Detecting pancreatic ductal adenocarcinoma in plasma

Abstract

To provide technology for pancreatic ductal adenocarcinoma (PDAC) screening and particularly, but not exclusively, methods, compositions, and related uses for detecting the presence of PDAC.SOLUTION: A method is provided, comprising: treating genomic DNA in a biological sample with a reagent that modifies DNA in a methylation-specific manner; amplifying the treated genomic DNA using a set of primers for one or more selected genes; and determining the methylation level of the one or more genes by polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nuclease, mass-based separation, and target capture, thereby measuring the methylation level of the one or more genes in the biological sample of the human individual.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] Provided herein are technologies for pancreatic ductal adenocarcinoma (PDAC) screening, particularly but not limited to methods, compositions and related uses for detecting the presence of PDAC. [Background technology]

[0002] Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid malignancies. Despite its very low incidence, mainly due to a disappointing diagnosis, pancreatic ductal adenocarcinoma remains the fourth leading cause of cancer-related deaths in the modern world (see Garrido-Laguna I., et al., Nat. Rev. Clin. Oncol. 2015; 12: 319-334). In the past decades, significant improvements in screening and therapy for various solid cancers have been realized, significantly increasing the chances of cure for patients. Nevertheless, despite advances in pancreatic cancer research, the ratio of mortality to incidence has not changed significantly over the past decades. The 5-year survival rate is still only around 5-7%, and 1-year survival is achieved in less than 20% of cases (see Vincent A., et al., Lancet. 2011; 378: 607-620). This grim prognosis is primarily due to the lack of visible and characteristic symptoms and reliable biomarkers for early diagnosis, as well as aggressive metastatic spread leading to poor response to treatment (see Maitra A., Hruban RH Annu. Rev. Pathol. 2008;3:157-188).

[0003] There is a need for improved methods for detecting PDAC and its various subtypes.

[0004] The present invention addresses these needs. Summary of the Invention

[0005] Methylated DNA has been investigated as a potential class of biomarker in tissues of most tumor types. In many cases, DNA methyltransferases add methyl groups to DNA at cytosine-phosphate-guanine (CpG) island sites as an epigenetic control of gene expression. In a biologically intriguing mechanism, acquired methylation events in the promoter regions of tumor suppressor genes are thought to silence expression, thereby contributing to carcinogenesis. DNA methylation may 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 colon cancer, methylation markers offer superior specificity, are more extensively informative, and are more sensitive than individual DNA mutations (Zou et al (2007) Cancer Epidemiol Biomarkers Prev 16:2686-96).

[0006] 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 about 0.3% of high CpG density promoters (HCPs, defined as having more than 7% CpG sequences within a 300 base pair region) were methylated, whereas low CpG density regions (LCPs, defined as having less than 5% CpG sequences within a 300 base pair region) tend to be highly 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 the HCP sites that were more than 50% methylated were several established markers such as Wnt2, NDRG2, SFRP2, and BMP3 (Meissner et al. (2008) Nature 454:766-70).

[0007] 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 intriguing mechanism, acquired methylation events in the promoter regions of tumor suppressor genes are thought to silence their expression and contribute to carcinogenesis. DNA methylation may be a more chemically and biologically stable diagnostic tool than RNA or protein expression. Furthermore, in other cancers, such as sporadic colon cancer, aberrant methylation markers are more informative and sensitive, providing better specificity than individual DNA mutations.

[0008] Several methods are available to search for novel methylation markers. Microarray-based interrogation of CpG methylation is a rational, high-throughput approach, but this strategy is biased towards known regions of interest, mainly established tumor suppressor promoters. Alternative methods for genome-wide analysis of DNA methylation have been developed in the last decade. Three basic approaches exist. The first uses digestion of DNA with restriction enzymes that recognize specific methylation sites, followed by several possible analytical techniques that provide methylation data limited to the enzyme recognition sites or primers used to amplify the DNA in a quantification step (such as methylation-specific PCR (MSP)). The second approach uses antibodies directed against methyl-cytosine or other methylation-specific binding domains to enrich the methylated fraction of genomic DNA, followed by microarray analysis or sequencing to map the fragments to a reference genome. This approach does not provide single-nucleotide resolution of all methylation sites within a fragment. The third approach begins with bisulfite treatment of DNA to convert all unmethylated tyrosines to uracils, followed by restriction enzyme digestion and full sequencing of all fragments after binding to adaptor ligands. The choice of restriction enzyme allows for enrichment of fragments in CpG-dense regions, reducing the number of redundant sequences that may map to multiple gene locations during analysis.

[0009] RRBS provides CpG methylation status data at single nucleotide resolution for 80-90% of all CpG islands and most tumor suppressor promoters at moderate to high read coverage. Analysis of these reads identifies variably methylated regions (DMRs) in cancer case-control studies. Previous RRBS analysis of pancreatic cancer specimens revealed hundreds of DMRs, many of which were never associated with carcinogenesis and many of which were never annotated. Further validation studies on an independent set of tissue samples confirmed marker CpGs that were 100% sensitive and specific in terms of performance.

[0010] Provided herein are technologies for PDAC screening, particularly but not limited to methods, compositions and related uses for detecting the presence of PDAC.

[0011] Indeed, as described in Example I, experiments conducted during the process of identifying embodiments of the present invention identified a novel set of variably methylated regions (DMRs) for distinguishing PDAC from non-neoplastic control DNA in tissue and plasma samples.

[0012] Such experiments have listed and described 13 DNA methylation markers (AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, MAX.chr5.4295, NTRK3, PRKCB, RYR2, SHISA9, and ZNF781) that a) distinguish between PDAC and non-neoplastic controls within plasma samples (see Table 3, Example I), and b) distinguish between PDAC tissue and benign pancreatic tissue (see Table 4, Example 1).

[0013] Such experiments have identified the following markers and / or panels of markers for detecting PDAC in blood samples (e.g., plasma samples, whole blood samples, white blood cell samples, serum samples): AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, MAX.chr5.4295, NTRK3, PRKCB, RYR2, SHISA9, and ZNF781 (see Table 3, Example 1).

[0014] Such experiments have identified the following markers and / or panels of markers that are capable of distinguishing PDAC tissue from benign pancreatic tissue: AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, MAX.chr5.4295, NTRK3, PRKCB, RYR2, SHISA9, and ZNF781 (see Table 4, Example 1).

[0015] As described herein, the technology provides a number of methylated DNA markers and subsets thereof (e.g., sets of 2, 3, 4, 5, 6, 7, 8, or 13 markers) that are highly discriminatory across PDAC. Experiments apply selection filters to candidate markers to identify markers that provide high signal-to-noise ratios and low background levels to provide high specificity for screening or diagnosing PDAC.

[0016] In some embodiments, the technology relates to assessing the presence and methylation status of one or more of the markers identified herein in a biological sample (e.g., pancreatic tissue sample, blood sample). These markers include one or more variably methylated regions (DMRs) discussed herein, e.g., as shown in Table 1. The methylation status is assessed in embodiments of the technology. Thus, the technology provided herein is not limited in the way in which the methylation status of a gene is measured. For example, in some embodiments, the methylation status is measured by a genome scanning method. For example, one method includes restriction enzyme 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 regions of interest (digestion-Southern). In some embodiments, analyzing changes in methylation patterns comprises a PCR-based process that includes digestion of genomic DNA with methylation-sensitive or methylation-dependent 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) as well as for the 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). Such techniques use an internal primer that anneals to a PCR-generated template and terminates immediately 5' of the single nucleotide being assayed. A method using the "quantitative Ms-SNuPE assay" described in U.S. Pat. No. 7,037,650 is used in some embodiments.

[0017] When assessing methylation status, methylation status is often expressed as the proportion or percentage of individual DNA strands that are methylated at a specific site (e.g., at a single nucleotide, at a specific region or locus, at a relatively long sequence of interest, e.g., up to about 100bp, 200bp, 500bp, 1000bp subsequence or more of DNA) compared to the total population of DNA in a sample that contains that specific site. Traditionally, the amount of unmethylated nucleic acid is determined by PCR using a calibrator. 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, such as the QuARTS assay (e.g., those provided by U.S. Patent No. 8,361,720, and U.S. Patent Application Publication Nos. 2012 / 0122088 and 2012 / 0122106, which are incorporated herein by reference).

[0018] For example, in some embodiments, the method includes generating a standard curve for an unmethylated target by using an external standard. The standard curve is composed of at least two points and relates to the real-time Ct value of unmethylated DNA to a known quantitative standard. Then, a second standard curve for a methylated target is generated from at least two points and an external standard. This second standard curve relates to the Ct of methylated DNA to a known quantitative standard. Then, the Ct values ​​of the test samples are determined for the methylated and unmethylated populations, and the genome equivalent of DNA is calculated from the standard curve generated by the first two steps. The percentage of methylation at the site of interest is calculated from the amount of methylated DNA relative to the total amount of DNA in the population, for example, (number of methylated DNA) / (number of methylated DNA+number of unmethylated DNA)×100.

[0019] Also provided herein are compositions and kits for carrying out the methods. For example, in some embodiments, reagents (e.g., primers, probes) specific for one or more markers are provided, either alone or in sets (e.g., a set of primer pairs for amplifying multiple markers). Additional reagents for carrying out detection assays (e.g., enzymes, buffers, positive and negative controls for carrying out QuARTS, PCR, sequencing, bisulfite, or other assays) may also be provided. In some embodiments, the kit contains reagents that can modify DNA in a methylation-specific manner (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents). In some embodiments, kits are provided that contain one or more reagents necessary, sufficient, or useful for carrying out the methods. Reaction mixtures containing the reagents are also provided. Also provided are master mix reagent sets that contain multiple reagents that can be added to each other and / or to a test sample to complete a reaction mixture.

[0020] In some embodiments, the technology described herein relates to a programmable machine designed to perform a sequence of arithmetic or logical operations 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 a computer that includes a form of memory, elements for performing arithmetic and logical operations, and a processing element (e.g., a microprocessor) for executing a sequence of instructions (e.g., the methods provided herein) to read, manipulate, and store data. In some embodiments, the microprocessor is part of a system for: determining the methylation status (e.g., of one or more DMRs, e.g., DMRs 1-13 as shown in Table 1); comparing the methylation status (e.g., of one or more DMRs, e.g., DMRs 1-13 as shown in Table 1); generating a standard curve; determining the Ct value; calculating the methylation rate, frequency, or percentage (e.g., of one or more DMRs, e.g., DMRs 1-13 as shown in Table 1); identifying CpG islands; determining the specificity and / or sensitivity of an assay or marker; calculating the ROC curve and associated AUC; and sequence analysis; all as described herein or known in the art.

[0021] In some embodiments, the microprocessor or computer uses the methylation status data in an algorithm to predict the site of cancer.

[0022] In some embodiments, the software or hardware component receives the results of the multiple assays and determines and reports to a user a single value result indicative of cancer risk based on the results of the multiple assays (e.g., determining the methylation status of multiple DMRs (e.g., as shown in Table 1)). 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 (such as multiple DMRs as shown in Table 1). 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.

[0023] Some embodiments include storage media and memory components. The memory components (e.g., volatile and / or non-volatile memory) are used to store instructions (e.g., process embodiments provided herein) and / or data (e.g., work pieces such as methylation measurements, sequences, and statistical descriptions associated therewith). Some embodiments relate to systems further including 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).

[0024] Programmable machines relevant to the present technology include conventional existing technologies as well as technologies under development or yet to be developed (eg, quantum computers, chemical computers, DNA computers, optical computers, spintronics-based computers, etc.).

[0025] In some embodiments, the technology involves wired (e.g., metal cable, fiber optics) 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 machine resides on the network as a peer, and in some embodiments, the programmable machine has a client / server relationship.

[0026] In some embodiments, the data is stored on a computer readable storage medium, such as a hard disk, flash memory, optical media, floppy disk, or the like.

[0027] In some embodiments, the technology provided herein relates to multiple programmable devices that operate together to perform the methods 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 (with on-board CPU, storage, power, network interfaces, etc.) connected to a network (private, public, or Internet) by traditional network interfaces such as Ethernet, fiber optics, etc., or by wireless network technology.

[0028] For example, some embodiments provide a computer including a computer-readable medium. An embodiment includes a random access memory (RAM) coupled to a processor. The processor executes computer-executable program instructions stored in the memory. Such a processor may include a microprocessor, an ASIC, a state machine, or other processor, and may be any of a number of computer processors, such as processors from Intel Corporation of Santa Clara, California and Motorola Corporation of Schaumburg, Illinois. Such a processor may include or be in communication with a medium, e.g., a computer-readable medium, that stores instructions that, when executed by the processor, cause the processor to perform the steps described herein.

[0029] Examples of computer-readable media include, but are not limited to, electronic, optical, magnetic, or other storage or transmission devices capable of providing computer-readable instructions to a processor. Other examples of suitable media include, but are not limited to, floppy disks, CD-ROMs, DVDs, magnetic disks, memory chips, ROMs, RAMs, ASICs, configured processors, any optical media, any magnetic tape or other magnetic media, or any other media from which a computer processor can read instructions. Various other forms of computer-readable media can also transmit or convey instructions to a computer, including routers, both wired and wireless, private or public networks, or other transmission devices or channels. Instructions can include code from any suitable computer programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, and JavaScript.

[0030] The computer is connected to a network in some embodiments. The computer may also include several external or internal devices, such as a mouse, CD-ROM, DVD, keyboard, display, or other input or output devices. Examples of computers include personal computers, digital assistants, personal digital assistants, cellular phones, mobile phones, and the like. Examples of computer-based applications include personal computers, mobile phones, smartphones, pagers, digital tablets, laptop computers, Internet appliances, and other processor-based devices. Generally, computers relevant to aspects of the technology provided herein may be any type of processor-based platform running any operating system capable of supporting one or more programs including the technology provided herein, e.g., Microsoft Windows, Linux, UNIX, Mac OS X, etc. Some embodiments include personal computers running other application programs (e.g., applications). Applications may be stored 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 executable by a client device.

[0031] All such components, computers, and systems described herein as relating to the present technology may be logical or virtual.

[0032] Thus, provided herein is technology relating to a method of screening for PDAC in a sample obtained from a subject, the method comprising assaying the methylation status of a marker in a sample (e.g., pancreatic tissue) obtained from a subject (e.g., a blood sample), and identifying the subject as having PDAC if the methylation status of the marker differs from the methylation status of the marker assayed in a subject not having PDAC, wherein the marker comprises a base in a variably methylated region (DMR) selected from the group consisting of DMRs 1 to 13 as shown in Table 1.

[0033] In some embodiments, the sample obtained from the subject is a blood sample (e.g., a plasma sample, a whole blood sample, a white blood cell sample, a serum sample), and the methylation status of one or more of the following markers differs from the methylation status of one or more markers assayed in a subject without PDAC, indicating that the subject has PDAC: AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, MAX.chr5.4295, NTRK3, PRKCB, RYR2, SHISA9, and ZNF781 (see Table 3, Example 1).

