Detecting prostate cancer

Abstract

To provide methods, compositions, and related uses for detecting the presence of prostate cancer.SOLUTION: There is provided a method for characterizing a sample from a human patient, comprising: a) obtaining DNA from a sample of a human patient; b) assaying a methylation state of a DNA methylation marker comprising a base in a differentially methylated region (DMR) selected from a group consisting of DMRs 1 to 140; and c) comparing the assayed methylation state of the one or more DNA methylation markers with methylation level references for the one or more DNA methylation markers for human patients not having prostate cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 464,800, filed February 28, 2017, the contents of which are incorporated by reference in their entirety.

[0002] Provided herein is the science and technology of prostate cancer screening, particularly, but not exclusively, methods, compositions, and related uses for detecting the presence of prostate cancer. [Background technology]

[0003] Prostate cancer (PCa) is the second most commonly diagnosed cancer in men, with 903,000 new cases and 258,000 deaths worldwide in 2008. While PCa is common, the disease is also heterogeneous in clinical behavior. It is estimated that approximately 1 in 6 American men will be diagnosed with PCa, yet the mortality rate for PCa among American men is only 2.8% (1 in 36 men) (see, e.g., Strand SH, et al., Int J Mol Sci 2014;15:16544-16576).

[0004] PCA survival rates depend on many factors. Earlier diagnosis of less advanced disease offers the most men the best chance for curative treatment. Indeed, earlier PCA diagnosis has been facilitated by the use of prostate-specific antigen (PSA) testing. The timing of PSA testing, during which PCA stage and grade transitions occur, leads to the identification of disease more amenable to curative treatment. While PSA is valuable in the diagnosis and management of PCA, PSA screening is also considered controversial. PSA screening may lead to the diagnosis of lower-grade, lower-risk tumors, and subsequent overtreatment may subject men to unnecessary quality-of-life harm (erectile dysfunction, incontinence). Consequently, novel biomarkers are needed to aid in the diagnosis of PCA. Novel tests are also needed to provide men with improved prognostic information about their cancer.

[0005] The present invention addresses these needs. Summary of the Invention

[0006] Methylated DNA has been investigated as a potential class of biomarker in tissues of most tumor types. In many instances, DNA methyltransferases add methyl groups to DNA at cytosine-phosphate-guanine (CpG) island sites as an epigenetic control of gene expression. In a biologically attractive mechanism, methylation events in the promoter regions of tumor suppressor genes are thought to silence their expression, thereby contributing to carcinogenesis. DNA methylation can be a more chemically and biologically stable diagnostic tool than RNA or protein expression (Laird (2010) Nat Rev Genet 11:191-203). Furthermore, in other cancers, such as sporadic colorectal cancer, methylation markers offer excellent specificity and are more broadly informative and sensitive than individual DNA mutations (Zou et al. (2007) Cancer Epidemiol Biomarkers Prev 16:2686-96).

[0007] Analysis of CpG islands has provided important insights when applied to animal models and human cell lines. For example, Zhang and colleagues found that amplicons from different parts of the same CpG island can have different levels of methylation (Zhang et al. (2009) PLoS Genet 5:e1000438). Furthermore, methylation levels were bimodally distributed between highly methylated and unmethylated sequences, further supporting a binary switch-like pattern of DNA methyltransferase activity (Zhang et al. (2009) PLoS Genet 5:e1000438). Analysis of mouse tissues in vivo and cell lines in vitro demonstrated that only approximately 0.3% of high CpG density promoters (HCPs, defined as having >7% CpG sequences within a 300 base pair region) were methylated, whereas low CpG density regions (LCPs, defined as having <5% CpG sequences within a 300 base pair region) tended to be frequently methylated in dynamic tissue-specific patterns (Meissner et al. (2008) Nature 454:766-70). HCPs include promoters for ubiquitous housekeeping genes and highly regulated developmental genes. Among HCP sites, several established markers were found to be >50% methylated, such as Wnt2, NDRG2, SFRP2, and BMP3 (Meissner et al. al. (2008) Nature 454:766-70).

[0008] Epigenetic methylation of DNA at cytosine-phosphate-guanine (CpG) island sites by DNA methyltransferases has been investigated as a potential class of biomarkers in tissues of most tumor types. In a biologically attractive mechanism, methylation events in the promoter regions of tumor suppressor genes are thought to silence their expression, contributing to carcinogenesis. DNA methylation can be a more chemically and biologically robust diagnostic tool than RNA or protein expression. Furthermore, in other cancers, such as sporadic colorectal cancer, aberrant methylation markers are more broadly informative, more sensitive, and offer superior specificity than individual DNA mutations.

[0009] Several methods are available for discovering novel methylation markers. Microarray-based interrogation of CpG methylation is a rational, high-throughput approach, but this strategy is biased toward known regions of interest, primarily established tumor suppressor promoters. Alternative methods for genome-wide analysis of DNA methylation have been developed over the past decade. There are three basic approaches. The first approach uses DNA digestion with restriction enzymes that recognize specific methylation sites, followed by amplification of the DNA in a quantification step using several possible analytical techniques (e.g., methylation-specific PCR; MSP). The second approach involves enriching the methylated fraction of genomic DNA using antibodies directed against methyl-cytosine or other methyl-specific binding domains, followed by microarray analysis or sequencing to map the fragments to a reference genome. This approach does not provide single-base resolution of all methylation sites within the fragments. The third approach begins with bisulfite treatment of DNA to convert all unmethylated cytosines to uracil, followed by restriction enzyme digestion and subsequent sequencing of all fragments after coupling to adaptor ligands. The choice of restriction enzyme can enrich for fragments with high CpG density, reducing the number of redundant sequences that can be mapped to multiple gene locations during analysis.

[0010] RRBS provides CpG methylation status data at single-base resolution for 80-90% of all CpG islands and most tumor suppressor promoters at medium to high read coverage. Analysis of these reads in cancer case-control studies leads to the identification of variably methylated regions (DMRs). In previous RRBS analyses of pancreatic cancer specimens, hundreds of DMRs were not covered, many of which had never been associated with oncogenesis, and many of these were unannotated. Furthermore, validation studies in independent tissue sample sets identified marker CpGs that performed with 100% sensitivity and specificity.

[0011] Provided herein is the science and technology of prostate cancer screening, particularly, but not exclusively, methods, compositions, and related uses for detecting the presence of prostate cancer.

[0012] Indeed, as described in Examples I-VIII, experiments conducted during the process of identifying embodiments of the present invention identified a novel set of 73 differentially methylated regions (DMRs) for distinguishing cancer from non-neoplastic control DNA in prostate-derived DNA. Additionally, we identified 10 novel DMRs that are methylated in prostate epithelium (cancer and normal) but unmethylated in normal leukocyte DNA samples. Both sets of these regions were identified from next-generation sequencing studies of CpGs enriched in bisulfite-converted tumor and normal DNA. Tumor samples contained low-grade Gleason 6 and high-grade Gleason 7+ patterns. DMRs were selected using proprietary filters and analysis and validated in an independent set of tissue samples using a novel methylation-specific PCR (MSP) assay. These 73 biomarker assays demonstrated excellent detection in tissues and had a wide range of clinical specificity (some for cancers across many different organ sites, others specific only to prostate cancer).

[0013] Experiments such as these led to the listing and description of 120 novel DNA methylation markers (Table 1) that distinguish prostate cancer tissue from benign prostate tissue. From these 120 novel DNA methylation markers, further experiments identified 73 markers that could distinguish high-grade prostate cancer tissue (e.g., Gleason score 7+) from benign prostate tissue. More specifically, markers and / or marker panels (e.g., chromosomal regions having annotations selected from ACOXL, AKR1B1_3644, ANXA2, CHST11_2206, FLJ45983, GAS6, GRASP, HAPLN3, HCG4P6, HES5_0822, ITPRIPL1, KCNK4, MAX.chr1.61519554-61519667, MAX.chr2.97193166-97193253, MAX.chr3.193, MAX.chr3.72788028-72788112, RAI1_7469, RASSF2, SERPINB9_3389, SLC4A11, and TPM4_8047) were identified that were able to distinguish prostate cancer tissue from benign prostate tissue (see Examples I-VI).

[0014] Additional experiments conducted during the course of developing embodiments of the present invention have identified markers (e.g., SERPINB9_3479, FLOT1_1665, HCG4P6_4618, CHST11_2206, MAX.chr12.485, GRASP_0932, GAS6_6425, MAX.chr3.193, MAX.chr2.971_3164, MAX.chr3.727_8028, HES5_0840, TPM4_8037, SLCO_0840 ... The present study was directed to identifying chromosomal regions having annotations selected from: 3A1_6187, ITPRIPL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487), which were capable of distinguishing prostate cancer tissue from benign prostate tissue (see Example VIII; Table 11).

[0015] Additional experiments conducted during the course of developing embodiments of the present invention were directed to identifying markers (e.g., chromosomal regions having annotations selected from SERPINB9_3479, GRASP_0932, SLCO3A1_6187, ITPRIPL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, WNT3A_5487) that could distinguish high-grade prostate cancer tissue (e.g., Gleason score 7+) from low-grade prostate cancer tissue (e.g., Gleason score 6), and these markers could distinguish prostate cancer tissue from benign prostate tissue (see Example VIII; Table 11).

[0016] Additional experiments conducted during the course of developing embodiments of the present invention were directed to identifying markers capable of detecting the presence or absence of prostate cancer in blood samples (e.g., blood plasma samples). Indeed, markers and / or marker panels (e.g., chromosomal regions with annotations selected from max.chr3.193, HES5, SLCO3A1, and TPM4_8047) were identified that were capable of detecting the presence or absence of prostate cancer tissue in blood plasma samples (see Examples I-VI).

[0017] As described herein, this technology provides multiple methylated DNA markers and subsets thereof (e.g., sets of 2, 3, 4, 5, 6, 7, or 8 markers) that have high discrimination for prostate cancer overall. Experiments have applied selection filters to candidate markers to identify markers that provide high signal-to-noise ratios and low background levels, providing high specificity for prostate cancer screening or diagnostic purposes.

[0018] In some embodiments, this technology relates to assessing the presence and methylation status of one or more of the markers identified herein in a biological sample (e.g., prostate tissue, plasma sample). These markers include one or more variably methylated regions (DMRs) as discussed herein, e.g., as provided in Tables 1 and 3. In embodiments of this technology, the methylation status is assessed. As such, the technology provided herein is not limited to methods of measuring the methylation status of genes. For example, in some embodiments, the methylation status is measured by genome scanning methods. For example, one method includes restriction landmark genome scanning (Kawai et al. (1994) Mol. Cell. Biol. 14:7421-7427), and another example includes methylation-sensitive arbitrarily primed PCR (Gonzalgo et al. (1997) Cancer Res. 57:594-599). In some embodiments, changes in methylation patterns at specific CpG sites are monitored by digestion of genomic DNA with methylation-sensitive restriction enzymes followed by Southern analysis of the region of interest (digestion-Southern method). In some embodiments, analyzing changes in methylation patterns involves a PCR-based process that involves digestion of genomic DNA with methylation-sensitive restriction enzymes prior to PCR amplification (Singer-Sam et al. (1990) Nucl. Acids Res. 18:687). In addition, other techniques have been reported that utilize bisulfite treatment of DNA as the starting point for methylation analysis. These include methylation-specific PCR (MSP) (Herman et al. (1992) Proc. Natl. Acad. Sci. USA 93:9821-9826) and restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA (Sadri and Hornsby (1996) Nucl. Acids Res. 24:5058-5059; and Xiong and Laird (1997) Nucl. Acids Res. 25:2532-2534).PCR techniques have been developed for the detection of genetic mutations (Kuppuswamy et al. (1991) Proc. Natl. Acad. Sci. USA 88:1143-1147) and quantification of allele-specific expression (Szabo and Mann (1995) Genes Dev. 9:3097-3108; and Singer-Sam et al. (1992) PCR Methods Appl. 1:160-163). Techniques such as these use internal primers that anneal to the PCR-generated template and terminate immediately 5' to the single nucleotide being assayed. In some embodiments, a method using a "quantitative Ms-SNuPE assay" such as that described in U.S. Pat. No. 7,037,650 is used.

[0019] Methylation status is often assessed as the proportion or percentage of individual strands of DNA that are methylated at a particular site (e.g., at a single nucleotide, at a specific region or locus, or in a longer sequence of interest, e.g., up to about 100-bp, 200-bp, 500-bp, or 1000-bp subsequences of one or more DNA fragments) compared to the total population of DNA in a sample that contains that site. Traditionally, the amount of unmethylated nucleic acid is determined by PCR using calibrators. A known amount of DNA is then bisulfite-treated, and the resulting methylation-specific sequence is determined using either real-time PCR or other exponential amplification techniques, such as the QuARTS assay (e.g., as provided by U.S. Pat. No. 8,361,720 and U.S. Patent Application Publication Nos. 2012 / 0122088 and 2012 / 0122106, both of which are incorporated herein by reference).

[0020] For example, in some embodiments, these methods comprise generating a standard curve for the unmethylated target by using an external standard. This standard curve is composed of at least two points and relates real-time Ct values for unmethylated DNA to the known quantitative standard. A second standard curve for the methylated target is then generated from at least two points and the external standard. This second standard curve relates Ct values for methylated DNA to the known quantitative standard. Ct values of test samples for the methylated and unmethylated populations are then determined, and the genome equivalents of DNA are calculated from the standard curves generated by the first two steps. The percentage of methylation at the site of interest is calculated from the amount of methylated DNA proportional to the total amount of DNA in the population, e.g., (number of methylated DNA) / (number of methylated DNA + number of unmethylated DNA) x 100.

[0021] Also provided herein are compositions and kits for carrying out these methods. For example, in some embodiments, reagents (e.g., primers, probes) specific to one or more markers are provided, either singly or in sets (e.g., sets of primer pairs for amplifying multiple markers). Additional reagents for performing detection assays (e.g., enzymes, buffers, positive and negative controls for performing QuARTS, PCR, sequencing, bisulfite, or other assays) can also be provided. In some embodiments, these kits are provided that contain one or more reagents necessary, sufficient, or useful for carrying out the methods. Also provided are reaction mixtures containing these reagents. Also provided are master mix reagent sets containing multiple reagents that can be added to each other and / or to a test sample to complete a reaction mixture.

[0022] In some embodiments, the technology described herein relates to programmable machines designed to perform sequences of arithmetic or logical operations, such as those provided by the methods described herein. For example, some embodiments of the technology relate to (e.g., are implemented in) computer software and / or computer hardware. In one aspect, the technology relates to computers that include some form of memory, elements for performing arithmetic and logical operations, and processing elements (e.g., microprocessors) for executing sequences of instructions (e.g., methods as provided herein), and that read, manipulate, and store data. In some embodiments, the microprocessor is part of a system that determines the methylation status (e.g., of one or more DMRs, e.g., DMRs 1-140 as provided in Tables 1 and 13); compares the methylation status (e.g., of one or more DMRs, e.g., DMRs 1-140 as provided in Tables 1 and 13); generates a standard curve; determines Ct values; calculates the methylation fraction, frequency, or percentage (e.g., of one or more DMRs, e.g., DMRs 1-140 as provided in Tables 1 and 13); identifies CpG islands; determines the specificity and / or sensitivity of an assay or marker; calculates ROC curves and associated AUCs; and performs sequencing analysis; all as described herein or known in the art.

[0023] In some embodiments, the microprocessor or computer uses the methylation status data in an algorithm to predict the site of the cancer.

[0024] In some embodiments, a software or hardware component receives results from multiple assays, determines a single-value result, and reports it to a user indicating cancer risk based on the results of the multiple assays (e.g., determining the methylation status of multiple DMRs, e.g., as provided in Tables 1 and 3). Related embodiments calculate a risk factor based on a mathematical combination (e.g., weighted combination, linear combination) of results from multiple assays, e.g., determining the methylation status of multiple markers (e.g., multiple DMRs, e.g., as provided in Tables 1 and 3). In some embodiments, the methylation status of the DMRs defines a dimension and can have values in a multidimensional space, and the coordinates defined by the methylation status of the multiple DMRs are a result, e.g., related to cancer risk, for reporting to a user, e.g.,

[0025] Some embodiments include storage media and memory components (e.g., volatile and / or non-volatile memory) that find use in storing instructions (e.g., process embodiments as provided herein) and / or data (e.g., workpieces such as methylation measurements, sequences, and their associated statistical descriptions). Some embodiments also relate to systems that include one or more of a CPU, a graphics card, and a user interface (e.g., including an output device such as a display, and an input device such as a keyboard).

[0026] The programmable machines associated with this technology include existing and emerging technologies, as well as technologies under development or already in development (e.g., quantum computers, chemical computers, DNA computers, optical computers, spintronics-based computers, etc.).

[0027] In some embodiments, the technology involves wired (e.g., metal cable, optical fiber) or wireless transmission media for transmitting data. For example, some embodiments relate to data transmission over a network (e.g., a local area network (LAN), a wide area network (WAN), an ad-hoc network, the Internet, etc.). In some embodiments, the programmable machines reside on the network as peers, and in some embodiments, the programmable machines have a client / server relationship.

[0028] In some embodiments, the data is stored on a computer-readable storage medium such as a hard disk, flash memory, optical media, or floppy disk.

[0029] In some embodiments, the technology provided herein involves multiple programmable devices that work in concert to perform the methods as described herein. For example, in some embodiments, multiple computers (e.g., connected by a network) can operate in parallel to collect and process data, for example, in an implementation of cluster computing or grid computing or some other distributed computer architecture that relies on complete computers (on-board CPU, storage, power, network interfaces, etc.) connected to a network (private, public, or the Internet) by traditional network interfaces such as Ethernet, fiber optics, etc., or by wireless networking technology.

[0030] For example, some embodiments provide a computer including a computer-readable medium. The embodiment includes a random access memory (RAM) coupled to a processor. The processor executes computer-executable program instructions stored in the memory. Processors such as these may include microprocessors, ASICs, state machines, or other processors, and may be any of a number of computer processors, such as processors from Intel Corporation of Santa Clara, California, or Motorola Corporation of Schaumburg, Illinois. Processors such as these may include or be in communication with a medium, such as a computer-readable medium, that stores instructions that, when executed by the processor, cause the processor to perform the steps described herein.

[0031] Embodiments of computer-readable media include, but are not limited to, electronic, optical, magnetic, or other storage or transmission devices capable of providing a processor with computer-readable instructions. Other examples of suitable media include, but are not limited to, floppy disks, CD-ROMs, DVDs, magnetic disks, memory chips, ROMs, RAM, ASICs, configured processors, all optical media, all magnetic tape or other magnetic media, or any other medium from which a computer processor can read instructions. Also, various other forms of computer-readable media can transmit or carry instructions to a computer, including routers, private or public networks, both wired and wireless, or other transmission devices or channels. These instructions can comprise code from any suitable computer programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, and JavaScript.

[0032] In some embodiments, the computer is connected to a network. The computer may also include multiple external or internal devices, such as a mouse, CD-ROM, DVD, keyboard, display, or other input or output devices. Examples of computers are personal computers, digital assistants, personal digital assistants, cellular phones, mobile phones, smartphones, pagers, digital tablets, laptop computers, Internet appliances, and other processor-based devices. Generally, these computers relevant to aspects of the technology provided herein can be any type of processor-based platform, running on any operating system, such as Microsoft Windows, Linux, UNIX, Mac OS X, or the like, and capable of supporting one or more programs, including the technology provided herein. Some embodiments include personal computers that execute other application programs (e.g., applications). These applications may be contained in memory and may include, for example, word processing applications, spreadsheet applications, email applications, instant messenger applications, presentation applications, Internet browser applications, calendar / organizer applications, and any other applications that can be executed by a client device.

[0033] All such components, computers, and systems described herein in connection with the technology can be logical or virtual.

[0034] Accordingly, provided herein is technology relating to a method of screening for prostate cancer in a sample obtained from a subject, the method comprising assaying the methylation state of a marker in a sample (e.g., prostate tissue) (e.g., a plasma sample) obtained from the subject, and identifying the subject as having prostate cancer when the methylation state of the marker differs from the methylation state of the marker assayed in a subject not having prostate cancer, wherein the marker comprises a base in a variably methylated region (DMR) selected from the group consisting of DMRs 1-140 as provided in Tables 1 and 13.

[0035] In some embodiments where the sample obtained from the subject is prostate cancer, the marker is selected from ACOXL, AKR1B1_3644, ANXA2, CHST11_2206, FLJ45983, GAS6, GRASP, HAPLN3, HCG4P6, HES5_0822, ITPRIPL1, KCNK4, MAX.chr1.61519554-61519667, MAX.chr2.97193166-97193253, MAX.chr3.193, MAX.chr3.72788028-72788112, RAI1_7469, RASSF2, SERPINB9_3389, SLC4A11, and TPM4_8047.

[0036] In some embodiments where the sample obtained from the subject is prostate cancer, the markers are selected from the group consisting of SERPINB9_3479, FLOT1_1665, HCG4P6_4618, CHST11_2206, MAX.chr12.485, GRASP_0932, GAS6_6425, MAX.chr3.193, MAX.chr2.971_3164, MAX.chr3.727_ 8028, HES5_0840, TPM4_8037, SLCO3A1_6187, ITPRIPL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487.

[0037] In some embodiments where the sample obtained from the subject is blood plasma, the marker is selected from max.chr3.193, HES5, SLCO3A1, and TPM4_8047.

[0038] This technology relates to identifying and differentiating prostate cancer. Some embodiments provide methods comprising assaying multiple markers, for example, assaying 2 to 11 to 100 or 140 markers.

[0039] This technology is not limited to the methylation state assayed. In some embodiments, assaying the methylation state of a marker in a sample comprises determining the methylation state of a single base. In some embodiments, assaying the methylation state of a marker in a sample comprises determining the degree of methylation at multiple bases. Further, in some embodiments, the methylation state of a marker comprises increased methylation of the marker compared to the normal methylation state of the marker. In some embodiments, the methylation state of a marker comprises decreased methylation of the marker compared to the normal methylation state of the marker. In some embodiments, the methylation state of a marker has a different pattern of methylation of the marker compared to the normal methylation state of the marker.

[0040] Furthermore, in some embodiments, the marker is a region of 100 bases or less, the marker is a region of 500 bases or less, the marker is a region of 1000 bases or less, the marker is a region of 5000 bases or less, or in some embodiments, the marker is 1 base. In some embodiments, the marker is in a promoter with high CpG density.

