occurs at, or downstream of, the kidneys. The cancer or neoplasia may be one whose cells may be released directly into the ureters or urethra. Urologic cancers within the scope of the present invention comprise, consist essentially of or consist of prostate, bladder, kidney and testicular cancer. In a most preferred embodiment, the methods of the invention are used to identify, diagnose, stage or otherwise characterise prostate cancer.

"Diagnosis" is defined herein to include monitoring the state and progression of the cancer or neoplasia, checking for recurrence of disease following treatment and monitoring the success of a particular treatment. The methods of the invention may also have prognostic value, and this is included within the definition of the term "diagnosis". The prognostic value of the methods may be used as a marker of potential susceptibility to cancer. Thus, patients at risk may be identified preferably before the disease has a chance to manifest itself in terms of symptoms identifiable in the patient. The methods of the invention may also be utilised to "stage" a cancer or neoplasia. By this is meant that the cancer or neoplasia is assigned a designated status, such as a histopathological status, based upon accepted standards. Using prostate cancer as an example, in stage I, cancer is found in the prostate only.. It cannot be felt during a digital rectal exam and is not visible by imaging. It is usually found accidentally during surgery for other reasons, such as benign prostatic hyperplasia. The Gleason score is low. Stage I prostate cancer may also be called stage Al prostate cancer. In stage II, cancer is more advanced than in stage I, but has not spread outside the prostate. The Gleason score can range from 2-10. Stage II prostate cancer may also be called stage A2, stage Bl, or stage B2 prostate cancer. In stage III, cancer has spread beyond the outer layer of the prostate to nearby tissues. Cancer may be found in the seminal vesicles. The Gleason score can range from 2- 10. Stage III prostate cancer may also be called stage C prostate cancer. In stage IV, cancer has metastasized

(spread) to lymph nodes near or far from the prostate or to other parts of the body, such as the bladder, rectum, bones, liver, or lungs. Metastatic prostate cancer often spreads to the bones. The Gleason score can range from 2-10. Stage IV prostate cancer may also be called stage Dl or stage D2 prostate cancer. Thus, the methods of the invention may be used to assign a stage to the cancer or neoplasia. These may be related to aggressiveness, maturity and capability of metastasis, for example.

The methods of the invention are most preferably in vitro methods carried out on an isolated urine sample. In one embodiment the method may also include the step of obtaining the urine sample from the subject under test.

In a most preferred embodiment, the subject is a human subject. Generally, the subject will be a patient wherein a urologic cancer or neoplasia is suspected or a potential cancer or neoplasia has been identified and the method may be used to determine if indeed there is a cancer or neoplasia present. The methods of the invention may be used in conjunction with other known methods for detecting cancer or neoplasias, and may improve the overall sensitivity and/or specificity of the methods.

As aforementioned, it has been found that cell-free DNA in urine is suitable for the analysis of epigenetic alteration- associated, in particular methylation-associated, gene silencing in human cancer cells, in particular in prostate cancer cells. The sensitivity obtained with the cell-free DNA fraction from urine has been shown to be higher than the sensitivity obtained with the traditionally used sediment fraction. Accordingly, central to the methods of the invention is the isolation of cell-free DNA from a urine sample under test. "Isolating" is defined herein to include any method by which cell-free DNA can be isolated, concentrated or otherwise purified from a urine sample.

Isolation of cell-free DNA allows the DNA to be analysed in accordance with the methods of the invention for identifying, diagnosing, staging or otherwise characterising a urologic cancer or neoplasia. The starting urine sample is preferably whole urine. In a most preferred embodiment, isolation of cell-free DNA involves (complete or partial) separation of cell-free DNA from cell-associated DNA. Thus, analysis of cell-free DNA may if required be separate from cell-associated DNA. Analysis results may be pooled as appropriate. Any suitable method for isolating cell-free DNA may be utilised. For example, centrifugation such as low speed centrifugation, may be utilised. Low speed centrifugation may be between approximately lOOOg and 400Og, preferably between approximately 150Og and 300Og for example. However, in a more preferred embodiment, cell-free DNA is isolated from the urine sample through use of a filter which retains cells from the urine sample, but allows the cell-free DNA to pass through the filter. As is shown in the experimental section below, use of suitable filters as opposed to the standard centrifugation methods leads to improved recovery of DNA. Any appropriate filter may be utilised in the methods of the invention provided it is effective to isolate cell-free DNA from a urine sample, in particular to achieve separation of cell-free DNA from the cell-associated DNA component of the urine sample. Suitable filters are commercially available and include the MINISART™ filters available from Sartorius.

The invention accordingly also relates to a method for isolating cell-free DNA from a urine sample, the isolated cell-free DNA being useful in the diagnostic methods of the invention, comprising, consisting essentially of or consisting of applying the urine sample to a filter which retains cells from the urine sample. Potential advantages compared to centrifugation include avoiding the need for centrifugation equipment, the possibility of freezing the cell-lysate obtained from the cells collected using the filter and storing these at the collection site before testing. The flow-through fraction of the urine passed on such filters can be further processed in similar fashion to the further processing of the supernatant fraction obtained by low-speed centrifugation.

In specific embodiments, the filter has a pore diameter of approximately 0.5 μm to approximately 10 μm. Such a pore diameter ensures that the filter retains cells from the urine sample but allows cell-free DNA to pass through, thus providing for isolation of cell-free DNA from the urine sample. In a preferred embodiment, the pore diameter is approximately 0.8 to approximately 5μm and in specific preferred embodiments, the pore diameter is approximately 0.8, approximately 1.2 or approximately 5μm with approximately 1.2 μm pores being most preferred. Filters including pores with multiple different shapes and sizes may be utilised as appropriate, provided the majority of pores are within the boundaries of the general dimensions referred to above. In particular, whilst the term "diameter" is used, pores may not always be circular in shape. In such cases, the longest dimension of the pore may be taken as the notional diameter. Similarly, filters may contain asymmetric pores to provide for filtration and more effective capture of cells from the urine.

In an alternative embodiment, cell-free DNA is isolated through an affinity capture process. Preferably, one or more surfaces are provided that have selective affinity for DNA and thus allow capture of the cell-free DNA from a urine sample. In a specific embodiment, the one or more surfaces comprises at least one bead, preferably at least one magnetic bead. In a preferred embodiment, the one or more surfaces has selective affinity for nucleic acid such as DNA under one set of conditions, but has lower affinity for the nucleic acid under a second set of conditions. This allows selective DNA capture to isolate the DNA and also, under the second set of conditions, allows the captured DNA to be eluted. The conditions may be pH, temperature, buffer type or buffer concentration for example. In a preferred embodiment, the one or more surfaces has differing affinity for nucleic acid based upon pH conditions. In a specific embodiment, magnetic beads are utilised which have high affinity for DNA under acidic conditions (preferably less than pH 6.5), for example through a positive charge on the surface of the beads. This allows DNA to be captured and non-nucleic acid components to be washed away. The pH conditions can be altered to alkaline conditions (preferably greater than pH 8.5) to allow elution of the DNA. In a specific application, following initial capture of DNA on the one or more surfaces, residual contaminants such as protein are digested and washed away to leave only pure intact DNA bound to the one or more surfaces. As specific examples of the techniques outlined above, the commercially available CHARGESWITCH® and GENECATCHER™ (Invitrogen) DNA purification technologies may be employed to isolate cell- free DNA. The CHARGESWITCH® system has been shown herein to be particularly effective at recovering large amounts of DNA. Alternative affinity capture processes may be utilised to isolate cell-free DNA, such as DNA capture using (specific) DNA probes and/or use of methyl binding proteins.

In one embodiment, the step of isolating cell-free DNA from the urine sample through use of an affinity capture process is carried out in combination with either centrifugation and/or use of a filter which retains cells found in the urine sample (as discussed in greater detail above) . Most preferably, however, the affinity capture process means that these additional steps are unnecessary.

In a further embodiment, cell-free DNA is isolated through use of a filter which retains the cell-free DNA. In a specific embodiment, the filter comprises, consists essentially of or consists of a molecular weight filter. Accordingly, in one aspect the invention relates to a method for concentrating cell-free DNA from a urine sample, in particular isolated cell-free DNA, comprising applying the cell-free DNA, in particular cell-free DNA previously isolated by an isolation technique described herein, to a filter which retains the isolated cell-free DNA. This method represents part of the preferred overall diagnostic methods of the invention.

Filters which isolate, purify or concentrate a desired species on the basis of size or molecular weight discrimination are known in the art and commercially available. For example, filters available from Millipore- such as the AMICON® Ultra-15, PLCC ϋltracel-PL Membrane filters (available in a range of molecular weight cut-offs) may be utilised. In one embodiment, Centricon Plus-70 filters (Millipore - in particular Cat No: UFC7 005 08 with a 5 kilodalton cut-off and Cat No: UFC7 010 08 with a 10 kilodalton cut-off) are utilised in the isolation of cell- free DNA. These filters are potentially advantageous compared to the AMICON® filters since they have a larger maximum volumetric capacity (70 ml) and also a minimal final concentration volume of 350 μl ("dead stop") which prevents the filter drying out. More generally, any suitable cellulose hydrate ultrafiltration membrane may be utilised. In a specific embodiment, the molecular weight filter has a cut off of less than or equal to around 10 kilodaltons, and most preferably the molecular weight filter has a cut off of approximately 5 kilodaltons.

In a particularly preferred embodiment, the step of isolating cell-free DNA from the urine sample through use of a molecular weight filter is carried out in combination with either centrifugation and/or use of a filter which retains cells found in the urine sample and/or use of an affinity capture process (as discussed in greater detail above) . Most preferably, cell-free DNA is concentrated utilising an appropriate filter that retains the cell-free DNA following an initial isolation step which separates the cell-free and cell-associated DNA components, preferably involving centrifugation and/or use of a filter which retains cells found in the urine sample and/or an appropriate affinity- capture process (as discussed in greater detail above) .

