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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/112—Disease subtyping, staging or classification
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/154—Methylation markers
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• 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.
• 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 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.
• 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.
• 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.
• 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.
• 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.
• Urologic cancer or neoplasia is meant a cancer or neoplasia of the urinary tract or the urogenital system.
• 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.
• 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.
• the cancer or neoplasia is assigned a designated status, such as a histopathological status, based upon accepted standards.
• a designated status such as a histopathological status
• 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.
• 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.
• 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
• Stage IV prostate cancer may also be called stage Dl or stage D2 prostate cancer.
• 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.
• the method may also include the step of obtaining the urine sample from the subject under test.
• the subject is a human subject.
• 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.
• 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.
• 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.
• isolation of cell-free DNA involves (complete or partial) separation of cell-free DNA from cell-associated DNA.
• 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.
• 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.
• a filter which retains cells from the urine sample, but allows the cell-free DNA to pass through the filter.
• 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 MINISARTTM 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.
• the filter has a pore diameter of approximately 0.5 ⁇ m to approximately 10 ⁇ m. 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.
• the pore diameter is approximately 0.8 to approximately 5 ⁇ m and in specific preferred embodiments, the pore diameter is approximately 0.8, approximately 1.2 or approximately 5 ⁇ m with approximately 1.2 ⁇ m 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.
• cell-free DNA is isolated through an affinity capture process.
• 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.
• the one or more surfaces comprises at least one bead, preferably at least one magnetic bead.
• 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.
• the one or more surfaces has differing affinity for nucleic acid based upon pH conditions.
• 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.
• residual contaminants such as protein are digested and washed away to leave only pure intact DNA bound to the one or more surfaces.
• CHARGESWITCH® and GENECATCHERTM (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.
• 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) .
• the affinity capture process means that these additional steps are unnecessary.
• cell-free DNA is isolated through use of a filter which retains the cell-free DNA.
• 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 ⁇ ltracel-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.
• Millipore- such as the AMICON® Ultra-15
• PLCC ⁇ ltracel-PL Membrane filters available in a range of molecular weight cut-offs
• Centricon Plus-70 filters are utilised in the isolation of cell- free DNA.
• 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.
• 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) .
• 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) .
• 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:
• purification involves alcohol precipitation of DNA.
• Preferred alcohols include ethanol and isopropanol.
• Suitable purification techniques also include salt-based precipitation methods.
• 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.
• the DNA purification technique is based upon use of organic solvents to extract contaminants from cell lysates.
• 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.
• extracted DNA is recovered through alcohol precipitation, such as ethanol or isopropanol precipitation.
• 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.
• a suitable purification technique 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.
• 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.
• the methods of the invention additionally comprise isolating and/or analysing cell- associated DNA from the urine sample.
• cell-associated DNA is isolated and/or analysed together with cell-free DNA.
• cell-associated DNA is isolated and/or analysed separately from the cell-free DNA.
• 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.
• 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
• the respective DNA components are (isolated separately and) pooled to provide methods of the invention with improved sensitivity.
• 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 may be analysed in any suitable fashion to identify, diagnose and/or stage or otherwise characterise the urologic cancer or neoplasia.
• 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 .
• 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.
• 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.
• promoter, exon and intron regions may be assessed to determine their methylation status.
• 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.
• 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.
• 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 .
• MSP methylation-specific PCR
• McMS-PCR melting curve methylation- specific PCR
• 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) .
• MBD methyl binding domain
• the MBD may be obtained from MBP, MBP2, MBP4 or poly-MBD (Jorgensen et al . , 2006).
• 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) .
• 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.
• an agent capable of modifying unmethylated cytosine residues are converted (through deamination) to uracil, whilst methylated residues remain unconverted.
• a most preferred reagent in this context is bisulfite, in particular sodium bisulfite.
• treatment of the DNA using the agent is carried out following isolation of the cell-free DNA.
• methylation is detected through use of methylation specific PCR (MSP) .
• MSP methylation specific PCR
• 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) .
• QMSP realtime quantitative MSP
• TAQMAN® realtime quantitative MSP
• PCR polymerase chain reaction
• a further technique which may be utilised is known as "Heavymethyl” .
• 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.
• methylation status is determined using real-time applications of methylation specific PCR.
• 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.
• 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.
• 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 ⁇ -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.
• absolute copy number of the methylated marker gene can be determined.
• 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.
• two standard curves are used, one for either the reference gene (such as ⁇ -actin) or the non-methylated marker and one for the methylated version of the marker ("m- gene") .