[0041] This technology is not limited by sample type, for example, in some embodiments, the sample is a fecal sample, a tissue sample (e.g., a prostate tissue sample), a blood sample (e.g., plasma, serum, whole blood), stool, or a urine sample.

[0042] Furthermore, the technology is not limited by the method used to determine methylation status. In some embodiments, the assay comprises using methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or target capture. In some embodiments, the assay comprises using methylation-specific oligonucleotides. In some embodiments, the technology uses massively parallel sequencing (e.g., next-generation sequencing), such as sequencing-by-synthesis, real-time (e.g., single-molecule) sequencing, bead emulsion sequencing, nanopore sequencing, etc., to determine methylation status.

[0043] This technology provides reagents for detecting DMRs, such as, in some embodiments, a set of oligonucleotides comprising the sequences provided by SEQ ID NOs: 1-146 and / or 147-234. In some embodiments, oligonucleotides are provided that comprise sequences complementary to chromosomal regions that contain bases in the DMR, e.g., oligonucleotides that are sensitive to the methylation status of the DMR.

[0044] This technology provides various panels of markers, for example, in some embodiments, the markers are ACOXL, AKR1B1_3644, ANXA2, CHST11_2206, FLJ45983, GAS6, GRASP, HAPLN3, HCG4P6, HES5_0822, ITPRIPL1, KCNK4, MAX.chr1.61519554-61519667, MAX.chr2.97193166-97193253, MAX.chr3.193, MAX.chr3.72788028-72788112, RAI1_7469, RASSF2, SERPINB9_3389, SLC4A11, and TPM4_8047, and includes chromosomal regions annotated with the markers (see Examples I-VI).

[0045] In some embodiments, the marker is selected from the group consisting of SERPINB9_3479, FLOT1_1665, HCG4P6_4618, CHST11_2206, MAX.chr12.485, GRASP_0932, GAS6_6425, MAX.chr3.193, MAX.chr2.971_3164, MAX.chr3.727_8028, HES5_0840, TPM4_8037, SLCO 3A1_6187, ITPRIPL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487, and also include chromosomal regions with annotations that include markers (see Example VIII).

[0046] In some embodiments, the sample obtained is a plasma sample, and the markers are max.chr3.193, HES5, SLCO3A1, and TPM4_8047, and include a chromosomal region annotated with the markers.

[0047] Kit embodiments are provided, for example, kits comprising a bisulfite reagent and a control nucleic acid, the control nucleic acid comprising a sequence from a DMR selected from the group consisting of DMRs 1-140 (from Tables 1 or 13) and having a methylation status associated with a subject without prostate cancer. In some embodiments, these kits comprise a bisulfite reagent and an oligonucleotide as described herein. In some embodiments, these kits comprise a bisulfite reagent and a control nucleic acid, the control nucleic acid comprising a sequence from a DMR selected from the group consisting of DMRs 1-140 (from Tables 1 or 13) and having a methylation status associated with a subject with prostate cancer. Some kit embodiments comprise a sampler for obtaining a sample from a subject (e.g., a stool sample, a prostate tissue sample, a plasma sample), reagents for isolating nucleic acids from the sample, a bisulfite reagent, and an oligonucleotide as described herein.

[0048] This technology relates to embodiments of compositions (e.g., reaction mixtures). In some embodiments, a composition is provided that includes a nucleic acid containing a DMR and a bisulfite reagent. Some embodiments provide a composition that includes a nucleic acid containing a DMR and an oligonucleotide as described herein. Some embodiments provide a composition that includes a nucleic acid containing a DMR and a methylation-sensitive restriction enzyme. Some embodiments provide a composition that includes a nucleic acid containing a DMR and a polymerase.

[0049] Additional related method embodiments are provided for screening for prostate cancer in a sample obtained from a subject (e.g., a prostate tissue sample, a plasma sample, a fecal sample), e.g., the method comprises determining the methylation state of a marker in the sample that includes a base in a DMR that is one or more of DMRs 1-140 (from Tables 1 or 13), comparing the methylation state of the marker from the subject sample with the methylation state of the marker from a normal control sample from a subject without prostate cancer, and determining a confidence interval and / or p-value for the difference in methylation states of the subject sample and the normal control sample. In some embodiments, the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99%, and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001. Some embodiments of these methods provide for reacting a nucleic acid comprising a DMR with a bisulfite reagent to produce a bisulfite-reacted nucleic acid; sequencing the bisulfite-reacted nucleic acid to provide a nucleotide sequence of the bisulfite-reacted nucleic acid; comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to the nucleotide sequence of a nucleic acid comprising a DMR from a subject without prostate cancer to identify differences in the two sequences; and identifying the subject as having prostate cancer when differences exist.

[0050] Systems for screening for prostate cancer in a sample obtained from a subject are provided by the technology. Exemplary embodiments of the systems include, for example, systems for screening for prostate cancer in a sample (e.g., a prostate tissue sample, a plasma sample, a fecal sample) obtained from a subject, the systems comprising: an analysis component configured to determine a methylation state of the sample; a software component configured to compare the methylation state of the sample with the methylation states of control or reference samples recorded in a database; and an alert component configured to alert a user of a methylation state associated with prostate cancer. In some embodiments, the software component receives results from multiple assays (e.g., determining the methylation states of multiple markers, e.g., DMRs, as provided in Tables 1 or 3), calculates a value or result for a report based on the multiple results, and determines an alert. Some embodiments provide a database of weighting parameters associated with each DMR provided herein for use in calculating a value or result and / or in alerting a user (e.g., a doctor, nurse, clinician, etc.). In some embodiments, all results from the multiple assays are reported, and in some embodiments, one or more results are used to provide a score, value, or result based on a composite of one or more results from the multiple assays that is indicative of cancer risk in the subject.

[0051] In some embodiments of the system, the sample includes a nucleic acid containing a DMR. In some embodiments, the system further includes a component for collecting the sample, such as a component for isolating the nucleic acid or a component for collecting a fecal sample. In some embodiments, the system includes a nucleic acid sequence containing a DMR. In some embodiments, the database includes nucleic acid sequences from subjects who do not have prostate cancer. Also provided are nucleic acids, e.g., a set of nucleic acids, each nucleic acid including a sequence containing a DMR. In some embodiments of the set of nucleic acids, each nucleic acid includes a sequence from a subject who does not have prostate cancer. Related system embodiments include a set of nucleic acids as described and a database of nucleic acid sequences associated with the set of nucleic acids. Some embodiments further include a bisulfite reagent. And, some embodiments further include a nucleic acid sequencer.

[0052] In certain embodiments, methods are provided for characterizing a sample (e.g., a prostate tissue sample, a plasma sample, a fecal sample) from a human patient. For example, in some embodiments, such embodiments comprise obtaining DNA from the human patient sample, assaying the methylation status of a DNA methylation marker comprising a base in a variably methylated region (DMR) selected from the group consisting of DMRs 1-140 from Tables 1 or 13, and comparing the assayed methylation status of the one or more DNA methylation markers to a reference methylation level for the one or more DNA methylation markers for human patients without prostate cancer.

[0053] These methods are not limited to a particular type of sample from a human patient. In some embodiments, the sample is a prostate tissue sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a fecal sample, a tissue sample, a prostate tissue sample, a blood sample, or a urine sample.

[0054] In some embodiments, such methods comprise assaying multiple DNA methylation markers. In some embodiments, such methods comprise assaying 2 to 11 DNA methylation markers. In some embodiments, such methods comprise assaying 12 to 140 DNA methylation markers. In some embodiments, such methods comprise assaying the methylation state of one or more DNA methylation markers in a sample and determining the methylation state of a single base. In some embodiments, such methods comprise assaying the methylation state of one or more DNA methylation markers in a sample and determining the degree of methylation at multiple bases. In some embodiments, such methods comprise assaying the methylation state of the forward strand or the methylation state of the reverse strand.

[0055] In some embodiments, the DNA methylation marker is a region of 100 bases or less. In some embodiments, the DNA methylation marker is a region of 500 bases or less. In some embodiments, the DNA methylation marker is a region of 1000 bases or less. In some embodiments, the DNA methylation marker is a region of 5000 bases or less. In some embodiments, the DNA methylation marker is a single base. In some embodiments, the DNA methylation marker is in a promoter with high CpG density.

[0056] In some embodiments, the assay comprises using methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or target capture.

[0057] In some embodiments, the assay comprises the use of a methylation-specific oligonucleotide, in some embodiments, the methylation-specific oligonucleotide is selected from the group consisting of SEQ ID NOs: 1-146 and / or SEQ ID NOs: 147-234.

[0058] In some embodiments, the chromosomal region having an annotation selected from the group consisting of ACOXL, AKR1B1_3644, ANXA2, CHST11_2206, FLJ45983, GAS6, GRASP, HAPLN3, HCG4P6, HES5_0822, ITPRIPL1, KCNK4, MAX.chr1.61519554-61519667, MAX.chr2.97193166-97193253, MAX.chr3.193, MAX.chr3.72788028-72788112, RAI1_7469, RASSF2, SERPINB9_3389, SLC4A11, and TPM4_8047 comprises a DNA methylation marker. In some embodiments, the DMR is from Table 3.

[0059] In some embodiments, SERPINB9_3479, FLOT1_1665, HCG4P6_4618, CHST11_2206, MAX.chr12.485, GRASP_0932, GAS6_6425, MAX.chr3.193, MAX.chr2.971_3164, MAX.chr3.727_8028, HES5_0840, TPM4_8037, SLCO3A1 _6187, ITPRIPL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487 comprise DNA methylation markers.

[0060] In some embodiments, the sample obtained is a plasma sample, and the markers are max.chr3.193, HES5, SLCO3A1, and TPM4_8047, and include a chromosomal region annotated with the markers.

[0061] In some embodiments, such methods comprise determining the methylation status of two DNA methylation markers. In some embodiments, such methods comprise determining the methylation status of a pair of DNA methylation markers provided in a row of Table 1 or 3.

[0062] In certain embodiments, this technology provides methods for characterizing a sample obtained from a human patient. In some embodiments, these methods comprise determining the methylation state of a DNA methylation marker in a sample containing a base in a DMR selected from the group consisting of DMRs 1-140 from Tables 1 and 13, comparing the methylation state of the DNA methylation marker from the patient sample with the methylation state of the DNA methylation marker from a normal control sample from a human subject without prostate cancer, and determining a confidence interval and / or p-value for the difference in methylation state between the human patient and normal control sample. In some embodiments, the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99%, and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001.

[0063] In certain embodiments, the technology provides a method for characterizing a sample (e.g., a prostate tissue sample, a plasma sample, a fecal sample) obtained from a human subject, the method comprising reacting a nucleic acid containing a DMR with a bisulfite reagent to produce a bisulfite-reacted nucleic acid; sequencing the bisulfite-reacted nucleic acid to provide a nucleotide sequence of the bisulfite-reacted nucleic acid; and comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to the nucleotide sequence of a nucleic acid containing a DMR from a subject without prostate cancer and identifying differences in the two sequences.

[0064] In certain embodiments, the technology provides a system for characterizing a sample (e.g., a prostate tissue sample, a plasma sample, a fecal sample) obtained from a human subject, the system comprising: an analytical component configured to determine the methylation state of the sample; a software component configured to compare the methylation state of the sample with the methylation states of control or reference samples recorded in a database; and an alert component configured to determine a single value based on a combination of the methylation states and alert a user of a methylation state associated with prostate cancer. In some embodiments, the sample comprises a nucleic acid containing a DMR.

[0065] In some embodiments, systems such as these further comprise a component for isolating nucleic acids, hi some embodiments, systems such as these further comprise a component for collecting samples.

[0066] In some embodiments, the sample is a stool sample, a tissue sample, a prostate tissue sample, a blood sample, or a urine sample. In some embodiments, the database comprises nucleic acid sequences that contain DMRs. In some embodiments, the database comprises nucleic acid sequences from subjects that do not have prostate cancer.

[0067] Additional embodiments will be apparent to those skilled in the art based on the teachings contained herein. DETAILED DESCRIPTION OF THE INVENTION

[0068] Provided herein is prostate cancer screening technology, particularly, but not exclusively, methods, compositions, and related uses for detecting the presence of prostate cancer. Where this technology is described herein, the section headings used are for organizational purposes only and should not be construed as limiting the subject matter of the invention in any way.

[0069] In this detailed description of various embodiments, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will understand that these various embodiments may be practiced with or without these specific details. In other instances, structures and devices are shown in block diagram form. Furthermore, those skilled in the art will readily appreciate that the specific order of presenting and executing these methods is exemplary, and it is contemplated that these orders can be changed and still remain within the spirit and scope of the various embodiments disclosed herein.

[0070] All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and Internet web pages, are expressly incorporated by reference in their entirety for all purposes. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the various embodiments described herein belong. When the definitions of these terms, when incorporated by reference, appear to differ from the definitions provided in the present teachings, the definitions provided in the present teachings shall control.

[0071] definition To facilitate understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0072] Throughout this specification and claims, the following terms shall take the meanings expressly associated therewith, unless the context clearly dictates otherwise. The phrase "in one embodiment," as used herein, does not necessarily refer to the same embodiment, although it may. Further, the phrase "in another embodiment," as used herein, does not necessarily refer to different embodiments, although it may. Thus, as described below, various embodiments of the invention can be readily combined without departing from the scope or spirit of the invention.

[0073] Additionally, as used herein, unless the context clearly dictates otherwise, the term "or" is an inclusive "or" operator and is equivalent to the term "and / or." The term "based on" is non-exclusive and allows for based on additional unlisted factors, unless the context clearly dictates otherwise. Additionally, throughout this specification, "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on."

[0074] As used herein, "nucleic acid" or "nucleic acid molecule" generally refers to any ribonucleic acid or deoxyribonucleic acid, which can be unmodified or modified DNA or RNA. "Nucleic acid" includes, but is not limited to, single-stranded and double-stranded nucleic acids. As used herein, the term "nucleic acid" also includes DNA, as described above, containing one or more modified bases. Thus, DNA containing a backbone modified for stability or for other reasons is a "nucleic acid." As used herein, the term "nucleic acid" encompasses chemically, enzymatically, or metabolically modified forms of nucleic acid, as well as chemical forms of DNA characteristic of viruses and cells, including, for example, simple and complex cells.

[0075] The term "oligonucleotide" or "polynucleotide" or "nucleotide" or "nucleic acid" refers to a molecule containing two or more, preferably more than three, and usually more than ten deoxyribonucleotides or ribonucleotides. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. Oligonucleotides can be produced in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides for DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides for RNA are uracil, adenine, cytosine, and guanine.

[0076] As used herein, the term "locus" or "region" of a nucleic acid refers to a small region of nucleic acid, for example, a gene on a chromosome, a single nucleotide, a CpG island, and the like.

[0077] The terms "complementary" and "complementarity" refer to nucleotides (e.g., single nucleotides) or polynucleotides (e.g., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence 5'-AGT-3' is complementary to the sequence 3'-TCA-5'. Complementarity can be "partial," in which only a few of the nucleic acids' bases match according to the base-pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid strands determines the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and in detection methods that depend on binding between nucleic acids.

[0078] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA or polypeptide or its precursor. A functional polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence, so long as the desired activity or functional property of the polypeptide (e.g., enzymatic activity, ligand binding, signal transduction, etc.) is retained. When used in reference to a gene, the term "portion" refers to fragments of that gene. These fragments can range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, "nucleotides comprising at least a portion of a gene" can include fragments of a gene or the entire gene.

[0079] The term "gene" also includes the coding region of a structural gene and sequences (e.g., including coding, regulatory, structural, and other sequences) located adjacent to the coding region at both the 5' and 3' ends, approximately 1 kb on either end, so that the gene corresponds to the length of the full-length mRNA. Sequences 5' of the coding region and present on the mRNA are referred to as 5' non-translated or untranslated sequences. Sequences 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated or untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. In some organisms (e.g., eukaryotes), genomic forms or gene clones contain coding regions interrupted by non-coding sequences called "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA) and can contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript and are therefore absent in the messenger RNA (mRNA) transcript, which functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0080] In addition to containing introns, genomic forms of a gene may also include sequences located both 5' and 3' to the sequences present in the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the untranslated sequences present in the mRNA transcript). The 5' flanking region may contain regulatory sequences, such as promoters and enhancers, that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage, and polyadenylation.

[0081] When referring to a gene, the term "wild-type" refers to a gene that has the characteristics of a gene isolated from a naturally occurring source. When referring to a gene product, the term "wild-type" refers to a gene product that has the characteristics of a gene product isolated from a naturally occurring source. The term "naturally occurring," when applied to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and has not been intentionally modified by human hands in a laboratory is naturally occurring. A wild-type gene is often that gene or allele that is most frequently observed in a population and is therefore arbitrarily referred to as the "normal" or "wild-type" form of the gene. In contrast, when referring to a gene or gene product, the terms "modified" or "mutant" refer to a gene or gene product, respectively, that exhibits modifications in sequence and / or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. Note that naturally occurring mutants can be isolated, and these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

[0082] The term "allele" refers to genetic variations, including, but not limited to, variants and mutations, polymorphic loci, and single nucleotide polymorphism loci, frameshifts, and splice variants. An allele can occur naturally in a population, or it can arise during the lifetime of any particular individual in this population.

[0083] Thus, when used in reference to a nucleotide sequence, the terms "variant" and "mutant" refer to a nucleic acid sequence that differs by one or more nucleotides from another, normally related, nucleotide sequence. A "variant" is the difference between two different nucleotide sequences, typically one sequence being a reference sequence.

[0084] "Amplification" is a special case of template-specific nucleic acid replication. It contrasts with non-specific template replication (e.g., replication that is template-dependent but not dependent on a specific template). Template specificity is distinguished herein from fidelity of replication (e.g., synthesis of the appropriate polynucleotide sequence) and nucleotide (ribonucleotide or deoxyribonucleotide) specificity. Template specificity is frequently described in terms of "target" specificity. Target sequences are "targets" in the sense that they are sought to be sorted out from other nucleic acids. Amplification techniques are primarily designed for this sorting.

[0085] Nucleic acid amplification generally refers to the production of multiple copies of a polynucleotide, or of a portion of this polynucleotide (e.g., single-stranded polynucleotide molecules that may or may not be exactly the same, 10 to 100 copies of a polynucleotide molecule), typically starting from a small amount of polynucleotide, where an amplification product or amplicon is generally detectable. Polynucleotide amplification encompasses a variety of chemical and enzymatic processes. The production of multiple DNA copies from one or a few copies of a target or template DNA molecule during polymerase chain reaction (PCR) or ligase chain reaction (LCR; see, e.g., U.S. Pat. No. 5,494,810; incorporated herein by reference in its entirety) is a form of amplification. Additional types of amplification include allele-specific PCR (see, e.g., U.S. Pat. No. 5,639,611; incorporated herein by reference in its entirety), assembly PCR (see, e.g., U.S. Pat. No. 5,965,408; incorporated herein by reference in its entirety), helicase-dependent amplification (see, e.g., U.S. Pat. No. 7,662,594; incorporated herein by reference in its entirety), hot-start PCR (see, e.g., U.S. Pat. Nos. 5,773,258 and 5,338,671; each of which is incorporated herein by reference in its entirety), inter-sequence-specific PCR, inverse PCR (see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186; incorporated herein by reference in its entirety), ligation-mediated PCR (see, e.g., Guilfoyle, R. et al., Nucleic Acids Res., 16:8186; incorporated herein by reference in its entirety), and ligation-mediated PCR (see, e.g., Guilfoyle, R. et al., Nucleic Acids Res., 16:8186; incorporated herein by reference in its entirety). Research, 25:1854-1858 (1997); U.S. Patent No. 5,508,169; each of which is incorporated herein by reference in its entirety), methylation-specific PCR (see, e.g., Herman, et al., (1996) PNAS 93(13) 9821-9826; which are incorporated herein by reference in their entirety), miniprimer PCR, multiplex ligation-dependent probe amplification (see, e.g., Schouten, et al., (2002) Nucleic Acids Research 30(12):e57; incorporated herein by reference in its entirety), multiplex PCR (see, e.g., Chamberlain, et al., (1988) Nucleic Acids Research 16(23)11141-11156; Ballabio, et al., (1990) Human Genetics 84(6)571-573; Hayden, et al., (2008) BMC Genetics 9:80; each of which is incorporated herein by reference in its entirety), nested PCR, overlap extension PCR (see, e.g., Higuchi, et al., (1988) Nucleic Acids Research 16(15)7351-7367; incorporated herein by reference in its entirety), real-time PCR (see, e.g., Higuchi, et al., (198 ... al., (1992) Biotechnology 10:413-417; Higuchi, et al., (1993) Biotechnology 11:1026-1030; each of which is incorporated herein by reference in its entirety), reverse transcription PCR (see, e.g., Bustin, SA (2000) J. Molecular Endocrinology 25:169-193; each of which is incorporated herein by reference in its entirety), solid-phase PCR, thermal asymmetric interlaced PCR, and touchdown PCR (see, e.g., Don, et al., Nucleic Acids Research (1991) 19(14) 4008; Roux, K. (1994) Biotechniques 16(5) 812-814; Hecker, et al., (1996) Biotechniques 20(3) 478-485; each of which is incorporated herein by reference in its entirety). It is also possible to achieve polynucleotide amplification using digital PCR (see, for example, Kalinina, et al., Nucleic. See Acids Research. 25; 1999-2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA. 96; 9236-41, (1999); International Patent Publication No. WO05023091A2; U.S. Patent Application Publication No. 20070202525; each of which is incorporated herein by reference in its entirety).

[0086] The term "polymerase chain reaction" ("PCR") refers to the method of K.B. Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, which describes a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying a target sequence involves introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of a double-stranded target sequence. To effect amplification, the mixture is denatured, and then the primers anneal to their complementary sequences within the target molecule. After annealing, the primers are extended by a polymerase, forming a new pair of complementary strands. The denaturation, primer annealing, and polymerase extension steps can be repeated many times (i.e., denaturation, annealing, and extension constitute one "cycle"; there can be multiple "cycles") to obtain highly concentrated amplified segments of the desired target sequence. The length of the amplified segments of the desired target sequence is determined by the relative positions of the primers with respect to each other, so this length is a controllable parameter. Due to the repetitive aspect of the process, this method is referred to as "polymerase chain reaction" ("PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are referred to as "PCR amplified" and are "PCR products" or "amplicons."