[0034] In some embodiments, where the sample obtained from the subject is pancreatic tissue, the methylation status of one or more of the following markers differs from the methylation status of one or more markers assayed in a subject without PDAC, indicating that the subject has PDAC: AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, MAX.chr5.4295, NTRK3, PRKCB, RYR2, SHISA9, and ZNF781 (see Table 4, Example 1).

[0035] The technology further relates to identifying and differentiating PDAC from blood and / or tissue samples. Some embodiments provide methods that include assaying a plurality of markers (e.g., including assaying 2-13, 3-13, 4-13, 5-13, 6-13, 7-13, 8-13, 9-13, 10-13, 11-13, 12-13) (e.g., including assaying 13 or fewer markers; including assaying 13 or fewer markers) (e.g., including assaying 12 or fewer markers, 11 or fewer markers, 10 or fewer markers, 9 or fewer markers, 8 or fewer markers, 7 or fewer markers, 6 or fewer markers, 5 or fewer markers, 4 or fewer markers, 3 or fewer markers, 2 or fewer markers).

[0036] The technology is not limited to the methylation state that is assessed. In some embodiments, assessing the methylation state of a marker in a sample includes determining the methylation state of one base. In some embodiments, assaying the methylation state of a marker in a sample includes determining the degree of methylation at a plurality of bases. Further, in some embodiments, the methylation state of a marker includes an increase in methylation of the marker compared to the normal methylation state of the marker. In some embodiments, the methylation state of a marker includes a decrease in methylation of the marker compared to the normal methylation state of the marker. In some embodiments, the methylation state of a marker includes a different pattern of methylation of the marker compared to the normal methylation state of the marker.

[0037] Further, 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 high CpG density promoter.

[0038] The present technology is not limited by the type of sample. For example, in some embodiments, the sample is a fecal sample, a tissue sample (e.g., a pancreatic tissue sample), a blood sample (e.g., plasma, white blood cells, serum, whole blood), feces, or a urine sample.

[0039] Furthermore, the present technology is not limited to the method used to determine the methylation status. In some embodiments, the assaying includes using methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or target capture. In some embodiments, the assaying includes using methylation-specific oligonucleotides. In some embodiments, the present 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 the methylation status.

[0040] The present technology provides reagents for detecting DMRs, for example, in some embodiments, a set of oligonucleotides is provided that includes sequences represented by SEQ ID NOs: 1-13 (see Table 1). In some embodiments, oligonucleotides are provided that include sequences complementary to chromosomal regions that have bases in the DMR, for example, oligonucleotides that are highly sensitive to the methylation status of the DMR.

[0041] The present technology provides a panel of various markers used to identify PDAC, for example, in some embodiments, the markers include chromosomal regions with the following annotations: AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, MAX.chr5.4295, NTRK3, PRKCB, RYR2, SHISA9, and ZNF781 (see Table 3 and / or Table 4, Example 1).

[0042] Kit embodiments are provided, for example, kits that include a reagent capable of modifying DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent) and a control nucleic acid that includes a sequence of a DMR selected from the group consisting of DMR1-13 (in Table 1) and has a methylation status associated with a subject without PDAC. In some embodiments, the kit includes a bisulfite reagent and an oligonucleotide described herein. In some embodiments, the kit includes a reagent capable of modifying DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent) and a control nucleic acid that includes a sequence of a DMR selected from the group consisting of DMR1-13 (in Table 1) and has a methylation status associated with a subject without PDAC. Some kit embodiments include a sample collector for obtaining a sample from a subject (e.g., a stool sample, a pancreatic tissue sample, a blood sample), a reagent capable of modifying DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent), and an oligonucleotide described herein.

[0043] The present technology relates to embodiments of compositions (e.g., reaction mixtures). In some embodiments, compositions are provided that include a nucleic acid that includes a DMR and a reagent that can modify DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent). Some embodiments provide compositions that include a nucleic acid that includes a DMR and an oligonucleotide described herein. Some embodiments provide compositions that include a nucleic acid that includes a DMR and a methylation-sensitive restriction enzyme. Some embodiments provide compositions that include a nucleic acid that includes a DMR and a polymerase.

[0044] Additional related method embodiments are provided for screening for PDAC in a sample (e.g., a pancreatic tissue sample, a blood sample, a fecal sample) obtained from a subject, for example, the method includes determining the methylation status of a marker in a sample that includes a base in a DMR that is one or more of DMRs 1-13 (in Table 1), comparing the methylation status of the marker in the subject sample to the methylation status of the marker in a normal control sample from a subject without PDAC, and determining a confidence interval and / or p-value for the difference in methylation status 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 the method provide for reacting a nucleic acid comprising a DMR with a reagent capable of modifying the nucleic acid in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent), e.g., to produce a methylation-specifically modified nucleic acid; sequencing the methylation-specifically modified nucleic acid to obtain a nucleotide sequence of the methylation-specifically modified nucleic acid; comparing the nucleotide sequence of the methylation-specifically modified nucleic acid to the nucleotide sequence of a nucleic acid comprising a DMR from a subject not having PDAC to identify differences between the two sequences; and, if differences are present, identifying the subject as having PDAC.

[0045] A system for screening for PDAC in a sample obtained from a subject is provided by the present technology. Exemplary embodiments of the system include, for example, a system for screening for PDAC in a sample obtained from a subject (e.g., a pancreatic tissue sample, a plasma sample, a fecal sample), which includes an analysis component configured to determine a methylation status of the sample, a software component configured to compare the methylation status of the sample with the methylation status of a control or reference sample recorded in a database, and an alert component configured to alert a user of a PDAC-associated methylation status. The alert, in some embodiments, is determined by a software component that receives results from a plurality of assays (e.g., determining the methylation status of a plurality of markers, e.g., DMRs (e.g., as shown in Table 1)) and calculates and reports a value or result based on the plurality of results. Some embodiments provide a database of weighting parameters associated with each DMR provided herein for use in calculating a value or result and / or alert reporting to a user (e.g., a physician, nurse, clinician, etc.). In some embodiments, the results from the multiple assays are all reported, and in some embodiments, one or more results are used to provide a score, value, or result based on a combined one or more results from the multiple assays that is indicative of cancer risk in a subject.

[0046] In some embodiments of the system, the sample comprises a nucleic acid that comprises a DMR. In some embodiments, the system further comprises a component for isolating the nucleic acid, a component for collecting the sample, e.g., a component for collecting a fecal sample. In some embodiments, the system comprises a nucleic acid sequence that comprises a DMR. In some embodiments, the database comprises nucleic acid sequences from subjects that do not have PDAC. Also provided are nucleic acids, e.g., sets of nucleic acids, each nucleic acid having a sequence that comprises a DMR. In some embodiments of the set of nucleic acids, each nucleic acid has a sequence from a subject that does not have PDAC. 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 comprise a reagent capable of modifying DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent). Also, some embodiments further comprise a nucleic acid sequencer.

[0047] In certain embodiments, methods are provided for characterizing a sample (e.g., a pancreatic tissue sample, a blood sample, a fecal sample) from a human patient. For example, in some embodiments, such embodiments include obtaining DNA from the human patient sample, assaying the methylation status of a DNA methylation marker that includes a base in a variably methylated region (DMR) selected from the group consisting of DMRs 1-13 of Table 1, and comparing the assayed methylation status of the one or more DNA methylation markers to a reference methylation level of the one or more DNA methylation markers from human patients without PDAC.

[0048] Such methods are not limited to a particular type of sample from a human patient. In some embodiments, the sample is a pancreatic tissue sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a fecal sample, a tissue sample, a pancreatic tissue sample, a blood sample (e.g., a white blood cell sample, a plasma sample, a whole blood sample, a serum sample), or a urine sample.

[0049] In some embodiments, such methods include assaying a plurality of DNA methylation markers (e.g., including assaying 2-13, 3-13, 4-13, 5-13, 6-13, 7-13, 8-13, 9-13, 10-13, 11-13, 12-13) (e.g., including assaying 13 or fewer markers; including assaying 13 or fewer markers) (e.g., including assaying 12 or fewer markers, 11 or fewer markers, 10 or fewer markers, 9 or fewer markers, 8 or fewer markers, 7 or fewer markers, 6 or fewer markers, 5 or fewer markers, 4 or fewer markers, 3 or fewer markers, 2 or fewer markers). In some embodiments, such methods include assaying the methylation status of one or more DNA methylation markers in a sample, including determining the methylation status of a single base. In some embodiments, such methods include assaying the methylation state of one or more DNA methylation markers in a sample, and determining the degree of methylation at a plurality of bases. In some embodiments, such methods include assaying the methylation state of the forward strand or assaying the methylation state of the reverse strand.

[0050] 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 high CpG density promoter.

[0051] In some embodiments, the assaying comprises using methylation-specific polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, or target capture.

[0052] In some embodiments, the assaying 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: 1-13 (Table 1).

[0053] In some embodiments, a chromosomal region having an annotation selected from the group consisting of AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, MAX.chr5.4295, NTRK3, PRKCB, RYR2, SHISA9, and ZNF781 (see Table 1, Example 1) comprises a DNA methylation marker.

[0054] In some embodiments, such methods include determining the methylation status of two DNA methylation markers. In some embodiments, such methods include determining the methylation status of a pair of DNA methylation markers shown in a row of Table 1.

[0055] In certain embodiments, the technology provides methods for characterizing a sample obtained from a human patient (e.g., a pancreatic tissue sample, a white blood cell sample, a plasma sample, a whole blood sample, a serum sample, a fecal sample). In some embodiments, such methods include determining the methylation status of a DNA methylation marker in a sample that includes a base in a DMR selected from the group consisting of DMRs 1-13 of Table 1, comparing the methylation status of the DNA methylation marker of the patient sample to the methylation status of the DNA methylation marker of a normal control sample from a human subject without PDAC, and determining a confidence interval and / or p-value of the difference in the methylation status of the human patient and normal control samples. 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.

[0056] In certain embodiments, the technology provides methods for characterizing a sample obtained from a human subject (e.g., a pancreatic tissue sample, a white blood cell sample, a plasma sample, a whole blood sample, a serum sample, a fecal sample), the methods comprising reacting a nucleic acid comprising a DMR with a reagent capable of modifying DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent) to produce a methylation-specifically modified nucleic acid, sequencing the methylation-specifically modified nucleic acid to obtain a nucleotide sequence of the methylation-specifically modified nucleic acid, and comparing the nucleotide sequence of the methylation-specifically modified nucleic acid to the nucleotide sequence of a nucleic acid comprising a DMR from a subject without PDAC to identify differences between the two sequences.

[0057] In certain embodiments, the technology provides a system for characterizing a sample (e.g., a pancreatic tissue sample, a plasma sample, a fecal sample) obtained from a human subject, the system including an analysis component configured to determine a methylation status of the sample, a software component configured to compare the methylation status of the sample with methylation statuses 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 statuses and alert a user of a PDAC-associated methylation status. In some embodiments, the sample includes a nucleic acid that includes a DMR.

[0058] In some embodiments, such systems further comprise a component for isolating nucleic acid, hi some embodiments, such systems further comprise a component for collecting the sample.

[0059] In some embodiments, the sample is a fecal sample, a tissue sample, a pancreatic tissue sample, a blood sample (eg, a plasma sample, a white blood cell sample, a whole blood sample, a serum sample), or a urine sample.

[0060] 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 PDAC.

[0061] Further embodiments will be apparent to those of ordinary skill in the relevant arts based on the teachings contained herein. [Brief description of the drawings]

[0062] [Figure 1] Marker chromosomal regions used for the 13 methylated DNA markers listed in Table 1, as well as information on associated primers and probes. [Diagram 2] Cross-validated sensitivity of the methylated DNA marker CA19-9 panel across stages of PDAC at a specificity of 92%. [Diagram 3] Cross-validated ROC curves of the methylated DNA marker panel alone, CA19-9 alone, and the combined panel for the discrimination of PDAC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] definition To facilitate understanding of the present technology, several terms and phrases are defined below. Further definitions are set forth throughout the detailed description.

[0064] Throughout this specification and claims, the following terms have the meanings expressly associated therewith, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used herein may refer to the same embodiment, but not necessarily the same embodiment. Additionally, the phrase "in another embodiment" as used herein may refer to different embodiments, but not necessarily the same embodiment. Thus, as described below, various embodiments of the invention can be readily combined without departing from the scope or spirit of the invention.

[0065] Additionally, as used herein, the term "or" is an inclusive "or" operator and is equivalent to the term "and / or" unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for based on additional unlisted factors unless the context clearly dictates otherwise. Additionally, throughout this specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on."

[0066] The transitional phrase "consisting essentially of," when used in the claims of this application, shall be construed as follows: In re Herz, 537 F.2d 549,551-52,190 USPQ As stated in 461, 463 (CCPA 1976), patent claims are limited to certain substances or steps "and which do not materially affect the basic and novel characteristic(s)" of the claimed invention. For example, a composition "consisting essentially of" recited elements may contain unrecited contaminants at levels that, although present, do not alter the function of the recited composition when compared to the pure composition, i.e., a composition "consisting of" the recited ingredients.

[0067] As used herein, "nucleic acid" or "nucleic acid molecule" generally refers to any ribonucleic acid or deoxyribonucleic acid, which may 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 that contains one or more modified bases. Thus, DNA with a backbone modified for stability or other reasons is a "nucleic acid". As used herein, the term "nucleic acid" encompasses such chemically, enzymatically, or metabolically modified forms of nucleic acid, as well as the chemical forms of DNA characteristic of viruses and cells (including, for example, simple and complex cells).

[0068] The term "oligonucleotide" or "polynucleotide" or "nucleotide" or "nucleic acid" refers to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. Oligonucleotides can be produced by any technique, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides of RNA are uracil, adenine, cytosine, and guanine.

[0069] As used herein, the term "locus" or "region" of a nucleic acid refers to a small region of a nucleic acid, e.g., a gene on a chromosome, a single nucleotide, a CpG island, and the like.

[0070] The terms "complementary" and "complementarity" refer to nucleotides (e.g., a single nucleotide) 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 may be "partial," where only a few of the bases of the nucleic acids match according to the base-pairing rules. Alternatively, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands determines the efficiency and strength of hybridization between the nucleic acid strands. This is particularly important in amplification reactions and in detection methods that depend on binding between nucleic acids.

[0071] 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 of a 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. The term "portion" when used in reference to a gene refers to a fragment of that gene. 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 a fragment of a gene or the entire gene.

