Detection of Hepatocellular Carcinoma

Abstract

A method for screening for hepatocellular carcinoma is provided. [Solution] A method for screening for hepatocellular carcinoma (HCC) in a sample taken from a subject, the method comprising: a) assaying the methylation status of a marker in the sample taken from the subject; and b) identifying the subject as having HCC if the methylation status of the marker is different from the methylation status of the marker assayed in a subject not having HCC, wherein the marker comprises a base in a differentially methylated region (DMR) selected from ACP1, BDH1, Chr12.133, CLEC11A, DAB2IP, DBNL, EMX1, EFNB2, HOXA1, LRRC4, SPINT2, TSPYL5, CCNJ_3707, CCNJ_3124, PFKP, SCRN1, and ECE1.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 383,165, filed on September 2, 2016. The provisional application is incorporated herein by reference in its entirety.

[0002] Provided herein is a technology for screening hepatocellular carcinoma, and specifically, but not limited to, methods, compositions, and related uses for detecting the presence of hepatocellular carcinoma.

Background Art

[0003] Hepatocellular carcinoma (HCC) is a primary malignant tumor of the liver and mainly occurs in patients with underlying chronic liver diseases and cirrhosis. The origin cell(s) is / are thought to be hepatic stem cells (see Alison MR. Stem Cell Rev. 2005. 1(3):253 - 60). The tumor progresses with local expansion, intrahepatic progression, and distant metastasis.

[0004] HCC is currently the third leading cause of cancer - related death worldwide, affecting more than 500,000 people. The incidence of HCC is highest in Asia and Africa, where high - infectivity hepatitis B and C make chronic liver diseases and subsequent HCC quite likely to occur.

[0005] The presentation of HCC has evolved significantly over the past few decades. In the past, HCC was generally found at an advanced stage with right upper quadrant pain, weight loss, and signs of decompensated liver disease, but now, due to the results of routine screening of known cirrhotic patients using cross - sectional imaging analysis and serum alpha - fetoprotein (AFP) measurement, recognition at a much earlier stage is increasing.

[0006] The threat of HCC is expected to continue to expand over the next few years (Llovet JM, et (See al., Liver Transpl. 2004 Feb. 10(2 Suppl 1): S115-20). Therefore, in order to increase the survival rate of these patients, there is a great demand for early detection of HCC.

Summary of the Invention

[0007] Hepatocellular carcinoma (HCC) is the second most lethal cancer worldwide. Survival rates improve with early detection, and accurate and non-invasive screening methods are needed. Technological innovation to provide accurate, inexpensive, and safe screening means for the pre-symptomatic detection of early HCC is essential.

[0008] The present invention addresses this need. In fact, the present invention provides novel methylated DNA markers that distinguish HCC from normal controls (cirrhosis-associated and non-cirrhosis-associated controls).

[0009] Methylated DNA has been studied as a potential biomarker type in most tumor types of tissue. In many cases, DNA methyltransferase adds a methyl group to DNA at cytosine-phosphate-guanine (CpG) island sites as an epigenetic control of gene expression. In a biologically attractive mechanism, acquired methylation events in the promoter region of tumor suppressor genes are thought to suppress expression and thus contribute to tumorigenesis. DNA methylation can be a more chemically and biologically stable diagnostic means than RNA or protein expression (Laird (2010) Nat Rev Genet 11: 191-203). Furthermore, in other cancers such as sporadic colorectal cancer, methylation markers give excellent specificity, provide more information than individual DNA mutations, and are highly sensitive (see Zou et al (2007) Cancer Epidemiol Biomarkers Prev 16: 2686-96).

[0010] Analysis of CpG islands has led to important discoveries when applied to animal models and human cell lines. For example, Zhang et al. 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, the levels of methylation were bimodally distributed between hypermethylated 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 showed that in dynamic tissue-specific patterns, promoters with high CpG density (HCP, defined as having >7% CpG sequences within a 300 base pair region) are only about 0.3% methylated, while regions with low CpG density (defined as having <5% CpG sequences within a 300 base pair region) tend to be methylated frequently (Meissner et al. (2008) Nature 454: 766 - 70). HCPs include promoters for ubiquitous housekeeping genes and highly regulated developmental genes. Among HCP sites, >50% methylation was found in several established markers such as Wnt2, NDRG2, SFRP2, and BMP3 (Meissner et al. (2008) Nature 454: 766 - 70).