[0087] Template specificity is achieved in most amplification techniques by the choice of enzyme. Amplification enzymes are enzymes that, under the conditions in which they are used, process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acids. For example, in the case of Q beta replicase, MDV-1 RNA is a specific template for the replicase (Kacian et al., Proc. Natl. Acad. Sci. USA, 69:3038

[1972] ). This amplification enzyme does not replicate other nucleic acids. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has stringent specificity for its own promoter (Chamberlin et al., Nature, 228:227

[1970] ). In the case of T4 DNA ligase, this enzyme will not join two oligonucleotides or polynucleotides, but rather will have a mismatch at the ligation junction between the oligonucleotide or polynucleotide substrate and the template (Wu and Wallace (1989) Genomics 4:560). Finally, thermostable template-dependent DNA polymerases (e.g., Taq and Pfu DNA polymerases) have been found to exhibit high specificity for sequences bounded by primers due to their ability to function at high temperatures, which create thermodynamic conditions that favor primer hybridization involving target sequences but not hybridization involving non-target sequences (H.A. Erlich (ed.), PCR Technology, Stockton Press

[1989] ).

[0088] As used herein, the term "nucleic acid detection assay" refers to any method for determining the nucleotide composition of a nucleic acid of interest. Nucleic acid detection assays include DNA sequencing methods, probe hybridization methods, structure-specific cleavage assays (e.g., INVADER assay, Hologic, Inc.), and methods described in, for example, U.S. Patent Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816; Lyamichev et al. al., Nat. Biotech., 17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000), and US2009 / 0253142, each of which is incorporated by reference in its entirety for all purposes; enzymatic mismatch cleavage methods (e.g., Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, each of which is incorporated by reference in its entirety); polymerase chain reaction; branched hybridization methods (e.g., Chiron, U.S. Pat. Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802, each of which is incorporated by reference in its entirety); rolling circle replication (e.g., U.S. Pat. No. 6,210,884, , 6,183,960, and 6,235,502, which are incorporated by reference in their entireties; NASBA (e.g., U.S. Pat. No. 5,409,818, which is incorporated by reference in its entirety), molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, which is incorporated by reference in its entirety), E-sensor technology (Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, which are incorporated by reference in their entireties); cycling probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769, and 5,660,988, which are incorporated by reference in their entireties); These include, but are not limited to, the Behring signal amplification method (e.g., U.S. Patent Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, which are incorporated by reference in their entireties); the ligase chain reaction (e.g., Barnay Proc. Natl. Acad. Sci. USA 88, 189-93 (1991)); and the sandwich hybridization method (e.g., U.S. Patent No. 5,288,609, which is incorporated by reference in its entirety).

[0089] The term "amplifiable nucleic acid" refers to a nucleic acid that can be amplified by any amplification method. It is intended that "amplifiable nucleic acid" typically includes a "sample template."

[0090] The term "sample template" refers to nucleic acid originating from a sample being analyzed for the presence of a "target" (defined below). In contrast, "background template" is used in reference to nucleic acid other than the sample template that may or may not be present in the sample. Background template is often largely accidental; it may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those being detected may be present as background in a test sample.

[0091] The term "primer" refers to an oligonucleotide, whether naturally occurring in purified restriction digestion or synthetically produced, that can act as a point of initiation of synthesis when placed under conditions that induce synthesis of primer extension products complementary to a strand of nucleic acid (e.g., at a suitable temperature and pH in the presence of nucleotides and an inducing agent such as DNA polymerase). The primer is preferably single-stranded for maximum efficiency in amplification, but can alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact length of the primer will depend on many factors, including temperature, source of primer, and the use of the method.

[0092] The term "probe" refers to an oligonucleotide (e.g., a sequence of nucleotides) capable of hybridizing to another oligonucleotide of interest, whether naturally occurring in a purified restriction digest, or produced synthetically, recombinantly, or by PCR amplification. Probes can be single-stranded or double-stranded. Probes are useful for the detection, identification, and isolation of specific gene sequences (e.g., "capture probes"). It is contemplated that any probe used in the present invention can, in some embodiments, be labeled with any "reporter molecule" and thus be detectable in any detection system, including, but not limited to, enzymatic (e.g., ELISA and enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.

[0093] As used herein, "methylation" refers to methylation of cytosine at the C5 or N4 position of cytosine, the N6 position of adenine, or other types of nucleic acid methylation. In vitro amplified DNA is usually unmethylated because typical in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" can also refer to amplified DNA in which the original template was unmethylated or methylated, respectively.

[0094] As a result, as used herein, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base, which is not present in recognized typical nucleotide bases. For example, cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at the 5-position of its pyrimidine ring. Thus, cytosine is not a methylated nucleotide, but 5-methylcytosine is a methylated nucleotide. In another example, thymine contains a methyl moiety at the 5-position of its pyrimidine ring, but for purposes of this specification, thymine is not considered a methylated nucleotide when present in DNA because thymine is a typical nucleotide base in DNA.

[0095] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule that includes one or more methylated nucleotides.

[0096] As used herein, the "methylation status," "methylation profile," and "methylation state" of a nucleic acid molecule refer to the presence or absence of one or more methylated nucleotide bases in a nucleic acid molecule. For example, a nucleic acid molecule that contains a methylated cytosine is considered to be methylated (e.g., the methylation state of the nucleic acid molecule is methylated). A nucleic acid molecule that does not contain any methylated nucleotides is considered to be unmethylated.

[0097] The methylation state of a particular nucleic acid sequence (e.g., a genetic marker, or a DNA region, as described herein) can indicate the methylation state of all bases in the sequence, or it can indicate the methylation state of a subset of these bases (e.g., one or more cytosines) within the sequence, or it can indicate information about the methylation density of a region within the sequence, with or without providing precise information about the position within the sequence where methylation occurs.

[0098] The methylation state of a nucleotide locus in a nucleic acid molecule refers to the presence or absence of a methylated nucleotide at a particular locus in the nucleic acid molecule. For example, the methylation state of a cytosine at the seventh nucleotide in a nucleic acid molecule is methylated when the nucleotide present at the seventh nucleotide in the nucleic acid molecule is 5-methylcytosine. Similarly, the methylation state of a cytosine at the seventh nucleotide in a nucleic acid molecule is unmethylated when the nucleotide present at the seventh nucleotide in the nucleic acid molecule is cytosine (and not 5-methylcytosine).

[0099] The methylation state can optionally be expressed or indicated by a "methylation value" (e.g., representing a methylation frequency, fraction, proportion, percent, etc.). Methylation values can be generated, for example, by quantifying the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, or by comparing amplification profiles after a bisulfite reaction, or by comparing sequences of nucleic acid that have been treated with bisulfite and sequences of nucleic acid that have not been treated with bisulfite. As a result, a value, e.g., a methylation value, represents the methylation state and can therefore be used as a quantitative indicator of methylation state across multiple copies of a locus. This is of particular application when it is desirable to compare the methylation state of sequences in a sample to a threshold or reference value.

[0100] As used herein, "methylation frequency" or "percent (%) methylation" refers to the number of times a molecule or locus is methylated compared to the number of times the molecule or locus is unmethylated.

[0101] As such, methylation state describes the methylation state of a nucleic acid (e.g., a genomic sequence). In addition, methylation state refers to properties of a nucleic acid segment at a particular genomic locus that relate to methylation. Such properties include, but are not limited to, whether any of the cytosine (C) residues within this DNA sequence are methylated, the location of the methylated C residue(s), the frequency or percentage of methylated Cs throughout any particular region of the nucleic acid, and allelic differences in methylation due to, for example, differences in allelic origin. The terms "methylation state," "methylation profile," and "methylation status" also refer to the relative concentration, absolute concentration, or pattern of methylated or unmethylated Cs throughout any particular region of the nucleic acid in a biological sample. For example, if a cytosine (C) residue(s) in a nucleic acid sequence are methylated, it can be referred to as "hypermethylated" or having "increased methylation," whereas if a cytosine (C) residue(s) in a DNA sequence are unmethylated, it can be referred to as "hypomethylated" or having "decreased methylation." Similarly, if a cytosine (C) residue(s) in a nucleic acid sequence are methylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual), the sequence is considered to be hypermethylated or have increased methylation compared to the other nucleic acid sequence. Alternatively, if a cytosine (C) residue(s) in a DNA sequence are unmethylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual), the sequence is considered to be hypomethylated or have decreased methylation compared to the other nucleic acid sequence. Additionally, as used herein, the term "methylation pattern" refers to the collection of methylated and unmethylated nucleotides across a nucleic acid region.Two nucleic acids can have the same or similar methylation frequency or percent methylation, or can have different methylation patterns when the numbers of methylated and unmethylated nucleotides are the same or similar throughout this region, but the positions of the methylated and unmethylated nucleotides are different. Sequences are referred to as having "variable methylation," "differential methylation," or "different methylation states" when they differ in the degree (e.g., one has increased or decreased methylation compared to the other), frequency, or pattern of methylation. The term "variable methylation" refers to the difference in the level or pattern of nucleic acid methylation in a cancer-positive sample compared to the level or pattern of nucleic acid methylation in a cancer-negative sample. It can also refer to the difference in the level or pattern between patients with cancer recurrence after surgery and patients without recurrence. Variable methylation and specific levels or patterns of DNA methylation are prognostic and predictive biomarkers, for example, by defining precise cutoffs or predictive characteristics.

[0102] Methylation state frequencies can be used to describe a population of individuals or a sample from a single individual. For example, a nucleotide locus with a 50% methylation state frequency is methylated in 50% of instances and unmethylated in 50% of instances. Such frequencies can be used, for example, to describe the degree to which a nucleotide locus or nucleic acid region is methylated in a population of individuals or a collection of nucleic acids. Thus, when the methylation in a first population or pool of nucleic acid molecules differs from the methylation in a second population or pool of nucleic acid molecules, the methylation state frequency of the first population or pool differs from the methylation state frequency of the second population or pool. Such frequencies can also be used, for example, to describe the degree to which a nucleotide locus or nucleic acid region is methylated in a single individual. For example, such frequencies can be used to describe the degree to which a group of cells from a tissue sample is methylated or unmethylated at a nucleotide locus or nucleic acid region.

[0103] As used herein, "nucleotide locus" refers to the position of a nucleotide in a nucleic acid molecule. The nucleotide locus of a methylated nucleotide refers to the position of the methylated nucleotide in a nucleic acid molecule.

[0104] Typically, methylation of human DNA occurs on dinucleotide sequences containing adjacent guanines and cytosines, where the cytosine is located 5' to the guanine (also called CpG dinucleotide sequences). Most cytosines within CpG dinucleotides are methylated in the human genome, but some remain unmethylated in genomic regions rich in specific CpG dinucleotides, known as CpG islands (e.g., Antequera (See, e.g., W. et al. (1990) Cell 62:503-514).

[0105] As used herein, "CpG island" refers to a G:C-rich region of genomic DNA that contains an increased number of CpG dinucleotides compared to the total genomic DNA. A CpG island can be at least 100, 200, or more base pairs in length, where the G:C content of the region is at least 50%, and the ratio of observed CpG frequency to desired frequency is 0.6; in some examples, a CpG island can be at least 500 base pairs in length, where the G:C content of the region is at least 55%, and the ratio of observed CpG frequency to desired frequency is 0.65. The observed CpG frequency to desired frequency can be calculated according to the method provided in Gardiner-Garden et al. (1987) J. Mol. Biol. 196:261-281. For example, the observed CpG frequency relative to the desired frequency can be calculated according to the formula R=(A×B) / (C×D), where R is the ratio of the observed CpG frequency to the desired frequency, A is the number of CpG dinucleotides in the analyzed sequence, B is the total number of nucleotides in the analyzed sequence, C is the total number of C nucleotides in the analyzed sequence, and D is the total number of G nucleotides in the analyzed sequence. Methylation status is typically determined in CpG islands, for example, in promoter regions. It will be appreciated that other sequences in the human genome are prone to DNA methylation, such as CpA and CpT (see Ramsahoye (2000) Proc. Natl. Acad. Sci. USA 97:5237-5242; Salmon and Kaye (1970) Biochim. Biophys. Acta. 204:340-351; Grafstrom (1985) Nucleic Acids Res. 13:2827-2842; Nyce (1986) Nucleic Acids Res. 14:4353-4367; Woodcock (1987) Biochem. Biophys. Res. Commun. 145:888-894).

[0106] As used herein, a reagent that modifies the nucleotides of a nucleic acid molecule as a function of the methylation state of the nucleic acid molecule, or a methylation-specific reagent, refers to a compound or composition or other agent capable of altering the nucleotide sequence of a nucleic acid molecule in a manner that reflects the methylation state of the nucleic acid molecule. Methods of treating a nucleic acid molecule with such a reagent can comprise contacting the nucleic acid molecule with the reagent, optionally combined with additional steps that achieve a desired change in the nucleotide sequence. Such changes in the nucleotide sequence of a nucleic acid molecule can result in a nucleic acid molecule in which each methylated nucleotide is modified to a different nucleotide. Such changes in the nucleotide sequence of a nucleic acid can result in a nucleic acid molecule in which each unmethylated nucleotide is modified to a different nucleotide. Such changes in the nucleotide sequence of a nucleic acid can result in a nucleic acid molecule in which each selected unmethylated nucleotide (e.g., each unmethylated cytosine) is modified to a different nucleotide. Use of such a reagent to alter the nucleotide sequence of a nucleic acid can result in a nucleic acid molecule in which each nucleotide that is a methylated nucleotide (e.g., each methylated cytosine) is modified to a different nucleotide. As used herein, the use of a reagent that modifies a selected nucleotide refers to a reagent that modifies one of the four typically occurring nucleotides in a nucleic acid molecule (C, G, T, and A for DNA and C, G, U, and A for RNA) such that the reagent modifies the one nucleotide without modifying the other three nucleotides. In one exemplary embodiment, such a reagent modifies an unmethylated selected nucleotide to generate a different nucleotide. In another exemplary embodiment, such a reagent is capable of deaminating an unmethylated cytosine nucleotide. An exemplary reagent is bisulfite.

[0107] As used herein, the term "bisulfite reagent" refers, in some embodiments, to a reagent comprising bisulfite, disulfite, hydrogen sulfite, or a combination thereof, which distinguishes between methylated and unmethylated cytidines, e.g., in CpG dinucleotide sequences.

[0108] The term "methylation assay" refers to any assay for determining the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence.

[0109] The term "MS AP-PCR" (methylation-sensitive arbitrary primer polymerase chain reaction) refers to an art-recognized technique that allows a global scan of the genome using CG-rich primers to focus on regions most likely to contain CpG dinucleotides, and is described in Gonzalgo et al. (1997) Cancer Research 57:594-599.

[0110] The term "MethyLight™" refers to an art-recognized fluorescence-based real-time PCR technique described in Eads et al. (1999) Cancer Res. 59:2302-2306.

[0111] The term "HeavyMethyl™" refers to an assay in which methylation-specific inhibitory probes (also referred to herein as inhibitors) that cover the CpG positions between or that are covered by the amplification primers enable methylation-specific selective amplification of a nucleic acid sample.

[0112] The term "HeavyMethyl™ MethyLight™" assay refers to a variant of the MethyLight™ assay, the HeavyMethyl™ MethyLight™ assay, in which the MethyLight™ assay is combined with a methylation-specific inhibitor probe that covers the CpG positions between the amplification primers.

[0113] The term "Ms-SNuPE" (Methylation-Sensitive Single-Base Primer Extension) refers to the art-recognized assay described in Gonzalgo & Jones (1997) Nucleic Acids Res. 25:2529-2531.

[0114] The term "MSP" (methylation-specific PCR) refers to the art-recognized methylation assay described in Herman et al. (1996) Proc. Natl. Acad. Sci. USA 93:9821-9826, and U.S. Pat. No. 5,786,146.

[0115] The term "COBRA" (Combined Bisulfite Restriction Analysis) refers to an art-recognized methylation assay described in Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534.

[0116] The term "MCA" (methylated CpG island amplification) refers to a methylation assay described in Toyota et al. (1999) Cancer Res. 59:2307-12, and WO00 / 26401A1.

[0117] As used herein, a "selected nucleotide" refers to one of the four nucleotides typically occurring in a nucleic acid molecule (C, G, T, and A for DNA and C, G, U, and A for RNA), and can include methylated derivatives of a typically occurring nucleotide (e.g., when C is a selected nucleotide, both methylated and unmethylated C are included in the meaning of the selected nucleotide), but a methylated selected nucleotide specifically refers to a methylated typically occurring nucleotide, and an unmethylated selected nucleotide specifically refers to an unmethylated typically occurring nucleotide.

[0118] The term "methylation-specific restriction enzyme" or "methylation-sensitive restriction enzyme" refers to an enzyme that selectively digests nucleic acids depending on the methylation state of its recognition site. In the case of a restriction enzyme that specifically cleaves when its recognition site is unmethylated or hemimethylated, this cleavage does not occur or occurs with significantly reduced efficiency when the recognition site is methylated. In the case of a restriction enzyme that specifically cleaves when its recognition site is methylated, this cleavage does not occur or occurs with significantly reduced efficiency when the recognition site is unmethylated. Preferably, the restriction enzyme is a methylation-specific restriction enzyme, the recognition sequence of which includes a CG dinucleotide (e.g., a recognition sequence such as CGCG or CCCGGG). More preferred in some embodiments is a restriction enzyme that does not cleave when the cytosine in this dinucleotide is methylated at the carbon atom C5.

[0119] As used herein, a "different nucleotide" refers to a nucleotide that is chemically different from a selected nucleotide, such that a typically occurring nucleotide complementary to a selected nucleotide is not identical to a typically occurring nucleotide complementary to a different nucleotide, due to the different nucleotide having different Watson-Crick base pairing properties than the typically selected nucleotide. For example, when C is a selected nucleotide, U or T can be a different nucleotide, as exemplified by the complementarity of C to G and U or T to A. As used herein, a nucleotide that is complementary to a selected nucleotide or a different nucleotide refers to a nucleotide containing the selected nucleotide or a different nucleotide that base pairs with a higher affinity under high stringency conditions than complementary nucleotides containing three of the four typically occurring nucleotides. An example of complementarity is Watson-Crick base pairing in DNA (e.g., AT and CG) and RNA (e.g., AU and CG). Thus, for example, G base pairs have a higher affinity for C under high stringency conditions than G base pairs for G, A, or T, so when C is the selected nucleotide, G is the complementary nucleotide to the selected nucleotide.

[0120] As used herein, the "sensitivity" of a given marker refers to the percentage of samples reporting DNA methylation values above a threshold that distinguishes between neoplastic and non-neoplastic samples. In some embodiments, a positive is defined as a histologically confirmed neoplasm reporting a DNA methylation value above a threshold (e.g., a range associated with disease), and a false negative is defined as a histologically confirmed neoplasm reporting a DNA methylation value below a threshold (e.g., a range associated with non-disease). Thus, the sensitivity value indicates the probability that a DNA methylation measurement value for a given marker obtained from a known affected sample falls within the range of disease-associated measurements. As defined herein, the clinical relevance of a calculated sensitivity value represents an estimate of the probability that a given marker will detect the presence of a clinical condition when applied to a subject with that clinical condition.

[0121] As used herein, the "specificity" of a given marker refers to the percentage of non-neoplastic samples reporting DNA methylation values below a threshold that distinguishes between neoplastic and non-neoplastic samples. In some embodiments, a negative is defined as a histologically confirmed non-neoplastic sample reporting a DNA methylation value below a threshold (e.g., a range associated with non-disease), and a false positive is defined as a histologically confirmed non-neoplastic sample reporting a DNA methylation value above a threshold (e.g., a range associated with disease). Thus, the specificity value indicates the probability that a DNA methylation measurement for a given marker obtained from a known non-neoplastic sample falls within the range of non-disease-associated measurements. As defined herein, the clinical relevance of a calculated specificity value represents an estimate of the probability that a given marker will detect the absence of a clinical condition when applied to subjects without that clinical condition.

[0122] As used herein, the term "AUC" is an abbreviation for "area under the curve." In particular, it refers to the area under the receiver operating characteristic (ROC) curve. The ROC curve is a plot of the true positive rate against the false positive rate for different possible cut points of a diagnostic test. The selected cut point shows the trade-off between sensitivity and specificity (any increase in sensitivity is accompanied by a decrease in specificity). The area under the ROC curve (AUC) is a measure of the accuracy of a diagnostic test (the larger the area, the better; the optimal value is 1; a randomized test has a diagonal ROC curve with an area of 0.5; see J.P. Egan. (1975) Signal Detection Theory and ROC Analysis, Academic Press, New York).

[0123] As used herein, the term "neoplasm" refers to "an abnormal mass of tissue whose growth exceeds and is uncoordinated with the growth of normal tissues." See, e.g., Willis RA, "The Spread of Tumors in See "The Human Body", London, Butterworth & Co, 1952.

[0124] As used herein, the term "adenoma" refers to a benign tumor of glandular origin. These growths are benign, although over time they can progress to become malignant.

[0125] The terms "precancerous" or "preneoplastic" and their equivalents refer to any cell proliferative disorder that is undergoing malignant transformation.

[0126] The "site" of a neoplasm, adenoma, cancer, etc. is the tissue, organ, cell type, anatomical region, body part, etc. in a subject in which the neoplasm, adenoma, cancer, etc. is located.

[0127] As used herein, the application of a "diagnostic" test comprises detecting or identifying a disease state or status in a subject, determining the likelihood that a subject will suffer from a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to treatment, determining the prognosis (or likelihood of progression or regression) of a subject with a disease or condition, and determining the effectiveness of a treatment for a subject with a disease or condition. For example, diagnostics can be used to detect the presence or likelihood of a subject suffering from a neoplasm, or the likelihood that such a subject will respond successfully to a compound (e.g., a pharmaceutical, e.g., a drug) or other treatment.

[0128] The term "marker," as used herein, refers to a substance (e.g., a nucleic acid or region of a nucleic acid) that can diagnose cancer by distinguishing cancer cells from normal cells, e.g., based on its methylation status.

[0129] The term "isolated," when used with reference to a nucleic acid, such as "isolated oligonucleotide," refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is normally associated in its natural source. An isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids, such as DNA and RNA, are found in the state in which they occur in nature. Examples of non-isolated nucleic acids include a given DNA sequence (e.g., a gene) found on a host cell chromosome adjacent to adjacent genes; and an RNA sequence, such as a specific mRNA sequence encoding a specific protein, found in a cell as a mixture containing multiple other mRNAs that encode multiple proteins. However, an isolated nucleic acid encoding a specific protein includes, for example, a nucleic acid in a cell that normally expresses that protein, where the nucleic acid is in a chromosomal location different from that of natural cells or is otherwise adjacent to nucleic acid sequences different from those found in nature. An isolated nucleic acid or oligonucleotide can exist in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is used to express a protein, the oligonucleotide will minimally contain a sense or coding strand (i.e., the oligonucleotide can be single-stranded), but can contain both a sense and an antisense strand (i.e., the oligonucleotide can be double-stranded). The isolated nucleic acid can be combined with other nucleic acids or molecules after isolation from its natural or typical environment. For example, the isolated nucleic acid can be present in a host cell in which it is placed, e.g., for heterologous expression.