[0072] The term "gene" also encompasses the coding region of a structural gene and includes sequences located adjacent to the coding region at both the 5' and 3' ends, e.g., at a distance of about 1 kb on either end, such that a gene corresponds to the length of a full-length mRNA (e.g., including coding, regulatory, structural and other sequences). Sequences located 5' of the coding region and present on the mRNA are referred to as 5' non-translated or 5' untranslated sequences. Sequences located 3' or downstream of the coding region and present on the mRNA are referred to as 3' non-translated or 3' untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. In some organisms (e.g., eukaryotes), genomic forms or clones of a gene contain the coding region interrupted by non-coding sequences, termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA), and introns may contain regulatory elements such as enhancers. Introns are removed or "spliced ​​out" from the nuclear or primary transcript, and therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

[0073] In addition to containing introns, genomic forms of a gene may also contain sequences located on both the 5' and 3' end of the sequences present on 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 on 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.

[0074] The term "wild type", when referring to a gene, refers to a gene that has the characteristics of a gene isolated from a naturally occurring source. The term "wild type", when referring to a gene product, refers to a gene product that has the characteristics of a gene product isolated from a naturally occurring source. The term "naturally occurring", when used with respect to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that can be isolated from a natural source and is present in an organism (including viruses) that has not been intentionally modified by the hand of man 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, the term "modified" or "mutant", when referring to a gene or gene product, refers to a gene or gene product that exhibits modifications (e.g., altered characteristics) in sequence and / or functional properties when compared to a wild type gene or wild type gene product, respectively. It is noted that naturally occurring mutants can be isolated and are identified by the fact that they have altered characteristics when compared to a wild type gene or wild type gene product.

[0075] The term "allele" refers to genetic variations, including, but not limited to, variants and mutations, polymorphic loci, and single nucleotide polymorphic loci, frameshifts, and splice mutations. Alleles may occur naturally in a population or may arise during the lifetime of any particular individual in a population.

[0076] Thus, the terms "variant" and "mutant," when used in reference to a nucleotide sequence, refer to a nucleic acid sequence that differs from another (usually related) nucleotide sequence by one or more nucleotides. "Diversity" is the difference between two different nucleotide sequences, usually one of which is a reference sequence.

[0077] "Amplification" is a special case of nucleic acid replication involving template specificity. It is in contrast to 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 often 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.

[0078] The term "amplifying" or "amplification" in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of a polynucleotide, usually starting from a small amount of polynucleotide (e.g., a single polynucleotide molecule), and the amplification product or amplicon is generally detectable. Amplification of polynucleotides 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 in a polymerase chain reaction (PCR) or ligase chain reaction (LCR, see, e.g., U.S. Patent No. 5,494,810, which is 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, which is incorporated by reference in its entirety), assembly PCR (see, e.g., U.S. Pat. No. 5,965,408, which is incorporated by reference in its entirety), helicase-dependent amplification (see, e.g., U.S. Pat. No. 7,662,594, which is incorporated 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 by reference in its entirety), inter-sequence specific PCR, inverse PCR (see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186, which is incorporated by reference in its entirety), ligation-mediated PCR (see, e.g., Guilfoyle, R. et al., Nucleic Acids Res., 16:8186, which is incorporated by reference in its entirety), and ligation-mediated PCR (see, e.g., Guilfoyle, R. et al., Nucleic Acids Res., 16:8186, which is incorporated by reference in its entirety). Research, 25:1854-1858 (1997); U.S. Pat. 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 is incorporated herein by reference in its entirety), miniprimer PCR, multiplex ligation-dependent probe amplification (see, e.g., Schouten, et al., (2002) Nucleic Acids Research 30(12):e57, which is 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, which is incorporated herein by reference in its entirety), real-time PCR (see, e.g., Higuchi, et al. 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, which is incorporated herein by reference in its entirety), solid-phase PCR, thermal asymmetric interlaced PCR, as well as 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. Polynucleotide amplification can also be performed using digital PCR (see, e.g., Kalinina, et al., Nucleic Acids Research.25;1999-2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA. 96;9236-41, (1999); International Patent Publication No. WO05023091A2; and U.S. Patent Application Publication No. 20070202525, each of which is incorporated herein by reference in its entirety.

[0079] 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 or other DNA or RNA without cloning or purification. This process for amplifying a target sequence consists of introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by thermal cycling in the presence of DNA polymerase in a precise order. The two primers are complementary to their respective strands of the double-stranded target sequence. To effect amplification, the mixture is denatured and the primers are then annealed to their complementary sequences within the target molecule. After annealing, the primers are extended with polymerase to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing, and extension constitute one "cycle" and there can be many "cycles") to obtain a highly concentrated amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore 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 they are "PCR products" or "amplicons". Those skilled in the art will understand that the term "PCR" encompasses many variations of the originally described method, such as using real-time PCR, nested PCR, reverse transcription PCR (RT-PCR), single primer and arbitrarily primed PCR.

[0080] 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, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. For example, in the case of Q-beta replicase, MDV-1 RNA is the specific template for the replicase (Kacian et al., Proc. Natl. Acad. Sci. USA, 69:3038

[1972] ). No other nucleic acid is replicated by the amplification enzyme. Similarly, in the case of T7 RNA polymerase, the amplification enzyme has stringent specificity for its own promoter (Chamberlin et al, Nature, 228:227

[1970] ). In the case of T4 DNA ligase, the enzyme will not ligate two oligonucleotides or polynucleotides if there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (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 a high degree of specificity for the sequences bound by and thereby defined by the primers, due to their ability to function at high temperatures, which provide thermodynamic conditions that favor primer hybridization with the target sequence but not with non-target sequences (HA Erlich (ed.), PCR Technology, Stockton Press

[1989] ).

[0081] 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, probe hybridization, 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., Nat. Biotech., 17:292 (1999); Hall et al., PNAS, USA, 97:8272 (2000), and U.S. Pat. No. 9,096,893, 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, and 5,851,770, each of which is incorporated by reference in its entirety); the polymerase chain reaction (PCR) described above; 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., Nos. 6,210,884, 6,183,960, and 6,235,502, all of which are incorporated herein by reference; NASBA (e.g., U.S. Pat. No. 5,409,818, all of which are incorporated herein by reference); molecular beacon technology (e.g., U.S. Pat. No. 6,150,097, all of which are incorporated herein by reference); E-sensor technology (Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, all of which are incorporated herein by reference); cycling probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769, and 5,660,988, all of which are incorporated herein by reference); Examples of suitable hybridization techniques include, but are not limited to, the Behring signal amplification method (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, which are incorporated herein by reference in their entireties); the ligase chain reaction (e.g., Baranay Proc. Natl. Acad. Sci USA 88,189-93 (1991)); and the sandwich hybridization method (e.g., U.S. Pat. No. 5,288,609, which is incorporated herein by reference in its entirety).

[0082] The term "amplifiable nucleic acid" refers to a nucleic acid that can be amplified by any amplification method. "Amplifiable nucleic acid" is generally intended to include a "sample template."

[0083] The term "sample template" refers to nucleic acid originating from a sample that is 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 most often accidental. Background template may be the result of carryover or it may be due to the presence of nucleic acid contaminants that are sought to be purified away from the sample. For example, nucleic acid from organisms other than those to be detected may be present as background in the test sample.

[0084] The term "primer" refers to an oligonucleotide, whether naturally occurring, for example as a nucleic acid fragment from a restriction digest, or synthetically produced, which can serve as a point of initiation of synthesis when placed under conditions that induce synthesis of a primer extension product complementary to a nucleic acid template strand (e.g., in the presence of nucleotides and an inducer such as DNA polymerase, and at a suitable temperature and pH). The primer is preferably single-stranded to maximize the efficiency of amplification, but may alternatively be double-stranded. If double-stranded, the primer is first treated to separate its strands and then used to prepare the extension product. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of an extension product in the presence of the inducer. The exact length of the primer will depend on many factors, including temperature, source of primer, and use of the method.

[0085] The term "probe" refers to an oligonucleotide (e.g., a sequence of nucleotides), whether naturally occurring, such as a purified restriction digest, or produced synthetically, recombinantly, or by PCR amplification, that can hybridize to another oligonucleotide of interest. 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" such that it is detectable by 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.

[0086] The term "target" as used herein refers to a nucleic acid that is sought to be sorted out from other nucleic acids, for example by probe binding, amplification, isolation, capture, etc. For example, when used in reference to the polymerase chain reaction, "target" refers to the region of nucleic acid bound by the primers used for the polymerase chain reaction, whereas when used in assays in which the target DNA is not amplified, for example, in some embodiments of an invasion cleavage assay, the target includes the site where the probe and the invading oligonucleotide (e.g., INVADER oligonucleotide) bind to form an invasion cleavage structure, which allows the presence of the target nucleic acid to be detected. A "segment" is defined as a region of nucleic acid within the target sequence.

[0087] As used herein, "methylation" refers to cytosine methylation 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 whose original template was unmethylated or amplified DNA whose original template was methylated, respectively.

[0088] Thus, 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 5th 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 5th position of its pyrimidine ring, but since thymine is a typical nucleotide base of DNA, for the purposes of this specification, thymine is not considered to be a methylated nucleotide when present in DNA.

[0089] As used herein, a "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more methylated nucleotides.

[0090] As used herein, the "methylation state," "methylation profile," and "methylation status" 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.

[0091] The methylation state of a particular nucleic acid sequence (e.g., a genetic marker or DNA region described herein) may indicate the methylation state of all bases in the sequence, or may indicate the methylation state of a subset of bases within the sequence (e.g., of one or more cytosines), or may indicate information about the local methylation density within the sequence, with or without providing information about the exact position within the sequence where methylation occurs.

[0092] 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 a nucleic acid molecule.For example, the methylation state of a cytosine at the 7th nucleotide in a nucleic acid molecule is methylated if the nucleotide present at the 7th nucleotide in the nucleic acid molecule is 5-methylcytosine.Similarly, the methylation state of a cytosine at the 7th nucleotide in a nucleic acid molecule is unmethylated if the nucleotide present at the 7th nucleotide in the nucleic acid molecule is cytosine (and is not 5-methylcytosine).

[0093] The methylation status can optionally be expressed or indicated by a "methylation value" (e.g., representing a methylation frequency, rate, ratio, percentage, 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 bisulfite-treated and untreated nucleic acid. Thus, the values, e.g., methylation values, represent the methylation status, such that they can be used as a quantitative indicator of the methylation status across multiple copies of a locus. This is of particular application when it is desirable to compare the methylation status of sequences in a sample to a threshold or reference value.

[0094] As used herein, "methylation frequency" or "percent (%) methylation" refers to the number of instances where a molecule or locus is methylated compared to the number of instances where the molecule or locus is unmethylated.

[0095] Thus, a methylation state represents the state of methylation of a nucleic acid (e.g., a genomic sequence). Moreover, a methylation state refers to the characteristics of a nucleic acid segment at a particular genomic locus that are related to methylation. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues in this DNA sequence are methylated, the location of the methylated C residue(s), the frequency or percentage of methylated C across 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 C or unmethylated C across any particular region of a nucleic acid in a biological sample. For example, if a cytosine (C) residue(s) in a nucleic acid sequence is methylated, it may be referred to as having "hypermethylation" or "increased methylation", whereas if a cytosine (C) residue(s) in a DNA sequence is unmethylated, it may be referred to as having "hypomethylation" or "decreased methylation". Similarly, if a cytosine (C) residue(s) in a nucleic acid sequence is methylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual), the sequence is considered to have hypermethylation or increased methylation compared to the other nucleic acid sequence. Alternatively, if a cytosine (C) residue(s) in a DNA sequence is unmethylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual), the sequence is considered to have hypomethylation or decreased methylation compared to the other nucleic acid sequence. Furthermore, as used herein, the term "methylation pattern" refers to the collection of sites of methylated and unmethylated nucleotides across a region of a nucleic acid. Two nucleic acids may have the same or similar methylation frequency or percent methylation, but different methylation patterns, if the number of methylated and unmethylated nucleotides is the same or similar across the region, but the positions of the methylated and unmethylated nucleotides are different.Sequences are referred to as being "variably methylated" or as having "differential methylation" or "differential methylation states" if 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 may also refer to the difference in the level or pattern between patients whose cancer has recurred after surgery and those whose cancer has not recurred. Variable methylation and specific levels or patterns of DNA methylation are prognostic and predictive biomarkers, for example, after precise cutoffs or predictive characteristics have been defined.

[0096] Methylation state frequencies can be used to describe a population of individuals or a sample derived from a single individual. For example, a nucleotide locus with a methylation state frequency of 50% is methylated in 50% of cases and unmethylated in 50% of cases. Such frequencies can be used, for example, to describe the extent to which a nucleotide locus or nucleic acid region is methylated in a population of individuals or a collection of nucleic acids. Thus, if the methylation in a first population or pool of nucleic acid molecules is different from the methylation in a second population or pool of nucleic acid molecules, the methylation state frequency of the first population or pool will be different from the methylation state frequency of the second population or pool. Such frequencies can also be used, for example, to describe the extent 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 extent to which a group of cells from a tissue sample is methylated or unmethylated at a nucleotide locus or nucleic acid region.

[0097] 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.

[0098] 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). Although many of the cytosines within CpG dinucleotides are methylated in the human genome, some remain unmethylated in specific CpG dinucleotide-rich genomic regions known as CpG islands (see, for example, Antequera et al. al. (1990) Cell 62:503-514).

[0099] 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 long, where the G:C content of the region is at least 50% and the ratio of observed CpG frequency to expected frequency is 0.6, and in some cases, a CpG island can be at least 500 base pairs long, where the G:C content of the region is at least 55% and the ratio of observed CpG frequency to expected frequency is 0.65. The observed CpG frequency to expected 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 to the expected 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 expected 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 usually determined in CpG islands, e.g., in promoter regions. However, it will be appreciated that other sequences in the human genome, such as CpA and CpT, are prone to DNA methylation (Ramsahoye (2000) Proc. Natl. Acad. Sci. USA 97:5237-5242; Salmon (see, for example, 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).

[0100] As used herein, a "methylation specific reagent" refers to a reagent that modifies the nucleotides of a nucleic acid molecule depending on the methylation state of the nucleic acid molecule, or a methylation specific reagent refers to a compound or composition or other agent that can change 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 reagents can include contacting the nucleic acid molecule with the reagent and optionally linking to additional steps to achieve the desired change in the nucleotide sequence. Such methods can be applied in a manner in which unmethylated nucleotides (e.g., each unmethylated cytosine) are modified into a different nucleotide. For example, in some embodiments, such reagents can deaminate unmethylated cytosine nucleotides to generate deoxyuracil residues. Examples of such reagents include, but are not limited to, methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents.

[0101] Alteration of a nucleic acid nucleotide sequence with a methylation specific reagent can also result in a nucleic acid molecule in which each methylated nucleotide is modified to a different nucleotide.

[0102] 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.

[0103] The term "MS AP-PCR" (methylation-sensitive arbitrarily primed polymerase chain reaction) refers to an art-recognized technique that uses CG-rich primers to globally scan the genome and focus on regions most likely to contain CpG dinucleotides, and is described by Gonzalgo et al. (1997) Cancer Research 57:594-599.