In a further embodiment of the methods of the invention, cell-free DNA is isolated through use of a DNA purification technique. Any suitable DNA purification technique may be utilised. Kits including suitable reagents are well known and commercially available. Preferred kits for use in the methods of the invention are as follows:

Examples of purification techniques may be found in standard texts such as Molecular Cloning - A Laboratory Manual (Third Edition) , Sambrook and Russell (see in particular Appendix 8 and Chapter 5 therein) . In one preferred embodiment, purification involves alcohol precipitation of DNA. Preferred alcohols include ethanol and isopropanol. Suitable purification techniques also include salt-based precipitation methods. Thus, in one specific embodiment the DNA purification technique comprises use of a high concentration of salt to precipitate contaminants. The salt may comprise, consist essentially of or consist of potassium acetate and/or ammonium acetate for example. The method may further include steps of removal of contaminants which have been precipitated, followed by recovery of DNA through alcohol precipitation.

In an alternative embodiment, the DNA purification technique is based upon use of organic solvents to extract contaminants from cell lysates. Thus, in one embodiment, the method comprises use of phenol, chloroform and isoamyl alcohol to extract the DNA. Suitable conditions are employed to ensure that the contaminants are separated into the organic phase and that DNA remains in the aqueous phase.

In preferred embodiments of these purification techniques, extracted DNA is recovered through alcohol precipitation, such as ethanol or isopropanol precipitation.

In a most preferred embodiment, the step of isolating cell- free DNA from the urine sample through a suitable purification technique is carried out in combination with any one or more of the additional DNA isolation techniques described herein. For example, centrifugation and/or use of a filter which retains cells found in the urine sample and/or an affinity capture process and/or use of a molecular weight filter (as discussed in greater detail above) may be utilised together with the DNA purification techniques. Most preferably, cell-free DNA is concentrated utilising an appropriate filter or affinity-capture process that retains the cell-free DNA following an initial isolation step which separates the cell-free and cell-associated DNA components, preferably involving centrifugation and/or use of a filter which retains cells found in the urine sample (as discussed in greater detail above) and is then purified. Purification is preferably carried out using one of the exemplary methods described herein. Thus, the purification step may be considered as the step of the isolation procedure which finally prepares the DNA for analysis.

As is shown in the experimental section herein, whilst analysis of the cell-free DNA component provides improved sensitivity as compared to analysis of the cell-associated DNA component for diagnosis of prostate cancer, sensitivity and specificity may be improved still further through a combination of both DNA components. Accordingly, in one preferred embodiment the methods of the invention additionally comprise isolating and/or analysing cell- associated DNA from the urine sample.

In one embodiment, cell-associated DNA is isolated and/or analysed together with cell-free DNA. In an alternative embodiment, cell-associated DNA is isolated and/or analysed separately from the cell-free DNA. Thus, certain DNA isolation techniques described herein for isolating cell- free DNA will consequentially also lead to isolation of cell-associated DNA. For example, (low speed) centrifugation of the urine sample produces a pellet fraction containing cell-associated DNA and a supernatant fraction containing cell-free DNA. Similarly, use of appropriate filters as described herein may lead to separate, but coincidental, isolation of cell-free and cell- associated DNA. Use of an affinity capture process may achieve isolation of cell-associated and cell-free DNA 93

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coincidentally provided the cells are lysed prior to the capture process. Accordingly, as appropriate, the embodiments described in respect of isolating cell-free DNA apply mutatis mutandis to the isolation of cell-associated DNA and are not repeated for reasons of conciseness.

Preferably, the respective DNA components are (isolated separately and) pooled to provide methods of the invention with improved sensitivity. In a preferred embodiment, the cell-associated DNA is isolated through use of a DNA purification technique. The discussion of DNA purification techniques herein applies mutatis mutandis to this embodiment of the invention.

The cell-free DNA component, optionally in addition to the separated cell-associated DNA component, may be analysed in any suitable fashion to identify, diagnose and/or stage or otherwise characterise the urologic cancer or neoplasia. In a most preferred embodiment, the analysis carried out comprises, consists essentially of or consists of determining whether the DNA contains an epigenetic modification. The epigenetic modification is indicative of the presence or stage of the cancer or neoplasia. Suitable epigenetic modifications known to be linked to cancers and neoplasias include histone acetylation and aberrant gene methylation. Typically deacetylation of histones leads to down regulation of gene expression and vice versa. Histone acetylation may be determined through any suitable means . In one preferred embodiment, histone acetylation of one or more appropriate genes or markers is determined through use of chromatin immunoprecipitation experiments. Aberrant gene methylation is generally manifested as promoter hypermethylation, often combined with hypomethylation elsewhere. Methylation of the promoter region causes down-regulation of gene expression, for example the expression of important tumour suppressor genes. In a most preferred embodiment, the analysis carried out on the DNA comprises, consists essentially of or consists of determining whether (or not) one or more genes are methylated/hypermethylated, in particular at one or more specific sites within the one or more genes. CpG dinucleotides susceptible to methylation are typically concentrated in the promoter region, exons and introns of human genes. Accordingly, promoter, exon and intron regions may be assessed to determine their methylation status. In a preferred embodiment, the methylation status of the promoter region of one or more appropriate genes is determined. A "promoter" is a region typically extending around 5000 bp upstream of the transcription start site, preferably less than or equal to around 1000 bp upstream of the transcription start site and more preferably 150 to 300 bp upstream from the transcription start site. Preferably, a CpG island positioned around the transcription start site of the one or more appropriate gene(s) is/are analysed to determine the methylation status. Alternatively or additionally, the methylation status of the exon and/or intron region of the appropriate gene(s) is/are determined.

Methods for determining methylation in a DNA sample are well known in the art and suitable reagents are commercially available. Any suitable assay may be used in the analysis step of the methods of the present invention. These assays generally rely upon two distinct approaches: a bisulfite conversion based approach or a non-bisulfite conversion based approach. Non-bisulfite conversion based methods for analysis of DNA methylation typically rely on the inability of methylation-sensitive enzymes, such as restriction enzymes, to cleave methylated cytosine residues. Bisulfite conversion relies on treatment of DNA samples with a reagent such as sodium bisulfite which converts unmethylated • cytosine to uracil, while methylated cytosines are maintained (Furuichi et al., 1970). This conversions result in a change in the sequence of the original DNA.

DNA methylation analysis has been performed successfully with a number of techniques including: sequencing, methylation-specific PCR (MSP) , melting curve methylation- specific PCR(McMS-PCR), MLPA with or without bisulfite treatment, QAMA (Zeschnigk et al, 2004), MSRE-PCR (Melnikov et al, 2005), MethyLight (Eads et al., 2000), ConLight-MSP (Rand et al . , 2002), bisulfite conversion-specific methylation-specific PCR (BS-MSP) (Sasaki et al., 2003), COBRA (which relies upon use of restriction enzymes to reveal methylation dependent sequence differences in PCR products of sodium bisulfite - treated DNA) , methylation- sensitive single-nucleotide primer extension conformation (MS-SNuPE) , methylation-sensitive single-strand conformation analysis (MS-SSCA) , Melting curve combined bisulfite restriction analysis (McCOBRA) (Akey et al., 2002), PyroMethA, HeavyMethyl (Cottrell et al. 2004), MALDI-TOF, MassARRAY, Quantitative analysis of methylated alleles (QAMA), enzymatic regional methylation assay (ERMA), QBSUPT, MethylQuant, Quantitative PCR sequencing and oligonucleotide-based microarray systems, Pyrosequencing, Meth-DOP-PCR and SYBRGreen-based PCR. A review of some useful techniques is provided in Nucleic acids research, 1998, Vol. 26, No. 10, 2255-2264, Nature Reviews, 2003, Vol.3, 253-266 and Oral Oncology, 2006, Vol. 42, 5-13, which references are incorporated herein in their entirety.

Additional methods for the identification of methylated CpG dinucleotides utilize the ability of the methyl binding domain (MBD) of the MeCP2 protein to selectively bind to methylated DNA sequences (Cross et al, 1994; Shiraishi et al, 1999) . Alternatively, the MBD may be obtained from MBP, MBP2, MBP4 or poly-MBD (Jorgensen et al . , 2006). In one method, restriction enconuclease digested genomic DNA is loaded onto expressed His-tagged methyl-CpG binding domain that is immobilized to a solid matrix and used for preparative column chromatography to isolate highly methylated DNA sequences. Such methylated DNA enrichment- steps may supplement the methods of the invention. Several other methods for detecting methylated CpG islands are well known in the art and include amongst others the methylated- CpG island recovery assay (MIRA) .

In a preferred embodiment, the methods of the invention comprise treatment of the DNA with an agent capable of modifying unmethylated cytosine residues in a detectable fashion but which is incapable of modifying methylated cytosine residues. Preferably, unmethylated residues are converted (through deamination) to uracil, whilst methylated residues remain unconverted. As mentioned above, a most preferred reagent in this context is bisulfite, in particular sodium bisulfite. In a specific embodiment, treatment of the DNA using the agent is carried out following isolation of the cell-free DNA. In a most preferred embodiment, methylation is detected through use of methylation specific PCR (MSP) . The MSP technique will be familiar to one of skill in the art. In the MSP approach, DNA may be amplified using primer pairs designed to distinguish methylated from unmethylated DNA by taking advantage of sequence differences as a result of sodium-bisulfite treatment (Herman et al.,1996; and WO 97/46705) .

A specific example of the MSP technique is designated realtime quantitative MSP (QMSP) , which permits reliable quantification of methylated DNA in real time. These methods are generally based on the continuous optical monitoring of an amplification procedure and utilise fluorescently labelled reagents whose incorporation in a product can be quantified and whose quantification is indicative of copy number of that sequence in the template. One such reagent is a fluorescent dye, called SYBR Green I that preferentially binds double-stranded DNA and whose fluorescence is greatly enhanced by binding of double- stranded DNA. Alternatively, labeled primers and/or labeled probes can be used. Such methods represent a specific application of the well known and commercially available real-time amplification techniques such as TAQMAN®,

MOLECULAR BEACONS®, AMPLIFLUOR®, SCORPION®, Plexor™ and DzyNA®, etc as described in more detail herein. Often, these real-time methods are used with the polymerase chain reaction (PCR) .

A further technique which may be utilised is known as "Heavymethyl" . Here, priming is methylation specific, but non-extendable oligonucleotide blockers provide specificity instead of the primers themselves. The blockers bind to bisulfite-treated DNA in a methylation-specific manner, and their binding sites overlap the primer binding sites. When the blocker is bound, the primer cannot bind and therefore no amplicon (amplification product) is generated. Heavymethyl can be used in combination with real-time detection, as required.