[0130] The term "purified" refers to either nucleic acid or amino acid sequence molecules that have been removed, isolated, or separated from their natural environment. Thus, an "isolated nucleic acid sequence" can be a purified nucleic acid sequence. "Substantially purified" molecules are at least 60%, preferably at least 75%, and more preferably at least 90% free from other components with which they are naturally associated. As used herein, the terms "purified" or "to purify" also refer to the removal of contaminants from a sample. Removal of contaminating proteins results in an increase in the percentage of the polypeptide or nucleic acid of interest in a sample. In another example, recombinant polypeptides are expressed in plant, bacterial, yeast, or mammalian host cells, and these polypeptides are purified by removal of host cell proteins, thereby increasing the percentage of recombinant polypeptide in a sample.

[0131] The term "composition comprising" a given polynucleotide sequence or polypeptide refers broadly to any composition that contains the given polynucleotide sequence or polypeptide. The composition can include aqueous solutions containing salts (e.g., NaCl), detergents (e.g., SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

[0132] The term "sample" is used in its broadest sense. In one sense, it can refer to animal cells or tissues. In another sense, it is meant to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from plants or animals (including humans) and can include fluids, solids, tissues, and gases. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a prostate tissue sample. In some embodiments, the sample is a fecal sample. Environmental samples include environmental materials such as surface material, soil, water, and industrial samples. These examples should not be construed as limiting the types of samples applicable to the present invention.

[0133] As used herein, a "remote sample," as used in some contexts, refers to a sample that is collected indirectly from a site that is not the sample source of the cells, tissues, or organs. For example, when evaluating sample material that originates from the pancreas in a fecal sample (e.g., not from a sample taken directly from the prostate), the sample is a remote sample.

[0134] As used herein, the term "patient" or "subject" refers to an organism that is the subject of various tests provided by this technology. The term "subject" includes animals, preferably mammals, including humans. In preferred embodiments, the subject is a primate. In even more preferred embodiments, the subject is human.

[0135] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of a reaction assay, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or supporting materials (e.g., buffers, written instructions for conducting the assay, etc.) from one location to another. For example, a kit may include one or more enclosed containers (e.g., boxes) containing relevant reaction reagents and / or supporting materials. As used herein, the term "fragmentation kit" refers to a delivery system that includes two or more separate containers, each containing a subportion of the overall kit components. The containers can be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container may contain an oligonucleotide. The term "fragmentation kit" is intended to encompass, but is not limited to, kits containing analyte-specific reagents (ASRs) as defined by Section 520(e) of the Federal Food, Drug, and Cosmetic Act. Indeed, any delivery system containing two or more separate containers, each containing a subportion of the total kit components, is encompassed by the term "fragmented kit." In contrast, a "combined kit" refers to a delivery system containing all components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term "kit" includes both fragmented and combined kits. Technology Embodied Provided herein is the science and technology of prostate cancer screening, particularly, but not exclusively, methods, compositions, and related uses for detecting the presence of prostate cancer.

[0136] Indeed, as described in Examples I-VI, experiments conducted during the process of identifying embodiments of the present invention identified a novel set of 73 variably methylated regions (DMRs) for distinguishing cancer from non-neoplastic control DNA in prostate-derived DNA. Additionally, we identified 10 novel DMRs that are methylated in prostate epithelium (cancer and normal) but unmethylated in normal leukocyte DNA samples. Both sets of these regions were identified from next-generation sequencing studies of CpGs enriched in bisulfite-converted tumor and normal DNA. Tumor samples contained low-grade Gleason 6 and high-grade Gleason 7+ patterns. DMRs were selected using proprietary filters and an analytical pipeline and validated in an independent set of tissue samples using a novel methylation-specific PCR (MSP) assay. These 73 biomarker assays demonstrated excellent detection in tissues and had a wide range of clinical specificity (some for cancers across many different organ sites, others specific only to prostate cancer).

[0137] Experiments such as these list and describe 120 novel DNA methylation markers (Table 1) that distinguish prostate cancer from benign prostate tissue. From these 120 novel DNA methylation markers, further experiments identified 73 markers that can distinguish high-grade prostate cancer tissue (e.g., Gleason score 7+) from benign prostate tissue. More specifically, markers and / or marker panels (e.g., chromosomal regions having annotations selected from ACOXL, AKR1B1_3644, ANXA2, CHST11_2206, FLJ45983, GAS6, GRASP, HAPLN3, HCG4P6, HES5_0822, ITPRIPL1, KCNK4, MAX.chr1.61519554-61519667, MAX.chr2.97193166-97193253, MAX.chr3.193, MAX.chr3.72788028-72788112, RAI1_7469, RASSF2, SERPINB9_3389, SLC4A11, and TPM4_8047) were identified that were able to distinguish prostate cancer tissue from benign prostate tissue (see Examples I-VI).

[0138] Additional experiments conducted during the course of developing embodiments of the present invention identified markers (e.g., SERPINB9_3479, FLOT1_1665, HCG4P6_4618, CHST11_2206, MAX.chr12.485, GRASP_0932, GAS6_6425, MAX.chr3.193, MAX.chr2.971_3164, MAX.chr3.727_8028, HES5_0840, TPM4_8037) that can distinguish prostate cancer tissue from benign prostate tissue. , SLCO3A1_6187, ITPRIPL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487), which were able to distinguish prostate cancer tissue from benign prostate tissue (see Example VIII; Table 11).

[0139] Additional experiments conducted during the course of developing embodiments of the present invention were directed to identifying markers (e.g., chromosomal regions having annotations selected from SERPINB9_3479, GRASP_0932, SLCO3A1_6187, ITPRIPL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487) that could distinguish high-grade prostate cancer tissue (e.g., Gleason score 7+) from low-grade prostate cancer tissue (e.g., Gleason score 6), and could distinguish prostate cancer tissue from benign prostate tissue (see Example VIII; Table 11).

[0140] Additional experiments conducted during the course of developing embodiments of the present invention were directed to identifying markers capable of detecting the presence or absence of prostate cancer in blood samples (e.g., blood plasma samples). Indeed, markers and / or marker panels (e.g., chromosomal regions with annotations selected from max.chr3.193, HES5, SLCO3A1, and TPM4_8047) were identified that were capable of detecting the presence or absence of prostate cancer tissue in blood plasma samples (see Examples I-VI).

[0141] While the disclosure herein refers to certain illustrated embodiments, it will be understood that these embodiments are presented by way of example and not by way of limitation.

[0142] In certain aspects, the present technology provides compositions and methods for identifying, determining, and / or classifying cancers, such as prostate cancer. These methods comprise determining the methylation status of at least one methylation marker in a biological sample (e.g., a stool sample, a prostate tissue sample, a plasma sample) isolated from a subject, wherein a change in the methylation status of the marker indicates the presence, classification, or location of prostate cancer. Certain embodiments relate to markers comprising variably methylated regions (DMRs, e.g., DMRs 1-140; see Tables 1 and 13) used for diagnosing (e.g., screening for) prostate cancer.

[0143] In addition to the embodiments provided herein that analyze the methylation analysis of at least one marker, region of a marker, or marker base that comprises a DMR listed in Tables 1 or 3 (e.g., a DMR, e.g., DMRs 1-140), the technology also provides panels of markers that include at least one marker, region of a marker, or marker base that comprises a DMR that have utility in detecting cancer, particularly prostate cancer.

[0144] Some embodiments of this technology are based on the analysis of the CpG methylation status of at least one marker, marker region, or marker base that comprises a DMR.

[0145] In some embodiments, the present technology provides for the use of bisulfite techniques in combination with one or more methylation assays to determine the methylation status of CpG dinucleotide sequences within at least one marker comprising a DMR (e.g., DMRs 1-140, see Tables 1 and 13). Genomic CpG dinucleotides can be methylated or unmethylated (alternatively known as hypermethylated and hypomethylated, respectively). However, these methods of the present invention are suitable for analyzing biological samples of heterogeneous nature, such as low concentrations of tumor cells or biological material therefrom, within a background of a distant sample (e.g., blood, organ effluent, or feces). Consequently, when analyzing the methylation status of CpG positions within such samples, one can use quantitative assays to determine the level (e.g., percent, fraction, proportion, ratio, or degree) of methylation at a particular CpG position.

[0146] In accordance with the science and technology of the present invention, determining the methylation status of CpG dinucleotide sequences in markers containing DMRs has utility in both the diagnosis and characterization of cancers such as prostate cancer. Marker Combinations In some embodiments, the technology involves assessing the methylation status of a combination of markers that include DMRs (DMR numbers 1-140) from Table 1, Table 3, or Table 13. In some embodiments, assessing the methylation status of more than one marker increases the specificity and / or sensitivity of a screen or diagnostic for identifying a neoplasm (e.g., prostate cancer) in a subject.

[0147] Different cancers are predicted by different combinations of markers, for example, as identified by statistical methods related to the specificity and sensitivity of the prediction. The technology provides methods for identifying predictive combinations and validated predictive combinations for several cancers. Methods for assaying methylation status The most frequently used method for analyzing nucleic acids for the presence of 5-methylcytosine is based on the bisulfite method described by Frommer et al. (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-31, expressly incorporated herein by reference in its entirety for all purposes) for detecting 5-methylcytosine, or variations thereof, in DNA. The bisulfite method for mapping 5-methylcytosine is based on the finding that cytosine, but not 5-methylcytosine, reacts with the ion of hydrogen sulfite (also known as bisulfite). This reaction is typically carried out according to the following steps: First, cytosine reacts with bisulfite to form sulfonated cytosine. Next, spontaneous deamination of the sulfonated reaction intermediate results in sulfonated uracil. Finally, this sulfonated uracil is desulfonated under alkaline conditions to form uracil. Detection is possible because uracil forms base pairs with adenine, whereas 5-methylcytosine forms base pairs with guanine (and thus behaves like cytosine), allowing discrimination between unmethylated and methylated cytosine, for example, by bisulfite genomic sequencing (Grigg G, & Clark S, Bioessays (1994) 16:431-36; Grigg G, DNA Seq. (1996) 6:189-98) or methylation-specific PCR (MSP), as disclosed, for example, in U.S. Pat. No. 5,786,146.

[0148] Some conventional techniques involve encapsulating the DNA to be analyzed in an agarose matrix, thereby preventing DNA diffusion and renaturation (bisulfite reacts only with single-stranded DNA), and replacing the precipitation and purification steps with high-speed dialysis (Olek A, et al. (1996) "A modified and improved method for bisulfite-based cytosine methylation analysis" Nucleic Acids Res. 24:5064-6). It is therefore possible to analyze individual cells for their methylation status, illustrating the usefulness and sensitivity of this method. An overview of conventional methods for detecting 5-methylcytosine is provided by Rein, T., et al. (1998) Nucleic Acids Res. 26:2255.

[0149] Bisulfite techniques typically involve bisulfite treatment followed by amplification of short, specific fragments of known nucleic acids, followed by assaying either the products by sequencing (Olek & Walter (1997) Nat. Genet. 17:275-6) or primer extension reactions (Gonzalgo & Jones (1997) Nucleic Acids Res. 25:2529-31; WO 95 / 00669; U.S. Patent No. 6,251,594) to analyze individual cytosine positions. Some methods use enzymatic digestion (Xiong & Laird (1997) Nucleic Acids Res. 25:2532-4). Detection by hybridization has also been described in the art (Olek et al., WO 99 / 28498). In addition, the use of bisulfite techniques for methylation detection of individual genes has been described (Grigg & Clark (1994) Bioessays 16:431-6; Zeschnigk et al. (1997) Hum Mol Genet. 6:387-95; Feil et al. (1994) Nucleic Acids Res. 22:695; Martin et al. (1995) Gene 157:261-4; WO9746705; WO9515373).

[0150] Various methylation assay techniques are known in the art and can be used in conjunction with bisulfite treatment in accordance with the present technology. These assays allow for the determination of the methylation status of one or more CpG dinucleotides (e.g., CpG islands) within a nucleic acid sequence. Assays such as these involve, among other techniques, sequencing of bisulfite-treated nucleic acids, PCR (for sequence-specific amplification), Southern blot analysis, and the use of methylation-sensitive restriction enzymes.

[0151] For example, genome sequencing has been simplified for analysis of methylation patterns and 5-methylcytosine distribution by using bisulfite treatment (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-1831). In addition, restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA finds use in assessing methylation status, for example, as described by Sadri & Hornsby (1997) Nucl. Acids Res. 24:5058-5059 or embodied in the method known as COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534).

[0152] The COBRA™ analysis is a quantitative methylation assay useful for determining DNA methylation levels at specific loci in small amounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997). Briefly, restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite-treated DNA. Methylation-dependent sequence differences are first introduced into genomic DNA by standard bisulfite treatment according to the procedure described by Frommer et al. (Proc. Natl. Acad. Sci. USA 89:1827-1831, 1992). PCR amplification of the bisulfite-converted DNA is then performed using primers specific for the CpG island of interest, followed by restriction endonuclease digestion, gel electrophoresis, and detection using specifically labeled hybridization probes. The relative amounts of digested and undigested PCR products represent the methylation level in the original DNA sample in a linearly quantitative manner across a wide range of DNA methylation levels. Additionally, this technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples.

[0153] Typical reagents for COBRA™ analysis (e.g., as may be found in a typical COBRA™-based kit) can include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, DMRs, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); restriction enzymes and appropriate buffers; gene hybridization oligonucleotides; control hybridization oligonucleotides; kinase labeling kits for oligonucleotide probes; and labeled nucleotides. In addition, bisulfite conversion reagents can include DNA denaturing buffers; sulfonation buffers; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns); desulfonation buffers; and DNA recovery components.

[0154] Preferably, assays such as "MethyLight™" (a fluorescence-based real-time PCR technique) (Eads et al., Cancer Res. 59:2302-2306, 1999), Ms-SNuPE™ (methylation-sensitive single-nucleotide primer expression) reactions (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997), methylation-specific PCR ("MSP"; Herman et al., Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; U.S. Patent No. 5,786,146), and methylated CpG island amplification ("MCA"; Toyota et al., Cancer Res. 59:2307-12, 1999) are used alone or in combination with one or more of these methods.

[0155] The "HeavyMethyl™" assay technique is a quantitative method for assessing methylation differences based on methylation-specific amplification of bisulfite-treated DNA. Methylation-specific inhibitor probes ("inhibitors") covering the CpG positions between or covered by the amplification primers allow for methylation-specific selective amplification of nucleic acid samples.

[0156] The term "HeavyMethyl™ MethyLight™" assay refers to a variation of the MethyLight™ assay, the HeavyMethyl™ MethyLight™ assay, in which the MethyLight™ assay is combined with a methylation-specific blocking probe that covers the CpG positions between the amplification primers. The HeavyMethyl™ assay can also be used in combination with methylation-specific amplification primers.

[0157] Typical reagents for HeavyMethyl™ analysis (e.g., as may be found in a typical MethyLight™-based kit) may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, DMRs, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, or bisulfite-treated DNA sequences or CpG islands, etc.); inhibitory oligonucleotides; optimized PCR buffer and deoxynucleotides; and Taq polymerase.

[0158] MSP (methylation-specific PCR) allows for the assessment of the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes (Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; U.S. Patent No. 5,786,146). Briefly, DNA is modified with sodium bisulfite, which converts unmethylated cytosines, but not methylated cytosines, to uracil, and these products are then amplified with primers specific for methylated versus unmethylated DNA. MSP requires only small amounts of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. Typical reagents for MSP analysis (e.g., as may be found in a typical MSP-based kit) may include, but are not limited to, methylated and unmethylated PCR primers for specific loci (e.g., specific genes, markers, DMRs, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); optimized PCR buffers and deoxynucleotides, and specificity probes.

[0159] The MethyLight™ assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (e.g., TaqMan®) and requires no further manipulation after the PCR step (Eads et al., Cancer Res. 59:2302-2306, 1999). Briefly, the MethyLight™ process begins with a mixed sample of genomic DNA that is converted into a mixed pool of methylation-dependent sequence differences in a sodium bisulfite reaction according to standard procedures (the bisulfite process converts unmethylated cytosine residues to uracil). Fluorescence-based PCR is then performed in a "biased" reaction, e.g., with PCR primers that overlap known CpG dinucleotides. Sequence discrimination occurs both at the level of the amplification process and at the level of the fluorescence detection process.

[0160] The MethyLight™ assay is used as a quantitative test for methylation patterns in nucleic acid, e.g., genomic DNA samples, in which sequence discrimination occurs at the level of probe hybridization. In the quantitative version, the PCR reaction provides methylation-specific amplification in the presence of fluorescent probes that overlap specific putative methylation sites. An unbiased control for input DNA amount is provided by a reaction in which neither the primers nor the probe cover any CpG dinucleotides. Alternatively, a quantitative test for genomic methylation is achieved by probing a biased PCR pool with either control oligonucleotides that do not cover known methylation sites (e.g., fluorescent-based versions of the HeavyMethyl™ and MSP techniques) or oligonucleotides that cover potential methylation sites.

[0161] The MethyLight™ process can be used with any suitable probe (e.g., TaqMan® probe, Lightcycler® probe, etc.). For example, in some applications, double-stranded genomic DNA is treated with sodium bisulfite and subjected to one of two PCR reactions using, for example, a TaqMan® probe with an MSP primer and / or a HeavyMethyl inhibitor oligonucleotide and a TaqMan® probe. The TaqMan® probe is dual-labeled with fluorescent reporter and quencher molecules and is designed to be specific for relatively GC-rich regions, so that it melts during PCR cycles at a temperature approximately 10°C higher than the forward or reverse primers. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing / extension step. When Taq polymerase enzymatically synthesizes a new strand during PCR, it ultimately reaches the annealed TaqMan® probe. The 5' to 3' endonuclease activity of Taq polymerase then displaces the TaqMan® probe by digesting it, releasing a fluorescent reporter molecule for quantitative detection of its unquenched signal using a real-time fluorescence detection system.

[0162] Typical reagents for MethyLight™ analysis (e.g., as may be found in a typical MethyLight™-based kit) may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, DMRs, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); TaqMan™ or Lightcycler™ probes; optimized PCR buffer and deoxynucleotides; and Taq polymerase.

[0163] The QM™ (Quantitative Methylation) Assay is an alternative quantitative test for methylation patterns in genomic DNA samples, in which sequence discrimination occurs at the level of probe hybridization. In this quantitative version, PCR reactions provide unbiased amplification in the presence of fluorescent probes that overlap specific putative methylation sites. An unbiased control for input DNA amount is provided by reactions in which neither the primers nor the probe cover any CpG dinucleotides. Alternatively, quantitative testing for genomic methylation is achieved by probing biased PCR pools with either control oligonucleotides that do not cover known methylation sites (fluorescence-based versions of HeavyMethyl™ and MSP techniques) or oligonucleotides that cover potential methylation sites.

[0164] The QM™ process can be used with any suitable probe, such as a TaqMan® probe, a Lightcycler® probe, or the like, in the amplification process. For example, double-stranded genomic DNA is treated with sodium bisulfite and subjected to unbiased primers and a TaqMan® probe. The TaqMan® probe is dual-labeled with fluorescent reporter and quencher molecules and is designed to be specific to relatively GC-rich regions, so it melts during PCR cycles at a temperature approximately 10°C higher than the forward or reverse primers. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing / extension step. When Taq polymerase enzymatically synthesizes a new strand during PCR, it ultimately reaches the annealed TaqMan® probe. The 5' to 3' endonuclease activity of Taq polymerase then displaces the TaqMan® probe by digesting it, releasing a fluorescent reporter molecule for quantitative detection of its unquenched signal using a real-time fluorescence detection system. Typical reagents for QM™ analysis (e.g., as may be found in a typical QM™-based kit) can include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, DMRs, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); TaqMan® or Lightcycler® probes; optimized PCR buffers and deoxynucleotides; and Taq polymerase.

[0165] The Ms-SNuPE™ technique is a quantitative method involving single-base primer extension, followed by bisulfite treatment of DNA to assess methylation differences at specific CpG sites (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997). Briefly, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosines to uracil, while leaving 5-methylcytosines unchanged. PCR primers specific to the bisulfite-converted DNA are then used to amplify the desired target sequence, and the resulting product is isolated and used as a template for methylation analysis at the CpG sites of interest. This allows for the analysis of small amounts of DNA (e.g., microdissected pathology slices), avoiding the use of restriction enzymes to determine the methylation status at CpG sites.

[0166] Typical reagents for Ms-SNuPE™ analysis (e.g., as may be found in a typical Ms-SNuPE™-based kit) can include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, DMRs, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.); optimized PCR buffers and deoxynucleotides; gel extraction kits; positive control primers; Ms-SNuPE™ primers for specific loci; reaction buffers (for the Ms-SNuPE reaction); and labeled nucleotides. In addition, bisulfite conversion reagents can include DNA denaturing buffers; sulfonation buffers; DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns); desulfonation buffers; and DNA recovery components.

[0167] Reduced Representation Bisulfite Sequencing (RRBS) begins with bisulfite treatment of nucleic acids to convert all unmethylated cytosines to uracil, followed by restriction enzyme digestion (e.g., with an enzyme that recognizes sites containing CG sequences, such as MspI) and completes fragment sequencing after coupling to an adaptor ligand. The restriction enzyme selection enriches for fragments in CpG-dense regions, reducing the number of redundant sequences that can be mapped to multiple gene locations during analysis. As such, RRBS reduces the complexity of a nucleic acid sample by selecting a subset of restriction fragments for sequencing (e.g., by size selection using preparative gel electrophoresis). In contrast to whole-genome bisulfite sequencing, all fragments generated by restriction enzyme digestion contain DNA methylation information for at least one CpG dinucleotide. As such, RRBS enriches samples for promoters, CpG islands, and other genomic features, including those with frequent restriction enzyme cleavage sites in these regions, providing an assay to assess the methylation status of one or more genomic loci.

[0168] A typical protocol for RRBS involves digesting a nucleic acid sample with a restriction enzyme such as MspI, filling in overhangs and A-tailing, ligating adapters, bisulfite conversion, and PCR. See, e.g., Meissner et al. (2005) "Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution" Nat Methods 7:133-6; Meissner et al. (2005) "Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis" Nucleic Acids Res. 33:5868-77.