[0104] The term "MethyLight™" refers to the art-recognized fluorescence-based real-time PCR technology described by Eads et al. (1999) Cancer Res. 59:2302-2306.

[0105] The term "HeavyMethyl™" refers to an assay in which methylation-specific blocking probes (also referred to herein as blockers) covering the CpG positions between or covered by the amplification primers enable methylation-specific selective amplification of a nucleic acid sample.

[0106] The term "HeavyMethyl™ MethyLight™" assay refers to the HeavyMethyl™ MethyLight™ assay, which is a variation of the MethyLight™ assay in which the MethyLight™ assay is combined with a methylation-specific blocking probe that covers the CpG positions between the amplification primers.

[0107] The term "Ms-SNuPE" (methylation-sensitive single nucleotide primer extension) refers to the art-recognized assay described by Gonzalgo & Jones (1997) Nucleic Acids Res. 25:2529-2531.

[0108] The term "MSP" (methylation specific PCR) refers to the art-recognized methylation assay described by Herman et al. (1996) Proc. Natl. Acad. Sci. USA 93:9821-9826 and by U.S. Patent No. 5,786,146.

[0109] The term "COBRA" (Combined Bisulfite Restriction Analysis) refers to an art-recognized methylation assay described by Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534.

[0110] The term "MCA" (Methylated CpG Island Amplification) refers to the methylation assay described by Toyota et al. (1999) Cancer Res. 59:2307-12 and in WO00 / 26401A1.

[0111] As used herein, a "selected nucleotide" refers to one of the four nucleotides normally occurring in a nucleic acid molecule (C, G, T, and A for DNA; C, G, U, and A for RNA) and can include methylated derivatives of the normally occurring nucleotide (e.g., if C is a selected nucleotide, then both methylated and unmethylated C are included in the meaning of the selected nucleotide), while a methylated selected nucleotide specifically refers to a methylated normally occurring nucleotide and an unmethylated selected nucleotide specifically refers to an unmethylated normally occurring nucleotide.

[0112] The term "methylation-specific restriction enzyme" refers to a restriction enzyme that selectively digests nucleic acids depending on the methylation state of its recognition site. For a restriction enzyme that specifically cleaves when its recognition site is unmethylated or hemimethylated (methylation-sensitive enzyme), cleavage will not occur (or will occur with significantly reduced efficiency) if the recognition site is methylated on one or both strands. For a restriction enzyme that specifically cleaves only when its recognition site is methylated (methylation-dependent enzyme), cleavage will not occur (or will occur with significantly reduced efficiency) if the recognition site is unmethylated. Preferably, the restriction enzyme is methylation-specific, and its recognition sequence contains a CG dinucleotide (e.g., a recognition sequence such as CGCG or CCCGGG). Further preferred for some embodiments are restriction enzymes that do not cleave when the cytosine in this dinucleotide is methylated at the carbon atom C5.

[0113] As used herein, "different nucleotide" refers to a nucleotide that is chemically different from the selected nucleotide, and typically results in that the different nucleotide has different Watson-Crick base pairing properties than the selected nucleotide, so that the commonly occurring nucleotide that is complementary to the selected nucleotide is not the same as the commonly occurring nucleotide that is complementary to the different nucleotide. For example, if C is the selected nucleotide, U or T can be the different nucleotide, as exemplified by the complementarity of C to G and the complementarity of 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 that base pairs with the selected nucleotide or the different nucleotide under high stringency conditions with a higher affinity than the base pairing of the complementary nucleotide with three of the four commonly occurring nucleotides. One example of complementarity is the Watson-Crick base pairing of DNA (e.g., AT and CG) and RNA (e.g., AU and CG). Thus, for example, G base pairs with C under high stringency conditions with greater affinity than G base pairs with G, A, or T, and thus, when C is a selected nucleotide, G is the complementary nucleotide to the selected nucleotide.

[0114] As used herein, the "sensitivity" of a given marker (or set of markers used together) 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 neoplasia reporting a DNA methylation value above the threshold (e.g., in the disease-associated range), and a false negative is defined as a histologically confirmed neoplasia reporting a DNA methylation value below the threshold (e.g., in the disease-unassociated range). Thus, the sensitivity value reflects the probability that a DNA methylation measurement value of a given marker obtained from a known diseased sample will be 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.

[0115] As used herein, the "specificity" of a given marker (or set of markers used together) refers to the percentage of non-neoplastic samples that report 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 that reports a DNA methylation value below the threshold (e.g., in the non-disease associated range), and a false positive is defined as a histologically confirmed non-neoplastic sample that reports a DNA methylation value above the threshold (e.g., in the disease associated range). Thus, the specificity value reflects the probability that a DNA methylation measurement of a given marker obtained from a known non-neoplastic sample will be in 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 patients who do not have that clinical condition.

[0116] The term "AUC" as used herein is an abbreviation for "area under the curve". In particular, AUC 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 various possible cut points of a diagnostic test. The ROC curve shows the trade-off between sensitivity and specificity depending on the cut point selected (any increase in sensitivity will be 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, with 1 being optimal, and a random test has a ROC curve that is located on the diagonal, with an area of ​​0.5. See: J.P.Egan. (1975) Signal Detection Theory and ROC Analysis, Academic Press, New York).

[0117] The term "neoplasm," as used herein, refers to any new abnormal growth of tissue. Thus, a neoplasm can be a premalignant or a malignant neoplasm.

[0118] The term "neoplasm-specific marker" as used herein refers to any biological material or element that can be used to indicate the presence of a neoplasm. Examples of biological materials include, but are not limited to, nucleic acids, polypeptides, carbohydrates, fatty acids, cellular components (e.g., cell membranes and mitochondria), and whole cells. In some cases, the marker is a specific nucleic acid region (e.g., a gene, an intragenic region, a specific locus, etc.). A region of a nucleic acid that is a marker may be referred to, for example, as a "marker gene," "marker region," "marker sequence," "marker locus," etc.

[0119] 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.

[0120] The terms "precancerous" or "preneoplastic" and their equivalents refer to any cell proliferative disorder that has undergone malignant transformation.

[0121] A "site" of a neoplasm, adenoma, cancer, etc. is a tissue, organ, cell type, anatomical region, body part, etc. in a subject's body in which the neoplasm, adenoma, cancer, etc. is located.

[0122] As used herein, the application of a "diagnostic" test includes detecting or identifying a disease state or condition 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 a therapy, determining the prognosis (or likelihood of progression or regression) of a subject with a disease or condition, and determining the effect of a treatment on 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 favorably to a compound (e.g., a pharmaceutical, e.g., a drug) or other treatment.

[0123] The term "isolated", when used with respect to a nucleic acid, such as "isolated oligonucleotide", refers to a nucleic acid sequence that is identified and separated from at least one contaminating nucleic acid with which it is normally associated in its natural source. An isolated nucleic acid exists in a form or context 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 naturally occur. 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, an RNA sequence, such as a particular mRNA sequence that encodes a particular protein, that is found in a cell as a mixture with many other mRNAs that encode many proteins, and the like. However, an isolated nucleic acid that encodes a particular protein includes, by way of example, such a nucleic acid in a cell that normally expresses that protein, where the nucleic acid is in a chromosomal location that is different from that of the natural cell, or is otherwise adjacent to a nucleic acid sequence that is different from that found in nature. An isolated nucleic acid or oligonucleotide may 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 may be single-stranded), but may also contain both a sense and an antisense strand (i.e., the oligonucleotide may be double-stranded). The isolated nucleic acid may be combined with other nucleic acids or molecules after isolation from its natural or normal environment. For example, the isolated nucleic acid may be present in a host cell, which may be placed therein, for example, for heterologous expression.

[0124] The term "purified" refers to molecules, either nucleic acid or amino acid sequences, 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% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated. As used herein, the term "purified" or "to purify" also refers to the removal of contaminants from a sample. The removal of contaminating proteins increases the percentage of the polypeptide or nucleic acid of interest in the sample. In another example, a recombinant polypeptide is expressed in a plant, bacterial, yeast, or mammalian host cell, and the polypeptide is purified by the removal of host cell proteins, thereby increasing the percentage of the recombinant polypeptide in the sample.

[0125] The term "composition comprising" a given polynucleotide sequence or polypeptide refers broadly to any composition that contains the given polynucleotide sequence or polypeptide. Compositions can include aqueous solutions containing salts (e.g., NaCl), detergents (e.g., SDS), and other components (e.g., Denhardt's solution, milk powder, salmon sperm DNA, etc.).

[0126] The term "sample" is used in its broadest sense. In one sense, a sample can refer to an animal cell or tissue. In another sense, a sample refers to a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from plants or animals (including humans) and encompass fluids, solids, tissues, and gases. Environmental samples include environmental materials such as surface materials, soil, water, and industrial samples. These examples should not be construed as limiting the types of samples applicable to the present invention.

[0127] As used herein, a "remote sample", when used in some contexts, relates to a sample that is collected indirectly from a site that is not the source of the sample's cells, tissues, or organs.

[0128] As used herein, the term "patient" or "subject" refers to an organism subjected to various tests provided by the present technology. The term "subject" includes animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human. Furthermore, with respect to the diagnostic method, the preferred subject is a vertebrate subject. The preferred vertebrates are warm-blooded, and the preferred warm-blooded vertebrates are mammals. The preferred mammal is most preferably a human. As used herein, the term "subject" includes both human and animal subjects. Thus, veterinary therapeutic uses are provided herein. Thus, the present technology allows for the diagnosis of mammals, such as humans, as well as mammals of importance because they are endangered, such as the Amur tiger, mammals of economic importance, such as animals raised on farms for human consumption, and / or animals of social importance 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, bulls, sheep, giraffes, deer, goats, bison, and camels; pinnipeds; and horses. Thus, diagnostics and treatments of livestock, including, but not limited to, domesticated pigs, ruminants, ungulates, horses (including race horses), and the like, are also provided. The subject matter disclosed herein further includes a system for diagnosing lung cancer in a subject. The system may be provided as a commercially available kit that can be used, for example, to screen for lung cancer risk or to diagnose lung cancer in a subject from whom a biological sample has been collected. An exemplary system provided according to the present technology includes evaluating the methylation status of the markers described herein.

[0129] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of a reaction assay, such a delivery system includes a system that allows for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides in appropriate containers, enzymes, etc.) and / or supporting materials (e.g., buffers, written instructions for performing the assay, etc.) from one location to another. For example, a kit includes one or more enclosed containers (e.g., boxes) that contain the relevant reaction reagents and / or supporting materials. As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separate containers, each of which contains a small portion of the total components of the kit. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in the assay, while a second container contains an oligonucleotide. The term "fragmented kit" is intended to encompass, but is not limited to, a kit that contains an analyte-specific reagent (ASR) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act. Indeed, any delivery system that includes two or more separate containers, each of which contains a small portion of all the components of the kit, is included in the term "fragmented kit." In contrast, a "composite kit" refers to a delivery system that contains all the components of a reaction assay in a single container (e.g., in a single box that houses each of the desired components). The term "kit" includes both fragmented and composite kits.

[0130] As used herein, the term "information" refers to any collection of facts or data. With respect to information stored or processed using a computer system(s), including but not limited to the Internet, the term refers to any data stored in any format (e.g., analog, digital, optical, etc.). As used herein, the term "information relating to a subject" refers to facts or data about a subject (e.g., a human, a plant, or an animal). The term "genomic information" refers to information about a genome, including but not limited to nucleic acid sequences, genes, methylation percentages, allele frequencies, RNA expression levels, protein expression, phenotypes associated with genotypes, and the like. "Allele frequency information" refers to facts or data about allele frequencies, including but not limited to allele identity information, statistical correlations between the presence of alleles and characteristics of a subject (e.g., a human subject), the presence or absence of alleles in an individual or population, the percentage likelihood of an allele being present in an individual with one or more particular characteristics, and the like.

[0131] Detailed Description In this detailed description of various embodiments, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will appreciate that these various embodiments may be practiced without or with these specific details. In other instances, structures and mechanisms are shown in block diagram form. Moreover, those skilled in the art will readily appreciate that the particular order in which the methods are presented and performed is illustrative, and that the order can be changed and still be within the spirit and scope of the various embodiments disclosed herein.

[0132] Provided herein is the technology for PDAC screening, particularly but not limited to, the method, composition and related applications for detecting the presence of PDAC.When describing this technology, the headings of sections used are for organizational purposes only and should not be interpreted as limiting the subject matter in any way.

[0133] Indeed, as described in Example 1, experiments conducted during the process of identifying embodiments of the present invention identified 13 variably methylated regions (DMRs) for distinguishing PDAC from non-neoplastic control DNA.

[0134] Such experiments have listed and described 13 DNA methylation markers (AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, MAX.chr5.4295, NTRK3, PRKCB, RYR2, SHISA9, and ZNF781) that a) distinguish between PDAC and non-neoplastic controls within plasma samples (see Table 3, Example I), and b) distinguish between PDAC tissue and benign pancreatic tissue (see Table 4, Example 1).

[0135] Such experiments have identified the following markers and / or panels of markers for detecting PDAC in blood samples (e.g., plasma samples, whole blood samples, white blood cell samples, serum samples): AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, MAX.chr5.4295, NTRK3, PRKCB, RYR2, SHISA9, and ZNF781 (see Table 3, Example 1).

[0136] Such experiments have identified the following markers and / or panels of markers that are capable of distinguishing PDAC tissue from benign pancreatic tissue: AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, MAX.chr5.4295, NTRK3, PRKCB, RYR2, SHISA9, and ZNF781 (see Table 4, Example 1).

[0137] Although the disclosure herein refers to certain illustrated embodiments, it is to be understood that these embodiments are presented by way of example and not by way of limitation.

[0138] In certain aspects, the technology provides compositions and methods for identifying, determining, and / or classifying cancers, such as PDAC. The methods include determining the methylation status of at least one methylation marker in a biological sample (e.g., a fecal sample, a pancreatic tissue sample, a plasma sample) isolated from a subject, where a change in the methylation status of the marker is indicative of the presence, class, or location of PDAC. Certain embodiments relate to markers that include variably methylated regions (DMRs, e.g., DMRs 1-13, see Table 1) that are used to diagnose (e.g., screen for) PDAC.

[0139] In addition to the embodiments provided herein in which methylation analysis of at least one marker, region of a marker, or marker base comprising a DMR (e.g., a DMR, e.g., DMRs 1-13) set forth in Table 1 is analyzed, the present technology also provides panels of markers comprising at least one marker, region of a marker, or marker base comprising a DMR useful for detecting cancer, particularly PDAC.

[0140] Some embodiments of the technology are based on the analysis of the CpG methylation status of at least one marker, region of a marker, or base of a marker that comprises a DMR.