In one preferred embodiment, methylation status is determined using real-time applications of methylation specific PCR. In specific embodiments, the real-time methylation specific PCR comprises use of TAQMAN® probes and/or MOLECULAR BEACONS probes and/or AMPLIFLUOR® primers and/or LIGHT-CYCLER® and/or FRET probes and/or SCORPION® primers and/or oligonucleotide blockers.

Real-time methods do not need to be utilised, however. Amplification products may simply be run on a suitable gel, such as an agarose gel, to determine if the expected sized products are present. This may involve use of ethidium bromide staining and visualisation of the DNA bands under a UV illuminator for example.

In real-time embodiments, quantitation may be on an absolute basis, or may be relative to a constitutively methylated DNA standard, or may be relative to an unmethylated DNA standard. Methylation status may be determined by using the ratio between the signal of the marker under investigation and the signal of a reference gene where methylation status is known (such as β-actin and/or tubilin for example) , or by using the ratio between the methylated marker and the sum of the methylated and the non-methylated marker. Alternatively, absolute copy number of the methylated marker gene can be determined.

In one embodiment, each sample is measured in duplicate and for both Ct values (cycles at which the amplification curves crossed the threshold value, set automatically by the relevant software) copy numbers are calculated. The average of both copy numbers (for each gene) is used for the result classification. To quantify the final results for each sample two standard curves are used, one for either the reference gene (such as β-actin) or the non-methylated marker and one for the methylated version of the marker ("m- gene") . The results of all samples (when m-Gene was detectable) are expressed as 1000 times the ratio of "copies m-Gene"/"copies β-actin" or "copies m-Gene"/"copies u- Gene+m-Gene" and then classified accordingly (methylated, non-methylated or invalid) (u=unmethylated; m=methylated) .

In a further embodiment, bisulfite sequencing is utilised in order to determine the methylation status of chosen genes in the sample. Primers may be designed for use in sequencing through the appropriate CpG islands in the gene or genes of interest. Thus, primers may be designed in both the sense and antisense orientation to direct sequencing across the promoter region of the relevant gene.

Other nucleic acid amplification techniques, in addition to PCR (which includes real-time versions thereof and variants such as nested PCR), may also be utilised, as appropriate, to detect the methylation status of the relevant gene or genes. Such amplification techniques are well known in the art, and include methods such as NASBA (Compton, 1991) , 3SR (Fahy et al., 1991 ) and Transcription Mediated Amplification (TMA) . Other suitable amplification methods include the ligase chain reaction (LCR) (Barringer et al, 1990), selective amplification of target polynucleotide sequences (US Patent No. 6,410,276), consensus sequence primed polymerase chain reaction (US Patent No 4,437,975), arbitrarily primed polymerase chain reaction (WO 90/06995) , invader technology, strand displacement technology, and nick displacement amplification (WO 2004/067726) . This list is not intended to be exhaustive; any nucleic acid amplification technique may be used provided the appropriate nucleic acid product is specifically amplified. Thus, these amplification techniques may be tied in to MSP and/or bisulfite sequencing techniques for example.

Sequence variation that reflects the methylation status at CpG dinucleotides in the original genomic DNA offers two approaches to primer design. Firstly, primers may be designed that themselves do not cover any potential sites of DNA methylation. Sequence variations at sites of differential methylation are located between the two primers. Such primers are used in bisulfite genomic sequencing, COBRA and Ms-SnuPE for example. Secondly, primers may be designed that anneal specifically with either the methylated or unmethylated version of the converted sequence. If there is a sufficient region of complementarity, e.g., 12, 15, 18, or 20 nucleotides, to the target, then the primer may also contain additional nucleotide residues that do not interfere with hybridization but may be useful for other manipulations. Examples of such other residues may be sites for restriction endonuclease cleavage, for ligand binding or for factor binding or linkers or repeats. The oligonucleotide primers may or may not be such that they are specific for modified methylated residues .

One way to distinguish between modified and unmodified DNA is to hybridize oligonucleotide primers which specifically bind to one form or the other of the DNA. After hybridization, an amplification reaction can be performed and amplification products assayed. The presence of an amplification product indicates that a sample hybridized to the primer. The specificity of the primer indicates whether the DNA had been modified or not, which in turn indicates whether the DNA had been methylated or not.

Another way to distinguish between modified and unmodified DNA is to use oligonucleotide probes which may also be specific for certain products. Such probes may be hybridized directly to modified DNA or to amplification products of modified DNA. Oligonucleotide probes can be labelled using any detection system known in the art. These include but are not limited to fluorescent moieties, radioisotope labelled moieties, bioluminescent moieties, luminescent moieties, chemiluminescent moieties, enzymes, substrates, receptors, or ligands.

In the MSP technique, amplification is achieved with the use of primers specific for the sequence of the gene whose methylation status is to be assessed. In order to provide specificity for the nucleic acid molecules, primer binding sites corresponding to a suitable region of the sequence may be selected. The skilled reader will appreciate that the nucleic acid molecules may also include sequences other than primer binding sites which are required for detection of the methylation status of the gene, for example RNA Polymerase binding sites or promoter sequences may be required for isothermal amplification technologies, such as NASBA, 3SR and TMA.

TMA (Gen-probe Inc.) is an RNA transcription amplification system using two enzymes to drive the reaction, namely RNA polymerase and reverse transcriptase. The TMA reaction is isothermal and can amplify either DNA or RNA to produce RNA amplified end products. TMA may be combined with Gen-probe's Hybridization Protection Assay (HPA) detection technique to allow detection of products in a single tube. Such single tube detection is a preferred method for carrying out the invention.

Any suitable marker with a link to a urologic cancer may be utilised in the methods of the invention. Particularly preferred are genes whose promoter is unmethylated in normal tissues and methylated in a urologic cancer or neoplasia. Detection of methylation of such genes provides a reliable signal which is readily observable as being significant in terms of cancer identification, diagnosis and staging. In a particularly preferred embodiment, the urologic cancer is prostate cancer and the methylation status of at least one of GST-Pi, APC, RARβ2, RASSFlA, P16 or P14 is determined. The methylation status of all these genes has been shown to be linked to the incidence of prostate cancer. Moreover, herein it is shown that the methylation status of these genes can be determined from a urine sample to provide improved methods of identifying, diagnosing, staging or otherwise characterising prostate cancer. In a specific embodiment, the methylation status of all of GST-Pi, APC, RARβ2, P16 and P14 is determined. Such a method provides improved specificity and sensitivity of detection. Additional methylation markers with a known link to a urologic cancer such as prostate cancer may be utilised as appropriate, either alone or in combination. A panel of genes may include 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more than 10 separate markers for example.

When determining methylation status, it may be beneficial to include suitable controls in order to ensure the method chosen to assess this parameter is working correctly and reliably. For example, suitable controls may include assessing the methylation status of a gene known to be methylated in the urine sample under test. This experiment acts as a positive control to ensure that false negative results are not obtained (i.e. a conclusion of a lack of methylation is made even though the gene or genes of interest may, in fact, be methylated) . The gene may be one which is known to be methylated in the sample under investigation or it may have been artificially methylated, for example by using a suitable methyltransferase enzyme, such as Sssl methyltransferase.

Additionally or alternatively, suitable negative controls may be employed with the methods of the invention. Here, suitable controls may include assessing the methylation status of a gene known to be unmethylated. This experiment acts as a negative control to ensure that false positive results are not obtained (i.e. a conclusion of methylation is made even though the gene or genes of interest may, in fact, be unmethylated) . The gene may be one which is known to be unmethylated in the sample under investigation or it may have been artificially demethylated, for example by using a suitable DNA methyltransferase inhibitor.

The application of the methods of present invention may, under certain circumstances, require the generation and amplification of a DNA library before testing for methylation of any specific gene. Suitable methods for whole genome amplification and library generation for such amplification (e.g. Methylplex and Enzyplex technology, Rubicon Genomics) are described in US2003/0143599, WO2004/081225 and WO2004/081183. In addition, WO2005/090507 describes library generation/amplification methods that require either bisulfite conversion or non-bisulfite based application. Bisulfite treatment may occur before or after library construction and may require the use of adaptors resistant to bisulfite conversion. Meth-DOP-PCR (Di Vinci et al, 2006) , a modified degenerate oligonucleotide-primed PCR amplification (DOP-PCR) that is combined with MSP, provides another suitable method for specific detection of methylation in small amount of DNA. Improved management of patient care may require these existing methods and techniques to supplement the methods of the invention.

Genes that become methylated early in the process of carcinogenesis are not only ideal for screening purposes, but also represent interesting targets for early cancer detection and for monitoring the progression or outcome of cancers. Moreover, urine based testing represents a noninvasive and potentially high throughput means for detecting cancers early in combination with epigenetic testing of urine DNA.

Early detection of epigenetic silencing of genes could provide justification for more definitive follow up of patients who have molecular, but not yet all the pathological or clinical, features associated with a urologic malignancy. Identification of cancer at its earliest stage while it is still localized and readily treatable may improve the clinical outcome in patients. Methods with a prognostic value should allow for the specific detection of urologic tumors.

As discussed herein, human total urine has been shown to possess two size categories of DNA: a larger species, heterogeneous in size, and typically greater than lkb and a smaller species generally less than lkb and mostly between 100 and 400bp, in particular between 150bp and 250bp. The large size class appears to be mainly derived from cells shed into the urine from the urinary tract. The kidney barrier prevents cells "upstream" of this point entering the urine. The small size class of DNA may be recovered from the supernatant following low-speed centrifugation of urine. It comprises, inter alia, cell-free circulating DNA from the blood circulation that passed the kidney barrier into urine. In a preferred embodiment, the cell-free DNA is predominantly, although not necessarily exclusively, less than or equal to 1 kb (1000 bp) in length. More preferably, the cell-free DNA is predominantly less than five hundred base pairs in length. Even more preferably, the cell free DNA is predominantly approximately 100 to 400 base pairs in length and most preferably approximately 150-250 base pairs in length. The cell-associated DNA is preferably (predominantly) greater than 1 kb (1000 bp) in length.