[0011] In the process of developing embodiments of the present invention, experiments were conducted to compare the methylation status of DNA markers derived from the plasma of subjects with HCC with the methylation status of the same DNA markers derived from control subjects (e.g., subjects with cirrhosis or normal subjects). Such experiments identified and confirmed candidates for methylated DNA markers that distinguish HCC from such control groups.

[0012] Accordingly, provided herein are techniques for screening (e.g., monitoring) HCC, and specifically, but not limited to, methods, compositions, and related uses for detecting the presence of HCC.

[0013] The marker and / or panel of markers has been determined to be able to detect HCC (see Examples I, II, and III) (ACP1, BDH1, Chr12.133, CLEC11A, DAB2IP, DBNL, EMX1, EFNB2, HOXA1, LRRC4, SPINT2, TSPYL5, CCNJ_3707, CCNJ_3124, PFKP, SCRN1, and ECE1).

[0014] As described herein, the technology provides a number of highly discriminatory methylated DNA markers and subsets thereof (e.g., sets of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 markers) for detecting the presence of HCC in a subject. Experiments applied selection filters to candidate markers to identify markers that give a high signal-to-noise ratio and low background level, and a high specificity, for example, when assaying a medium (e.g., plasma) for the purpose of screening or diagnosis (e.g., screening or diagnosis of HCC).

[0015] In some embodiments, the technology relates to assessing the presence and methylation status of one or more markers identified herein in a biological sample (e.g., a plasma sample). These markers include, for example, one or more differentially methylated regions (DMRs) discussed herein as shown in Tables 1 and 4. The methylation status is evaluated in embodiments of the technology. Thus, the technology provided herein is not limited to methods of measuring the methylation status of genes. For example, in some embodiments, the methylation status is measured by genomic scanning methods. For example, one method includes the restriction landmark genomic scanning method (see Kawai et al. (1994) Mol. Cell. Biol. 14:7421-7427), and another example includes methylation-sensitive arbitrarily primed PCR (see Gonzalgo et al. (1997) Cancer Res. 57:594-599). In some embodiments, changes in the methylation pattern at specific CpG sites are observed by digestion of genomic DNA with methylation-sensitive restriction enzymes followed by Southern analysis of the region of interest (the digestion-Southern method). In some embodiments, analysis of changes in the methylation pattern includes PCR-based methods that include digestion of genomic DNA with methylation-sensitive restriction enzymes prior to PCR amplification (see Singer-Sam et al. (1990) Nucl. Acids Res. 18:687). Additionally, other techniques that utilize bisulfite treatment of DNA as a starting point for methylation analysis have been reported. These include methylation-specific PCR (MSP) (see Herman et al. (1992) Proc. Natl. Acad. Sci. USA 93:9821-9826) and restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA (see Sadri and Hornsby (1996) Nucl. Acids Res. 24:5058-5059, and Xiong and Laird (1997) Nucl. Acids Res. 25:2532-2534).The PCR technique was developed for the detection of gene mutations (see Kuppuswamy et al. (1991) Proc. Natl. Acad. Sci. USA 88:1143-1147) and 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 internal primers that anneal to the PCR-generated template and directly terminate at the 5' of a single nucleotide to be assayed. Methods using the "quantitative Ms-SNuPE assay" described in U.S. Patent No. 7,037,650 are used in some embodiments.

[0016] In assessing the methylation state, the methylation state is often expressed as the ratio or percentage of the individual strands of methylated DNA at a particular site (e.g., at a single nucleotide, in a particular region or locus, in a longer target sequence, e.g., a sub-sequence of DNA up to about 100 bp, 200 bp, 500 bp, 1000 bp, or more) to the total population of DNA in a sample containing that particular site. Traditionally, the amount of unmethylated nucleic acid is measured by PCR using a standard. Subsequently, a known amount of DNA is treated with bisulfite, and the resulting methylation-specific sequences are measured using either real-time PCR or other exponential amplification, e.g., the QuARTS assay (e.g., as provided in U.S. Patent Nos. 8,361,720, 8,715,937, and 8,916,344).

[0017] For example, in some embodiments, the method includes creating a standard curve for unmethylated targets using an external standard. The standard curve is composed of at least two points and associates the real-time Ct value of unmethylated DNA with a known quantitative standard. Thereafter, a second standard curve for methylated targets is composed of at least two points and an external standard. This second standard curve associates the Ct of methylated DNA with a known quantitative standard. Next, the Ct values of the test sample are measured for methylated and unmethylated populations, and the genomic equivalent of the DNA is calculated from the standard curves created in the previous 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 DNAs) / (number of methylated DNAs + number of unmethylated DNAs)×100.