[0169] In some embodiments, quantitative allele-specific real-time target and signal amplification (QuARTS) assays are used to assess methylation status. Three reactions occur sequentially in each QuARTS assay: amplification in the primary reaction (reaction 1) and target probe cleavage (reaction 2), and FRET cleavage and fluorescent signal generation in the secondary reaction (reaction 3). When a target nucleic acid is amplified with specific primers, a specific detection probe containing a flap sequence loosely binds to the amplicon. The presence of a specific invasive oligonucleotide at the target binding site causes cleavage between the detection probe and the flap sequence, resulting in the release of the flap sequence by the cleavage reaction. The flap sequence is complementary to the non-hairpin portion of the corresponding FRET cassette. As a result, the flap sequence functions as an invasive oligonucleotide on this FRET cassette, resulting in cleavage between the FRET cassette fluorophore and quencher, generating a fluorescent signal. This cleavage reaction cleaves multiple probes per target, thereby releasing multiple fluorophores per flap and providing exponential signal amplification. QuARTS is capable of detecting multiple targets in a single reaction well by using FRET cassettes containing different dyes (see, e.g., Zou et al. (2010) "Sensitive quantification of methylated markers with a novel methylation-specific technology" Clin Chem 56:A199; U.S. Patent Application Nos. 12 / 946,737, 12 / 946,745, 12 / 946,752, and 61 / 548,639).

[0170] The term "bisulfite reagent" refers to a reagent containing bisulfite, disulfite, hydrogen sulfite, or a combination thereof, which is useful as disclosed herein to distinguish between methylated and unmethylated CpG dinucleotide sequences. Methods for this treatment are known in the art (e.g., PCT / EP2004 / 011715, incorporated by reference in its entirety). It is preferred to perform the bisulfite treatment in the presence of a denaturing solvent, such as, but not limited to, n-alkylene glycol or diethylene glycol dimethyl ether (DME), or in the presence of dioxane or a dioxane derivative. In some embodiments, the denaturing solvent is used at a concentration (v / v) between 1% and 35%. In some embodiments, the bisulfite reaction is carried out in the presence of a scavenger, such as, but not limited to, a chroman derivative, e.g., 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, or trihydroxybenzoic acid and its derivatives, e.g., gallic acid (see PCT / EP2004 / 011715, incorporated by reference in its entirety). The bisulfite conversion is preferably carried out at a reaction temperature between 30°C and 70°C, with the temperature rising to above 85°C for a short period during the reaction (see PCT / EP2004 / 011715, incorporated by reference in its entirety). Prior to quantification, the bisulfite-treated DNA is preferably purified. This can be done by any means known in the art, such as, but not limited to, ultrafiltration, e.g., by means of a Microcon™ column (manufactured by Millipore™). This purification is carried out according to a modified manufacturer's protocol (see, eg, PCT / EP2004 / 011715, which is incorporated by reference in its entirety).

[0171] In some embodiments, fragments of the treated DNA are amplified using a set of primer oligonucleotides (see, for example, Tables 3 and / or 5) according to the present invention and an amplification enzyme. Amplification of several DNA segments can be carried out simultaneously in one and the same reaction vessel. Typically, amplification is carried out using the polymerase chain reaction (PCR). Amplicons are generally 100 to 2000 base pairs in length.

[0172] In another embodiment of this method, the methylation status of CpG positions within or near markers containing DMRs (e.g., DMRs 1-140 as provided in Tables 1 and 13) can be detected by using methylation-specific primer oligonucleotides. This technique (MSP) is described in U.S. Patent No. 6,265,171 to Herman. The use of methylation-status-specific primers for the amplification of bisulfite-treated DNA allows differentiation between methylated and unmethylated nucleic acids. MSP primer pairs contain at least one primer that hybridizes to a bisulfite-treated CpG dinucleotide. Thus, the sequences of these primers contain at least one CpG dinucleotide. MSP primers specific for unmethylated DNA contain a "T" at the C position in the CpG.

[0173] These fragments obtained by amplification can carry directly or indirectly detectable labels.In some embodiments, these labels are fluorescent labels, radionuclides, or detachable molecular fragments, which have a typical mass that can be detected in mass spectrometer.When these labels are mass labels, some embodiments provide that labeled amplicons have a single positive or negative net charge, which allows better detectability in mass spectrometer.For example, this detection can be performed and visualized by matrix-assisted laser desorption / ionization mass spectrometry (MALDI) or by using electron spray mass spectrometry (ESI).

[0174] Methods for isolating DNA suitable for these assay technologies are known in the art. In particular, some embodiments comprise isolating nucleic acids as described in U.S. Patent Application No. 13 / 470,251 ("Isolation of Nucleic Acids"), which is incorporated herein by reference in its entirety. method In some embodiments of this technology, the steps of: 1) contacting nucleic acid obtained from a subject (e.g., genomic DNA isolated from a stool sample or a bodily fluid such as a prostate tissue or plasma sample) with at least one reagent, or a set of reagents, that distinguishes between methylated and unmethylated CpG dinucleotides within at least one marker that comprises a DMR (e.g., DMRs 1-140, as provided in Tables 1 and 13); and 2) detecting prostate cancer (e.g., provided by a sensitivity of 80% or greater and a specificity of 80% or greater); The present invention provides a method comprising:

[0175] In some embodiments of this technology, the steps of: 1)ACOXL, AKR1B1_3644, ANXA2, CHST11_2206, FLJ45983, GAS6, GRASP, HAPLN3, HCG4P6, HES5_0822, ITPRIPL1, KCNK4, MAX.chr1. 61519554-61519667, MAX.chr2.97193166-97193253, MAX.chr3.193, MAX.chr3.72788028-72788112, RAI1_7469, RASSF2, SERPI contacting nucleic acid obtained from a subject (e.g., genomic DNA isolated from a bodily fluid such as a fecal sample or prostate tissue) with at least one reagent, or a set of reagents, that distinguishes between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of NB9_3389, SLC4A11, and TPM4_8047; and 2) detecting prostate cancer (e.g., provided by a sensitivity of 80% or greater and a specificity of 80% or greater); The present invention provides a method comprising:

[0176] In some embodiments of this technology, the steps of: 1)SERPINB9_3479, FLOT1_1665, HCG4P6_4618, CHST11_2206, MAX.chr12.485, GRASP_0932, GAS6_6425, MAX.chr3.193, MAX.chr2.971_3164, M AX.chr3.727_8028, HES5_0840, TPM4_8037, SLCO3A1_6187, ITPRIPL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALN contacting nucleic acid obtained from the subject (e.g., genomic DNA isolated from a bodily fluid such as a fecal sample or prostate tissue) with at least one reagent, or a set of reagents, that distinguishes between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of AC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487; and 2) detecting prostate cancer (e.g., provided by a sensitivity of 80% or greater and a specificity of 80% or greater); The present invention provides a method comprising:

[0177] In some embodiments of this technology, the steps of: 1) contacting nucleic acid obtained from a subject (e.g., genomic DNA isolated from a plasma sample) with at least one reagent or set of reagents that distinguish between methylated and unmethylated CpG dinucleotides within at least one marker selected from a chromosomal region having an annotation selected from the group consisting of max.chr3.193, HES5, SLCO3A1, and TPM4_8047; and 2) detecting prostate cancer (e.g., provided by a sensitivity of 80% or greater and a specificity of 80% or greater); The present invention provides a method comprising:

[0178] Preferably, the sensitivity is about 70% to about 100%, or about 80% to about 90%, or about 80% to about 85%. Preferably, the specificity is about 70% to about 100%, or about 80% to about 90%, or about 80% to about 85%.

[0179] Genomic DNA can be isolated by any means, including the use of commercially available kits. Briefly, a biological sample in which the DNA of interest is encapsulated by a cell membrane must be disrupted and lysed by enzymatic, chemical, or mechanical means. The DNA solution can then be freed of proteins and other contaminants, for example, by digestion with proteinase K. The genomic DNA is then recovered from this solution. This can be accomplished by a variety of methods, including salting out, organic extraction, or binding of the DNA to a solid support. The choice of method is influenced by several factors, including time, cost, and the amount of DNA required. All clinical sample types are suitable for use in the methods of the present invention, including neoplastic or pre-neoplastic material, such as cell lines, tissue slides, biopsies, paraffin-embedded tissue, body fluids, feces, prostate tissue, colonic effluent, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, and combinations thereof.

[0180] This technology is not limited to the method used to prepare the sample and provide nucleic acids for testing. For example, in some embodiments, DNA is isolated from a fecal sample, or from a blood or plasma sample using direct gene capture, e.g., as detailed in U.S. Patent Application No. 61 / 485,386, or related methods.

[0181] The genomic DNA sample is then treated with at least one reagent, or a series of reagents, that distinguishes between methylated and unmethylated CpG dinucleotides within at least one marker that includes a DMR (e.g., DMRs 1-140, as provided in Tables 1 and 13).

[0182] In some embodiments, the reagent converts unmethylated cytosine bases at the 5' position to uracil, thymine, or another base that differs from cytosine in hybridization behavior, although in some embodiments the reagent can be a methylation-sensitive restriction enzyme.

[0183] In some embodiments, the genomic DNA sample is treated in such a way as to convert cytosine bases that are not methylated at the 5' position to uracil, thymine, or another base that differs from cytosine in terms of hybridization behavior. In some embodiments, this treatment is carried out with bisulfite (hydrogen sulfite), followed by alkaline hydrolysis.

[0184] The treated nucleic acid is then analyzed to determine the methylation status of the target gene sequence (at least one gene, genomic sequence, or nucleotide from a marker comprising a DMR, e.g., at least one DMR selected from DMRs 1-140, as provided in Tables 1 and 13). The analytical method can be selected from those known in the art, including analytical methods listed herein, e.g., QuARTS and MSP, as described herein.

[0185] Aberrant methylation, and more specifically hypermethylation of markers that comprise DMRs (eg, DMRs 1-140, as provided in Tables 1 and 13), is associated with prostate cancer.

[0186] This technology relates to the analysis of any sample associated with prostate cancer. For example, in some embodiments, the sample includes tissue and / or biological fluid obtained from a patient. In some embodiments, the sample includes secretions. In some embodiments, the sample includes blood, serum, plasma, gastric secretions, pancreatic juice, a gastrointestinal biopsy sample, cells microdissected from a prostate biopsy, and / or prostate cells recovered from feces. In some embodiments, the subject is human. The sample can include cells, secretions, or tissue from the prostate, liver, bile duct, pancreas, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyps, gallbladder, anus, and / or peritoneum. In some embodiments, the sample includes cellular fluid, ascites, urine, feces, pancreatic juice, fluid obtained during endoscopy, blood, mucus, or saliva. In some embodiments, the sample is a fecal sample.

[0187] Samples such as these can be obtained by any number of means known in the art, as will be apparent to those skilled in the art. For example, urine and fecal samples are readily achievable, while blood, ascites, serum, or pancreatic juice samples can be obtained parenterally, for example, by using a needle and syringe. Cell-free or substantially cell-free samples can be obtained by subjecting the sample to a variety of techniques known to those skilled in the art, including, but not limited to, centrifugation and filtration. While obtaining samples using non-invasive techniques is generally preferred, obtaining samples such as tissue homogenates, tissue sections, and biopsy specimens remains preferred.

[0188] In some embodiments, this technology relates to methods of treating a patient (e.g., a patient who has prostate cancer, has early stage prostate cancer, or is at risk of developing prostate cancer) by determining the methylation status of one or more DMRs as provided herein and administering a treatment to the patient based on the results of determining the methylation status. The treatment can be the administration of a pharmaceutical compound, a vaccine, performing surgery, imaging the patient, or performing another test. Preferably, this application is in methods of clinical screening, prognostic evaluation, monitoring the outcome of treatment, identifying patients most likely to respond to a particular therapeutic treatment, imaging patients or subjects, and drug screening and development methods.

[0189] In some embodiments of this technology, a method for diagnosing prostate cancer in a subject is provided. As used herein, the terms "diagnosing" and "diagnosis" refer to a method that enables a skilled artisan to estimate, and even determine, whether a subject is suffering from a given disease or condition, or whether a subject is likely to suffer from a given disease or condition in the future. Those skilled in the art often make a diagnosis based on one or more diagnostic indicators, such as biomarkers (e.g., DMRs as disclosed herein), the methylation status of which indicates the presence, severity, or absence of the condition.

[0190] In addition to diagnosis, clinical cancer prognosis involves determining the aggressiveness of cancer and the likelihood of tumor recurrence, and planning the most effective treatment. If a more accurate prognosis can be made, or even if the potential risk of developing cancer can be assessed, it is possible to select appropriate treatment, and in some cases, less harsh treatment, for the patient. Assessment of cancer biomarkers (e.g., determining methylation status) is useful for separating subjects with a good prognosis and / or low risk of developing cancer who do not require treatment, or who are more likely to develop cancer, into treatment-limited subjects or subjects who will experience cancer recurrence and may benefit from more intensive treatment.

[0191] As such, "making a diagnosis" or "diagnosing," as used herein, further includes determining the risk of developing cancer or determining a prognosis, which can be provided to predict a clinical outcome (with or without medical treatment), select an appropriate treatment (or whether the treatment is effective), or monitor a current treatment to potentially modify the treatment, based on measurements of a diagnostic biomarker (e.g., DMR) disclosed herein. Furthermore, in some embodiments of the disclosed subject matter, multiple determinations of biomarkers over time can be made to facilitate diagnosis and / or prognosis. Changes in biomarkers over time can be used to predict clinical regression, monitor the progression of prostate cancer, and / or monitor the effectiveness of appropriate cancer-directed treatments. In example such embodiments, one can expect to see changes in the methylation status of one or more biomarkers (e.g., DMR) disclosed herein (and potentially one or more additional biomarker(s) if monitored) in biological samples over time during the course of effective treatment.

[0192] The disclosed subject matter further provides, in some embodiments, a method for determining whether to initiate or continue prevention or treatment of cancer in a subject. In some embodiments, the method comprises providing a series of biological samples from the subject over a period of time; analyzing the series of biological samples to determine the methylation status of at least one biomarker disclosed herein in each of the biological samples; and comparing any measurable changes in the methylation status of one or more of the biomarkers in each of the biological samples. Any changes in the methylation status of the biomarkers over a period of time can be used to predict the risk of developing cancer, predict clinical regression, determine whether to initiate or continue cancer prevention or treatment, or determine whether a current treatment is effectively treating the cancer. For example, a first time point can be selected before the start of treatment, and a second time point can be selected at a time after the start of treatment. The methylation status can be measured in each of the samples taken from the different time points, and qualitative and / or quantitative differences can be noted. Changes in the methylation status of biomarker levels from the different samples can be correlated with prostate cancer risk, prognosis, treatment efficacy, and / or cancer progression in the subject.

[0193] In preferred embodiments, the methods and compositions of the invention are for the treatment or diagnosis of disease at an early stage, e.g., before disease symptoms appear, hi some embodiments, the methods and compositions of the invention are for the treatment or diagnosis of disease at a clinical stage.

[0194] As described, in some embodiments, multiple determinations of one or more diagnostic or prognostic biomarkers can be made, and changes in the markers over time can be used to determine a diagnosis or prognosis. For example, a diagnostic marker can be determined a first time and again a second time. In these embodiments, an increase in a marker from the first to the second time can be diagnostic of a particular type or severity of cancer, or a given prognosis. Similarly, a decrease in a marker from the first to the second time can indicate a particular type or severity of cancer, or a given prognosis. Furthermore, the degree of change in one or more markers can be related to the severity of cancer and future adverse events. Those skilled in the art will understand that in certain embodiments, comparative measurements of the same biomarkers can be made at multiple time points, and a given biomarker can be measured at one time point and a second biomarker at a second time point, and comparison of these markers can provide diagnostic information.

[0195] As used herein, the phrase "determining a prognosis" refers to a method by which a person skilled in the art can predict the course or outcome of a condition in a subject. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, or even the ability to predict that a given course or outcome will occur more or less likely based on the methylation status of a biomarker (e.g., DMR). Alternatively, a person skilled in the art will understand that the term "prognosis" refers to the increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a subject who exhibits a given condition, compared to those individuals who do not exhibit this condition. For example, in individuals who do not exhibit this condition (e.g., who have normal methylation status of one or more DMRs), the chance of a change in a given outcome (e.g., suffering from prostate cancer) is very low.

[0196] In some embodiments, statistical analysis correlates prognostic indicators with predisposition to adverse outcomes. For example, in some embodiments, a methylation state that differs from that in a normal control sample obtained from a patient without cancer can indicate that the subject is more likely to suffer from cancer than a subject having a methylation state more similar to that in the control sample, as determined by the level of statistical significance. In addition, the change in methylation state from the baseline (e.g., "normal") level can reflect the subject's prognosis, and the degree of change in methylation state can be related to the severity of an adverse event. Statistical significance is often determined by comparing two or more populations and determining a confidence interval and / or p-value. See, for example, Dowdy and Wearden, Statistics, 2009, pp. 111-114, which is incorporated herein by reference in its entirety. See "Scientific Remarks for Research," John Wiley & Sons, New York, 1983. Exemplary confidence intervals for the present subject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, and 99.99%, and exemplary p-values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

[0197] In other embodiments, a threshold degree of change in the methylation status of a prognostic or diagnostic biomarker (e.g., DMR) disclosed herein can be established, and the degree of change in the methylation status of this biomarker in a biological sample can be simply compared to the threshold degree of change in methylation status. Preferred threshold changes in methylation status for the biomarkers provided herein are about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, and about 150%. In yet other embodiments, a "nomogram" can be established, which directly relates the methylation status of a prognostic or diagnostic indicator (biomarker, or combination of biomarkers) to a property associated with a given outcome. Those skilled in the art are familiar with the use of such nomograms and relate the two values, understanding that because measurements refer to individual samples, rather than population averages, the uncertainty in the measurement is the same as the uncertainty in the marker concentration.

[0198] In some embodiments, a control sample is analyzed simultaneously with the biological sample so that results obtained from the biological sample can be compared to results obtained from the control sample. Additionally, it is contemplated that a standard curve can be provided to compare assay results for the biological sample. Standard curves such as these present the methylation status of biomarkers as a function of assay unit, e.g., fluorescent signal intensity when fluorescent labels are used. Samples from multiple donors can be used to provide a standard curve for the control methylation status of one or more biomarkers in normal tissue and a standard curve for the "at-risk" level of one or more biomarkers in tissue from donors with metaplasia or from donors with prostate cancer. In certain embodiments of this method, a subject is identified as having metaplasia based on identifying an aberrant methylation status of one or more DMRs provided herein in a biological sample obtained from the subject. In other embodiments of this method, detecting an aberrant methylation status of one or more such biomarkers in a biological sample obtained from the subject results in the subject being identified as having cancer.

[0199] Analysis of markers can be performed separately or simultaneously with additional markers within a single test sample. For example, several markers can be combined in a single test for efficient processing of multiple samples and potentially to provide greater diagnostic and / or prognostic accuracy. In addition, those skilled in the art will recognize the value of testing multiple samples (e.g., at successive time points) from the same subject. Testing such successive samples can enable the identification of changes in the methylation status of markers over time. Changes in methylation status, and the absence of changes in methylation status, can provide useful information about the disease state, including, but not limited to, revealing the approximate time since the occurrence of the event, the presence and amount of recoverable tissue, the suitability of drug therapy, the effectiveness of various therapies, and the identification of the subject's outcome, including the risk of future events.

[0200] Biomarker analysis can be performed in a variety of physical formats. For example, microtiter plates or automated applications can be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats can be developed to facilitate immediate treatment and diagnosis in a timely manner, for example, in a mobile transport or emergency room setting.

[0201] In some embodiments, a subject is diagnosed with prostate cancer when there is a measurable difference in the methylation status of at least one biomarker in the sample compared to a control methylation status. Conversely, when no change in methylation status is identified in the biological sample, the subject can be identified as not having prostate cancer, not at risk for cancer, or at low risk for cancer. In this regard, subjects with cancer or at risk for cancer can be distinguished from subjects with substantial cancer or low to substantially no risk of cancer. Those subjects at risk for prostate cancer can be placed on a more intensive and / or regular screening schedule, including endoscopy. Meanwhile, those subjects at low to substantially no risk can avoid undergoing endoscopy until a future screening, such as a screening performed according to the technology of the present invention, indicates that a risk of prostate cancer has emerged in these subjects.

[0202] As described above, detecting a change in the methylation state of one or more biomarkers according to embodiments of the present technology methods can be a qualitative determination, or it can be a quantitative determination. As such, diagnosing a subject having or at risk of developing prostate cancer involves making a certain threshold measurement, e.g., indicating that the methylation state of one or more biomarkers in a biological sample differs from a predetermined control methylation state. In some embodiments of this method, the control methylation state is a detectable methylation state of any of the biomarkers. In other embodiments of the method, in which a control sample is tested simultaneously with the biological sample, the predetermined methylation state is the methylation state in the control sample. In other embodiments of this method, the predetermined methylation state is based on and / or identified by a standard curve. In other embodiments of this method, the predetermined methylation state is a specific state or range of states. As such, the predetermined methylation state can be selected based in part on the method being performed and the specificity desired, within acceptable limits apparent to one of skill in the art.

[0203] Further, with respect to diagnostic methods, preferred subjects are vertebrate subjects. Preferred vertebrates are warm-blooded animals, and preferred warm-blooded animals are mammals. Preferred mammals are most preferably humans. As used herein, the term "subject" includes both human and animal subjects. Accordingly, veterinary therapeutic applications are provided herein. As such, the present technology provides for the diagnosis of mammals, such as humans, and those mammals that are economically important, such as endangered and therefore important animals raised on farms for human consumption, such as the Amur tiger, and / or animals that are socially important to humans, such as animals kept as pets or in zoos. Examples of such animals include, but are not limited to, carnivores, such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and / or ungulates, such as cows, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. Accordingly, also provided are diagnostics and treatments for livestock, including, but not limited to, domesticated pigs, ruminants, ungulates, horses (including racehorses), and the like. The disclosed subject matter further includes a system for diagnosing prostate cancer in a subject. The system can be provided, for example, as a commercially available kit, and can be used to screen for prostate cancer risk or diagnose prostate cancer in a subject from whom a biological sample is obtained. An exemplary system provided in accordance with the present technology comprises assessing the methylation status of DMRs as provided in Tables 1 and 3.