Enhanced sensitivity of tumor marker detection may in addition be achieved through improved urine sample quality. Optimized preservation of the nucleic acid in particular cell-free DNA, either isolated from or found in the urine, provides a second improvement to traditional sample collection methods. Optimized preservation can be achieved by reducing DNA degradation at the moment of collection and/or by creating an optimal stabilizing environment during storage, before sample processing. This can be achieved through the addition of stabilizing agents (e.g. EDTA) and/or the addition of antibiotics (AB) in order to prevent bacterial growth in the urine sample. Alternatively, optimized preservation of the DNA, especially cell-free DNA, can be achieved through storage at optimal temperature and/or through an optimal pH environment. Accordingly, in one embodiment, the methods of the invention additionally comprises stabilising the urine sample. It is shown herein that stabilising the urine sample, by adding a suitable stabilizing buffer to the urine, may avoid the need for centrifugation of the urine sample shortly after obtaining the sample. Typically centrifugation occurs within 4 hours of obtaining the urine sample in order to maintain the integrity of the DNA (in particular in the sediment fraction) . The samples can be maintained at room temperature for up to 48 or 72 hours following addition of a stabilizing buffer, without the requirement for centrifugation. This advantageously permits home collection of urine samples and also removes the necessity for centrifugation equipment at each collection site. Thus, the invention provides a method for conveniently storing urine samples for a period of up to 72 (or 48) hours at room temperature, such as at least 4, 12, 24, 36 or 48 hours up to 72 hours, comprising adding a stabilising buffer to the urine sample, with the proviso that the urine sample is not centrifuged or otherwise fractionated prior to or during the storage period and storing the urine for this period. Suitable stabilizing buffers for use in these methods are described herein.

In specific embodiments, the isolated cell-free DNA portion of the urine sample is stabilised following an isolation procedure.

Whilst stabilization may be achieved via any suitable means, in a preferred embodiment, stabilisation occurs through addition of a stabilising buffer. The stabilising buffer incorporates suitable components to maintain DNA integrity in the urine sample and/or to maintain the quality of the urine sample as a whole. Thus, in a further aspect, the invention provides a stabilising buffer solution for storing urine samples comprising EDTA, an antibacterial and optionally a STABILUR™ tablet. This solution is preferably for storing a urine sample at a temperature of around 4⁰C. In a related aspect, the invention provides a stabilising buffer for storing urine samples comprising EDTA, DMSO and an antibacterial. The solution is preferably for storing a urine sample under freezing conditions. These buffer solutions are particularly designed for storing the cell- free DNA component from a urine sample. This cell-free DNA component may be produced by any of the isolation techniques referred to above. For example, it may represent the supernatant portion of a urine sample following low speed centrifugation or the filtrate following use of a filter as defined herein, or the eluted fraction where an affinity capture process has been used.

Accordingly, in one embodiment, the stabilising buffer for use in methods of the invention comprises, consists essentially of or consists of at least one component selected from EDTA, an antibacterial, DMSO and STABILUR™ tablets. STABILUR tablets are available from Cargille Labs and contain appropriate mixtures of buffering and osmolarity adjustment ingredients. Suitable equivalents to this product may be utilised as appropriate, such as preservative tubes available from CellSave (CellSave Preservative Tubes, see http://www.veridex.com/Svstems/Systems.aspx?id=1&section=cellSaveTube) .

The term "antibacterial" is intended to cover any compound, molecule or otherwise which has an inhibitory effect on the growth or viability of one or more bacteria. Both biological and non-biological molecules are intended to fall within the definition. In a preferred embodiment, the antibacterial comprises, consists essentially of or consists of an antibiotic. Many antibiotics are well known in the art and commercially available. Mixtures of antibiotics may be utilised as appropriate, such as the Antibiotic-Antimycotic A5955-100ml antibiotic mix available from Sigma-Aldrich.

Suitable anti-bacterials may include cytokines such as interferons and interleukins and derivatives and mimetics thereof, for example as described in WO 2006/123164 (which reference is incorporated herein in its entirety) and "small molecules". A small molecule is defined as a molecular entity with a molecular weight of less than 1500 daltons, preferably less than 1000 daltons. The small molecule may for example be an organic, inorganic or organometallic molecule, which may also be in the form or a suitable salt, such as a water-soluble salt; and may also be a complex, chelate and/or a similar molecular entity, as long as its (overall) molecular weight is within the range indicated above .

In a further embodiment, the stabilising buffer comprises, consists essentially of or consists of EDTA, DMSO and an antibacterial and wherein the sample may be frozen for storage prior to the analysis carried out on the isolated cell-free DNA. In a still further embodiment, the stabilising buffer comprises a STABILUR™ tablet, EDTA and an antibacterial and the sample may be stored at a temperature of around 4⁰C prior to the analysis. In specific embodiments of the stabilising buffers, the EDTA is present at a final concentration of around 1OmM and/or the DMSO is present at around 10% of the final stabilising buffer volume .

Samples (to which a stabilising buffer of the invention has been added) may be stored at any suitable temperature, including room temperature. For example, the storage temperature may be anywhere between approximately -5O⁰C and approximately 37°C , preferably approximately -1O⁰C to -30⁰C, such as approximately -2O⁰C or approximately 1°C to 1O⁰C, such as approximately 4°C. By "freezing" is meant a temperature at or below O⁰C, preferably approximately -2O⁰C. In one specific embodiment, the methods of the invention comprise freezing the urine sample taken from the subject for storage prior to isolating cell-free DNA from the urine sample. In a further embodiment, the methods of the invention comprise freezing the urine sample taken from the subject for storage prior to analysis of the cell-free DNA. The methods may additionally or alternatively comprise freezing the urine sample taken from the subject prior to stabilising the urine sample.

In a related "stabilization" aspect, the invention provides a method for storing urine, in particular the cell-free DNA component of urine, comprising adding a stabilising buffer of the invention to the urine and storing the mixture at a suitable temperature. The temperature is preferably around 4⁰C. Similarly, the invention also provides a method for storing urine, in particular the cell-free DNA component of urine, comprising adding a stabilising buffer of the invention to the urine sample and storing the mixture under freezing conditions. The freezing conditions may be around -20⁰C. As mentioned above, mixtures may be stored at other temperatures, such as room temperature for example.

Taken together, the various aspects of the invention may be combined as appropriate to improve the methods of the invention relating to identifying, diagnosing, staging or otherwise characterising a urologic cancer such as prostate cancer. Thus, DNA isolation steps may be combined with one another as appropriate and may also be combined with stabilization of the urine sample at any suitable juncture. Other preferred processing steps apply in particular where both cell-free and cell-associated DNA are obtained and analysed in the methods of the invention. Both fractions may need to be processed separately to isolate DNA. Subsequent pooling of the DNA obtained from each fraction may be helpful to increase the sensitivity to detect tumor markers. Standard processing steps typically involve collecting a urine samples as a whole, sedimenting the cells by centrifugation, freezing the pellet which includes the cell-associated DNA component and discarding the supernatant (incorporating the cell-free DNA) .

Possible processing steps useful in the methods of the present invention include, prior to the analysis step: 1) - Collect urine sample as a whole, separate cell-free DNA from cell-associated DNA (for example by centrifugation or through use of a filter) , store the cell-associated DNA (pellet) , add a stabilizing buffer (of the invention) to the cell-free DNA component (supernatant or filtrate) , and store the cell-free DNA component (supernatant or filtrate) .

Optionally, the cell-associated DNA (pellet) is then processed in standard fashion. The cell-free DNA may be concentrated using any one or more of the various isolation methods of the invention (in particular use of a molecular weight filter or an affinity-capture process) . DNA may then be further isolated through use of an appropriate DNA purification technique as discussed above. For methylation detection, DNA may be suitably modified (for example using bisulfite) and an appropriate detection method, such as real-time MSP, carried out.

2) - Collect urine sample as a whole, - add stabilizing buffer before separation (centrifugation or use of a filter etc.), separate cell-free DNA from cell-associated DNA (for example by centrifugation or use of a filter etc.), store the cell-associated DNA (pellet or captured on filter) , and store the cell-free DNA component (supernatant or filtrate) .

3) - Collect urine samples as a whole, - separate cell-free DNA from cell-associated DNA (for example by centrifugation or use of a filter etc.), store the cell-associated DNA (pellet or captured on filter) , and store the cell-free DNA component (supernatant or filtrate) .

Stabilizing buffer may be added once the respective DNA- containing components have been thawed.

4) - Collect urine samples as a whole, - optionally add stabilizing buffer, and store sample as a whole.

Here, any desired separation of cell-associated and cell- free DNA components occurs downstream.

5) - Prepare stabilizing buffer,

Collect urine sample as a whole into the buffer, separate cell-free DNA from cell-associated DNA (for example by centrifugation, use of a filter etc.), store the cell-associated DNA (pellet or captured on filter) , and - store the cell-free DNA component (supernatant or filtrate) .

Finally, it may also be possible to simply collect urine samples as a whole (for example in the clinic) and then store the whole urine prior to further processing in accordance with the methods of the invention.

In all of the embodiments above, storage of the whole urine, cell-associated DNA or cell-free DNA respectively may be under any suitable conditions. Samples may be stored at any suitable temperature, including room temperature. For example, the storage temperature may be anywhere between approximately -50⁰C and approximately 37⁰C, preferably approximately -1O⁰C to -3O⁰C, such as approximately -20⁰C or approximately 1°C to 10⁰C, such as approximately 4°C. By "freezing" is meant a temperature at or below 0⁰C, preferably approximately -20⁰C.

Optionally, in each case, the cell-associated DNA (pellet or captured on a filter or other surface etc.) is then processed in standard fashion. The cell-free DNA may be concentrated using any one or more of the various isolation methods of the invention (in particular use of a molecular weight filter) . DNA may then be further isolated through use of an appropriate DNA purification technique as discussed above. DNA level may be quantitated using any suitable means, such as through use of PICOGREEN for example. For methylation detection, DNA may be suitably modified (for example using bisulfite) and an appropriate detection method, such as real-time MSP, carried out.

The invention also relates to kits for identifying, diagnosing and/or staging or otherwise characterising a urological cancer or neoplasia in a subject. The kits comprise a filter for isolating cell-free DNA from a urine sample taken from the subject; and means for analysing the isolated cell-free DNA.

All aspects of the methods of the invention apply mutatis mutandis to the kits of the invention since the kits of the invention are adapted to facilitate carrying out these methods.