[0018] Also provided herein are compositions and kits for performing the method. For example, in some embodiments, reagents specific for one or more markers (e.g., primers, probes) are provided alone or in sets (e.g., sets of primer pairs for amplifying multiple markers). Additional reagents for performing detection assays (e.g., enzymes, buffers, positive and negative controls for performing QuARTS, PCR, sequencing, bisulfite, or other assays) may also be provided. In some embodiments, a kit is provided that includes one or more reagents necessary, sufficient, or useful for performing the method. A reaction mixture containing the reagents is also provided. Further, a set of master mix reagents is provided that includes a plurality of reagents that can be added to each other and / or to a test sample to complete the reaction mixture.

[0019] In some embodiments, the techniques described herein are related to a programmable machine designed to perform a series of arithmetic or logical operations provided by the methods described herein. For example, some embodiments of the techniques are related to (e.g., are implemented by) computer software and / or computer hardware. In one aspect, the techniques are related 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 series of instructions (e.g., the methods provided herein) for reading, manipulating, and storing data. In some embodiments, the microprocessor is part of a system for measuring methylation states (e.g., one or more DMRs, e.g., those of DMR1 - 400 shown in Tables 1 and 4), comparing methylation states (e.g., one or more DMRs, e.g., those of DMR1 - 400 shown in Tables 1 and 4), creating standard curves, measuring Ct values, calculating ratios, frequencies, or percentages of methylation (e.g., one or more DMRs, e.g., those of DMR1 - 400 shown in Tables 1 and 4), identifying CpG islands, measuring the specificity and / or sensitivity of an assay or marker, calculating ROC curves and associated AUCs, and performing array analysis, all as described herein or known in the art.

[0020] In some embodiments, the microprocessor or computer uses methylation state data in an algorithm for predicting the site of cancer.

[0021] In some embodiments, a software or hardware component receives the results of multiple assays and determines a single-valued result indicative of the risk of cancer based on the results of the multiple assays for reporting to the user (e.g., determining the methylation status of multiple DMRs, e.g., the DMRs shown in Tables 1 and 4). Related embodiments calculate a risk factor based on a mathematical combination (e.g., weighted combination, linear combination) of results from multiple assays that measure the methylation status of multiple markers (e.g., multiple DMRs, e.g., those shown in Tables 1 and 4). In some embodiments, the methylation status of a DMR may define a dimension and have a value in a multi-dimensional space, and the coordinates defined by the methylation status of multiple DMRs are, for example, the results for reporting to the user.

[0022] Some embodiments include a storage medium and memory elements. The memory elements (e.g., volatile and / or non-volatile memory) find use in storing instructions (e.g., embodiments of the methods provided herein) and / or data (e.g., workpieces such as methylation measurements, arrays, and statistical descriptions related thereto). Some embodiments relate to a system that also includes one or more of a CPU, a graphics card, and a user interface (e.g., including output devices such as a display and input devices such as a keyboard).

[0023] The programmable machines associated with the technology include conventional existing technologies and technologies that are under development or not yet developed (e.g., quantum computers, chemical computers, DNA computers, optical computers, spintronics-based computers, etc.).

[0024] In some embodiments, the technology includes a wired (e.g., metal cable, optical fiber) or wireless transmission medium for data transmission. For example, some embodiments relate to data transmission through a network (e.g., local area network (LAN), wide area network (WAN), ad hoc network, Internet, etc.). In some embodiments, the programmable machine is included in such a network as a peer, and in some embodiments, the programmable machine has a client / server relationship.

[0025] In some embodiments, the data is stored in a computer-readable storage medium such as a hard disk, flash memory, optical medium, floppy disk, etc.

[0026] In some embodiments, the technology provided herein is associated with a plurality of programmable devices that cooperate to perform the methods described herein. For example, in some embodiments, in the execution of, for example, conventional network interfaces, such as Ethernet®, optical fiber, or wireless network technology, a network (private, public, or Internet), cluster computing or grid computing, or some other distributed computer architecture that depends on a complete computer (equipped with a CPU, storage device, power supply, network interface, etc.), a plurality of computers (e.g., connected by a network) can operate in parallel to collect and process data.