[0141] In some embodiments, the technology allows for the use of reagents that modify DNA in a methylation-specific manner (e.g., methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents) in combination with one or more methylation assays to determine the methylation status of CpG dinucleotide sequences within at least one marker that comprises a DMR (e.g., DMRs 1-13, see Table 1). Genomic CpG dinucleotides can be methylated or unmethylated (alternatively known as up-methylated and down-methylated, respectively). However, the methods of the invention are suitable for the analysis of biological samples of heterogeneous nature, such as low concentrations of tumor cells, or biological material therefrom, within a background of distant samples (e.g., blood, organ waste, or feces). Thus, when analyzing the methylation status of CpG positions within such samples, quantitative assays can be used to determine the level (e.g., percentage, proportion, ratio, proportion, or degree) of methylation at a particular CpG position.

[0142] According to the present technology, determining the methylation status of CpG dinucleotide sequences in markers containing DMRs is useful for both diagnosing and characterizing cancers such as PDAC.

[0143] Marker Combinations In some embodiments, the technology relates to assessing the methylation status of a combination of markers that include the DMRs of Table 1 (e.g., DMR numbers 1-13). In some embodiments, assessing the methylation status of multiple markers improves the specificity and / or sensitivity of screening or diagnosis for identifying a neoplasm (e.g., PDAC) in a subject.

[0144] Different cancers are predicted by different combinations of markers, for example as identified by statistical techniques for the specificity and sensitivity of prediction. The present technology provides methods for identifying predictive combinations and validated predictive combinations for several cancers.

[0145] Methods for assaying methylation status In certain embodiments, methods of analyzing nucleic acids for the presence of 5-methylcytosine include treating DNA with reagents that modify DNA in a methylation-specific manner, examples of such reagents include, but are not limited to, methylation-sensitive restriction enzymes, methylation-dependent restriction enzymes, and bisulfite reagents.

[0146] A frequently used method for analyzing nucleic acids for the presence of 5-methylcytosine is based on the bisulfite method described by Frommer, et al. for the detection of 5-methylcytosine in DNA (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-31, expressly incorporated herein by reference in its entirety for all purposes) or their variations. The bisulfite method for mapping 5-methylcytosine is based on the finding that cytosine, but not 5-methylcytosine, reacts with bisulfite ions (also known as bisulfite). The reaction is usually carried out according to the following steps: first, cytosine reacts with bisulfite to form sulfonated cytosine. Then, spontaneous deamination of the sulfonated reaction intermediate gives rise to sulfonated uracil. Finally, the sulfonated uracil is desulfonated under alkaline conditions to form uracil. Uracil base pairs with adenine (and thus behaves like thymine), whereas 5-methylcytosine base pairs with guanine (and thus behaves like cytosine), allowing detection, for example, by bisulfite genomic sequencing (Grigg G, & Clark S, Bioessays (1994) 16:431-36; Grigg G, DNA Seq. (1996) 6:189-98), methylation-specific PCR (MSP), as disclosed, for example, in U.S. Pat. No. 5,786,146, or by assays involving sequence-specific probe cleavage, for example, the QuARTS flap endonuclease assay (see, for example, Zou et al. (2010) “Sensitive quantification of methylated markers With the use of a novel methylation specific technology" Clin Chem 56:A199, and see U.S. Pat. Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392, it is possible to distinguish between methylated and unmethylated cytosines.

[0147] Some prior art 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 by rapid 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, demonstrating the usefulness and sensitivity of the method. An overview of prior methods for detecting 5-methylcytosine is provided by Rein, T., et al. (1998) Nucleic Acids Res. 26:2255.

[0148] Bisulfite technology usually involves amplifying short specific fragments of known nucleic acids after bisulfite treatment, then assaying the products to analyze individual cytosine positions either by sequencing (Olek & Walter (1997) Nat. Genet. 17:275-6) or primer extension reaction (Gonzalgo & Jones (1997) Nucleic Acids Res. 25:2529-31, WO95 / 00669, U.S. Patent No. 6,251,594). 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., WO99 / 28498). Additionally, the use of bisulfite technology for methylation detection on 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).

[0149] Various methylation assay procedures can be used in conjunction with bisulfite treatment according to the present technology. These assays can determine the methylation state of one or more CpG dinucleotides (e.g., CpG islands) in a nucleic acid sequence. Such assays include, among other techniques, sequencing of bisulfite-treated nucleic acid, PCR (for sequence-specific amplification), Southern blot analysis, and the use of methylation-specific restriction enzymes, e.g., methylation-sensitive or methylation-dependent enzymes.

[0150] For example, genome sequencing has been simplified for the analysis of methylation patterns and 5-methylcytosine distribution by using bisulfite treatment (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-1831). Furthermore, restriction enzyme digestion of PCR products amplified from bisulfite converted DNA is used to assess methylation status, for example as described by Sadri & Hornsby (1997) Nucl. Acids Res. 24:5058-5059, or as embodied in the method known as COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534).

[0151] COBRA™ analysis is a quantitative methylation assay that is useful for determining DNA methylation levels at specific loci with 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 specific labeled hybridization probes. Methylation levels in the original DNA samples are represented by the relative amounts of digested and undigested PCR products in a linear manner over a wide range of DNA methylation levels. Moreover, this technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples.

[0152] Typical reagents for COBRA™ analysis (e.g., those that may be found in a typical COBRA™-based kit) may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, DMRs, regions of genes, regions of markers, 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. Additionally, bisulfite conversion reagents may include DNA denaturation buffers, sulfonation buffers, DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns), desulfonation buffers, and DNA recovery components.

[0153] Assays such as "MethyLight™" (fluorescence-based real-time PCR technology) (Eads et al., Cancer Res. 59:2302-2306, 1999), Ms-SNuPE™ (methylation-sensitive single nucleotide primer extension) 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) may be used alone or in combination with one or more of these methods.

[0154] The "HeavyMethyl™" assay technology is a quantitative method to assess methylation differences based on methylation-specific amplification of bisulfite-treated DNA. Methylation-specific blocking probes ("blockers") covering the CpG positions between or covered by the amplification primers allow for methylation-specific selective amplification of nucleic acid samples.

[0155] The term "HeavyMethyl™ MethyLight™" assay refers to the HeavyMethyl™ MethyLight™ assay, which is a variation of the 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.

[0156] Typical reagents for HeavyMethyl™ analysis (e.g., those that might be found in a typical MethyLight™-based kit) can include, but are not limited to, PCR primers for a specific locus (e.g., a specific gene, marker, region of a gene, region of a marker, bisulfite-treated DNA sequence, CpG island, or bisulfite-treated DNA sequence or CpG island, etc.), blocking oligonucleotides, optimized PCR buffer and deoxynucleotides, and Taq polymerase.

[0157] MSP (methylation specific PCR) allows 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 to convert unmethylated cytosines to uracils (but not methylated cytosines), and the product is then amplified with primers specific for methylated DNA compared to 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., those that might be found in a typical MSP-based kit) can include, but are not limited to, methylated and unmethylated PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.), optimized PCR buffers and deoxynucleotides, and specific probes.

[0158] The MethyLight™ assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (e.g., TaqMan®) without the need for 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 by standard procedures into a mixed pool of methylation-dependent sequence differences during a sodium bisulfite reaction (the bisulfite process converts unmethylated cytosine residues to uracil). Fluorescence-based PCR is then performed in a "biased" reaction, for example, with PCR primers that overlap known CpG dinucleotides. Sequence discrimination is performed both at the level of the amplification process and at the level of the fluorescence detection process.

[0159] The MethyLight™ assay is used as a quantitative test of methylation patterns in nucleic acids, e.g., genomic DNA samples, where sequence discrimination is performed at the level of probe hybridization. In the quantitative version, the PCR reaction results in methylation-specific amplification in the presence of fluorescent probes overlapping specific putative methylation sites. An unbiased control for input DNA amount is provided by a reaction in which neither the primers nor the probe overlap any CpG dinucleotides. Alternatively, a qualitative test of genomic methylation is performed by probing biased PCR pools with either control oligonucleotides that do not cover known methylation sites (e.g., fluorescent-based versions of HeavyMethyl™ and MSP technologies) or oligonucleotides that cover potential methylation sites.

[0160] The MethyLight™ process is 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 sets of PCR reactions using, for example, MSP primers and / or HeavyMethyl blocker oligonucleotides and TaqMan® probes using TaqMan® probes. The TaqMan® probes are dual-labeled with fluorescent “reporter” and “quencher” molecules and are designed to be specific for relatively high GC content regions such that the probe melts at a temperature about 10° C. higher than the forward or reverse primers during the PCR cycle. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing / extension step. The Taq polymerase will eventually reach the annealed TaqMan® probe to enzymatically synthesize new strands during PCR. The 5' to 3' endonuclease activity of Taq polymerase then removes the TaqMan® probe by digesting it to release the fluorescent reporter molecule for quantitative detection of the now unquenched signal of the fluorescent reporter molecule using a real-time fluorescence detection system.

[0161] Typical reagents for MethyLight™ analysis (e.g., those that 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, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.), TaqMan® or Lightcycler® probes, optimized PCR buffer and deoxynucleotides, and Taq polymerase.

[0162] The QM™ (Quantitative Methylation) assay is an alternative quantitative test of methylation patterns in genomic DNA samples, where sequence discrimination is performed at the level of probe hybridization. In this quantitative version, the PCR reaction results in unbiased amplification in the presence of fluorescent probes overlapping specific putative methylation sites. An unbiased control for the input DNA amount is provided by a reaction in which neither the primers nor the probe overlap any CpG dinucleotides. Alternatively, a qualitative test of genomic methylation is performed by probing the biased PCR pool with either control oligonucleotides that do not cover known methylation sites (e.g. fluorescent-based versions of HeavyMethyl™ and MSP technologies) or oligonucleotides that cover potential methylation sites.

[0163] The QM™ process can be used with any suitable probe in the amplification process, e.g., "TaqMan®" probe, Lightcycler® probe. For example, double-stranded genomic DNA is treated with sodium bisulfite and subjected to unbiased primers and TaqMan® probe. The TaqMan® probe is dual-labeled with fluorescent "reporter" and "quencher" molecules and is designed to be specific for relatively high GC content regions such that the probe melts at a temperature about 10° C. higher than the forward or reverse primers during the PCR cycle. This allows the TaqMan® probe to remain fully hybridized during the PCR annealing / extension step. Taq polymerase will eventually reach the annealed TaqMan® probe to enzymatically synthesize new strands during PCR. The 5' to 3' endonuclease activity of Taq polymerase then removes the TaqMan® probe by digesting it to release the fluorescent reporter molecule in order to quantitatively detect the now unquenched signal of the fluorescent reporter molecule using a real-time fluorescence detection system. Typical reagents for QM™ analysis (e.g., those that might 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, regions of genes, regions of markers, bisulfite-treated DNA sequences, CpG islands, etc.), TaqMan® or Lightcycler® probes, optimized PCR buffers and deoxynucleotides, and Taq polymerase.

[0164] Ms-SNuPE™ technology is a quantitative method for evaluating methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by single nucleotide primer extension (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. Amplification of the desired target sequence is then performed using PCR primers specific for bisulfite-converted DNA, and the resulting products are isolated and used as templates for methylation analysis at the CpG sites of interest. Small amounts of DNA can be analyzed (e.g., microdissected pathology sections), which avoids the use of restriction enzymes to determine methylation status at CpG sites.

[0165] Typical reagents for Ms-SNuPE™ analysis (e.g., those that may be found in a typical Ms-SNuPE™-based kit) may include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, regions of genes, regions of markers, 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 Ms-SNuPE reactions), and labeled nucleotides. Additionally, bisulfite conversion reagents may include DNA denaturation buffers, sulfonation buffers, DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns), desulfonation buffers, and DNA recovery components.

[0166] Reduced Representation Bisulfite Sequencing (RRBS) begins with bisulfite treatment of nucleic acids to convert all unmethylated cytosines to uracils, followed by restriction enzyme digestion (e.g., with an enzyme that recognizes sites containing CG sequences, such as MspI), and full sequencing of the fragments after binding to an adaptor ligand. The choice of restriction enzyme enriches for fragments in CpG-dense regions, reducing the number of redundant sequences that may map to multiple gene locations during the analysis. Thus, RRBS reduces the complexity of the 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. Thus, RRBS enriches samples for promoters, CpG islands, and other genomic traits that have a high frequency of restriction enzyme cleavage sites in these regions, thereby providing an assay for assessing the methylation status of one or more genomic loci.

[0167] A typical protocol for RRBS includes the steps of digesting a nucleic acid sample with a restriction enzyme such as MspI, filling in the overhangs and A-tailing, ligating adapters, bisulfite conversion, and a PCR step. See, for example, 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.

[0168] In some embodiments, a quantitative allele-specific real-time target and signal amplification (QuARTS) assay is used to evaluate the methylation status. In each QuARTS assay, three reactions occur in succession, the primary reaction includes amplification (reaction 1) and target probe cleavage (reaction 2), and the secondary reaction includes FRET cleavage and fluorescent signal generation (reaction 3). When the target nucleic acid is amplified with a specific primer, a specific detection probe with a flap sequence is loosely bound to the amplicon. The presence of a specific invading oligonucleotide at the target binding site releases the flap sequence by a 5' nuclease, e.g., FEN-1 endonuclease, cleaving between the detection probe and the flap sequence. The flap sequence is complementary to the non-hairpin portion of the corresponding FRET cassette. Thus, the flap sequence functions as an invading oligonucleotide on the FRET cassette, resulting in cleavage between the FRET cassette fluorophore and the quencher, generating a fluorescent signal. The cleavage reaction can cleave multiple probes per target, thereby releasing multiple fluorophores per flap, resulting in exponential signal amplification. QuARTS can detect multiple targets in a single reaction well by using FRET cassettes with different dyes. See, for example, Zou et al. (2010) “Sensitive quantification of methylated "Clin Chem 56:A199," as well as U.S. Pat. Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392, each of which is incorporated by reference herein for all purposes.

[0169] The term "bisulfite reagent" refers to a reagent that includes bisulfite, disulfite, hydrogen sulfite, or a combination thereof, and is useful for distinguishing between methylated and unmethylated CpG dinucleotide sequences, as disclosed herein. Methods of such treatment are known in the art (e.g., PCT / EP2004 / 011715 and WO2013 / 116375, each of which is incorporated herein by reference in its entirety). In some embodiments, the bisulfite treatment is carried out 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 of 1% to 35% (v / v). In some embodiments, the bisulfite reaction is carried out in the presence of a scavenger, such as, but not limited to, a chroman derivative, such as 6-hydroxy-2,5,7,8,-tetramethylchroman 2-carboxylic acid or trihydroxybenzoic acid and its derivatives, such as gallic acid (see PCT / EP2004 / 011715, which is incorporated by reference in its entirety). In certain preferred embodiments, the bisulfite reaction includes treatment with ammonium bisulfite, such as, for example, as described in WO2013 / 116375.