The means for analysing the isolated cell-free DNA may include any suitable reagents, alone or in combination, which permit the identification, diagnosis and/or staging or otherwise characterising of a urological cancer or neoplasia in a subject. In one specific embodiment, the means for analysing the isolated cell-free DNA comprises specific primers for amplification of a DNA sequence to produce an amplification product, wherein the amplification product is an indicator of a urologic cancer or neoplasia or a specific stage or characteristic thereof. Various expression markers are known to be linked to the incidence of various urologic cancers and suitable primers are known or may be designed by one of skill in the art. Preferred markers are those whose expression is epigenetically regulated. Thus, the means for analysing cell-free DNA preferably includes means for determining whether the DNA contains an epigenetic modification. In particular, as discussed above, methylation markers are preferably investigated to facilitate identification or diagnosis. However, methylation leads to, as a direct consequence, down-regulation of gene expression and so primers for determining whether a gene is expressed may be usefully included in the kits of the invention.

In a particularly preferred embodiment, the primers are methylation specific PCR primers. Preferably, the urologic cancer is prostate cancer. Preferably the methylation specific PCR primers allow the methylation status of at least one of GST-Pi, APC, RARβ2, RASSSFlA, P16 or P14 to be determined. Even more preferably, the methylation specific PCR primers allow the methylation status of all of GST-Pi, APC, RARβ2, P16 and P14 to be determined. Primers for determining expression levels of these genes may be included in the kits of the invention as appropriate.

In a still further embodiment, the means for analysing the isolated cell-free DNA comprises an agent capable of modifying unmethylated cytosine residues but which is incapable of modifying methylated cytosine residues. Preferably, this agent comprises bisulfite, in particular sodium bisulfite.

The means for analysing the cell-free DNA may alternatively include appropriate methylation sensitive restriction enzymes. Preferably, the urologic cancer is prostate cancer and the methylation sensitive restriction enzymes allow the methylation status of at least one of GST-Pi, APC, RARβ2, RASSSFlA, P16 or P14 to be determined. Even more preferably, the methylation sensitive restriction enzymes allow the methylation status of all of GST-Pi, APC, RARβ2, P16 and P14 to be determined. Specific examples of restriction enzymes include Ava I, Hha I, HinPl I, Hpa II, Acil, HpyCH4IV, BsaHI, Nrul, BspDI and McrB. Others would be well known to the skilled person (see for example http : //rebase . neb . com) .

In a preferred embodiment, the filter included in the kits of the invention retains cells from the urine sample but allows cell-free DNA to pass through. Preferably, the filter has a pore diameter of approximately 0.5 to approximately 10 μm, more preferably approximately 0.8 to approximately 5μm and most preferably approximately 0.8, 1.2 or 5μm.

In one alternative embodiment, suitable affinity-capture means are incorporated into the kits of the invention. They may be suitable magnetic beads for example. The discussion of affinity capture techniques above applies equally to the kits of the invention, in terms of the components which may be included.

The kits of the invention may additionally comprise a stabilising buffer solution of the invention as defined hereinabove.

Similarly, the kits of the invention may additionally comprise a suitable molecular weight filter for concentrating the isolated cell-free DNA. Appropriate filters are described in greater detail above. Preferably, the molecular weight filter has a cut off of less than or equal to approximately 10 kilodaltons and more preferably approximately 5 kilodaltons.

Finally, the kits of the invention may further comprise one or more reagents for purification of DNA. Details of the various DNA purification protocols are outlined above and the various components may be incorporated into the kits of the invention as appropriate.

The invention will now be described in the following non- limiting experimental examples with reference to the accompanying drawings .

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 presents results of a comparison between filtration and centrifugation for recovery of DNA from urine:

A Evaluation of β-Actin recovery with the 0.8, 1.2 & 5 μm

Sartorius filters compared to direct spin B Evaluation of GST-Pi recovery with the 0.8, 1.2 & 5 μm

Sartorius filters compared to direct spin

The Y axis represents the number of copies of the gene under evaluation

Figure 2 presents the effects of various stabilising agents on DNA recovery from urine samples:

A: β~Actin copy number recovery when processed directly (MCF7 spiked urine) B: β-Actin copy number recovery when processed directly (unspiked urine) C: GST-Pi copy number recovery when processed directly (MCF7 spiked urine)

D: β-Actin copy number recovery when stored for 2 days (MCF7 spiked urine) E: β-Actin copy number recovery when stored for 2 days

(unspiked urine)

F: GST-Pi copy number recovery when stored for 2 days (MCF7 spiked urine)

G: β-Actin copy number recovery when stored for 5 days (MCF7 spiked urine)

H: β-Actin copy number recovery when stored for 5 days

(unspiked urine)

I: GST-Pi copy number recovery when stored for 5 days (MCF7 spiked urine

Figure 3 presents the effects of various storage conditions on DNA recovery from urine samples:

A: β-Actin copy number recovery for different storage conditions (SW48 spiked urine) B: P16 copy number recovery for different storage conditions

(SW48 spiked urine)

Figure 4 shows the results of an evaluation of different urine sampling methods in terms of DNA recovery. A: β-Actin recovered in different fractions of the urine

B: β-Actin total copy number (supernatant + pellet fraction or whole urine) recovered in urine.

Figure 5 presents a schematic overview on the different steps used to isolate and analyse DNA from various urine samples from prostate cancer patients. Figure 6 shows DNA recovery of the APC gene in the pellet and supernatant fractions for various non-cancer (A) and cancer (B) samples.

Figure 7 shows the Receiver Operating Characteristics (ROC) curves calculated for a 3-marker combination GST-Pi, RARβ2 and APC. The true positive rate (sensitivity) is plotted in function of the false positive rate (100-specificity) A: ROC curve analysis on urine sediment fraction from 13 cancer samples and 14 controls for GST-Pi, RARβ2 and APC marker combination

B: ROC curve analysis on urine supernatant fraction from 13 cancer samples and 14 controls for GST-Pi, RARβ2 and APC marker combination

Figure 8 : Decision tree for sample classification (Methylated, Non-Methylated or Invalid)

DETAILED DESCRIPTION - EXPERIMENTAL SECTION

METHODS

Sample collection and processing of the pellet

Fresh collected urine samples were low-speed centrifuged at

300Og at room temperature for 10 minutes. The supernatant was separated from the sediment fraction leaving about 5 ml of supernatant on top of the cell pellet. Both fractions were stored at -2O⁰C until further processing. Prior to DNA isolation of the pellet fraction, the frozen sample was thawed at room temperature and centrifuged at 300Og for 5 minutes to separate the remaining supernatant (~5 ml) from the cell debris pellet. The supernatant was collected and pooled with the corresponding supernatant fraction derived from the first centrifugation step.

Genomic DNA was extracted from the sediment fraction using the PUREGENE® DNA Purification Kit from Gentra. 700 μl of Cell Lysis Solution (provided with kit) was added to the pellet and further processed according to manufacturer' s instructions. DNA was rehydrated by adding 45 μl of LoTE buffer and was incubated during 1 hour shaking at 65⁰C followed by overnight shaking at 20⁰C.

Processing of urine supernatant samples through Amicon-15 filter device

Cell pellets and corresponding supernatant from urine samples following low-speed centrifugation were kept at - 20⁰C from patients with suspected prostate cancer. Supernatant was thawed and EDTA (10 mM final) & DMSO (10% final) were added. The supernatant was subsequently concentrated using the Millipore Amicon Ultra-15 filter device (5K) . In parallel, positive controls (supernatants from healthy urine spiked with PCR products (10,000 copies for GST-Pi, APC, pl4 and plβ) ) and negative controls (supernatants from healthy urine) were processed. Briefly, available binding sites of the plastic filter were blocked with passivation solution according to the instructions of the Manufacturer

(http: //www.millipore. com/publications . nsf/docs/pelOOlenOO) . 45 ml of urine was filtered, first 15 ml and subsequently 30 ml using the same filter. Using the PUREGENE® DNA Purification Kit from Gentra 700 μl of cell lysis solution was added and DNA was extracted. Processing of urine supernatant samples through affinity capture process

The ChargeSwitch® gDNA 1 ml serum kit from Invitrogen (cat# CS11040) with either 300 μl or 150 μl magnetic bead volume was used to extract free floating DNA from the urine supernatant fraction as an alternative to the Amicon-15 filter device combined with Puregene DNA extraction. 100 ml of morning urine containing EDTA (10 mM final) and DMSO (10% final) was divided in 10 ml aliquots. Half of the aliquots were spiked with 10,000 copies of a DNA library, consisting of modified DNA from SW480 cell line, linear plasmid or PCR product as indicated in Table 1, the other half was left unspiked and was used as negative control. The samples were spun down; supernatant fraction was recovered and further processed through the ChargeSwitch® gDNA 1 ml serum kit according to the manufacturer' s manual with following modifications :

- use an up-scaled volume of 10 ml

- Step 1.4: no RNAse treatment - Step 2.2: Place the tube in the MagnaRack for 15 min (instead of 3 min)

- Step 4.3: Place the tube in the MagnaRack for 2 min (instead of 1 min)

Subsequently the DNA was bisulfite treated and processed further as described hereafter. Methylation levels of β-

Actin, APC, GST-Pi, P14 and Plδ were determined by real-time MSP.

Table 1 : DNA library material

Quantification, bisulfite treatment and amplification DNA was quantified using the PicoGreen dsDNA quantitation kit from Molecular Probes followed by sodium bisulfite treatment (BT) using the EZ DNA Methylation kit from Zymo Research. Briefly, up to 2 μg of genomic DNA was denatured and incubated with 100 μl of CT conversion reagent (provided in kit) shaking at 70⁰C for 3 hours. The modified DNA was further desalted and desulfonated according to manufacturer' s instructions and eluted in 50 μl Tris-HCl 1 mM pH 8.0. The modified DNA was stored at -80⁰C until further processing.

The chemically treated DNA was used as template for realtime MSP. Details of this method have previously been provided in International Publication WO97/46705 for example. Methylation levels of the GST-Pi, RARβ2, RASSFlA, Plδ and P14 gene promoter were determined by real-time MSP.

Relative levels of methylated promoter DNA in each sample was determined by comparing the values of each gene of interest with the values of the internal reference gene to obtain a ratio that was then multiplied by 1000 for easier tabulation.