[0027] For example, some embodiments provide a computer that includes a computer-readable medium. The embodiments include 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 processors, and may be any of several computer processors such as those manufactured by Intel Corporation of Santa Clara, California and Motorola Corporation of Schaumburg, Illinois. Such a processor may comprise, or be in communication with, a computer-readable medium that stores instructions that, when executed by the processor, cause the processor to perform the steps described herein.

[0028] Embodiments of computer-readable media include, but are not limited to, electronic, optical, magnetic, or other storage or transmission devices that can provide computer-readable instructions to a processor. Examples of other suitable media include, but are not limited to, floppy disks, CD-ROMs, DVDs, magnetic disks, memory chips, ROMs, RAMs, ASICs, pre-programmed processors, all optical media, all magnetic tapes or other magnetic media, or any other media from which a computer processor can read instructions. Also, various other forms of computer-readable media may transmit or deliver instructions to a computer, including routers, private or public networks, and other transmission devices or channels both wired and wireless. The instructions may include code in any suitable computer programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, and JavaScript.

[0029] In some embodiments, the computer is connected to a network. The computer may also include some external or internal devices such as a mouse, CD-ROM, DVD, keyboard, display, or other input or output devices. Examples of computers are personal computers, digital assistants, portable terminals, cellular phones, mobile phones, smartphones, pocket bells, digital tablets, laptop computers, Internet appliances, and other processor-based devices. Generally, the computer related to the technical aspects provided herein may be any type of processor-based platform operating on any operating system such as Microsoft Windows, Linux (registered trademark), UNIX (registered trademark), Mac OS X, etc. that can support one or more programs including the technology provided herein. Some embodiments include personal computers that execute other application programs (e.g., applications). These applications may be included in the memory and may also include, for example, a document creation application, a spreadsheet application, an email application, an instant messenger application, a presentation application, an Internet browser application, a calendar / schedule management application, and any other application that can be executed by the client device.

[0030] All such components, computers, and systems described herein related to the technology may be logical or virtual.

[0031] Provided herein is a technology related to a method for screening HCC in a sample collected from a subject, the method comprising assaying the methylation status of a marker in a sample collected from the subject, and identifying that the subject has HCC when the methylation status of the marker is different from the methylation status of the marker assayed in a subject without HCC (e.g., a subject without HCC) (e.g., a subject with cirrhosis but without HCC), wherein the marker comprises bases of one or more differentially methylated regions (DMRs) selected from ACP1, BDH1, Chr12.133, CLEC11A, DAB2IP, DBNL, EMX1, EFNB2, HOXA1, LRRC4, SPINT2, TSPYL5, CCNJ_3707, CCNJ_3124, PFKP, SCRN1, and ECE1 shown in Tables 1 and 4.

[0032] The technology is not limited to the methylation status to be evaluated. In some embodiments, evaluating the methylation status of a marker in a sample comprises measuring the methylation status of one base. In some embodiments, evaluating the methylation status of a marker in a sample comprises measuring the degree of methylation with multiple bases. Further, in some embodiments, the methylation status of a marker comprises an increase in methylation of the marker relative to the normal methylation status of the marker. In some embodiments, the methylation status of a marker comprises a decrease in methylation of the marker relative to the normal methylation status of the marker. In some embodiments, the methylation status of a marker comprises a different methylation pattern of the marker relative to the normal methylation status of the marker.

[0033] Furthermore, in some embodiments, the marker is a region of 100 bases or less, the marker is a region of 500 bases or less, the marker is a region of 1000 bases or less, the marker is a region of 5000 bases or less, or, in some embodiments, the marker is one base. In some embodiments, the marker is within a promoter with a high CpG density.

[0034] The technology is not limited by the type of sample. For example, in some embodiments, the sample is a blood sample (e.g., plasma, serum, whole blood), a fecal sample, a tissue sample (e.g., gastric tissue, pancreatic tissue, bile duct / liver tissue, pancreatic juice, and colorectal tissue), excreta, or a urine sample.

[0035] Furthermore, the technology is not limited to the methods used to measure the methylation state. In some embodiments, the assay 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 assay includes the use of methylation-specific oligonucleotides. In some embodiments, the technology measures the methylation state using next-generation sequencing (e.g., sequencing by synthesis), real-time (e.g., single molecule) sequencing, bead emulsion sequencing, nanopore sequencing, etc.