[0170] In some embodiments, the treated DNA fragments are amplified using a set of primer oligonucleotides according to the invention (see, for example, Table 10, Table 19 and Table 20) and an amplification enzyme. Amplification of several DNA segments can be carried out simultaneously in one and the same reaction vessel. Usually, the amplification is carried out using the polymerase chain reaction (PCR). The amplicons are usually 100 to 2000 base pairs long.

[0171] In another embodiment of the method, the methylation status of CpG positions in or near markers containing DMRs (e.g., DMRs 1-13, Table 1) can be detected 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 for the discrimination between methylated and unmethylated nucleic acids. MSP primer pairs contain at least one primer that hybridizes to bisulfite-treated CpG dinucleotides. Thus, the sequence of the primer contains at least one CpG dinucleotide. MSP primers specific for unmethylated DNA contain a "T" at the C position in the CpG.

[0172] The fragments obtained by amplification can carry a label that can be detected directly or indirectly. In some embodiments, the label is a fluorescent label, a radionuclide, or a detachable molecular fragment that has a typical mass that can be detected by mass spectrometer. When the label is a mass label, some embodiments allow good detectability in mass spectrometer by having a single positive or negative net charge of the labeled amplicon. Detection can be performed and visualized, for example, by matrix-assisted laser desorption ionization mass spectrometry (MALDI) or using electron spray mass spectrometry (ESI).

[0173] Methods for isolating DNA suitable for these assay techniques are known in the art. In particular, some embodiments involve the isolation of nucleic acids as described in U.S. Patent Application Serial No. 13 / 470,251 ("Isolation of Nucleic Acids"), which is incorporated herein by reference in its entirety.

[0174] In some embodiments, the markers described herein are used in a QUARTS assay performed on a stool sample. In some embodiments, methods are provided for generating DNA samples, particularly DNA samples that contain small volumes (e.g., less than 100 microliters, less than 60 microliters) of highly purified low abundance nucleic acids and that are substantially and / or effectively free of substances that inhibit assays (e.g., PCR, INVADER, QuARTS assays, etc.) used to test the DNA sample. Such DNA samples are used in diagnostic assays that qualitatively detect the presence or quantitatively measure the activity, expression, or amount of genes, genetic variants (e.g., alleles), or genetic modifications (e.g., methylation) present in a sample taken from a patient. For example, some cancers are correlated with the presence of certain mutant alleles or certain methylation states, and thus detection and / or quantification of such mutant alleles or methylation states has predictive value in cancer diagnosis and treatment.

[0175] Many useful genetic markers are present in samples in very small amounts, and many of the events that generate such markers are rare.As a result, even highly sensitive detection methods such as PCR require a large amount of DNA to provide enough low-abundance targets to meet or invalidate the detection threshold of the assay.Furthermore, the presence of even small amounts of inhibitors impairs the accuracy and precision of these assays aimed at detecting such low-abundance targets.Therefore, provided herein is a method for generating such DNA samples that provides the necessary volume and concentration control.

[0176] In some embodiments, the sample comprises blood, serum, white blood cells, plasma, or saliva. In some embodiments, the subject is a human. Such samples can be obtained by any number of means known in the art, such as those that will be apparent to those of skill in the art. Acellular or substantially acellular samples can be obtained by subjecting the sample to various techniques known to those of skill in the art, including, but not limited to, centrifugation and filtration. Although it is generally preferred to obtain samples without using invasive techniques, it may still be preferred to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens. The techniques are not limited to the methods used to prepare the sample and provide nucleic acids for testing. For example, in some embodiments, DNA is isolated from a stool sample, or from a blood sample, or from a plasma sample, using direct gene capture, such as those detailed in U.S. Pat. Nos. 8,808,990 and 9,169,511, and WO2012 / 155072, or by related methods.

[0177] The analysis of markers may be performed separately or simultaneously with additional markers in one test sample. For example, some markers may be combined in one test to efficiently process multiple samples and potentially provide higher accuracy of diagnosis and / or prognosis. Furthermore, those skilled in the art will recognize the value of testing multiple samples from the same subject (e.g., at successive time points). Such testing of a series of samples allows for the identification of changes in the methylation status of markers over time. The absence of changes in methylation status, in addition to changes in methylation status, may provide useful information regarding disease status, including but not limited to identifying the approximate time from the onset of an event, the presence and amount of recoverable tissue, the appropriateness of drug therapy, the effectiveness of various therapies, and the subject's outcome, including the risk of future events.

[0178] Analysis of biomarkers can be performed in a variety of physical formats. For example, the use of microtiter plates or automation 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 an outpatient or emergency room setting.

[0179] It is contemplated that embodiments of the present technology are provided in the form of a kit. The kit includes embodiments of the compositions, devices, apparatus, etc. described herein, and instructions for use of the kit. Such instructions describe appropriate methods for preparing an analyte from a sample, e.g., methods for collecting a sample and preparing nucleic acid from the sample. Individual components of the kit are packaged in suitable containers and packaging (e.g., vials, boxes, blister packs, ampoules, jars, bottles, tubes, etc.), and the components are packaged together in a suitable container (e.g., box(es)) for convenient storage, transportation, and / or use by the user of the kit. It is understood that liquid components (e.g., buffers) may be provided in a lyophilized form that is reconstituted by the user. The kit may include controls or references to evaluate, verify, and / or ensure the performance of the kit. For example, a kit for assaying the amount of nucleic acid present in a sample may include a control that includes a known concentration of the same or another nucleic acid for comparison, and in some embodiments, may include a detection reagent (e.g., primers) specific for the control nucleic acid. The kit is suitable for use in a clinical setting, and in some embodiments, for use in a user's home. The components of the kit, in some embodiments, provide the functionality of a system for preparing a nucleic acid solution from a sample. In some embodiments, certain components of the system are provided by the user.

[0180] method In some embodiments of the present technology, the following steps are performed: 1) contacting nucleic acid obtained from a subject (e.g., genomic DNA isolated from a blood sample (e.g., plasma sample, whole blood sample, white blood cell sample, serum 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 AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, MAX.chr5.4295, NTRK3, PRKCB, RYR2, SHISA9, and ZNF781; and 2) detecting PDAC (e.g., with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0181] In some embodiments of the present technology, the following steps are performed: 1) contacting nucleic acid obtained from a subject (e.g., genomic DNA isolated from pancreatic tissue) 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 AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, MAX.chr5.4295, NTRK3, PRKCB, RYR2, SHISA9, and ZNF781; and 2) detecting PDAC (e.g., with a sensitivity of 80% or greater and a specificity of 80% or greater).

[0182] In some embodiments of the present technology, the following steps are performed: 1) measuring the methylation level of one or more genes in a biological sample of a human individual through treating genomic DNA in the biological sample with a reagent that modifies DNA in a methylation-specific manner (e.g., where the reagent is a bisulfite reagent, a methylation-sensitive restriction enzyme, or a methylation-dependent restriction enzyme), wherein the one or more genes are selected from one of AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, MAX.chr5.4295, NTRK3, PRKCB, RYR2, SHISA9, and ZNF781; 2) amplifying the treated genomic DNA using a set of primers for one or more selected genes; and 3) determining the methylation level of one or more genes by polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation specific nucleases, mass-based separation, and target capture.

[0183] In some embodiments of the present technology, the following steps are performed: 1) measuring the amount of at least one methylation marker gene in DNA from a sample, where the one or more genes are selected from AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, MAX.chr5.4295, NTRK3, PRKCB, RYR2, SHISA9, and ZNF781; 2) measuring the amount of at least one reference marker in the DNA; and 3) calculating a value of the amount of at least one methylation marker gene measured in the DNA as a percentage of the amount of a reference marker gene measured in the DNA, said value being indicative of the amount of the at least one methylation marker DNA measured in the sample.

[0184] In some embodiments of the present technology, the following steps are performed: 1) measuring the methylation level of CpG sites of one or more genes in a biological sample from a human individual by treating genomic DNA in the biological sample with bisulfite, a reagent that can modify DNA in a methylation-specific manner (e.g., a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and a bisulfite reagent); 2) amplifying the modified genomic DNA using a set of primers for one or more selected genes; and 3) 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; Here, a method is provided, wherein the one or more genes are selected from AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, MAX.chr5.4295, NTRK3, PRKCB, RYR2, SHISA9, and ZNF781.

[0185] Preferably, the sensitivity of such a method 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%.

[0186] Genomic DNA can be isolated by any means, including the use of commercially available kits. Briefly, if the DNA of interest is encapsulated by a cell membrane, the biological sample must be disrupted and dissolved by enzymatic, chemical or mechanical means. Proteins and other contaminants can then be removed from the DNA solution, for example, by digestion with proteinase K. The genomic DNA is then recovered from the solution. This can be done by a variety of methods, including salting out, organic extraction, or binding of 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, including neoplastic or pre-neoplastic material, are suitable for use in the method, such as cell lines, histological slides, biopsies, paraffin-embedded tissues, body fluids, feces, breast tissue, pancreatic tissue, white blood cells, colonic effluent, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, and combinations thereof.

[0187] The present technology is not limited to the method used to prepare samples and provide nucleic acid for testing.For example, in some embodiments, DNA is isolated from fecal samples, or from blood, or from plasma samples by direct gene capture, for example, as described in US Patent Application No. 61 / 485386, or related methods.

[0188] 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 comprises a DMR (e.g., DMRs 1-13, as shown in Table 1).

[0189] In some embodiments, the reagent converts 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, however, in some embodiments, the reagent may be a methylation-sensitive restriction enzyme.

[0190] In some embodiments, the genomic DNA sample is treated in such a way that cytosine bases that are not methylated at the 5' position are converted 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, disulfite) followed by alkaline hydrolysis.

[0191] 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 that includes at least one DMR, e.g., a DMR selected from DMRs 1-13 as shown in Table 1). Methods of analysis can be selected from those known in the art, including those described herein, e.g., QuARTS and MSP as described herein.

[0192] Aberrant methylation, and more specifically, hypermethylation of markers including the DMRs (eg, DMRs 1-13 as shown in Table 1), is associated with PDAC.

[0193] The technology relates to the analysis of any sample related to PDAC. For example, in some embodiments, the sample comprises tissue and / or bodily fluid obtained from a patient. In some embodiments, the sample comprises a secretion. In some embodiments, the sample comprises blood, serum, plasma, gastric secretions, pancreatic juice, gastrointestinal biopsy samples, microdissected cells from breast biopsies, and / or cells recovered from feces. In some embodiments, the sample comprises pancreatic tissue. In some embodiments, the subject is a human. The sample may include cells, secretions, or tissue from the endometrium, breast, liver, bile duct, pancreas, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyps, gallbladder, anus, and / or peritoneum. In some embodiments, the sample comprises cell fluid, ascites, urine, feces, pancreatic juice, fluid obtained during endoscopy, blood, mucus, or saliva. In some embodiments, the sample is a fecal sample. In some embodiments, the sample is a pancreatic tissue sample.

[0194] Such samples can be obtained by any number of means known in the art, including those that will be apparent to those of skill in the art. For example, urine and fecal samples are readily available, while blood, ascites, serum, or pancreatic juice samples can be obtained parenterally, for example, by using a needle and syringe. Acellular or substantially acellular samples can be obtained by subjecting the sample to various techniques known to those of skill in the art, including, but not limited to, centrifugation and filtration. Although it is generally preferred to obtain samples without the use of invasive techniques, it may still be preferable to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens.

[0195] In some embodiments, the technology relates to a method of treating a patient (e.g., a patient with PDAC), the method comprising determining the methylation status of one or more DMRs provided herein, and administering a treatment to the patient based on the results of determining the methylation status. The treatment can be administering a pharmaceutical compound, administering a vaccine, performing a surgical procedure, imaging the patient, performing another test. Preferably, the use is in a method of clinical screening, a method of prognostic evaluation, a method of monitoring the outcome of a therapy, a method of identifying patients most likely to respond to a particular therapeutic treatment, a method of imaging a patient or subject, and a method for screening and developing drugs.

[0196] In some embodiments of the present technology, a method for diagnosing PDAC in a subject is provided. The terms "diagnose" and "diagnosis" as used herein refer to a method that allows a person skilled in the art to estimate and even determine whether a subject suffers from a given disease or condition, or whether the subject may develop a given disease or condition in the future. A person skilled in the art often performs a diagnosis based on one or more diagnostic indicators, such as biomarkers (e.g., DMRs disclosed herein), whose methylation status indicates the presence, severity, or absence of a condition.

[0197] In addition to diagnosis, clinical cancer prognosis is concerned with determining the aggressiveness of cancer and the likelihood of tumor recurrence, and planning the most effective therapy. If a more accurate prognosis can be made, or even the potential risk of developing cancer can be evaluated, appropriate therapy, and in some cases, a therapy that is less harsh for the patient, can be selected. Assessment of cancer biomarkers (e.g., determining methylation status) is useful for separating subjects who have a good prognosis and / or are at a low risk of developing cancer and do not require therapy or require limited therapy from subjects who are more likely to develop cancer or have cancer recurrence and may benefit from more aggressive treatment.

[0198] Thus, "making a diagnosis" or "diagnosing" as used herein further includes determining the risk of developing cancer or determining a prognosis, which may allow predicting a clinical outcome (with or without medical treatment), selecting an appropriate treatment (or whether a treatment is effective), or monitoring a current treatment and possibly modifying the treatment based on a measure of a diagnostic biomarker (e.g., DMR) disclosed herein. Furthermore, in some embodiments of the subject matter disclosed herein, multiple determinations of biomarkers over time can be made to facilitate diagnosis and / or prognosis. The temporal changes in the biomarkers can be used to predict clinical outcomes, monitor the progression of PDAC, and / or monitor the effectiveness of an appropriate therapy directed against the cancer. In such embodiments, for example, it may be expected to ascertain changes in the methylation status of one or more biomarkers (e.g., DMR) disclosed herein (and optionally one or more additional biomarker(s) if monitored) in a biological sample over time during the course of an effective therapy.

[0199] The subject matter disclosed herein 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 includes obtaining a series of biological samples from a subject over a period of time, analyzing the series of biological samples, determining 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 outcomes, determine whether to initiate or continue cancer prevention or therapy, and whether a current therapy is effectively treating 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 point after the start of treatment. The methylation status can be measured in each of the samples taken at different time points, and qualitative and / or quantitative differences are recorded. Changes in the methylation status of the biomarker levels from the different samples can be correlated with PDAC risk, prognosis, determining treatment efficacy, and / or cancer progression in the subject.

[0200] 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 symptoms of the disease are manifest, hi some embodiments, the methods and compositions of the invention are for the treatment or diagnosis of disease at a clinical stage.

[0201] As already mentioned, in some embodiments, multiple determinations of one or more diagnostic or prognostic biomarkers can be made, and the change in the marker over time can be used to determine the diagnosis or prognosis. For example, the diagnostic marker can be determined a first time and again a second time. In such embodiments, an increase in the marker from the first time to the second time can diagnose a particular type or severity of cancer, or a given prognosis. Similarly, a decrease in the marker from the first time 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, but also a given biomarker can be measured at one time point and a second biomarker at a second time point, and the comparison of these markers can provide diagnostic information.