Sample preparation with use of Sartorius Urine filters Sartorius filters (Minisart, Sartorius Inc.) with either 0.8 μm, 1.2 μm or 5 μm pore diameters were used to separate the cells from the urine samples as an alternative to low speed centrifugation. 40 ml morning urine containing DMSO and EDTA as stabilizing buffer was spiked with 12,000 MCF7 cells (fresh culture) . The spiked urine samples were filtered with use of the Sartorius filters. Cells retained on the filtering membrane were eluted with 1400 μl PureGene Lysis Buffer (buffer is added in the opposite flow direction to that of the sample) . DNA was extracted following the Puregene method for urine from the retentate of the filters. DNA was bisulfite modified and processed through β-Actin and GST-Pi real-time MSP assays as described.

RESULTS

Filtration compared to centrifugation We investigated whether filtration is an alternative to low- speed centrifugation for recovering cancerous cells in urine spiked with MCF7 cells. Cells from urine were collected with use of various Sartorius filters with either 0.8 μm, 1.2 μm or 5 μm pore diameters (Minisart, Sartorius Inc.) or by low-speed centrifugation. The ability of the filters to retain cells as well as the absence of clogging was evaluated. Results are presented in figure 1. The sensitivity obtained with the 0.8, 1.2 & 5 μm filters appeared improved compared to that obtained with low-speed centrifugation. Sartorius filters are therefore a reasonable alternative to centrifugation and provide a particularly useful alternative for processing urine samples at collection sites that do not possess centrifuge equipment.

Effect of stabilizing environment

Morning urine from different healthy volunteers was collected and pooled to a total volume of 900 ml. This volume was split in half: one half was spiked with 450,000 freshly cultured MCF7 cells, the other half was left unchanged and used as a negative control. Both volumes were aliquoted (5 ml) and tested for different conditions. Three aliquots of spiked and unspiked urine sample were processed through 6 different stabilizing mix compositions all or not comprising EDTA (10 mM final), DMSO (10% final), 1 STABILUR® tablet (commercially available at Cargille Labs) , or 50 μl of a 10Ox diluted Sigma A5955: Antibiotic Antimycotic Solution, stabilized (100^χ)

A) EDTA , DMSO, Antibiotics (AB)

B) EDTA, AB

C) EDTA, 500 μl glycerol, AB

D) 1 STABILUR® tablet , AB E) 1 STABILUR® tablet, lOOμl EDTA, AB

F) no stabilizing agent added: native urine

Each stabilizing condition was stored at different temperatures as follows: 1) 2 days at 4⁰C

2) 2 days at -2O⁰C

3) 5 days at 4⁰C

4) 5 days at -2O⁰C

5) directly spun down and further processed

Subsequently all samples were spun down, sediment was recovered and processed further using a PUREGENE® DNA Purification Kit and EZ DNA Methylation kit. The chemically modified DNA was used as input material for GST-Pi and β- Actin real-time MSP. Recovered copy numbers of the GST-Pi gene promoter and β- Actin reference gene were calculated and compared for each different condition.

Figures 2A to 21 show that stabilizing agents add a protective effect compared to native urine, except for glycerol for which cell recovery is not optimal due to phase formation during centrifugation. Frozen samples are preserved best when adding EDTA+DMSO+AB to urine while samples kept at 4⁰C show a higher recovery yield when adding STABILUR® tablet+EDTA+AB.

Evaluation of different urine storage conditions (temperature and time) To reduce transport costs, urine samples are often grouped at the collection site before sending to the lab for further processing. In practice, it occurs that urine samples are stored for several days before arriving at the lab. We investigated how different storage conditions affect the urine DNA quality.

Morning urine from healthy volunteers containing DMSO, EDTA and antibiotics as stabilizing buffer was aliquoted over 39 falcon tubes containing each 10 ml of urine mix. Each tube was spiked with 20,000 copies of fresh cultured SW48 cells and was tested for different storage conditions:

1) 15 tubes were stored at room temperature (RT) of which 3 tubes were processed immediately, 3 were stored for 2 days, 6 days and 8 days respectively

2) 12 tubes were stored at 4°C (of which 3 tubes were stored for 2, 6 and 8 days respectively)

3) 12 tubes were stored at -20⁰C (of which 3 tubes were stored for 2, 6 and 8 days respectively) Subsequently all samples were spun down, sediment was recovered and processed further using a PUREGENE® DNA Purification Kit and EZ DNA Methylation kit. The chemically modified DNA was used as input material for P16 and β-Actin real-time MSP.

Recovered copy numbers of the P16 gene promoter and β-Actin reference gene were calculated and compared for each different condition.

Figure 3A and 3B show that urine samples are preserved best when they are stored at -20⁰C, particularly when stored over a longer period.

Affinity capture process compared to Amicon Filter device Spiked urine material

We investigated whether the ChargeSwitch® gDNA kit from Invitrogen can offer a valid alternative to the Amicon Filter device for recovering cell-free DNA in spiked urine samples. Supernatant from DNA library spiked urine samples were processed side by side through the ChargeSwitch® gDNA kit and the Amicon filter combined with Puregene kit for DNA extraction. Methylation levels of β-Actin, APC, GST-Pi, P14 and P16 were determined by real-time MSP to quantify the number of DNA copies recovered for both methods tested.

A summary is presented in Table 2: the magnetic bead system shows a considerable increase in DNA recovery (for all DNA fragment sizes) , compared to the Amicon filter system.

Table 2: Copy number recovery ChargeSwitch® versus Amicon (spiked urine)

Clinical material 5 clinical samples (post-massage urine samples from the prostate trial) were processed in parallel through the ChargeSwitch® gDNA kit and the Amicon filter/Puregene method. Approximately 50 ml of supernatant could be recovered (pooling the supernatant from both spins, according to Figure 5); after the addition of DMSO and EDTA, the sample was split in 2 (≤ 25 ml) and processed simultaneously through both methods. The ChargeSwitch method was adapted in-house to handle a volume of 25 ml. Healthy urine sample material was used as negative control. β-Actin methylation levels were determined by real-time MSP to quantify the number of copies recovered for both methods. Details are presented in Table 3.

Based on the results, it can be concluded that the ChargeSwitch® gDNA kit offers a valid alternative to the Amicon filter/Puregene method for the recovery of cell free DNA from urine samples, the method is less time consuming and particularly suitable in automation setup.

Table 3 : β-Aσtin copy number recovery ChargeSwitαh® versus Atαicon (clinical material)

Evaluation of different urine sampling methods

Different urine sampling methods were evaluated as specified below:

900 ml of non-morning urine were collected from healthy volunteers and pooled.

Pooled urine was aliquoted in 18 x 45 ml and processed following the 6 different conditions set out below (and labelled A, B, C, D, E and F)

Stabilizing buffer compositions were:

- 90 ml DMSO 100%

- 18 ml EDTA 0.5M

- 9 ml antibiotics (Sigma A5955)

Sample collection methods:

A) 3 x 45 ml of urine were spun down for 5 min at 4500 RPM.

Supernatant :

- Supernatants were recovered and frozen at -2O⁰C until further use.

- Supernatants were defrosted and added with 5.8 ml of stabilizing buffer. - Supernatants were processed through Amicon-15 filters as described by the manufacturer until 300 μl of sample was left.

- DNA from the remaining 300 μl of supernatant was extracted using the Puregene protocol for urine

- DNA was bisulfite modified

Pellet:

- Pellets were frozen at -2O⁰C until further use. - DNA from the pellets was extracted using the Puregene protocol for urine

- DNA was bisulfite modified

B) 3 x 45 ml of urine was directly frozen at -20⁰C until further use.

- The samples were defrosted and added with 5.8 ml of stabilizing buffer.

- Samples were processed through Amicon-15 filters as described by the manufacturer until 300 μl of sample was left.

- DNA was bisulfite modified

C) 3 x 45 ml of urine were spun for 5 min at 4500 RPM. Supernatant :

- Supernatants were recovered and frozen at -20⁰C until further use.

Pellet:

- Pellets were frozen at -20⁰C until further use.

- DNA from the pellets was extracted using the Puregene protocol for urine - DNA from corresponding pellet and supernatant are pooled and bisulfite modified as described

D) 3 x 45 ml of urine were directly frozen at -20⁰C until further use. - The samples were defrosted and spun down for 5 min at 4500 RPM. Supernatant :

- Supernatants were added with 5.8 ml of stabilizing buffer.

- Supernatants were processed through Amicon-15 filters as described by the manufacturer until 300 μl of sample was left.

- DNA was bisulfite modified as described Pellet:

- DNA from the pellets was extracted using the Puregene protocol for urine

- DNA was bisulfite modified as described

E) 3 x 45 ml of urine were added with 5.8 ml stabilizing buffer then directly frozen at -20°C until further use. - The samples were defrosted and processed through Amicon-15 filters as described by the manufacturer until 300 μl of sample was left.

- DNA was bisulfite modified as described

F) 3 x 45 ml of urine were added with 5.8 ml stabilizing buffer then directly frozen at -20⁰C until further use. - The samples were defrosted and spun down for 5 min at 4500 RPM. Supernatant :

- DNA was bisulfite modified as described Pellet: - DNA from the pellets was extracted using the Puregene protocol for urine

- DNA was bisulfite modified as described

- All samples were processed in duplicate through the β-Actin QMSP assay - Recovered copy numbers for each condition were compared.

Results are presented in Fig. 4A and B. Most of the DNA was recovered from the supernatant fraction. Highest copies numbers in the supernatant phase were recovered for stabilizing conditions A (samples spun down, frozen,

Stabilizing buffer added then processed on Amicon) and D (samples are frozen, spun down, buffer added then processed on Amicon) . Separating the supernatant and the pellet before processing on the Amicon filters gives better results (B, E compared to the remaining)

Sensitivity supernatant and pellet fraction of urine

In an initial experiment, urine spiked with template DNA including short and long size PCR products with molecular weight above and under the cutoff limit of the Millipore membrane was used to evaluate the performance of different Amicon filters 5K, 3OK and 10OK. The Amicon 5K filter was used in subsequent experiments.