[0204] The present invention can also be configured as follows.

[0205] [1] 1. A method for characterizing a sample from a human patient, comprising: a) obtaining DNA from a human patient sample; b) assaying the methylation status of a DNA methylation marker comprising a base in a variably methylated region (DMR) selected from the group consisting of DMRs 1 to 140 from Tables 1 or 13; c) comparing the assayed methylation status of the one or more DNA methylation markers with reference methylation levels for the one or more DNA methylation markers for human patients without prostate cancer; The method comprising:

[0206] [2] The method of claim 1, wherein the sample is a fecal sample, a tissue sample, a prostate tissue sample, a blood sample, a plasma sample, or a urine sample.

[0207] [3] the sample is a prostate tissue sample and the DMR is selected from DMR numbers 63, 3, 64, 70, 7, 39, 8, 10, 11, 12, 14, 41, 81, 16, 17, 18, 20, 21, 44, 25, and 47; or The sample is a plasma sample, and the DMR is selected from DMR numbers 17, 12, 45, and 47. The method described in [1].

[0208] [4] The method of [1], comprising assaying multiple DNA methylation markers.

[0209] [5] The method of [1], comprising assaying 2 to 11 DNA methylation markers.

[0210] [6] The method of [1], comprising assaying 12 to 140 DNA methylation markers.

[0211] [7] The method of claim 1, wherein assaying the methylation status of the one or more DNA methylation markers in the sample comprises determining the methylation status of a single base.

[0212] [8] 2. The method of claim 1, wherein assaying the methylation status of the one or more DNA methylation markers in the sample comprises determining the degree of methylation at a plurality of bases.

[0213] [9] The method of [1], comprising assaying the methylation state of the forward strand or assaying the methylation state of the reverse strand.

[0214]

[10] The method described in [1], wherein the DNA methylation marker is a region of 100 bases or less.

[0215]

[11] The method described in [1], wherein the DNA methylation marker is a region of 500 bases or less.

[0216]

[12] The method described in [1], wherein the DNA methylation marker is a region of 1,000 bases or less.

[0217]

[13] The method described in [1], wherein the DNA methylation marker is a region of 5,000 bases or less.

[0218]

[14] The method described in [1], wherein the DNA methylation marker is a single base.

[0219]

[15] The method described in [1], wherein the DNA methylation marker is located in a high CpG density promoter.

[0220]

[16] The method of claim 1, wherein the assay comprises using methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or target capture.

[0221]

[17] The method of [1], wherein the assay comprises the use of a methylation-specific oligonucleotide selected from SEQ ID NOs: 1 to 234.

[0222]

[18] The method of claim 1, wherein the chromosomal region having an annotation selected from the group consisting of ACOXL, AKR1B1_3644, ANXA2, CHST11_2206, FLJ45983, GAS6, GRASP, HAPLN3, HCG4P6, HES5_0822, ITPRIPL1, KCNK4, MAX.chr1.61519554-61519667, MAX.chr2.97193166-97193253, MAX.chr3.193, MAX.chr3.72788028-72788112, RAI1_7469, RASSF2, SERPINB9_3389, SLC4A11, and TPM4_8047 comprises the DNA methylation marker.

[0223]

[19] SERPINB9_3479, FLOT1_1665, HCG4P6_4618, CHST11_2206, MAX.chr12.485, GRASP_0932, GAS6_6425, MAX .chr3.193, MAX.chr2.971_3164, MAX.chr3.727_8028, HES5_0840, TPM4_8037, SLCO3A1_6187, ITPRIPL1 The method of [1], wherein a chromosomal region having an annotation selected from the group consisting of: _1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487 comprises the DNA methylation marker.

[0224]

[20] The method described in [1], wherein a chromosomal region having an annotation selected from the group consisting of max.chr3.193, HES5, SLCO3A1, and TPM4_8047 includes the DNA methylation marker.

[0225] 〔twenty one〕 1. A method for characterizing a sample obtained from a human patient, comprising: a) determining the methylation status of a DNA methylation marker in said sample comprising a base in a DMR selected from the group consisting of DMRs 1 to 140 from Tables 1 and 13; b) comparing the methylation status of the DNA methylation markers from the patient sample with the methylation status of the DNA methylation markers from a normal control sample from a human subject without prostate cancer; c) determining a confidence interval and / or p-value for the difference in methylation status of the human patient and the normal control sample; The method comprising:

[0226] 〔twenty two〕

[21] The method of

[21] , wherein the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99%, and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001.

[0227] 〔twenty three〕 1. A method for characterizing a sample obtained from a human subject, comprising: reacting a nucleic acid containing a DMR with a bisulfite reagent to produce a bisulfite-reacted nucleic acid; sequencing said bisulfite-reacted nucleic acid to provide a nucleotide sequence of said bisulfite-reacted nucleic acid; comparing the nucleotide sequence of the bisulfite-reacted nucleic acid with the nucleotide sequence of a nucleic acid containing the DMR from a subject without prostate cancer and identifying differences in the two sequences; The method comprising:

[0228] 〔twenty four〕 1. A system for characterizing a sample obtained from a human subject, comprising: an analytical component configured to determine the methylation state of the sample; a software component configured to compare the methylation state of the sample with a control or reference sample methylation state recorded in a database; and an alert component configured to determine a single value based on the combination of methylation states and alert a user of a prostate cancer-associated methylation state; The system comprising:

[0229] 〔twenty five〕 The system of claim 24, wherein the sample contains a nucleic acid containing a DMR.

[0230]

[26] The system of claim 24, further comprising a component for isolating nucleic acids.

[0231]

[27] The system of claim 24, further comprising a component for collecting a sample.

[0232]

[28] The system of claim 24, wherein the sample is a fecal sample, a tissue sample, a prostate tissue sample, a blood sample, a plasma sample, or a urine sample.

[0233]

[29] The system of claim 24, wherein the database contains nucleic acid sequences containing DMRs.

[0234]

[30] The system of claim 24, wherein the database includes nucleic acid sequences from subjects who do not have prostate cancer.

[0235]

[31] 1. A method of screening for prostate cancer in a sample obtained from a subject, comprising: 1) assaying the methylation status of a marker in a sample obtained from a subject; and 2) identifying the subject as having prostate cancer when the methylation status of the marker differs from the methylation status of the marker assayed in subjects without prostate cancer; Equipped with The marker comprises a base in a variably methylated region (DMR) selected from the group consisting of DMRs 1 to 140 from Tables 1 or 13, The method.

[0236]

[32] The method of

[31] , comprising assaying multiple markers.

[0237]

[33] 32. The method of claim 31, wherein assaying the methylation status of the marker in the sample comprises determining the methylation status of a single base.

[0238]

[34] 32. The method of claim 31, wherein assaying the methylation state of the marker in the sample comprises determining the degree of methylation at a plurality of bases.

[0239]

[35] The method of claim 31, wherein the methylation status of the marker comprises increased or decreased methylation of the marker compared to a normal methylation status of the marker.

[0240]

[36] The method of claim 31, wherein the methylation status of the marker has a different pattern of methylation of the marker compared to a normal methylation status of the marker.

[0241]

[37] The method of claim 31, further comprising assaying the methylation status of the forward strand or the reverse strand.

[0242]

[38] The method of claim 31, wherein the marker is a region of 100 bases or less.

[0243]

[39] The method of claim 31, wherein the marker is a region of 500 bases or less.

[0244]

[40] The method of

[31] , wherein the marker is a region of 1000 bases or less.

[0245]

[41] The method of

[31] , wherein the marker is a region of 5,000 bases or less.

[0246]

[42] The method according to

[31] , wherein the marker is a single base.

[0247]

[43] The method of

[31] , wherein the marker is in a high CpG density promoter.

[0248]

[44] The method of claim 31, wherein the sample is a fecal sample, a tissue sample, a prostate tissue sample, a blood sample, a plasma sample, or a urine sample.

[0249]

[45] The method of claim 31, wherein the assay comprises using methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or target capture.

[0250]

[46] The method of

[31] , wherein the assay comprises the use of a methylation-specific oligonucleotide selected from the group consisting of SEQ ID NOs: 1 to 234.

[0251]

[47] The method of claim 31, wherein a chromosomal region having an annotation selected from the group consisting of ACOXL, AKR1B1_3644, ANXA2, CHST11_2206, FLJ45983, GAS6, GRASP, HAPLN3, HCG4P6, HES5_0822, ITPRIPL1, KCNK4, MAX.chr1.61519554-61519667, MAX.chr2.97193166-97193253, MAX.chr3.193, MAX.chr3.72788028-72788112, RAI1_7469, RASSF2, SERPINB9_3389, SLC4A11, and TPM4_8047 comprises the marker.

[0252]

[48] SERPINB9_3479, FLOT1_1665, HCG4P6_4618, CHST11_2206, MAX.chr12.485, GRASP_0932, GAS6_6425, M AX.chr3.193, MAX.chr2.971_3164, MAX.chr3.727_8028, HES5_0840, TPM4_8037, SLCO3A1_6187, ITPRI The method of claim 31, wherein a chromosomal region having an annotation selected from the group consisting of PL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487 comprises the marker.

[0253]

[49] The method of

[31] , wherein a chromosomal region having an annotation selected from the group consisting of max.chr3.193, HES5, SLCO3A1, and TPM4_8047 includes the marker.

[0254]

[50] 1) Bisulfite reagent, and 2) a control nucleic acid comprising a sequence from a DMR selected from the group consisting of DMRs 1-140 from Tables 1 and 13, and having a methylation status associated with a subject without prostate cancer; Includes a kit.

[0255]

[51] A kit comprising a bisulfite reagent and an oligonucleotide according to

[47] .

[0256]

[52] a sample collector for obtaining a sample from a subject; Reagents for isolating nucleic acids from said sample; Bisulfite reagent, and oligonucleotides, A kit comprising the above in accordance with

[47] .

[0257]

[53] The kit of claim 52, wherein the sample is a fecal sample, a tissue sample, a prostate tissue sample, a blood sample, a plasma sample, or a urine sample.

[0258]

[54] A composition comprising a nucleic acid containing a DMR and a bisulfite reagent.

[0259]

[55] A composition comprising a nucleic acid containing a DMR and an oligonucleotide according to

[47] .

[0260]

[56] A composition comprising a nucleic acid containing a DMR and a methylation-sensitive restriction enzyme.

[0261]

[57] A composition comprising a nucleic acid containing a DMR and a polymerase.

[0262]

[58] 1. A method of screening for prostate cancer in a sample obtained from a subject, comprising: a) determining the methylation status of a marker in said sample comprising a base in a DMR selected from the group consisting of DMRs 1 to 140 from Tables 1 and 13; b) comparing the methylation status of the markers from the subject sample with the methylation status of the markers from a normal control sample from a subject not having prostate cancer; c) determining a confidence interval and / or p-value for the difference in methylation status between the subject sample and the normal control sample; The method comprising:

[0263]

[59] 59. The method of claim 58, wherein the confidence interval is 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, or 99.99%, and the p-value is 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, or 0.0001.

[0264]

[60] 1. A method of screening for prostate cancer in a sample obtained from a subject, comprising: reacting a nucleic acid containing a DMR with a bisulfite reagent to produce a bisulfite-reacted nucleic acid; sequencing said bisulfite-reacted nucleic acid to provide a nucleotide sequence of said bisulfite-reacted nucleic acid; comparing the nucleotide sequence of the bisulfite-reacted nucleic acid to the nucleotide sequence of a nucleic acid containing the DMR from a subject without prostate cancer and identifying differences in the two sequences; and identifying the subject as having prostate cancer when a difference exists; The method comprising:

[0265]

[61] 1. A system for screening for prostate cancer in a sample obtained from a subject, comprising: an analytical component configured to determine the methylation state of the sample; a software component configured to compare the methylation state of the sample with a control or reference sample methylation state recorded in a database; and an alert component configured to determine a single value based on the combination of methylation states and alert a user of a prostate cancer-associated methylation state; The system comprising:

[0266]

[62] The system described in

[61] , wherein the sample contains a nucleic acid containing a DMR.

[0267]

[63] The system of claim 61, further comprising a component for isolating nucleic acids.

[0268]

[64] The system of claim 61, further comprising a component for collecting a sample.

[0269]

[65] The system of claim 61, further comprising components for collecting a fecal sample, a prostate tissue sample, a blood sample, and / or a plasma sample.

[0270]

[66] The system described in

[61] , wherein the database contains nucleic acid sequences containing DMRs.

[0271]

[67] The system of claim 61, wherein the database includes nucleic acid sequences from subjects who do not have prostate cancer.

[0272]

[68] A set of isolated nucleic acids, each nucleic acid comprising a sequence containing a DMR.

[0273]

[69] The set of nucleic acids according to

[68] , wherein each nucleic acid comprises a sequence from a subject who does not have prostate cancer.

[0274]

[70] A system comprising the set of nucleic acids according to

[68] or

[69] and a database of nucleic acid sequences associated with the set of nucleic acids.

[0275]

[71] The system of claim 70, further comprising a bisulfite reagent.

[0276]

[72] The system described in

[70] further comprising a nucleic acid sequencer.

[0277]

[73] 1. A method for characterizing a biological sample, comprising: (a) measuring the methylation levels of CpG sites for two or more genes selected from ACOXL, AKR1B1_3644, ANXA2, CHST11_2206, FLJ45983, GAS6, GRASP, HAPLN3, HCG4P6, HES5_0822, ITPRIPL1, KCNK4, MAX.chr1.61519554-61519667, MAX.chr2.97193166-97193253, MAX.chr3.193, MAX.chr3.72788028-72788112, RAI1_7469, RASSF2, SERPINB9_3389, SLC4A11, and TPM4_8047 in a biological sample from a human individual; treating genomic DNA in said biological sample with bisulfite; The following sets of primers for the two or more selected genes: A set of primers consisting of SEQ ID NOs: 93 and 94 for ACOXL; one set of primers consisting of SEQ ID NOs: 27 and 28 for AKR1B1_3644; One set of primers consisting of SEQ ID NOs: 89 and 90 for ANXA2; One set of primers consisting of SEQ ID NOs: 85 and 86 for CHST11_2206; One set of primers consisting of SEQ ID NOs: 31 and 32 for FLJ45983; one set of primers consisting of SEQ ID NOs: 117 and 118 for GAS6; a set of primers consisting of SEQ ID NOs: 33 and 34 for GRASP; one set of primers consisting of SEQ ID NOs: 37 and 38 for HAPLN3; One set of primers consisting of SEQ ID NOs: 39 and 40 for HCG4P6; One set of primers consisting of SEQ ID NOs: 41 and 42 for HES5_0822; One set of primers consisting of SEQ ID NOs: 45 and 46 for ITPRIPL1; One set of primers consisting of SEQ ID NOs: 125 and 126 for KCNK4; One set of primers consisting of SEQ ID NOs: 91 and 92 for MAX.chr1.61519554-61519667; One set of primers consisting of SEQ ID NOs: 49 and 50 for MAX.chr2.97193166-97193253; One set of primers consisting of SEQ ID NOs: 51 and 52 for MAX.chr3.193; One set of primers consisting of SEQ ID NOs: 53 and 54 for MAX.chr3.72788028-72788112, One set of primers consisting of SEQ ID NOs: 55 and 56 for RAI1_7469; one set of primers consisting of SEQ ID NOs: 57 and 58 for RASSF2; one set of primers consisting of SEQ ID NOs: 129 and 130 for SERPINB9_3389; A set of primers consisting of SEQ ID NOs: 59 and 60 for SLC4A11, and One set of primers consisting of SEQ ID NOs: 123 and 124 for TPM4_8047; amplifying the bisulfite-treated genomic DNA using determining the methylation level of the CpG sites by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR; Through measuring, (b) comparing the methylation level with the methylation level of a corresponding set of genes in a control sample without prostate cancer; and (c) determining that the individual has prostate cancer when the methylation levels measured in the two or more genes are higher than the methylation levels measured in each of the control samples; The method comprising:

[0278]

[74] The method of claim 73, wherein the biological sample is a blood sample or a tissue sample.

[0279]

[75] The method of claim 74, wherein the tissue is prostate tissue.

[0280]

[76] The method of

[73] , wherein the CpG site is present in a coding region or a regulatory region.

[0281]

[77] The method of claim 73, wherein measuring the methylation levels of CpG sites for two or more genes comprises determining a methylation score for the CpG sites and determining the methylation frequency for the CpG sites.

[0282]

[78] 1. A method for characterizing a plasma sample, comprising: (a) measuring the methylation levels of CpG sites for two or more genes selected from max.chr3.193, HES5, SLCO3A1, and TPM4_8047 in a plasma sample from a human individual; treating genomic DNA in said biological sample with bisulfite; A set of primers for the two or more selected genes: One set of primers consisting of SEQ ID NOs: 174 and 175 for max.chr3.193; One set of primers consisting of SEQ ID NOs: 180 and 181 for HES5; A set of primers consisting of SEQ ID NOs: 171 and 172 for SLCO3A1, and One set of primers consisting of SEQ ID NOs: 189 and 190 for TPM4_8047; amplifying the bisulfite-treated genomic DNA using determining the methylation level of the CpG sites by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR; Through measuring, (b) comparing the methylation level with the methylation level of a corresponding set of genes in a control sample without prostate cancer; and (c) determining that the individual has prostate cancer when the methylation levels measured in the two or more genes are higher than the methylation levels measured in each of the control samples; The method comprising:

[0283]

[79] The method of

[78] , wherein the CpG site is present in a coding region or a regulatory region.

[0284]

[80] The method of claim 78, wherein measuring the methylation levels of CpG sites for two or more genes comprises determining a methylation score for the CpG sites and determining the methylation frequency for the CpG sites.

[0285]

[81] 1. A method for characterizing a biological sample, comprising: ACOXL, AKR1B1_3644, ANXA2, CHST11_2206, FLJ45983, GAS6, GRASP, HAPLN3, HCG4P6, HES5_0822, ITPRIPL1, KCNK4, MAX.chr1.61519554-61519667, MAX.chr2.97193166-97193253, MAX.chr3.193, MAX.chr3.72788028-72788112, RAI1_7469, RASSF2, SERPINB9_3389, SLC4A11, and TPM4_8047 in a human biological sample, when the biological sample is a prostate tissue sample from said human individual; when the biological sample is a plasma sample from a human individual, max.chr3.193, HES5, SLCO3A1, and TPM4_8047 in the biological sample from the human individual; The methylation levels of CpG sites for two or more genes selected from either treating genomic DNA in said biological sample with bisulfite; amplifying the bisulfite-treated genomic DNA using a set of primers for the two or more selected genes; and determining the methylation level of the CpG sites by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR; Through measuring, The method comprising:

[0286]

[82] comparing the methylation level with the methylation level of a corresponding set of genes in a control sample that does not have prostate cancer; and determining that the individual has prostate cancer when the methylation levels measured in the two or more genes are higher than the methylation levels measured in each of the control samples; The method of claim 81, further comprising:

[0287]

[83] When the biological sample is a tissue sample, a set of primers for the two or more selected genes: A set of primers consisting of SEQ ID NOs: 93 and 94 for ACOXL; one set of primers consisting of SEQ ID NOs: 27 and 28 for AKR1B1_3644; One set of primers consisting of SEQ ID NOs: 89 and 90 for ANXA2; One set of primers consisting of SEQ ID NOs: 85 and 86 for CHST11_2206; One set of primers consisting of SEQ ID NOs: 31 and 32 for FLJ45983; one set of primers consisting of SEQ ID NOs: 117 and 118 for GAS6; a set of primers consisting of SEQ ID NOs: 33 and 34 for GRASP; one set of primers consisting of SEQ ID NOs: 37 and 38 for HAPLN3; One set of primers consisting of SEQ ID NOs: 39 and 40 for HCG4P6; One set of primers consisting of SEQ ID NOs: 41 and 42 for HES5_0822; One set of primers consisting of SEQ ID NOs: 45 and 46 for ITPRIPL1; One set of primers consisting of SEQ ID NOs: 125 and 126 for KCNK4; One set of primers consisting of SEQ ID NOs: 91 and 92 for MAX.chr1.61519554-61519667; One set of primers consisting of SEQ ID NOs: 49 and 50 for MAX.chr2.97193166-97193253; One set of primers consisting of SEQ ID NOs: 51 and 52 for MAX.chr3.193; One set of primers consisting of SEQ ID NOs: 53 and 54 for MAX.chr3.72788028-72788112, One set of primers consisting of SEQ ID NOs: 55 and 56 for RAI1_7469; one set of primers consisting of SEQ ID NOs: 57 and 58 for RASSF2; one set of primers consisting of SEQ ID NOs: 129 and 130 for SERPINB9_3389; One set of primers consisting of SEQ ID NOs: 59 and 60 for SLC4A11; One set of primers consisting of SEQ ID NOs: 123 and 124 for TPM4_8047;

[81] The method according to

[81] , wherein

[0288]

[84] When the biological sample is a plasma sample, a set of primers for the two or more selected genes is: One set of primers consisting of SEQ ID NOs: 174 and 175 for max.chr3.193; One set of primers consisting of SEQ ID NOs: 180 and 181 for HES5; A set of primers consisting of SEQ ID NOs: 171 and 172 for SLCO3A1, and One set of primers consisting of SEQ ID NOs: 189 and 190 for TPM4_8047;

[81] The method according to

[81] , wherein

[0289]

[85] The method of claim 81, wherein the tissue sample is a prostate tissue sample.

[0290]

[86] The CpG site is present in a coding region or a regulatory region. method.

[0291]

[87]

[81] The method of

[81] , wherein measuring the methylation levels of CpG sites for two or more genes comprises determining the methylation score of the CpG sites and determining the methylation frequency of the CpG sites. method.

[0292]

[88] 1. A method for characterizing a biological sample, comprising: (a) SERPINB9_3479, FLOT1_1665, HCG4P6_4618, CHST11_2206, MAX.chr12.485, GRASP_0932, GAS6_6425, MAX.chr3.193, MAX.chr2.971_3164, MAX.chr3.727_8028, HES5_0840, TPM4_8037, S in biological samples from human individuals The methylation levels of CpG sites for two or more genes selected from LCO3A1_6187, ITPRIPL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487 are measured. treating the genetic DNA in said biological sample with bisulfite; amplifying the bisulfite-treated genomic DNA using a set of primers for the two or more selected genes: determining the methylation level of the CpG sites by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR; Through measuring, (b) comparing the methylation level with the methylation level of a corresponding set of genes in a control sample without prostate cancer; and (c) determining that the individual has prostate cancer when the methylation levels measured in the two or more genes are higher than the methylation levels measured in each of the control samples; The method comprising:

[0293]

[89] The method of claim 88, wherein the biological sample is a blood sample or a tissue sample.