• 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.
• primers may be designed in both the sense and antisense orientation to direct sequencing across the promoter region of the relevant gene.
• 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.
• 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) .
• suitable amplification methods include the ligase chain reaction (LCR) (Barringer et al, 1990), selective amplification of target polynucleotide sequences (US Patent No.
• 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.
• 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.
• 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.
• 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.
• amplification is achieved with the use of primers specific for the sequence of the gene whose methylation status is to be assessed.
• primer binding sites corresponding to a suitable region of the sequence may be selected.
• 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.
• HPA Hybridization Protection Assay
• 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.
• the urologic cancer is prostate cancer and the methylation status of at least one of GST-Pi, APC, RAR ⁇ 2, 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.
• 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.
• the methylation status of all of GST-Pi, APC, RAR ⁇ 2, 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.
• 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.
• suitable negative controls may be employed with the methods of the invention.
• suitable controls may include assessing the methylation status of a gene known to be unmethylated. 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
• 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.
• 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.
• 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.
• Optimized preservation of the nucleic acid in particular cell-free DNA 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.
• 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.
• 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.
• 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.
• 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.
• a stabilising buffer for use in these methods are described herein.
• the isolated cell-free DNA portion of the urine sample is stabilised following an isolation procedure.
• 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.
• the invention provides a stabilising buffer solution for storing urine samples comprising EDTA, an antibacterial and optionally a STABILURTM tablet. This solution is preferably for storing a urine sample at a temperature of around 4 0 C.
• 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.
• This 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.
• 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 STABILURTM tablets.
• 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.
• 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 .
• 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.
• the stabilising buffer comprises a STABILURTM tablet, EDTA and an antibacterial and the sample may be stored at a temperature of around 4 0 C prior to the analysis.
• 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 may be stored at any suitable temperature, including room temperature.
• the storage temperature may be anywhere between approximately -5O 0 C and approximately 37°C , preferably approximately -1O 0 C to -30 0 C, such as approximately -2O 0 C or approximately 1°C to 1O 0 C, such as approximately 4°C.
• freezing is meant a temperature at or below O 0 C, preferably approximately -2O 0 C.
• 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.
• 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.
• 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 4 0 C.
• 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 -20 0 C.
• mixtures may be stored at other temperatures, such as room temperature for example.
• 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) .
• 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.
• DNA may be suitably modified (for example using bisulfite) and an appropriate detection method, such as real-time MSP, carried out.
• Stabilizing buffer may be added once the respective DNA- containing components have been thawed.
• 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) .
• samples may be stored at any suitable temperature, including room temperature.
• the storage temperature may be anywhere between approximately -50 0 C and approximately 37 0 C, preferably approximately -1O 0 C to -3O 0 C, such as approximately -20 0 C or approximately 1°C to 10 0 C, such as approximately 4°C.
• freezing is meant a temperature at or below 0 0 C, preferably approximately -20 0 C.
• 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.
• DNA may be suitably modified (for example using bisulfite) and an appropriate detection method, such as real-time MSP, carried out.
• kits for identifying, diagnosing and/or staging or otherwise characterising a urological cancer or neoplasia in a subject 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.
• 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.
• 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.
• the means for analysing cell-free DNA preferably includes means for determining whether the DNA contains an epigenetic modification.
• methylation markers are preferably investigated to facilitate identification or diagnosis.
• 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.
• the primers are methylation specific PCR primers.
• the urologic cancer is prostate cancer.
• the methylation specific PCR primers allow the methylation status of at least one of GST-Pi, APC, RAR ⁇ 2, 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, RAR ⁇ 2, 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.
• 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.
• this agent comprises bisulfite, in particular sodium bisulfite.
• the means for analysing the cell-free DNA may alternatively include appropriate methylation sensitive restriction enzymes.
• the urologic cancer is prostate cancer and the methylation sensitive restriction enzymes allow the methylation status of at least one of GST-Pi, APC, RAR ⁇ 2, 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, RAR ⁇ 2, 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) .
• the filter included in the kits of the invention retains cells from the urine sample but allows cell-free DNA to pass through.
• the filter has a pore diameter of approximately 0.5 to approximately 10 ⁇ m, more preferably approximately 0.8 to approximately 5 ⁇ m and most preferably approximately 0.8, 1.2 or 5 ⁇ m.
• suitable affinity-capture means are incorporated into the kits of the invention. They may be suitable magnetic beads for example.
• affinity capture techniques applies equally to the kits of the invention, in terms of the components which may be included.