[0202] As used herein, the phrase "determine prognosis" refers to a method by which a person skilled in the art can predict the course or outcome of a condition of 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 to predict that a given course or outcome is more or less likely to occur based on the methylation status of a biomarker (e.g., DMR). Instead, a person skilled in the art will understand that the term "prognosis" refers to an increased probability of a certain course or outcome occurring, i.e., a course or outcome is more likely to occur in a subject that exhibits a given condition compared to an individual that does not exhibit the condition. For example, an individual that does not exhibit a condition (e.g., has normal methylation status of one or more DMRs) may have a very low probability of a given outcome (e.g., suffer from PDAC).

[0203] In some embodiments, the statistical analysis correlates the prognostic indicator with a predisposition to an adverse outcome. For example, in some embodiments, a methylation status that differs from that in a normal control sample obtained from a patient without cancer may indicate that the subject is more likely to suffer from cancer than a subject having a level more similar to the methylation status in the control sample, as determined by the level of statistical significance. Furthermore, the change in methylation status from the baseline (e.g., "normal") level may reflect the subject's prognosis, and the degree of change in methylation status may 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 for Research, John Wiley & Sons, New York, incorporated herein by reference in its entirety. See York, 1983. Exemplary confidence intervals of the present subject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while exemplary p-values ​​are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

[0204] 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 the biomarker in a biological sample is simply compared to the threshold degree of change in the methylation status. Preferred threshold changes in the methylation status of 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, whereby the methylation status of a prognostic or diagnostic indicator (biomarker or combination of biomarkers) is directly related to the predisposition associated with a given outcome. Because reference is made to individual sample measurements rather than population averages, one skilled in the art is familiar with using such nomograms to relate the two numerical values, with the understanding that the uncertainty of this measurement is the same as the uncertainty of the marker concentration.

[0205] 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. It is further contemplated that a standard curve can be provided and the assay results of the biological sample can be compared to the standard curve. Such a standard curve presents the methylation status of the biomarker according to the assay unit, e.g., the fluorescent signal intensity if a fluorescent label is used. Samples taken from multiple donors can be used to obtain standard curves for the control methylation status of one or more biomarkers in normal tissue and for the "at risk" level of one or more biomarkers in tissue taken from a donor with dysplasia or from a donor with PDAC. In certain embodiments of the method, a subject is identified as having dysplasia upon identification of an abnormal methylation status of one or more DMRs provided herein in a biological sample taken from the subject. In other embodiments of the method, a subject is identified as having cancer upon detection of an abnormal methylation status of one or more of such biomarkers in a biological sample taken from the subject.

[0206] The analysis of markers may be performed separately or simultaneously with additional markers in one test sample. For example, some markers may be combined in one test to efficiently process multiple samples and potentially provide higher accuracy of diagnosis and / or prognosis. Furthermore, those skilled in the art will recognize the value of testing multiple samples from the same subject (e.g., at successive time points). Such testing of a series of samples allows for the identification of changes in the methylation status of markers over time. The absence of changes in methylation status, in addition to changes in methylation status, may provide useful information regarding disease status, including but not limited to identifying the approximate time from the onset of an event, the presence and amount of recoverable tissue, the appropriateness of drug therapy, the effectiveness of various therapies, and the subject's outcome, including the risk of future events.

[0207] Analysis of biomarkers can be performed in a variety of physical formats. For example, the use of microtiter plates or automation 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 an outpatient or emergency room setting.

[0208] In some embodiments, a subject is diagnosed as having PDAC when there is a measurable difference in the methylation status of at least one biomarker in the sample compared to the control methylation status. Conversely, if no change in the methylation status in the biological sample is identified, the subject may be identified as not having PDAC, not having risk of cancer, or having low risk of cancer. In this regard, subjects with cancer or risk of cancer may be differentiated from subjects with low risk of cancer or substantially no risk of cancer. Those subjects with risk of developing PDAC may be placed on a more intensive and / or regular screening schedule, including endoscopic surveillance. On the other hand, subjects with low risk to substantially no risk may be avoided from being subjected to additional tests (e.g., invasive procedures) for PDAC until future screening, e.g., screening performed according to the present technology, indicates that the risk of PDAC has appeared in those subjects.

[0209] As mentioned above, depending on the embodiment of the method of the present technology, detecting a change in the methylation state of one or more biomarkers can be a qualitative determination, or it can be a quantitative determination. Thus, the step of diagnosing a subject as having or at risk of developing PDAC indicates that a certain threshold measurement is made, e.g., that the methylation state of one or more biomarkers in a biological sample is different from a predetermined control methylation state. In some embodiments of the present method, the control methylation state is any detectable methylation state of the biomarker. In other embodiments of the method, where 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 the present method, the predetermined methylation state is identified based on and / or by a standard curve. In other embodiments of the present method, the predetermined methylation state is a specific state or range of states. Thus, the predetermined methylation state can be selected, in part, based on the embodiment of the method to be performed and the desired specificity, etc., within acceptable limits that will be apparent to those skilled in the art.

[0210] Further with respect to the diagnostic method, the preferred subject is a vertebrate subject. The preferred vertebrate is warm-blooded, and the preferred warm-blooded vertebrate is a mammal. The preferred mammal is most preferably a human. As used herein, the term "subject" includes both human and animal subjects. Thus, veterinary therapeutic uses are provided herein. Thus, the present technology allows for the diagnosis of mammals such as humans, as well as mammals of importance due to being endangered, such as the Amur tiger, mammals of economic importance, such as animals raised on farms for human consumption, and / or animals of social importance 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, bulls, sheep, giraffes, deer, goats, bison, and camels; and horses. Thus, diagnosis and treatment of livestock, including but not limited to domesticated pigs, ruminants, ungulates, horses (including racehorses), and the like, is also provided.

[0211] The subject matter disclosed herein further comprises a system for diagnosing PDAC in a subject.This system can be provided as a commercially available kit, which can be used to screen for the risk of PDAC in the subject whose biological sample is collected, or to diagnose PDAC.The exemplary system provided according to the present technology comprises evaluating the methylation status of DMR as shown in table 1. EXAMPLES

[0212] Example I This example describes the identification of plasma markers for detecting pancreatic ductal adenocarcinoma (PDAC).

[0213] Thirteen methylated DNA markers (MDMs) were utilized to identify plasma markers for detecting pancreatic ductal adenocarcinoma (PDAC) (see Table 1).

[0214] [Table 1]

[0215] The 13 MDMs shown in Table 1 arose from early stage pancreatic cancer tissue experiments using next generation bisulfite sequencing (Kisiel JB, et al., Clin Cancer Res.2015 Oct 1;21(19):4473-81). Briefly, from remining these data, hundreds of variably methylated regions (DMRs) were identified based on a combination of selection criteria including area under the ROC curve (AUC), false positives, relative and absolute methylation rate differences between cases and controls, CpG density within the DMR, and the presence of uniform sequential comethylation of adjacent residues (in cases). Subsequent validation using targeted chemistries (e.g., quantitative methylation-specific PCR) with high sensitivity and specificity on a larger set of independent tissue samples allowed further refinement of the markers. Such selection yielded 20–30 potential MDMs, the majority of which were mapped to putative or known regulatory regions determined from genome browser tracks. Many of the gene products acted in defined tumorigenic pathways and had functions as promoter-binding transcription factors, enhancers, cell signaling mediators, growth factors, and ion channel proteins. Additional experiments were performed to define a subset of highly capable discriminatory assays (individually and complementary) that could be used for formal plasma testing. For this purpose, additional tests were performed on a pool of neoplasia-free control plasma, removing the amplified MDMs from steady-state circulating cfDNA (a crucial step). This resulted in the 13 MDMs shown in Table 1. Table 2 shows primer and probe information for the 13 MDMs listed in Table 1, and Figure 1 further shows the marker chromosomal regions used for the 13 MDMs listed in Table 1 as well as the associated primer and probe information.

[0216] [Table 2] JPEG2025063204000004.jpg199169 JPEG2025063204000005.jpg65169

[0217] This panel of 13 MDMs was tested on a set of plasma samples from 26 patients diagnosed with PDAC (N=26; 4 SI, 11 S-II, 6 S-III, 5 S-IV) and normal EDTA plasma samples (N=26). Table 3 shows the area under the curve (AUC), fold change, p-value, and methylation percentage for each marker. The 13 marker panel detected all stage 1 and stage 4 PDAC cancers, and all but one each of PDAC stage 2 and PDAC stage 3 cancers, with 100% specificity. Additionally, this panel of 13 MDMs was tested on a set of PDAC tissue samples compared to benign tissue (Table 4) and a set of PDAC tissue samples compared to buffy coat (Table 5).

[0218] [Table 3]

[0219] [Table 4]

[0220] [Table 5]

[0221] The only clinically available blood biomarker for detecting PDAC is CA19-9. CA19-9 is unreliable for early PDAC detection and may be normal in advanced disease. Next, we performed an experiment to test the accuracy of the 13 markers shown in Table 1 with or without CA19-9 to distinguish PDAC cases from age- and sex-matched control patients.

[0222] All assays with the 13 markers were performed blindly by the Target Enrichment Long Probe Quantitative Amplification Signal (TELQAS) test (see Kisiel JB, et al., Hepatology. 2018 Aug 31). Briefly, TELQAS oligos (forward invasion primer, reverse primer, flap probe) were designed for CpG motifs within each of the 13 DMRs (IDT, Coralville IA). 12 cycles of multiplex amplification of the markers as well as B3GALT6 (reference gene) and RASSF1 (zebrafish processing control) were performed. The products were then diluted 10-fold in TE buffer. 10 μL of the diluted amplicon was used in triplex format (FAM, HEX, Quasar 670) to amplify and quantify the two markers plus the B3GALT6 reference gene. TELQAS reactions were performed using the ABI 7500DX instrument (Applied Biosystems, Foster City, The study was conducted in California.

[0223] CA19-9 was quantified from plasma samples using the MILLIPLEX® Map Kit (EMD Millipore) on a Luminex® MAGPIX® analyzer. Briefly, plasma samples were diluted 1:6 using the serum matrix provided in the kit as the diluent. Only CA19-9 antibody-immobilized magnetic beads were used in the immunoassay. The assay was completed using the protocol provided with the kit reagents. Quantitative results for each sample were generated from the median fluorescence intensity signal using Luminex® xPONENT® software.

[0224] From 340 plasma samples (170 PDAC cases, 170 controls), the experiment first used quantitative MDM and CA19-9 levels in 120 advanced stage PDAC cases (60 stage 3, 60 stage 4) and 120 healthy controls to train a predictive algorithm with random forest (rForest) modeling at 97.5% specificity. The locked algorithm was then applied to an independent blinded test set of 50 early stage PDAC cases (5 stage 1, 45 stage 2) and 50 controls. Data from all 340 patients were then aggregated and refit using rForest. The MDM panel was cross-validated by randomly splitting the entire dataset 2:1 for training and testing. The fitted rForest model from the training set was used to predict the disease status of the test set. Median AUC after 500 iterations was reported.

[0225] The area under the curve results for the 13 markers are shown in Table 6. In the initial training set, the MDM-CA19-9 panel detected 54 / 60 (90%) stage 3 and 59 / 60 (98%) stage 4 PDAC with a specificity of 97.5%. The area under the curve MDM cutoff values ​​obtained from these advanced cases and applied to stage 1 and 2 PDAC as well as controls yielded an AUC of 0.84 (95% CI 0.76-0.92) for the MDM panel alone versus 0.91 (0.84-0.97) for the combined MDM-CA19-9 panel (p=0.038). For all 340 cases and controls combined, the cross-validated sensitivity of the MDM-CA19-9 panel was 79% for stage 1 PDAC, 82% for stage 2 PDAC, 94% for stage 3 PDAC, and 99% for stage 4 PDAC, with a specificity of 92% (81-100%) (Figure 2). The cross-validated AUC was 0.9 (0.85-0.94) for the MDM panel alone, compared with 0.97 (0.94-0.99) for the combined MDM-CA19-9 panel (p=<0.0001) (Figure 3). Overall, the sensitivity for PDAC was 92% (83-98%) with a specificity of 92%. These results indicate that the 13 MDMs shown in Table 1, combined or not combined with CA19-9, detect PDAC across all stages with moderate to high accuracy.

[0226] [Table 6]

[0227] Ten cc of blood from each subject was collected in K2EDTA vacutainers (BD, Franklin Lakes NJ). Within 4 hours, the tubes were centrifuged at 1500×G (10 min), plasma was removed and centrifuged a second time, aliquoted into 2 mL cryotubes, and stored at −80°C without intermittent thawing. cfDNA was purified and bisulfite converted using an automated silica bead method. A non-human DNA spike was used to control for processing anomalies. For all samples, 3.8 mL of plasma was first subjected to proteinase K treatment, followed by lysis with detergent and chaotropic agents. Silica-coated binding beads and isopropyl alcohol-containing lysis buffer were added to each sample for DNA capture and DNA precipitation. All samples were washed multiple times with a Hamilton STARlet liquid handling system (Hamilton Company, Reno NV), and the binding beads were dried before eluting the DNA samples with elution buffer. The samples were then bisulfite converted using a Hamilton STARlet liquid handling system as previously described (see Lidgard, et al., 2013;11:1313-1318). Briefly, the samples were first denatured with sodium hydroxide. Ammonium bisulfite was added to each sample for deamination. The samples were then bound to silica-coated binding beads and washed multiple times before being desulfonated. The sample was washed repeatedly and the purified sample was eluted with elution buffer.

[0228] Sample cfDNA was tested using TELQAS (Target Enrichment by Long Probe Quantitative Amplification Signal), a highly sensitive multiplex assay format. (See Kisiel JB, et al., Hepatology. 2018 Aug 31). Briefly, TELQAS oligos (forward invasion primer, reverse primer, flap probe) were designed for CpG motifs within each of the 13 DMRs (IDT, Coralville IA). 12 cycles of multiplex amplification of the markers as well as B3GALT6 (reference gene) and RASSF1 (zebrafish processing control) were performed. The products were then diluted 10-fold in TE buffer. 10 μL of the diluted amplicon was used in triplex format (FAM, HEX, Quasar 670) to amplify and quantify the two markers plus the B3GALT6 reference gene. TELQAS reactions were performed on an ABI 7500DX instrument (Applied Biosystems, Foster City Calif.). Table 7 shows the nine LQAS assays that were performed. All LQAS assays were set up and performed under standard conditions previously published.

[0229] [Table 7]

[0230] Example II. Testing of the panel of 13 MDMs listed in Table 1 was further tested on LBgard (Biomatrica, San Diego, CA) plasma samples comprised of 12 patients diagnosed with PDAC (N=12; 3 S-II, 1 S-III, 8 S-IV) and a collection of 27 normal non-PDAC plasma samples (N=27). Table 8 shows the nominal logistic fit to assess whether a sample is from a control or a PDAC case.