[0294]

[90] The method of claim 89, wherein the tissue is prostate tissue.

[0295]

[91] The method of

[88] , wherein the CpG site is present in a coding region or a regulatory region.

[0296]

[92] The method of claim 88, wherein measuring the methylation levels of CpG sites for two or more genes comprises determining a methylation score for the CpG sites and determining the methylation frequency for the CpG sites.

[0297]

[93] The set of primers for the two or more selected genes comprises: one set of primers consisting of SEQ ID NOs: 177 and 178 or 177 and 219 for MAX.chr3.727_8028; one set of primers consisting of SEQ ID NOs: 220 and 221 for RASGRF2_6325; One set of primers consisting of SEQ ID NOs: 210 and 211 for ZNF655_6075; One set of primers consisting of SEQ ID NOs: 223 and 224 for PAMR1_7364; One set of primers consisting of SEQ ID NOs: 216 and 217 for ST6GALNAC2_1113; One set of primers consisting of SEQ ID NOs: 229 and 230 for CCNJL_9070; One set of primers consisting of SEQ ID NOs: 213 and 214 for KCNB2_9128; One set of primers consisting of SEQ ID NOs: 226 and 227 for IGFBP7_6412; One set of primers consisting of SEQ ID NOs: 232 and 233 for WNT3A_5487; One set of primers consisting of SEQ ID NOs: 147 and 148 for SERPINB9_3479; one set of primers consisting of SEQ ID NOs: 150 and 151 for FLOT1_1665; One set of primers consisting of SEQ ID NOs: 153 and 154 for HCG4P6_4618; One set of primers consisting of SEQ ID NOs: 156 and 157 for CHST11_2206; one set of primers consisting of SEQ ID NOs: 159 and 160 for MAX.chr12.485; One set of primers consisting of SEQ ID NOs: 162 and 163 for GRASP_0932; One set of primers consisting of SEQ ID NOs: 165 and 166 for GAS6_6425; One set of primers consisting of SEQ ID NOs: 174 and 175 for MAX.chr3.193; One set of primers consisting of SEQ ID NOs: 198 and 199 for MAX.chr2.971; One set of primers consisting of SEQ ID NOs: 180 and 181 for HES5_0840; One set of primers consisting of SEQ ID NOs: 189 and 190 for TPM4_8037; One set of primers consisting of SEQ ID NOs: 171 and 172 for SLCO3A1_6187; A set of primers consisting of SEQ ID NOs: 195 and 196 for ITPRIPL1_1244, and one set of primers consisting of SEQ ID NOs: 201 and 202 for AKR1B1_3644; The method according to

[88] , wherein the compound is selected from the group consisting of:

[0298]

[94] 1. A method for characterizing cancerous prostate tissue, comprising: (a) measuring the methylation levels of CpG sites for two or more genes selected from SERPINB9_3479, GRASP_0932, SLCO3A1_6187, ITPRIPL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487 in a biological sample from a human individual; treating genomic DNA in said biological sample with bisulfite; amplifying the bisulfite-treated genomic DNA using a set of primers for the two or more selected genes: determining the methylation level of the CpG sites by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, or bisulfite genomic sequencing PCR; Through measuring, (b) comparing the methylation level with the methylation level of a corresponding set of genes from prostate cancer tissue with a Gleason score of 6; and (c) determining that the individual contains prostate cancer tissue with a Gleason score greater than 7 when the methylation level measured in the two or more genes is higher than the methylation level measured in the corresponding set of genes from prostate cancer tissue with a Gleason score of 6; The method comprising:

[0299]

[95] The method of

[94] , wherein the CpG site is present in a coding region or a regulatory region.

[0300]

[96] The method of claim 94, wherein measuring the methylation levels of CpG sites for two or more genes comprises determining a methylation score for the CpG sites and determining the methylation frequency for the CpG sites.

[0301]

[97] The set of primers for the two or more selected genes comprises: One set of primers consisting of SEQ ID NOs: 147 and 148 for SERPINB9_3479; One set of primers consisting of SEQ ID NOs: 162 and 163 for GRASP_0932; One set of primers consisting of SEQ ID NOs: 171 and 172 for SLCO3A1_6187; One set of primers consisting of SEQ ID NOs: 195 and 196 for ITPRIPL1_1244; one set of primers consisting of SEQ ID NOs: 201 and 202 for AKR1B1_3644; one set of primers consisting of SEQ ID NOs: 220 and 221 for RASGRF2_6325; One set of primers consisting of SEQ ID NOs: 210 and 211 for ZNF655_6075; One set of primers consisting of SEQ ID NOs: 223 and 224 for PAMR1_7364; One set of primers consisting of SEQ ID NOs: 216 and 217 for ST6GALNAC2_1113; One set of primers consisting of SEQ ID NOs: 229 and 230 for CCNJL_9070; One set of primers consisting of SEQ ID NOs: 213 and 214 for KCNB2_9128; A set of primers consisting of SEQ ID NOs: 226 and 227 for IGFBP7_6412, and One set of primers consisting of SEQ ID NOs: 232 and 233 for WNT3A_5487; The method according to

[94] , wherein the compound is selected from the group consisting of:

[0302]

[98] SERPINB9_3479, FLOT1_1665, HCG4P6_4618, CHST11_2206, MAX.chr12.485, GRASP_0932, GAS6_6425 , MAX.chr3.193, MAX.chr2.971_3164, MAX.chr3.727_8028, HES5_0840, TPM4_8037, SLCO3A1_6187, I 1. A method for measuring the methylation level of one or more CpG sites in a gene selected from TPRIPL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487, comprising: a) extracting genomic DNA from a biological sample of a human individual suspected of having or having prostate cancer; b) treating the extracted genomic DNA with bisulfite; c)SERPINB9_3479, FLOT1_1665, HCG4P6_4618, CHST11_2206, MAX.chr12.485, GRASP_0932, GAS6_6425, MAX.chr 3.193, MAX.chr2.971_3164, MAX.chr3.727_8028, HES5_0840, TPM4_8037, SLCO3A1_6187, ITPRIPL1_1244, AKR1 amplifying the bisulfite-treated genomic DNA with primers consisting of a primer pair specific for the one or more CpG sites in a gene selected from B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487; and d) SERPINB9_3479, FLOT1_1665, HCG4P6_4618, CHST11_2206, MAX.chr12.485, GRASP_0932, GAS6_6425, MAX.chr3.193, MAX.chr2.971_3164, MAX.chr3.727_80 by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, or bisulfite genomic sequencing PCR. measuring the methylation level of one or more CpG sites in genes selected from: 28, HES5_0840, TPM4_8037, SLCO3A1_6187, ITPRIPL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487; The method comprising:

[0303]

[99] The method of claim 98, wherein the sample is a prostate tissue sample.

[0304]

[100] SERPINB9_3479, FLOT1_1665, HCG4P6_4618, CHST11_2206, MAX.chr12.485, GRASP_0932, GA S6_6425, MAX.chr3.193, MAX.chr2.971_3164, MAX.chr3.727_8028, HES5_0840, TPM4_8037 , SLCO3A1_6187, ITPRIPL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487. one set of primers consisting of SEQ ID NOs: 177 and 178 or 177 and 219 for MAX.chr3.727_8028; one set of primers consisting of SEQ ID NOs: 220 and 221 for RASGRF2_6325; One set of primers consisting of SEQ ID NOs: 210 and 211 for ZNF655_6075; One set of primers consisting of SEQ ID NOs: 223 and 224 for PAMR1_7364; One set of primers consisting of SEQ ID NOs: 216 and 217 for ST6GALNAC2_1113; One set of primers consisting of SEQ ID NOs: 229 and 230 for CCNJL_9070; One set of primers consisting of SEQ ID NOs: 213 and 214 for KCNB2_9128; One set of primers consisting of SEQ ID NOs: 226 and 227 for IGFBP7_6412; One set of primers consisting of SEQ ID NOs: 232 and 233 for WNT3A_5487; One set of primers consisting of SEQ ID NOs: 147 and 148 for SERPINB9_3479; one set of primers consisting of SEQ ID NOs: 150 and 151 for FLOT1_1665; One set of primers consisting of SEQ ID NOs: 153 and 154 for HCG4P6_4618; One set of primers consisting of SEQ ID NOs: 156 and 157 for CHST11_2206; one set of primers consisting of SEQ ID NOs: 159 and 160 for MAX.chr12.485; One set of primers consisting of SEQ ID NOs: 162 and 163 for GRASP_0932; One set of primers consisting of SEQ ID NOs: 165 and 166 for GAS6_6425; One set of primers consisting of SEQ ID NOs: 174 and 175 for MAX.chr3.193; One set of primers consisting of SEQ ID NOs: 198 and 199 for MAX.chr2.971; One set of primers consisting of SEQ ID NOs: 180 and 181 for HES5_0840; One set of primers consisting of SEQ ID NOs: 189 and 190 for TPM4_8037; One set of primers consisting of SEQ ID NOs: 171 and 172 for SLCO3A1_6187; A set of primers consisting of SEQ ID NOs: 195 and 196 for ITPRIPL1_1244, and one set of primers consisting of SEQ ID NOs: 201 and 202 for AKR1B1_3644; The method according to

[98] , comprising:

[0305]

[101] 1. A method for measuring the methylation level of one or more CpG sites in a gene selected from ACOXL, AKR1B1_3644, ANXA2, CHST11_2206, FLJ45983, GAS6, GRASP, HAPLN3, HCG4P6, HES5_0822, ITPRIPL1, KCNK4, MAX.chr1.61519554-61519667, MAX.chr2.97193166-97193253, MAX.chr3.193, MAX.chr3.72788028-72788112, RAI1_7469, RASSF2, SERPINB9_3389, SLC4A11, and TPM4_8047, comprising: a) extracting genomic DNA from a biological sample of a human individual suspected of having or having prostate cancer; b) treating the extracted genomic DNA with bisulfite; c) amplifying the bisulfite-treated genomic DNA with primers consisting of a primer pair specific for the one or more CpG sites in a gene selected from ACOXL, AKR1B1_3644, ANXA2, CHST11_2206, FLJ45983, GAS6, GRASP, HAPLN3, HCG4P6, HES5_0822, ITPRIPL1, KCNK4, MAX.chr1.61519554-61519667, MAX.chr2.97193166-97193253, MAX.chr3.193, MAX.chr3.72788028-72788112, RAI1_7469, RASSF2, SERPINB9_3389, SLC4A11, and TPM4_8047; and d) ACOXL, AKR1B1_3644, ANXA2, CHST11_2206, FLJ45983, GAS6, GRASP, HAPLN3, HCG4P6, HES5_0822, ITPRIPL1, KCNK4, MAX.chr1 by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis or bisulfite genomic sequencing PCR. measuring the methylation level of one or more CpG sites in genes selected from: MAX.chr2.61519554-61519667, MAX.chr2.97193166-97193253, MAX.chr3.193, MAX.chr3.72788028-72788112, RAI1_7469, RASSF2, SERPINB9_3389, SLC4A11, and TPM4_8047; The method comprising:

[0306]

[102] The method of claim 101, wherein the sample is a prostate tissue sample.

[0307]

[103] The primer pairs specific for ACOXL, AKR1B1_3644, ANXA2, CHST11_2206, FLJ45983, GAS6, GRASP, HAPLN3, HCG4P6, HES5_0822, ITPRIPL1, KCNK4, MAX.chr1.61519554-61519667, MAX.chr2.97193166-97193253, MAX.chr3.193, MAX.chr3.72788028-72788112, RAI1_7469, RASSF2, SERPINB9_3389, SLC4A11, and TPM4_8047 are: A set of primers consisting of SEQ ID NOs: 93 and 94 for ACOXL; one set of primers consisting of SEQ ID NOs: 27 and 28 for AKR1B1_3644; One set of primers consisting of SEQ ID NOs: 89 and 90 for ANXA2; One set of primers consisting of SEQ ID NOs: 85 and 86 for CHST11_2206; One set of primers consisting of SEQ ID NOs: 31 and 32 for FLJ45983; one set of primers consisting of SEQ ID NOs: 117 and 118 for GAS6; a set of primers consisting of SEQ ID NOs: 33 and 34 for GRASP; one set of primers consisting of SEQ ID NOs: 37 and 38 for HAPLN3; One set of primers consisting of SEQ ID NOs: 39 and 40 for HCG4P6; One set of primers consisting of SEQ ID NOs: 41 and 42 for HES5_0822; One set of primers consisting of SEQ ID NOs: 45 and 46 for ITPRIPL1; One set of primers consisting of SEQ ID NOs: 125 and 126 for KCNK4; One set of primers consisting of SEQ ID NOs: 91 and 92 for MAX.chr1.61519554-61519667; One set of primers consisting of SEQ ID NOs: 49 and 50 for MAX.chr2.97193166-97193253; One set of primers consisting of SEQ ID NOs: 51 and 52 for MAX.chr3.193; One set of primers consisting of SEQ ID NOs: 53 and 54 for MAX.chr3.72788028-72788112, One set of primers consisting of SEQ ID NOs: 55 and 56 for RAI1_7469; one set of primers consisting of SEQ ID NOs: 57 and 58 for RASSF2; one set of primers consisting of SEQ ID NOs: 129 and 130 for SERPINB9_3389; A set of primers consisting of SEQ ID NOs: 59 and 60 for SLC4A11, and One set of primers consisting of SEQ ID NOs: 123 and 124 for TPM4_8047; The method according to

[101] , comprising:

[0308]

[104] 1. A method for measuring the methylation level of one or more CpG sites in a gene selected from max.chr3.193, HES5, SLCO3A1, and TPM4_8047, comprising: a) extracting genomic DNA from a plasma sample of a human individual suspected of having or having prostate cancer; b) treating the extracted genomic DNA with bisulfite; c) amplifying the bisulfite-treated genomic DNA with primers consisting of a primer pair specific for the one or more CpG sites in a gene selected from max.chr3.193, HES5, SLCO3A1, and TPM4_8047; and d) measuring the methylation level of one or more CpG sites in a gene selected from max.chr3.193, HES5, SLCO3A1, and TPM4_8047 by methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, or bisulfite genomic sequencing PCR; The method comprising:

[0309]

[105] The primer pairs specific for max.chr3.193, HES5, SLCO3A1, and TPM4_8047 are: One set of primers consisting of SEQ ID NOs: 174 and 175 for max.chr3.193; A set of primers consisting of SEQ ID NOs: 180 and 181 for HES5, a set of primers consisting of SEQ ID NOs: 171 and 172 for SLCO3A1, and One set of primers consisting of SEQ ID NOs: 189 and 190 for TPM4_8047; The method according to

[98] , comprising: [Example]

[0310] Example I This example provides materials and methods for Examples II, III, IV, V, and VI.

[0311] Examples II, III, IV, V, and VI demonstrate that methylated DNA markers can distinguish prostate tissue (e.g., cancerous prostate tissue and / or non-cancerous prostate tissue) from non-prostate tissue (e.g., white blood cells), that methylated DNA markers can distinguish cancerous prostate tissue from non-cancerous prostate tissue, that methylated DNA markers can distinguish very high-grade cancerous prostate tissue (e.g., Gleason score of 7.0 or greater (e.g., 7, 8, 9, 10)) from low-grade cancerous prostate tissue (e.g., Gleason score of less than 7 (e.g., 6)), and that methylated DNA markers can detect PCa in blood samples.

[0312] These experiments involved five stages. First, DNA methylation marker discovery was performed using Reduced Representation Bisulfite Sequencing (RRBS) (see, e.g., Gu H, et al., Nat Methods 2010;7:133-6) on DNA extracted from prostate cancer (PCa) tissues (both Gleason score 6 and 7+), normal prostates, and buffy coat samples from healthy volunteers. Second, discriminant variably methylated regions (DMRs) were identified using strict filtering criteria, and these sequences were used to develop real-time methylation-specific PCR assays (qMSP). These assays were then applied to the original sample set to ensure reproducibility of results (technical validation). Third, the best candidate markers were selected for qMSP biological validation on DNA extracted from independent archival case and control tissues. Fourth, candidate marker sequences were compared in silico across the pan-cancer RRBS sequencing dataset, and the extent of site-specific methylation was measured for each marker. Fifth, a set of high-performing PCa markers was selected for testing in blinded, independent plasma samples to evaluate PCa detection in clinical media. Research subjects and samples This study was approved by the Mayo Clinic Institutional Review Board (Rochester, MN). Fresh-frozen (FF) tissue, plasma, and buffy coat samples were provided by an IRB-approved patient biobank. Tumor tissue sections were reviewed twice by an expert GI pathologist to confirm the diagnosis and estimate neoplastic cellularity. These sections were then macrodissected. Genomic DNA was purified using a QiaAmp Mini kit (Qiagen, Valencia, CA) and then repurified using an AMPure XP kit (Beckman Coulter, Brea, CA). Reduced Representation Bisulfite Sequencing Library Preparation 150 ng of DNA from each sample was diluted in 26 μl of Te buffer (5.77 ng / μl). This was digested overnight with 1 μl (20 units) of MspI in a 1x final concentration of CutSmart buffer (New England Biolabs). 3' overhangs were end-repaired and A-tailed with a mixture of 0.6 μl of 100 mM dATP, 0.06 μl of 100 mM dCTP, and 0.06 μl of dGTP containing 2 μl (10 units) of Klenow DNA polymerase (New England Biolabs). The product was incubated for 20 minutes at 30°C, 20 minutes at 37°C, and held at 4°C. After end-repair, the product was purified with 2x Agencourt Ampure XP beads (Beckman Coulter), washed twice with 70% EtOH, and eluted in 20 μl of water. Illumina adapters were ligated to this product using 1 μl of T4 ligase (400 units) in 1x T4 ligase buffer, incubated overnight at 16°C. The product was then heat-inactivated at 65°C for 20 minutes. After ligation, the product was purified with 2x Agencourt Ampure XP beads (Beckman Coulter), washed twice with 70% EtOH, and eluted in 47 μl of water. 45 μl of the product was bisulfite converted using the EZ-96 DNA Methylation Kit (Zymo Research) as described in their protocol. The converted product was purified with 2x Agencourt Ampure XP beads (Beckman Coulter), washed twice with 70% EtOH, and eluted in 22 μl of water. Illumina index was added via PCR using 16ul of bisulfite converted product, 1ul (2.5 units) of PfuTurbo Cx hotstart DNA polymerase, 0.5ul of dNTPs (25mM each), 6ul of Illumina index (2.5uM each), and 1x PfuTurbo Cx hotstart DNA polymerase buffer in a total volume of 50ul.The product was dual-sized by using Ampure XP at 0.7x and then collecting the supernatant and pooling the bead-bound product with Ampure XP at 1.2x. The final product was eluted in 40 μl. DNA molecular weight yield was determined by Pico Green (Molecular Probes) and measured on a Tecan fluorometer. DNA size was determined on an Agilent 2100 (Agilent) with a High Sensitivity DNA chip. The molar concentration of the product was calculated, and these samples were pooled equimolarly at 10 nM, with 4 samples / pool. Massively Parallel Sequencing and Bioinformatics These samples were loaded onto flow cells according to randomized lane assignments. Sequencing was performed on an Illumina HiSeq2000 by the next-generation sequencing core at the Mayo Clinic Medical Genome Facility. Reads were unidirectional for 101 cycles. Each flow cell lane generated 100 to 120 million reads, sufficient for a median coverage of 30 to 50x sequencing depth for aligned sequences. Standard Illumina pipeline software called bases and generated reads in fastq format. SAAP-RRBS (Streamline Analysis and Annotation Pipeline for Reduced Representation Bisulfite Sequencing) was used for sequence read evaluation and cleanup, alignment to the reference genome, methylation status extraction, and CpG reporting and annotation. CpGs with low coverage (≤10) were excluded. Tertiary analysis consisted of filtering out uninformative or low-sample coverage CpGs and identifying methylated CpG regions with low background and high-density clusters within a sliding 100-bp window. The read depth criterion was based on the desired statistical power to detect a 10% difference in percent methylation between cases and controls. Statistical significance was determined by logistic regression of the methylation percentage per DMR based on read counts. To account for varying read depth across individual subjects, an overdispersed logistic regression model was used, where the dispersion parameter was estimated using the Pearson chi-square statistic of the residuals from the fitted model. DMRs were ranked according to their significance level, and additionally considered when percent methylation in the control group was ≤1% and ≥10% in the cancer. For most organ sites, this resulted in hundreds of potential candidates. Additional filters utilized were the receiver operating characteristic curve (AUC), % methylation case / control fold change (FC), and the extent under co-methylation between CpG positive samples throughout the DMR (and their absence in controls). Technical and biological tissue validation Methylation-specific PCR (MSP) marker assays were developed for the 120 most promising DMRs from the PCa discovery dataset, as determined by the criteria listed above. Primers were designed either by software (Methprimer - University of California, San Francisco, CA) or manually. These assays were rigorously tested and optimized by SYBR Green qPCR on bisulfite-converted controls (methylated and unmethylated genomic DNA), unconverted controls, and non-template controls. Assays that cross-reacted with the negative controls were either redesigned or discarded. Additionally, melting curve analysis was performed to ensure specific amplification had occurred. For the technical validation phase, the same samples used for RRBS discovery were retested by qMSP. A β-actin assay, designed to be methylation-blind, was used as a common factor representing total DNA copies. This data was analyzed by logistic regression, comparing the AUC and signal to background results with the discovery values. Approximately 27% of markers were below expectations and were removed. The remainder (N=72) were tested on an extended set of independent tissue samples by qMSP. These results were analyzed logistically, and outcome metrics were AUC, FC, and % methylation of robust case samples. Whole organ validation To evaluate how the best methylation markers performed outside the prostate, a comparative CpG % methylation matrix was constructed using sequencing reads for the validation DMRs across prostate samples compared to other major cancers previously sequenced (colon, pancreas, esophagus, liver, and stomach). A final panel of markers was selected and tested in plasma based on 1) overall performance in the tissue validation phase and 2) the site-specific properties of these markers across other cancers. To best detect PCa in blood, a robust marker panel was selected that exhibited both a general and prostate-specific cancer signal given the excess of non-PCa DNA. QuART assay design and plasma validation DNA was extracted from 3-4 mL of deposited, frozen plasma by the following automated silica bead method (see, e.g., U.S. Patent Application No. 15 / 335,111):

[0313] [Table 1]

[0314] The DNA was then converted to bisulfite and purified using the following method:

[0315] [Table 2]

[0316] Samples (10 μL) were then run on an ABI real-time PCR instrument in QuARTs-X (see, e.g., U.S. Patent Application No. 15 / 335,096) format using primers and probes developed from the DMR sequences. Plasmids containing the marker sequences of interest were obtained from Genscript and diluted in 1X QuART reagent to a nominal concentration of 1 copy per 15 μL reaction. This reaction mixture was dispensed into each of 96 wells and cycled for 45 cycles on a LightCycler, during which data was collected. Wells were requested to either contain or not contain sample. A Poisson random variable was set to 1, and mean success rate values were entered by trial and error. These values were used to calculate the cumulative probability for that value. The correct mean success rate, in this case the copy number, was found when the cumulative probability was equal to the percentage of wells containing a signal. These plasmids were diluted and used as assay standards.