Samples from a prospective prostate trial were used to evaluate whether urine samples from prostate cancer patients contain free floating tumor DNA. Figure 5 presents a schematic overview on the different steps used. Supernatant was separated from the sediment fraction (leaving about 5 ml of supernatant on top of the cell pellet) by low-speed centrifugation of fresh collected urine samples at the collection site. Both fractions were stored at -20 °C until further processing. Genomic DNA was extracted from the sediment fraction using the PUREGENE® DNA Purification Kit from Gentra. Supernatant was concentrated using the Millipore Amicon Ultra-15 filter device (5K) and DNA was extracted using the PUREGENE® DNA Purification Kit. After bisulfite modification, the samples were assessed for the presence of methylated β-actin, GST-Pi, RARβ2, RASSFlA, pl4, APC and pl6 by real-time PCR.

Table 4 and Figure 6 (APC) provide an overview of the results obtained. The data confirm the presence of tumor DNA in the pellet fraction of urine as well as in the supernatant fraction. For nearly all markers tested, the sensitivity in the supernatant fraction was higher than in the pellet fraction: GST-Pi was detected in 39% of the supernatant samples compared to 29% of the pellet; RAR2beta samples was detected in 33% of the supernatant samples compared to 12% of the pellet; for both markers a good specificity was obtained in pellet and supernatant. When combining GST-Pi, RARβ2, pl4 and pl6, an improved sensitivity of 20% was obtained in the supernatant compared to the pellet fraction (55% sensitivity in the supernatant vs 35% sensitivity in the pellet fraction) , whereas the specificity of the pellet was slightly better in the pellet (10%) when compared to the supernatant. Based on the results obtained, we calculated that a combination of pellet and supernatant DNA would lead to a theoretical 83% specificity and a 61% sensitivity.

The present results show that cell-free DNA in urine is suitable for the analysis of methylation-associated gene silencing in human cancer cells, in particular in prostate cancer cells. Unexpectedly, the sensitivity obtained with cell-free DNA fraction from urine is higher when compared to the sensitivity obtained with the traditionally used sediment fraction. The use of the cell-free DNA fraction thus leads to improvement of sensitivity and specificity of tumor marker detection, in particular methylation marker detection, in urine for cancers that release their cells and cellular components directly in the urethra. Table 4: Sensitivity and specificity of supernatant fraction and pellet fraction from urine.

Valid: β-Actin > 2

To confirm the results obtained in table 4, an additional sample set of 27 post prostate massage urine samples was investigated. This independent sample set included 14 urine samples from patients without cancer and 13 urine samples from patients with prostate cancer. The supernatant was separated from the sediment fraction by low-speed centrifugation of fresh collected urine samples (50 ml) at the collection site. Both fractions were stored at -20⁰C until further processing. Sediment and supernatant fraction were separately processed as described above (Figure 5) and were simultaneously tested for different methylation markers and several combinations of markers. Results were analysed with Receiver Operating Characteristics (ROC) curves. The ROC curve was calculated by plotting the true positive rate (sensitivity) in function of the false positive rate (100- specificity) . An example for the combination of GST-Pi, RARβ2 and APC markers is shown in Figure 7A (sediment fraction) and 7B (supernatant) . This comparative study confirms previous obtained results: the sensitivity obtained with the supernatant fraction from urine (61.5%) is higher compared to the sensitivity from the traditionally used sediment fraction (52.2%).

Storage of urine samples and its effect on DNA degradation

Materials and methods Urine sample collection and processing: In this study, voided urine samples were collected from multiple centers. Symptomatic patients, attending a urology clinic and diagnosed with primary bladder transitional cell carcinoma (cancer cases) or other non-malignant urological disorders (control cases) provided a urine sample for use in real-time MSP.

Within 4h from collection, the urine samples were divided in 2 identical portions. One portion was directly centrifuged at 300Og at room temperature for 10 minutes, the other portion was stored with stabilizing buffer (Stabilur® tablets, Cargille Laboratories, #40050, 5 tablets per 50 ml urine) for up to 48h at room temperature before centrifugation (72h if urine was collected on Fridays) . The urine sediment fractions from both procedures were stored at -2O⁰C until further processing.

Genomic DNA was extracted from the sediment fraction using the PUREGENE^® DNA Purification Kit from Qiagen (i.e. #158908 and #158912) . Briefly, 700 μl of Cell Lysis Solution (provided with kit) was added to the pellet and further processed according to manufacturer's instructions. DNA was rehydrated adding 45 μl of LoTE buffer and was incubated during Ih shaking at 65⁰C followed by overnight shaking at 20⁰C.

Quantification, bisulfite treatment and amplification: DNA was quantified using the PicoGreen dsDNA quantitation reagent kit (Molecular Probes, #P7589) followed by sodium bisulfite treatment (BT) using the EZ-96 DNA Methylation kit from Zymo Research (Cat# D5003) performed on a pipetting robot (Tecan Freedom EVOII, Roma, Liha, Mca, Te-Vacs) . Briefly, up to 1 μg of genomic DNA was denatured and incubated with 100 μl of CT conversion reagent (provided in kit) shaking at 70⁰C for 3h. The modified DNA was further desalted and desulfonated according to manufacturer' s instructions and eluted in 25 μl Tris-HCl 1 mM pH8.0. The modified DNA was stored at -80⁰C until further processing.

Real-time MSP was done on a 7900HT fast real-time PCR cycler from Applied Biosystems. The analytes defined in real-time

MSP were TWISTl, RUNX3, NID2 and ACTB (β-Actin) .

2.4 μl of the modified DNA was added to a PCR mix (total volume 12.5 μl) containing home-made buffer solution (final concentrations are summarized: 16.6 mM (NH₄) ₂SO4, 67 mM Tris (pH 8.8), 6.7 mM MgCl₂, 10 mM β-mercaptoethanol) , dNTPs (5 mM; Amersham Biosciences cat# 27-2035-02), methylation specific forward primer (6 ng) , methylation specific reverse primer (18 ng) , molecular beacon (0.16 μM) and Jumpstart DNA Taq polymerase (0.4 units; Sigma Cat# D9307) .Cycling conditions are specified in Table 5. Results were generated using the SDS 2.2 software (Applied Biosystems) , exported as Ct values (cycle number at which the amplification curves cross the threshold value, set automatically by the software) , and then used to calculate copy numbers based on a linear regression of the values plotted on a standard curve of 20 - 2 x 10^Aβ gene copy equivalents, using plasmid DNA containing the bisulfite modified sequence of interest. Cell lines were included in each run as positive and negative controls, and entered the procedure at the DNA extraction step.

Table 5 : Cycling profile

Results : Stabilization procedure compared to direct centrifugation

We investigated whether adding stabilizing buffer (Stabilur® tablets) to freshly collected urine samples is a possible alternative to direct centrifugation. Our current bladder protocol states that urine samples have to be centrifuged within 4h of collection, this to minimize the DNA degradation. A disadvantage of this procedure is the rather short timeframe before centrifugation (4h) (in particular when patients collect the urine at home) and the requirement of centrifugation equipment at each collection site. 08 002093

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Therefore the use of stabilizing buffer could be a reasonable and particularly useful alternative.

DNA degradation was investigated in voided urine samples from 3 bladder cancer patients and 7 control patients collected at multiple centers. The collected urine samples were divided in 2 equal portions and processed side by side through the direct centrifugation and the stabilized method as described above.

Methylation levels of TWISTl, RUNX3, NID2 and ACTB were determined by real-time MSP to quantify the number of DNA copies recovered for both methods tested. Samples were classified as methylated, non-methylated, or invalid based on the decision tree shown in Figure 8.

The results presented in Table 6, show that comparable results are obtained independent of which procedure is applied.

Table 6: Direct comparison between direct spinning and Stabilur method

The present results indicate that the DNA quality is not affected when urine samples are stored for up to 72h at room temperature in the presence of a stabilizing buffer. Consequently, collection centers do not require special centrifugation equipment at their site and can easily transport the samples to the designated processing lab. Therefore one can conclude that the addition of stabilizer (Stabilur® tablets) provides a useful alternative to the direct spinning method.

References Das PM, Singal R: DNA methylation and cancer. J Clin Oncol. 2004; 22: 4632-42.

Cairns P, Esteller M, Herman JG, Schoenberg M, Jeronimo C, Sanchez-Cespedes M, Chow NH, Grasso M, Wu L, Westra WB et al. 2001 Molecular detection of prostate cancer in urine by GST-Pi hypermethylation. Clinical Cancer Research 7 2727- 2730.

Ying-Hsiu Su, Mengjun Wang, Dean E. Brenner ,Alan Ng, Hovsep Melkonyan, Samuil Umansky, Sapna Syngal, and

Timothy M. Block. Journal of Molecular Diagnostics, Vol. 6, No. 2, May 2004

Botezatu, I. et al . 2000. Genetic analysis of DNA excreted in urine: a new approach for detecting specific genomic DNA sequences from cells dying in an organism. Clin. Chem. 46: 1078-1084. Ying-hsiu, Su. et al . 2004. Transrenal DNA as a diagnostic tool: important technical notes. Ann. N. Y. Acad. Sci. 1022: 81-89.

Bryzgunova et al. Ann. N. Y. Acad. Sci. 1075: 334-340 (2006) .

ϋtting M., et al . Clinical Cancer Research. Vol. 8, 35-40, January 2002.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all embodiments described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, as appropriate.

Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties .

Claims

1. A method for identifying, diagnosing and/or staging or otherwise characterising a urologic cancer or neoplasia in a subject the method comprising isolating cell-free DNA from a urine sample taken from the subject and analysing the DNA to determine whether the DNA contains an epigenetic modification, wherein said epigenetic modification is indicative of the presence or stage or other characteristic of the urologic cancer or neoplasia and thus facilitates diagnosis, staging or otherwise characterising the urologic cancer or neoplasia.

2. The method of claim 1 wherein the urologic cancer comprises prostate and/or bladder and/or kidney cancer.

3. A method for identifying, diagnosing and/or staging or otherwise characterising prostate cancer in a subject the method comprising isolating cell-free DNA from a urine sample taken from the subject and analysing the isolated cell-free DNA to identify, diagnose, stage or otherwise characterise the prostate cancer.

4. The method of claim 3 wherein the analysis carried out on the isolated cell-free DNA comprises determining whether the DNA contains an epigenetic modification wherein said epigenetic modification is indicative of the presence or stage of the prostate cancer.