[0231] [Table 8]

[0232] Although the present invention has now been fully described, it will be understood by those skilled in the art that the same can be practiced within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any of its embodiments. All patents, patent applications, and publications cited herein are hereby fully incorporated by reference in their entirety.

[0233] Incorporation by Reference The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

[0234] Equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects as illustrative rather than limiting the invention described herein. The scope of the invention is therefore indicated by the appended claims rather than the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. A method for assessing the methylation level of one or more genes in a biological sample from a human individual having or suspected of having pancreatic ductal adenocarcinoma (PDAC), said method comprising: amplifying DNA from said biological sample using a set of primers to RYR2, SHISA9 and / or MAX.chr5.4295; and measuring the methylation level of at least one CpG site in RYR2, SHISA9 and / or MAX.chr5.4295 using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, and / or target capture.

2. The method of claim 1, further comprising a step of comparing the methylation level of one or more CpG sites with the methylation level of one or more CpG sites in one or more corresponding genes from a control or reference sample.

3. The method of claim 2, wherein the control or reference sample is derived from a subject who does not have PDAC.

4. A method described in any one of claims 1 to 3, wherein the step of measuring the methylation level of one or more CpG sites includes measuring at least one reference marker.

5. The method described in claim 4, wherein the at least one reference marker is B3GALT6 and / or β-actin.

6. A method described in any one of claims 1 to 5, further comprising measuring the level of carbohydrate antigen 19-9 (CA19-9) from the biological sample.

7. A method according to any one of claims 1 to 6, wherein the at least one CpG site is present in a coding region or a regulatory region.

8. A method described in any one of claims 1 to 7, wherein the step of measuring the methylation level of at least one CpG site includes determining a methylation score of the CpG site and / or determining a methylation frequency of the CpG site.

9. A method according to any one of claims 1 to 8, wherein the biological sample comprises one or more of a plasma sample, a blood sample and / or a pancreatic tissue sample.

10. The method described in claims 1 to 9, wherein the step of measuring the methylation level of at least one CpG site comprises using methylation-specific PCR, quantitative methylation-specific PCR, methylation-specific DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, flap endonuclease assay, PCR-flap assay and / or bisulfite genomic sequencing PCR.

11. A method described in any one of claims 1 to 10, further comprising a step of treating the DNA in the biological sample with a reagent that modifies the DNA in a methylation-specific manner.

12. The method of claim 11, wherein the reagent comprises one or more of a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and / or a bisulfite reagent.

13. A method described in any one of claims 1 to 12, comprising the steps of amplifying the DNA from the biological sample using a set of primers for RYR2 and measuring the methylation level of at least one CpG site in RYR2.

14. A method described in any one of claims 1 to 12, comprising the steps of amplifying the DNA from the biological sample using a set of primers for SHISA9 and measuring the methylation level of at least one CpG site in SHISA9.

15. The method of any one of claims 1 to 12, comprising amplifying the DNA from the biological sample using a set of primers directed to MAX.chr5.4295, and measuring the methylation level of at least one CpG site in MAX.chr5.4295.

16. A method according to any one of claims 1 to 12, comprising the steps of amplifying the DNA from the biological sample using a set of primers for each of RYR2 and SHISA9, and measuring the methylation level of at least one CpG site in each of RYR2 and SHISA9.

17. The method of any one of claims 1 to 12, comprising the steps of amplifying the DNA from the biological sample using a set of primers for each of RYR2 and MAX.chr5.4295, and measuring the methylation level of at least one CpG site in each of RYR2 and MAX.chr5.4295.

18. The method according to any one of claims 1 to 12, comprising the steps of amplifying the DNA from the biological sample using a set of primers for each of SHISA9 and MAX. chr5.4295, and measuring the methylation level of at least one CpG site in each of SHISA9 and MAX. chr5.4295.

19. The method of any one of claims 1 to 12, comprising the steps of amplifying the DNA from the biological sample using a set of primers for each of RYR2, SHISA9, and MAX.chr5.4295, and measuring the methylation level of at least one CpG site in each of RYR2, SHISA9, and MAX.chr5.4295.

20. The set of primers for RYR2 comprises SEQ ID NOs: 31 and 32, or comprises a set of primers that specifically bind to at least a portion of a gene region comprising coordinates 237205577-237205684 on chromosome 1; The set of primers for SHISA9 includes a set of primers that specifically bind to at least a portion of a gene region including SEQ ID NOs: 34 and 35, or including coordinates 12995930-12996219 on chromosome 16; and The method of any one of claims 1 to 19, wherein the set of primers for MAX.chr5.4295 comprises a set of primers that specifically bind to at least a portion of a gene region comprising SEQ ID NOs: 22 and 23, or comprising coordinates 42951691-42951760 on chromosome 5.

21. The method of any one of claims 1 to 20, further comprising a step of measuring the methylation level of one or more additional genes selected from AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, NTRK3, PRKCB and ZNF781.

22. The set of primers for AK055957 comprises a set of primers that specifically bind to at least a portion of a gene region including SEQ ID NOs: 1 and 2, or including coordinates 133484978-133485739 on chromosome 12; The set of primers for CD1D comprises a set of primers that specifically bind to at least a portion of a gene region comprising SEQ ID NOs: 4 and 5, or comprising coordinates 158150797-158151205 on chromosome 1; The set of primers for CLEC11A comprises a set of primers that specifically bind to at least a portion of the gene region comprising SEQ ID NOs: 7 and 8, or the region comprising coordinates 51228217-51228732 on chromosome 19; The set of primers for FER1L4 comprises a set of primers that specifically bind to at least a portion of the gene region comprising SEQ ID NOs: 10 and 11, or the region comprising coordinates 34189488-34189566 on chromosome 20; The set of primers for GRIN2D comprises a set of primers that specifically bind to at least a portion of the gene region comprising SEQ ID NOs: 13 and 14, or the region comprising coordinates 48917755-48918477 on chromosome 19; The set of primers for HOXA1 includes a set of primers that specifically bind to at least a portion of a gene region comprising SEQ ID NOs: 16 and 17, or the region comprising coordinates 27136145-27136425 on chromosome 7; The set of primers for LRRC4 comprises SEQ ID NOs: 19 and 20, or a set of primers that specifically bind to at least a portion of a gene region comprising coordinates 127671993-127672310 on chromosome 7; The set of primers for NTRK3 comprises SEQ ID NOs: 25 and 26, or a set of primers that specifically bind to at least a portion of the gene region comprising coordinates 88800287-88800464 on chromosome 15; The set of primers for PRKCB comprises a set of primers that specifically bind to at least a portion of the gene region comprising SEQ ID NOs: 28 and 29, or the region comprising coordinates 23846964-23848168 on chromosome 16; and The method of claim 21, wherein the set of primers for ZNF781 comprises a set of primers that specifically bind to at least a portion of the gene region comprising SEQ ID NOs: 37 and 38, or comprising coordinates 38182950-38183127 on chromosome 19.

23. A method for assessing the methylation level of one or more genes in a biological sample from a human individual having or suspected of having pancreatic ductal adenocarcinoma (PDAC), said method comprising the steps of amplifying DNA from said biological sample using a set of primers for one or more genes; and measuring the methylation level of at least one CpG site in said one or more genes based on at least one reference marker, wherein said one or more genes are selected from RYR2, SHISA9, MAX. chr5.4295, AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, NTRK3, PRKCB, and / or ZNF781.

24. The method described in claim 23, wherein the at least one reference marker is B3GALT6 and / or β-actin.

25. A method according to claim 23 or claim 24, wherein the step of measuring the methylation level of the at least one CpG site in the one or more genes comprises using polymerase chain reaction, nucleic acid sequencing, mass spectrometry, methylation-specific nucleases, mass-based separation, and / or target capture.

26. A method described in any one of claims 23 to 25, further comprising a step of comparing the methylation level of the at least one CpG site in the one or more genes with the methylation level of at least one CpG site in one or more corresponding genes from a control or reference sample.

27. The method of claim 26, wherein the control or reference sample is derived from a subject who does not have PDAC.

28. A method according to any one of claims 23 to 27, wherein the biological sample comprises one or more of a plasma sample, a blood sample and / or a pancreatic tissue sample.

29. A method described in any one of claims 23 to 28, further comprising the step of treating the DNA in the biological sample with a reagent that modifies the DNA in a methylation-specific manner.

30. A method described in any one of claims 23 to 29, wherein the one or more genes include RYR2, and the method comprises the steps of amplifying the DNA from the biological sample using a set of primers for RYR2, and measuring the methylation level of at least one CpG site in RYR2.

31. A method described in any one of claims 23 to 29, wherein the one or more genes include SHISA9, and the method comprises the steps of amplifying the DNA from the biological sample using a set of primers for SHISA9, and measuring the methylation level of at least one CpG site in SHISA9.

32. The method of any one of claims 23 to 29, wherein the one or more genes include MAX.chr5.4295, and the method comprises amplifying the DNA from the biological sample using a set of primers to MAX.chr5.4295, and measuring the methylation level of at least one CpG site in MAX.chr5.4295.

33. A method according to any one of claims 23 to 29, wherein the one or more genes include RYR2 and SHISA9, and the method comprises the steps of amplifying the DNA from the biological sample using a set of primers for each of RYR2 and SHISA9, and measuring the methylation level of at least one CpG site in each of RYR2 and SHISA9.

34. The method of any one of claims 23 to 29, wherein the one or more genes include RYR2 and MAX.chr5.4295, and the method comprises amplifying the DNA from the biological sample using a set of primers for each of RYR2 and MAX.chr5.4295, and measuring the methylation level of at least one CpG site in each of RYR2 and MAX.chr5.4295.

35. The method of any one of claims 23 to 29, wherein the one or more genes include SHISA9 and MAX. chr5.4295, and the method comprises the steps of amplifying the DNA from the biological sample using a set of primers for each of SHISA9 and MAX. chr5.4295, and measuring the methylation level of at least one CpG site in each of SHISA9 and MAX. chr5.4295.

36. The method of any one of claims 23 to 29, wherein the one or more genes include RYR2, SHISA9, and MAX.chr5.4295, and the method comprises the steps of amplifying the DNA from the biological sample using a set of primers for each of RYR2, SHISA9, and MAX.chr5.4295, and measuring the methylation level of at least one CpG site in each of RYR2, SHISA9, and MAX.chr5.4295.

37. The set of primers for RYR2 comprises SEQ ID NOs: 31 and 32, or a set of primers that specifically bind to at least a portion of a gene region comprising coordinates 237205577-237205684 on chromosome 1; The set of primers for SHISA9 includes a set of primers that specifically bind to at least a portion of a gene region including SEQ ID NOs: 34 and 35, or including coordinates 12995930-12996219 on chromosome 16; and The method of any one of claims 23 to 36, wherein the set of primers for MAX.chr5.4295 comprises a set of primers that specifically bind to at least a portion of a gene region comprising SEQ ID NOs: 22 and 23, or comprising coordinates 42951691-42951760 on chromosome 5.

38. The one or more genes further include AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, NTRK3, PRKCB, and / or ZNF781, and the method further comprises administering to at least one of AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, NTRK3, PRKCB, and / or ZNF781.

38. The method of any one of claims 30 to 37, comprising amplifying said DNA from said biological sample using a set of primers directed against: AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, NTRK3, PRKCB, and / or ZNF781; and measuring the methylation level of at least one CpG site in at least one of AK055957, CD1D, CLEC11A, FER1L4, GRIN2D, HOXA1, LRRC4, NTRK3, PRKCB, and / or ZNF781.

39. The set of primers for AK055957 includes a set of primers that specifically bind to at least a portion of a gene region including SEQ ID NOs: 1 and 2, or including coordinates 133484978-133485739 on chromosome 12; The set of primers for CD1D comprises a set of primers that specifically bind to at least a portion of a gene region comprising SEQ ID NOs: 4 and 5, or comprising coordinates 158150797-158151205 on chromosome 1; The set of primers for CLEC11A comprises a set of primers that specifically bind to at least a portion of the gene region comprising SEQ ID NOs: 7 and 8, or the region comprising coordinates 51228217-51228732 on chromosome 19; The set of primers for FER1L4 comprises a set of primers that specifically bind to at least a portion of the gene region comprising SEQ ID NOs: 10 and 11, or the region comprising coordinates 34189488-34189566 on chromosome 20; The set of primers for GRIN2D comprises a set of primers that specifically bind to at least a portion of the gene region comprising SEQ ID NOs: 13 and 14, or the region comprising coordinates 48917755-48918477 on chromosome 19; The set of primers for HOXA1 includes a set of primers that specifically bind to at least a portion of a gene region comprising SEQ ID NOs: 16 and 17, or the region comprising coordinates 27136145-27136425 on chromosome 7; The set of primers for LRRC4 comprises SEQ ID NOs: 19 and 20, or a set of primers that specifically bind to at least a portion of a gene region comprising coordinates 127671993-127672310 on chromosome 7; The set of primers for NTRK3 comprises SEQ ID NOs: 25 and 26, or a set of primers that specifically bind to at least a portion of the gene region comprising coordinates 88800287-88800464 on chromosome 15; The set of primers for PRKCB comprises a set of primers that specifically bind to at least a portion of the gene region comprising SEQ ID NOs: 28 and 29, or the region comprising coordinates 23846964-23848168 on chromosome 16; and The method of claim 38, wherein the set of primers for ZNF781 comprises a set of primers that specifically bind to at least a portion of the gene region comprising SEQ ID NOs: 37 and 38, or comprising coordinates 38182950-38183127 on chromosome 19.

40. A reagent for modifying DNA in a methylation-specific manner; a set of primers directed to at least one CpG site in RYR2, SHISA9 and / or MAX.chr5.4295; and instructions for assessing methylation levels for RYR2, SHISA9 and / or MAX.chr5.4295 in a biological sample from a human individual having or suspected of having pancreatic ductal adenocarcinoma (PDAC).

41. The kit described in Claim 40, further comprising reagents for evaluating at least one reference marker, wherein the at least one reference marker is B3GALT6 and / or β-actin.

42. The set of primers for RYR2 comprises SEQ ID NOs: 31 and 32, or a set of primers that specifically bind to at least a portion of a gene region comprising coordinates 237205577-237205684 on chromosome 1; The set of primers for SHISA9 includes a set of primers that specifically bind to at least a portion of a gene region including SEQ ID NOs: 34 and 35, or including coordinates 12995930-12996219 on chromosome 16; and The kit of claim 40 or claim 41, wherein the set of primers for MAX.chr5.4295 comprises a set of primers that specifically bind to at least a portion of a gene region comprising SEQ ID NOs: 22 and 23, or comprising coordinates 42951691-42951760 on chromosome 5.

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