[0317] QuARTs-X was performed by first creating a preamplification plate of samples run with primers for up to 12 targets that underwent 11 cycles of amplification. This product was then diluted 1:9 and used as template for subsequent QuART reactions involving only three targets in a triplicate reaction. The standards used to calculate strand number were not preamplified. Preamplifying the samples, but not the strands, increased the sensitivity of the assay.

[0318] Results were analyzed by regression partitioning (rPart). Combining multiple methylation markers into a single risk score using logistic regression is standard practice. However, it is difficult to identify and / or model higher-order interactions between markers in a logistic model. This limits the predictive power of a panel of markers when these effects are present. Regression partitioning trees (rPart) are a decision tree approach that can identify higher-order interactions between markers in a way that maximizes the predictive accuracy of the marker panel. Example II. This example describes the RRBS results and technical validation results.

[0319] PCa yields numerous discriminatory DMRs, many of which have not been previously identified. Comparing methylation in normal prostate and PCa samples, we identified 256 regions that met the cutoffs of AUC > 0.85, FC > 20, and p-value < 0.05. 22 of these regions had an AUC of 1. When comparing methylation in PCa and normal prostate with its buffy coat samples, 1,895 regions exceeded the cutoff, and 827 had perfect AUC scores. FC in both comparisons extended into the hundreds and thousands, respectively. We searched for potential DMRs that distinguished Gleason 7+ PCa (high-grade, treatment indicated cancer) versus Gleason 6 PCa (low-grade, treatment usually not required). We observed 129 DMRs with FC > 2 (7+ / 6), with the highest FC = 72.

[0320] The second step in the biomarker development process was to address the uncertainties arising from the relatively small sample sizes in the initial discovery phase. Retesting the same samples using different technological platforms on a smaller number of DMRs or candidate markers was the first step toward this goal. Real-time or quantitative methylation-specific PCR (qMSP) using SYBR Green is an easy-to-use method with high analytical sensitivity and specificity.

[0321] DMRs were selected by taking the top candidates from all three comparisons by increasing the cutoff until a manageable number of regions (N=120) was obtained (see Table 1).

[0322] [Table 3] JPEG2025114618000004.jpg229169JPEG2025114618000005.jpg238169JPEG2025114618000006.jpg239169JPEG2025114618000007.jpg62169

[0323] DMR sequences also had to show significant co-methylation or adjacent methylation throughout the addressed CpGs on a strand-by-strand basis. qMSP and other amplification-based methods work best when all addressed CpGs are methylated (in cases) and unmethylated (in controls). After QC testing on strands (bisulfite-treated universally methylated genomic DNA) and control samples (bisulfite-treated unmethylated genomic DNA, unconverted genomic DNA, etc.), 99 regions were implemented with sufficient linearity, specificity, and robustness to be used to retest Phase 1 samples. Logistically analyzed results for most assays were comparable to the percent methylation derived from the sequencing step. Total DNA strand methylation in all samples exceeded 100, with averages within 1,000 numbers. Z markers (see, e.g., U.S. Patent Application No. 14 / 966,617) continued to demonstrate an AUC of 1 and very high FC compared to normal buffy coat samples. When examining the cancer-to-benign ratio for candidate Z markers, approximately half were 1:1, with the remainder having a ratio between 2 and 10 (median Z). Of the cancer-to-benign markers, 34 contained an AUC within the range of 0.95 to 1. All were negative for buffy coat samples. Markers that distinguished Gleason 7+ from Gleason 6 cancers in the phase 1 results generally continued their performance in validation studies. Twenty-six markers had FCs greater than 2, with a maximum of 292.

[0324] Table 2 shows the DMRs identified in Table 1: 1) the area under the curve (AUC) for prostate cells with a Gleason score of 6 or greater versus benign prostate cells, 2) the fold change (FC) for prostate cells with a Gleason score of 6 or greater versus benign prostate cells, and 3) the fold change (FC) for prostate cells with a Gleason score of 6 or greater versus buffy (standard).

[0325] [Table 4] JPEG2025114618000009.jpg238169JPEG2025114618000010.jpg235169JPEG2025114618000011.jpg165169

[0326] Example III. The best-performing candidate markers, identified through the experiments described in Example II, were selected for qMSP biological validation on DNA extracted from independent archival case and control tissues.

[0327] From the Phase 2 study, 73 markers were selected (see Table 3) and run on an independent set of prostate tissue (N=35 normal prostate, 19 Gleason score 6, 31 Gleason score 7+) and normal buffy coat (N=36) samples. 27 markers were removed that either had a sub-0.85 AUC primarily in the cancer vs. benign set or less-than-perfect positive methylation in the Z marker set. The majority of Gleason 7+ vs. Gleason 6 markers were carried forward. All samples were assayed by qMSP as before. The DMR genomic coordinates and AUCs for Gleason score 7+ vs. normal benign prostate tissue for the 73 assays are listed in Table 3, and the respective primer sequences are provided in Table 4.

[0328] [Table 5] JPEG2025114618000013.jpg236169JPEG2025114618000014.jpg82169

[0329] [Table 6] JPEG2025114618000016.jpg234169JPEG2025114618000017.jpg234169JPEG2025114618000018.jpg238169JPEG20251146180 00019.jpg237169JPEG2025114618000020.jpg239169JPEG2025114618000021.jpg237169JPEG2025114618000022.jpg227169

[0330] The AUC was overall excellent, but not as good as would have been expected in this extended, independent set in earlier validation. As shown in Table 3, the 20 markers had AUC values (Gleason 7+ vs. normal prostate) in the range of 0.95-0.99 and FC17-164. The % methylation in the buffy coat samples was negligible except for GRASP, which had a single outlier.

[0331] Because clinical follow-up data were available for prostate cancer cases, it was decided to explore the prognostic aspects of candidate epigenetic markers. Using regression partitioning (rPart), a mathematical method for discovering and / or modeling higher-order interactions between markers within a logistic model, five prognostic markers (FAM78A, WNT3A, GAS6, LOC100129726, and MAX.chr3.727) were selected. Risk groupings were defined by methylated DNA markers, which added significant prognostic content in predicting progression-free survival compared with Gleason scoring (p<0.0001), whereas Gleason scoring did not contain any additional value compared with methylated DNA marker risk groupings (p=0.2174). Example IV. Additional experiments were conducted to identify markers that can distinguish between PCa Gleason scores above 7 versus Gleason scores of 6 in prostate tissue. Experiments such as these utilized QuARTs-X (Quantitative Allele-Specific Real-Time Target and Signal Assay) (see, e.g., U.S. Patent Application No. 15 / 335,096). Table 5 shows the marker sensitivity at 100% and fold change for PCa Gleason 7+ versus Gleason 6 in prostate tissue (oligo sequences are provided in Table 6).

[0332] [Table 7]

[0333] [Table 8] JPEG2025114618000025.jpg215169JPEG2025114618000026.jpg232169JPEG2025114618000027.jpg74169

[0334] Example V We performed experiments in which candidate marker sequences were compared in silico across pan-cancer RRBS sequencing datasets and measured the degree of site-specific methylation for each marker.

[0335] We demonstrate DNA methylation signatures that accurately predict tumor location within human anatomy. To better define organ-site-associated specificity, we constructed an in silico CpGx sample matrix of RRBSs, derived from methylation values for each of 73 DMRs / markers across multiple cancers and organ tissues. These included prostate, liver, colon, pancreas, lung, esophagus, stomach, and bile duct tissues. Location specificity could be modeled by quantitative methylation differences between organ sites and the degree of adjacent methylation (defined by pattern recognition) across DMRs. As shown in Table 7, eight markers demonstrated specificity exclusively for prostate cancer, while 11 markers were universal across all cancers and tissues. Among them, there were clusters of similar or different degrees of specificity, such as prostate / liver, prostate / colon / liver, etc. One subset of prostate markers remains undefined due to missing DMR sequences in aligned reads from other cancers.

[0336] [Table 9] JPEG2025114618000029.jpg239169JPEG2025114618000030.jpg232169JPEG2025114618000031.jpg237169JPEG2025114618000032.jpg121169

[0337] Example VI Experiments were performed to select a set of high performance PCa markers to be tested in blinded independent plasma samples and evaluated for PCa detection in clinical media.

[0338] For the development of the multiplexed QuARTs-X (quantitative allele-specific real-time target and signal assay), we selected 25 DMRs / markers (see Table 8) that demonstrated the best combination of performance metrics suitable for analyte detection in complex biological media (e.g., blood plasma), a testing platform frequently used by other cancer plasma researchers. After initial in silico testing for DMR sequence uniqueness, design filters, and QC testing on pooled plasma controls, 17 designs were developed for testing on retrospectively collected and frozen plasma samples from the Mayo Prostate Cancer Biobank. Nine three-stage reactions and one two-stage reaction were developed, each of which included a control β-actin assay, unaffected by methylation. Two additional process controls were also tested. The QuARTs-X assay is listed in Table 8 (see Table 6 for primer and probe information). Final marker strands (copies) were normalized to the β-actin control and expressed as % methylation. The top four markers were max.chr3.193, HES5, SLCO3A1, and TPM4_8047. In conjunction with rPart modeling, the sensitivity and specificity for detecting PCa in blood samples were 78% and 91%, respectively.

[0339] [Table 10]

[0340] Example VII. Treatment decisions for prostate cancer are often guided by Gleason grade, which is subjective and lacks precision. During discovery and early validation, methylated DNA markers (MDMs) were identified with prognostic relevance (see Example I). Further experiments were performed to evaluate the value of novel MDMs in predicting biochemical recurrence after radical prostatectomy (RP) using archival tissue from an independent group with >12-year follow-up.

[0341] Of 737 men who underwent radical prostatectomy (RP) in 2004, 446 were randomly selected, and 155 met quality criteria. Formalin-fixed, paraffin-embedded (FFPE) tissue blocks were utilized. Expert pathologists reviewed all specimens in a blinded manner using the latest Gleason criteria and marked tumors for macrodissection. Genomic DNA was purified using the QiaAmp FFPE Tissue Kit (Qiagen) and quantified by Picogreen fluorescence. Because FFPE DNA can be highly degraded, samples were tested for amplifiable genomic equivalents using a 100-bp β-actin amplification assay. DNA was then treated with sodium bisulfite and purified (Zymo Research).

[0342] Twenty-three MDMs were selected and tested for the samples. The 23 MDMs were arrived at by performing recursive partitioning analysis (rPART) on independent tissue validation results using 73 MDMs. All patients used in that study (Example 1) included outcome data in their clinical records. Specifically, the experiment performed rPART (in silico) on 1,000 bootstrap samples to find the MDMs that appeared most frequently in the modeling tree. These were then ranked by frequency (high to low), and the top MDMs were selected (see Table 10). Blinded MSP assays were performed as before against dilutions of universal methylation standards and appropriate negative and positive controls. Raw counts were normalized to the overall β-actin count for each sample. Recurrence was defined as a PSA >0.4 ng / mL. To assign recurrence risk, the top MDMs were selected by a regression partitioning tree model and grouped by quartile (M1 (lowest) to M4 (highest)). The prognostic value of MDM and Gleason grade group (GGG) was evaluated and compared based on their concordance with outcomes after RP. The following markers were identified as optimal for predicting recurrence rate: WNT3A, LOC100129726, FNBP1, GSDMD, ITPRIPL1, Chr1.61519554, and Chr17.77786040.

[0343] [Table 11] JPEG2025114618000035.jpg223169

[0344] Example VIII. Additional experiments were performed to identify markers that could distinguish 1) PCa in prostate tissue with a Gleason score greater than 7 versus a Gleason score of 6, and 2) PCa with a Gleason score greater than 6 versus non-cancerous prostate tissue. Experiments such as these utilized QuARTs-X (Quantitative Allele-Specific Real-Time Target and Signal Assay) (see, e.g., U.S. Patent Application No. 15 / 335,096). DNA extraction Frozen DNA tissue samples with known clinical information were obtained from the Mayo Clinic repository. DNA was extracted from the tissue using the DNeasy Blood & Tissue kit from Qiagen according to the manufacturer's protocol. Approximately 100 ng of extracted DNA was subjected to a bisulfite conversion reaction. Bisulfite conversion and purification of DNA

[0345] [Table 12]

[0346] [Table 13]

[0347] The following procedures were followed for bisulfite conversion and purification of DNA. 1. Add 10 μL of inhibitor solution to each well in a deep well plate (DWP). 2. Add 80 μL of each sample into DWP. 3. Mix carefully by pipetting with a pipette set to 30-40 µL, avoiding foaming. 4. Seal and centrifuge the DWP at 3000 x g for 1 minute. 5. Incubate at 42°C for 20 minutes. 6. Add 120 μL of BIS SLN to each well. Cool for 7-8 minutes. 8. Incubate at 65°C for 75 minutes, mixing during the first 3 minutes. 9. Add 750 μL of BND SLN. 10. Premixing silica beads (BND BDS) and adding 50 μL of silica beads (BND BDS) to the wells of the DWP. 11. Mix on a heater shaker at 30°C and 1,200 rpm for 30 minutes. After 12.5 minutes on the plate magnet to allow the beads to bind, the solution is aspirated and discarded. After adding 13.1 mL of wash buffer (CNV WSH), the plate is transferred to a heater shaker and mixed at 1,200 rpm for 3 minutes. After 14.5 minutes on the plate magnet to allow the beads to bind, the solution is aspirated and discarded. 15. After adding 0.25 mL of wash buffer (CNV WSH), move the plate to a heater shaker and mix at 1,200 rpm for 3 minutes. 16. After 2 minutes on the magnet to bind the beads, aspirate and discard the solution. 17. Add 0.2 mL of desulfonation buffer (DES-SLN) and mix at 1,200 rpm for 7 minutes at 30°C. 18. After 2 minutes on the magnet to bind the beads, aspirate and discard the solution. 19. After adding 0.25 mL of wash buffer (CNV WSH), move the plate to a heater shaker and mix at 1,200 rpm for 3 minutes. 20. After 2 minutes on the magnet to bind the beads, aspirate and discard the solution. 21. After adding 0.25 mL of wash buffer (CNV WSH), move the plate to a heater shaker and mix at 1,200 rpm for 3 minutes. 22. After 2 minutes on the magnet to bind the beads, aspirate and discard the solution. 23. Allow plates to dry by transferring to a heater shaker and incubating at 70°C for 15 minutes with mixing at 1,200 rpm. 24. Add 80 μL of elution buffer (ELU BFR) across all samples in DWP. 25. Incubate at 65°C for 25 minutes while mixing at 1,200 rpm. 26. Manually transfer the eluate to a 96-well plate and store at -80°C after sealing the plate with a foil seal. 27. The recoverable / transferable volume is approximately 65 μL. QuARTS-X for methylated DNA detection and quantification Multiplex PCR (mPCR) setup: 1. Prepare a 10x primer mix containing forward and reverse primers for each methylated marker of interest to a final concentration of 750 nM each. Use 10 mM Tris-HCl, pH 8, 0.1 mM EDTA as the diluent. 2. Prepare a 10x mPCR buffer containing 100 mM MOPS, pH 7.5, 75 mM MgCl2, 0.08% Tween 20, 0.08% IGEPAL CA-630, and 2.5 mM dNTPs. 3- Prepare the mPCR master mix as follows:

[0348] [Table 14]

[0349] 4- Thaw DNA and spin down plates. Add 5-25 μL of the master mix to a 96-well ABI Veriti plate. 6- Transfer 50 μL of each sample to each well and mix each sample by pipetting up and down several times. 7- The plate is sealed with an aluminum foil seal. 8- Place heated lid in thermal cycler and proceed to cycle using the following profile "QX12 cycle":

[0350] [Table 15]

[0351] 9- After the incubation was completed, 1 to 10-fold dilutions of the amplicon were performed as follows: a. Obtain a deep well plate and transfer 180 μL of 10 mM Tris-HCl, pH 8, 0.1 mM EDTA to each well. b. Carefully punch holes in the foil seal of the amplified plate with a 96-well stamp. c. Using a new tip and a pipettor set to 200 μL for 50 μL (do not generate aerosols), mix 75 μL of the amplified sample by repeated pipetting (do not use a shaker for mixing). d. Using a new tip and a pipettor set to 20 μL for 20 μL (does not generate aerosols), add 20 μL of amplified sample to each pre-filled well. e. Using a new tip and a pipettor set to 200 μL for 100 μL (do not generate aerosols), mix the diluted sample by repeated pipetting (do not use a shaker for mixing). f. Seal the diluted plate with a plastic seal. g. Centrifuge the diluted plate at 1,000 rpm for 1 minute. h. Seal any remaining undiluted mPCR product with a new aluminum foil seal. Place at -80°C. Manual QuARTS Assay Setup: 1 - Thaw fish DNA dilution solution (20 ng / µL) and use it to dilute the plasmid calibrators required for this assay. Use the following table as a dilution guideline:

[0352] [Table 16]

[0353] 2- Prepare a 10x triplex QuARTS oligo mix using the following table for markers A, B, and C:

[0354] [Table 17]

[0355] 3- Prepare the QuARTS Master Mix using the following table:

[0356] [Table 18]

[0357] Using four 96-well ABI plates, pipette 20 µL of QuARTS Master Mix into each well. Add 5-10 μL of the appropriate calibrator or diluted mPCR sample. 6- The plate is sealed with an ABI clear plastic seal. 7- Centrifuge the plate using 3000 rpm for 1 minute. 8 - After placing the plate in an ABI thermal cycler programmed to carry out the following thermal protocol "Quarts 5+40", the instrument is started.

[0358] [Table 19]

[0359] A. Automated QuARTS Setup: Thaw 1 tube of diluted fish DNA (20 ng / µL), 2 tubes of 1.62x oligo mix, and prepare the calibrator series required to place it on the Hamilton STARlet Deck. 2- Vortex and centrifuge all reagents before loading onto the Hamilton STARlet Deck. 3- Place the deep well plate containing the samples on the magnet. 4- Place a full tray of 50 μL CORE tips on the deck as shown in the diagram below. 5- Place 1000 μL CORE tips on the deck for at least one full row as shown in the diagram below. 6- With the barcode facing the front of the machine (with respect to the A1 well in the rear left corner), place an empty ABI 96 well plate onto the STARlet deck as shown in the diagram below. 7-Follow the on-screen deck layout and software instructions to load reagents into the indicated carrier positions (see Figure 2 below). 8- Place two empty, uncapped barcoded tubes on the deck as shown in the diagram below. Run the "QuARTSONLYV4.0_BA_20160127" method on 9-Hamilton. 10- Upon completion of the method, the 96-well QuARTS plate is removed and sealed with a clear plastic cover. 11- Centrifuge the plate using 3000 rpm for 1 minute. 12-The instrument is then started after placing the plate in an ABI thermal cycler which is programmed to carry out the following thermal protocol: "Quarts5+40". result These experiments 1) differentially methylated DNA markers (SERPINB9_3479, GRASP_0932, SLCO3A1_6187, ITPRIPL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487) distinguish very high-grade cancerous prostate tissue (e.g., Gleason score of 7.0 or higher (e.g., 7, 8, 9, 10)) from low-grade cancerous prostate tissue (e.g., Gleason score of less than 7 (e.g., 6)), and 2) Differentially methylated DNA markers (SERPINB9_3479, FLOT1_1665, HCG4P6_4618, CHST11_2206, MAX.chr12.485, GRASP_0932, GAS6_6425, MAX.chr3.193, MAX.chr2.971_3164, MAX.chr3.727_8028, HES5_0840, TPM4_8037, SLCO3A1_6187, ITP RIPL1_1244, AKR1B1_3644, RASGRF2_6325, ZNF655_6075, PAMR1_7364, ST6GALNAC2_1113, CCNJL_9070, KCNB2_9128, IGFBP7_6412, and WNT3A_5487) to distinguish cancerous prostate tissue (e.g., a Gleason score of 6.0 or greater (e.g., 6, 7, 8, 9, 10)) from non-cancerous prostate tissue; was identified.

[0360] Table 11 shows the % methylation for normal tissue, the % methylation for prostate tissue with a Gleason score of 6, and the % methylation for prostate tissue with a Gleason score between 7 and 10 (Table 12 provides oligo sequences; Table 13 provides DMR information).

[0361] [Table 20]

[0362] Using a logistic regression fit of percent methylation compared to ACTB for this data with a 100% cutoff, two markers, FLOT1 and MAX.Chr3.193, with an AUC of 0.99, allowed for the prediction of cancer from normal with 98.5% sensitivity. For the prediction of 6 vs. 6+, a logistic regression fit of percent methylation compared to ACTB for the data with nine markers (AUC = 0.96) predicted Gleason 6+ with 92.8% sensitivity at 91.7% specificity. The markers were GRASP, GAS6, MAX.chr3.193, MAX.chr2.971, TPM4, ITPRIPL2, AKR1B1, ZNF655, and WNT3A.

[0363] [Table 21] JPEG2025114618000046.jpg215169JPEG2025114618000047.jpg238169JPEG2025114618000048.jpg233169JPEG2025114618000049.jpg39169

[0364] [Table 22]

[0365] All publications and patents mentioned in the above specification are incorporated herein by reference in their entirety for all purposes. Various modifications and variations of the compositions, methods, and applications of the described technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in pharmacology, biochemistry, medicine, or related fields are intended to be within the scope of the following claims.

Claims

[Claim 1] 1. A method for characterizing a sample from a human patient, comprising: a) obtaining DNA from a human patient sample; b) assaying the methylation status of a DNA methylation marker comprising a base in a variably methylated region (DMR) selected from the group consisting of DMRs 1-140 from Tables 1 or 13; c) comparing the assayed methylation status of the one or more DNA methylation markers with reference methylation levels for the one or more DNA methylation markers for human patients without prostate cancer; The method comprising:

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