5. The method of any of claims 1, 2 or 4 wherein the epigenetic modification comprises methylation of one or more genes .

6. The method of claim 5 which comprises treatment of the DNA with an agent capable of modifying unmethylated cytosine residues but which is incapable of modifying methylated cytosine residues.

7. The method of claim β wherein the treatment of the DNA is carried out following isolation of the cell-free DNA.

8. The method of any of claims 5 to 7 wherein methylation is detected through use of methylation specific PCR.

9. The method of any of claims 5 to 8 when dependent from claim 1 or 2 wherein the urologic cancer is prostate cancer or of any of claims 5 to 8 when dependent from claim 4 wherein the methylation status of at least one of GST-Pi, APC, RARβ2, RASSFlA, P16 or P14 is determined.

10. The method of claim 9 wherein the methylation status of all of GST-Pi, APC, RARβ2, P16 and P14 is determined.

11. The method of any preceding claim wherein the cell-free DNA is predominantly less than five hundred base pairs in length.

12. The method of claim 11 wherein the cell free DNA is predominantly 150-250 base pairs in length.

13. The method of any preceding claim wherein isolation of cell-free DNA involves separation of cell-free DNA from cell-associated DNA.

14. The method of any preceding claim wherein cell-free DNA is isolated from the urine sample through use of a filter which retains cells from the urine sample.

15. The method of claim 14 wherein the filter has a pore diameter of approximately 0.5 to approximately 10 μm.

16. The method of claim 15 wherein the pore diameter is approximately 0.8 to approximately 5μm.

17. The method of claim 16 wherein the pore diameter is approximately 0.8, 1.2 or 5μm.

18. The method of any one of claims 1 to 13 wherein cell- free DNA is isolated through use of an affinity capture process .

19. The method of claim 18 wherein the affinity capture process involves use of magnetic beads.

20. The method of any one of claims 1 to 13 wherein cell- free DNA is isolated from the urine sample through use of low speed centrifugation.

21. The method of any one of claims 1 to 13 wherein cell- free DNA is isolated through use of a filter which retains the cell-free DNA.

22. The method of claim 21 wherein the filter comprises a molecular weight filter.

23. The method of claim 22 wherein the molecular weight filter has a cut off of less than or equal to approximately 10 kilodaltons.

24. The method of claim 23 wherein the molecular weight filter has a cut off of approximately 5 kilodaltons.

25. The method of any one of claims 14 to 20 which additionally comprises carrying out the method of any one of claims 21 to 24 in order to further concentrate the cell- free DNA.

26. The method of any one of claims 1 to 13 wherein cell- free DNA is isolated through use of a DNA purification technique.

27. The method of claim 26 wherein the DNA purification technique comprises use of a high concentration of salt to precipitate contaminants, removal of contaminants and recovery of DNA through alcohol precipitation.

28. The method of claim 26 wherein the DNA purification technique comprises use of phenol, chloroform and isoamyl alcohol.

29. The method of claim 26 wherein purified DNA is recovered through alcohol precipitation.

30. The method of any one of claims 14 to 25 which additionally comprises carrying out the method of any one of claims 26 to 29 in order to further purify the cell-free DNA.

31. The method of any preceding claim which additionally comprises isolating and/or analysing cell-associated DNA from the urine sample.

32. The method of claim 31 wherein cell-associated DNA is isolated and/or analysed together with cell-free DNA.

33. The method of claim 31 wherein cell-associated DNA is . isolated and/or analysed separately from the cell-free DNA.

34. The method of any of claims 31 to 33 wherein cell- associated DNA is isolated through use of a DNA purification technique.

35. The method of claim 34 wherein the DNA purification technique is as defined in any of claims 26 to 29.

36. The method of any preceding claim which additionally comprises stabilising the urine sample.

37. The method of claim 36 wherein the urine sample is stabilised prior to isolation of cell-free DNA.

38. The method of claim 36 wherein the isolated cell-free

DNA portion of the urine sample is stabilised following the isolation procedure.

39. The method of any of claims 36 to 38 wherein stabilisation occurs through addition of a stabilising buffer. 8 002093

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40. The method of claim 39 wherein the stabilising buffer comprises at least one component selected from EDTA, an antibacterial, DMSO and STABILUR™ tablets.

41. The method of claim 39 or 40 wherein the stabilising buffer comprises EDTA, DMSO and an antibacterial and wherein the sample may be frozen for storage or stored at room temperature prior to the analysis carried out on the isolated cell-free DNA.

42. The method of claim 39 or 40 wherein the stabilising buffer comprises a STABILUR™ tablet, EDTA and an antibacterial and the sample may be stored at a temperature of around 4°C or at room temperature prior to the analysis.

43. The method of any of claims 40 to 42 wherein the EDTA is present at a final concentration of around 1OmM.

44. The method of any of claims 40 to 43 wherein the DMSO is present at around 10% of the final stabilising buffer volume .

45. The method of any preceding claim which comprises freezing the urine sample taken from the subject for storage prior to isolating cell-free DNA from the urine sample.

46. The method of any preceding claim which comprises freezing the urine sample taken from the subject for storage prior to analysis of the DNA.

47. The method of any of claims 36 to 44 which comprises freezing the urine sample taken from the subject prior to stabilising the urine sample.

48. A stabilising buffer solution for storing urine samples at a temperature of around 4⁰C, in particular the cell-free DNA component from a urine sample, comprising EDTA, an antibacterial and a STABILUR™ tablet.

49. A stabilising buffer for storing urine samples under freezing conditions, in particular the cell-free DNA component of a urine sample, comprising EDTA, DMSO and an antibacterial .

50. The stabilising buffer solution of claim 48 or 49 wherein the EDTA is present at a final concentration of around 1OmM.

51. The stabilising buffer solution of claim 49 wherein the DMSO is present at around 10% of the final stabilising buffer volume.

52. A method for storing urine, in particular the cell-free DNA component of urine, comprising adding a stabilising buffer as claimed in any one of claims 48, 50 or 51 to the urine and storing the mixture at a temperature of around 4⁰C or at room temperature.

53. A method for storing urine, in particular the cell-free DNA component of urine, comprising adding the stabilising buffer as defined in any one of claims 49 to 51 to the urine T/GB2008/002093

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sample and storing the mixture under freezing conditions or at room temperature.

54. A method for isolating cell-free DNA from a urine sample, the isolated cell-free DNA being useful in the method of any one of claims 1 to 47, comprising applying the urine sample to a filter which retains cells from the urine sample but allows the cell-free DNA to pass through.

55. The method of claim 54 wherein the filter comprises pores having a diameter of approximately 0.5 to approximately lOμm.

56. The method of claim 55 wherein the pore diameter is approximately 0.8 to approximately 5μm.

57. The method of claim 56 wherein the pore diameter is approximately 0.8, 1.2, or 5μm.

58. The method as claimed in any one of claims 54 to 57 which additionally comprises carrying out the steps of the method as defined in claim 52 or 53.

59. A method for concentrating isolated cell-free DNA from a urine sample comprising applying the isolated cell-free DNA to a filter which retains the isolated cell-free DNA.

60. The method of claim 59 wherein the filter is a molecular weight filter.

61. The method of claim 60 wherein the molecular weight filter has a molecular weight cut off of less than or equal to approximately 10 kilodaltons.

62. The method of claim 61 wherein the molecular weight filter has a molecular weight cut off of approximately 5 kilodaltons.

63. The method of any one of claims 54 to 58 which additionally comprises carrying out the method of any one of claims 59 to 63 to concentrate the isolated cell-free DNA.

64. The method of any of claims 52 to 63 further comprising DNA purification.

65. The method of claim 64 wherein a DNA purification technique as defined in any of claims 27 to 29 is utilised.

66. A kit for identifying, diagnosing and/or staging or otherwise characterising an urological cancer or neoplasia in a subject comprising;

(i) a filter for isolating cell-free DNA from a urine sample taken from the subject; and

(ii) means for analysing the isolated cell-free DNA.

67. The kit of claim 66 wherein the means for analysing the isolated cell-free DNA comprises specific primers for amplification of a DNA sequence to produce an amplification product, wherein the amplification product is an indicator of an urologic cancer or neoplasia or a specific stage or characteristic thereof.

68. The kit of claim 67 wherein the primers are methylation specific PCR primers.

69. The kit of claim 68 wherein the urologic cancer is prostate cancer and the methylation specific PCR primers allow the methylation status of at least one of GST-Pi, APC,RARβ2, RASSSFlA, P16 or P14 to be determined.

70. The kit of claim 69 wherein the methylation specific PCR primers allow the methylation status of all of GST-Pi, APC,

RARβ2, P16 and P14 to be determined.

71. The kit of any one of claims 66 to 70 wherein the means for analysing the isolated cell-free DNA comprises an agent capable of modifying unmethylated cytosine residues but which is incapable of modifying methylated cytosine residues .

72. The kit of claim 71 wherein the agent comprises bisulfite.

73. The kit of any of claims 66 to 72 wherein the filter retains cells from the urine sample but allows cell-free DNA to pass through.

74. The kit of claim 73 wherein the filter has a pore diameter of approximately 0.5 to approximately 10 μm.

75. The kit of claim 74 wherein the pore diameter is approximately 0.8 to approximately 5μm.

76. The kit of claim 75 wherein the pore diameter is approximately 0.8, 1.2 or 5μm.

77. The kit of any claims 66 to 72 wherein the filter comprises an affinity-capture surface for capturing cell- free DNA.

78. The kit of claim 76 wherein the affinity-capture surface comprises one or more magnetic beads.

79. The kit of any of claims 66 to 78 additionally comprising a stabilising buffer solution as defined in any of claims 48 to 51.

80. The kit of any of claims 66 to 77 additionally comprising a molecular weight filter for concentrating the isolated cell-free DNA.

81. The kit of claim 80 wherein the molecular weight filter has a cut off of less than or equal to approximately 10 kilodaltons .

82. The kit of claim 81 wherein the molecular weight filter has a cut off of approximately 5 kilodaltons.

83. The kit of any of claims 66 to 82 further comprising reagents for purification of DNA.

84. A method for storing urine samples for a period of up to 72 hours at room temperature comprising adding a stabilising buffer to the urine sample, with the proviso that the urine sample is not centrifuged or otherwise fractionated prior to or during the storage period.