• kits of the invention may additionally comprise a stabilising buffer solution of the invention as defined hereinabove.
• kits of the invention may additionally comprise a suitable molecular weight filter for concentrating the isolated cell-free DNA.
• a suitable molecular weight filter for concentrating the isolated cell-free DNA.
• Appropriate filters are described in greater detail above.
• the molecular weight filter has a cut off of less than or equal to approximately 10 kilodaltons and more preferably approximately 5 kilodaltons.
• 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.
• Figure 1 presents results of a comparison between filtration and centrifugation for recovery of DNA from urine:
• the Y axis represents the number of copies of the gene under evaluation
• FIG. 1 presents the effects of various stabilising agents on DNA recovery from urine samples:
• FIG. 3 presents the effects of various storage conditions on DNA recovery from urine samples:
• Figure 4 shows the results of an evaluation of different urine sampling methods in terms of DNA recovery.
• A ⁇ -Actin recovered in different fractions of the 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.
• FIG. 7 shows the Receiver Operating Characteristics (ROC) curves calculated for a 3-marker combination GST-Pi, RAR ⁇ 2 and APC.
• the true positive rate (sensitivity) is plotted in function of the false positive rate (100-specificity)
• Figure 8 Decision tree for sample classification (Methylated, Non-Methylated or Invalid)
• Genomic DNA was extracted from the sediment fraction using the PUREGENE® DNA Purification Kit from Gentra. 700 ⁇ l 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 ⁇ l of LoTE buffer and was incubated during 1 hour shaking at 65 0 C followed by overnight shaking at 20 0 C.
• the ChargeSwitch® gDNA 1 ml serum kit from Invitrogen (cat# CS11040) with either 300 ⁇ l or 150 ⁇ l 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 :
• 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)
• Actin, APC, GST-Pi, P14 and Pl ⁇ were determined by real-time MSP.
• 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 ⁇ g of genomic DNA was denatured and incubated with 100 ⁇ l of CT conversion reagent (provided in kit) shaking at 70 0 C for 3 hours. The modified DNA was further desalted and desulfonated according to manufacturer' s instructions and eluted in 50 ⁇ l Tris-HCl 1 mM pH 8.0. The modified DNA was stored at -80 0 C 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, RAR ⁇ 2, RASSFlA, Pl ⁇ 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.
• 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 ⁇ l of a 10Ox diluted Sigma A5955: Antibiotic Antimycotic Solution, stabilized (100 ⁇ )
• Each stabilizing condition was stored at different temperatures as follows: 1) 2 days at 4 0 C
• 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 4 0 C show a higher recovery yield when adding STABILUR® tablet+EDTA+AB.
• 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:
• Figure 3A and 3B show that urine samples are preserved best when they are stored at -20 0 C, particularly when stored over a longer period.
• 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.
• ⁇ -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.
• 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.
• Stabilizing buffer compositions were:
• FIG. 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 ⁇ -actin, GST-Pi, RAR ⁇ 2, RASSFlA, pl4, APC and pl6 by real-time PCR.
• Table 4 and Figure 6 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.
• 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.
• 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.
• 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.
• 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.
• cancer cases primary bladder transitional cell carcinoma
• control cases non-malignant urological disorders
• 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 -2O 0 C until further processing.
• stabilizing buffer Stabilur® tablets, Cargille Laboratories, #40050, 5 tablets per 50 ml urine
• Genomic DNA was extracted from the sediment fraction using the PUREGENE ® DNA Purification Kit from Qiagen (i.e. #158908 and #158912) . Briefly, 700 ⁇ l 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 ⁇ l of LoTE buffer and was incubated during Ih shaking at 65 0 C followed by overnight shaking at 20 0 C.
• 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 ⁇ g of genomic DNA was denatured and incubated with 100 ⁇ l of CT conversion reagent (provided in kit) shaking at 70 0 C for 3h. The modified DNA was further desalted and desulfonated according to manufacturer' s instructions and eluted in 25 ⁇ l Tris-HCl 1 mM pH8.0. The modified DNA was stored at -80 0 C until further processing.
• PicoGreen dsDNA quantitation reagent kit Molecular Probes, #P7589
• BT sodium bisulfite treatment
• EZ-96 DNA Methylation kit from Zymo
• Real-time MSP was done on a 7900HT fast real-time PCR cycler from Applied Biosystems.
• MSP were TWISTl, RUNX3, NID2 and ACTB ( ⁇ -Actin) .
• 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 A ⁇ 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.
• 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.

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