[0036] The technology provides reagents for detecting DMRs. For example, in some embodiments, it provides a series of oligonucleotides including the sequences shown in SEQ ID NOs: 1-94 (Tables 2 and 5). In some embodiments, it provides oligonucleotides that include sequences complementary to chromosomal regions having the bases of the DMR, e.g., oligonucleotides sensitive to the methylation state of the DMR.

[0037] The technology provides panels of various markers. For example, in some embodiments, the markers are shown in Table 1 or 3 and include chromosomal regions having annotations containing the markers. Furthermore, the embodiments provide methods for analyzing DMRs from Tables 1 and / or 4 that are one or more of DMR numbers 1-400.

[0038] Embodiments of the kit, such as bisulfite reagents, and control nucleic acids comprising sequences derived from DMRs selected from the group consisting of DMR1-400 (from Tables 1 and 4), having a methylation status associated with a subject without HCC (e.g., a subject without HCC and without cirrhosis) (e.g., a subject without HCC but with cirrhosis) are provided. Embodiments of the kit, such as bisulfite reagents, and control nucleic acids comprising sequences derived from DMRs selected from the group consisting of DMR1-400 (from Tables 1 and 4), having a methylation status associated with a subject without HCC are provided.

[0039] Some embodiments of the kit include a sample collector for collecting a sample (e.g., a fecal sample) from a subject, reagents for isolating nucleic acids from the sample, bisulfite reagents, and the oligonucleotides described herein.

[0040] The technology relates to embodiments of compositions (e.g., reaction mixtures). In some embodiments, compositions comprising nucleic acids containing DMRs and bisulfite reagents are provided. Some embodiments provide compositions comprising nucleic acids containing DMRs and the oligonucleotides described herein. Some embodiments provide compositions comprising nucleic acids containing DMRs and methylation-sensitive restriction enzymes. Some embodiments provide compositions comprising nucleic acids containing DMRs and polymerases.

[0041] Embodiments of further related methods for screening for HCC in a sample (e.g., a plasma sample) collected from a subject are provided. For example, one method includes measuring the methylation status of a marker in a sample containing bases of one or more DMRs of DMR1-400 (from Tables 1 and 4), comparing the methylation status of the marker from the subject's sample with the methylation status of the marker from a normal control sample from a subject without HCC, and measuring the confidence interval and / or p-value of the difference in methylation status between the subject's sample and the normal control sample.

[0042] 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 method embodiments provide for reacting a nucleic acid containing a DMR with a bisulfite reagent to produce a bisulfite-reacted nucleic acid, determining the sequence of the bisulfite-reacted nucleic acid to provide the nucleotide sequence of the bisulfite-reacted nucleic acid, comparing the nucleotide sequence of the bisulfite-reacted nucleic acid with the nucleotide sequence of a nucleic acid containing a DMR from a subject without cancer to identify the difference between these two sequences, and identifying that the subject has a tumor if a difference exists.

[0043] A system for screening HCC in a sample collected from a subject is provided by this technology. As an exemplary system embodiment, for example, a system for screening HCC in a sample collected from a subject is included. The system includes an analysis component configured to measure the methylation state of the sample, a software component configured to compare the methylation state of the sample with the methylation state of a control sample or reference sample recorded in a database, and a warning component configured to warn the user of the methylation state related to HCC (e.g., the methylation state in the absence of HCC, the methylation state of HCC). In some embodiments, the warning is determined by a software component that receives results from multiple assays (e.g., measures the methylation state of multiple markers, e.g., DMRs, e.g., those shown in Tables 1 and 4) and calculates a reported value or result based on these multiple results. Some embodiments provide a database of weighting parameters related to each DMR provided herein for use in calculating the values or results to be reported to the user (e.g., physicians, nurses, clinicians, etc.) and / or for warnings. In some embodiments, all results from multiple assays are reported, and in some embodiments, one or more results are used to provide a score, value, or result based on a mixture of one or more results from multiple assays, which indicates the risk of HCC in the subject.

[0044] In some embodiments of the system, the sample contains nucleic acids including DMR. In some embodiments, the system further includes components for isolating nucleic acids, components for collecting samples, for example, components for collecting plasma samples. In some embodiments, the system includes nucleic acid sequences including DMR. In some embodiments, the database includes nucleic acid sequences from subjects without HCC. A set of nucleic acids is also provided, for example, nucleic acids each having a sequence including DMR. In some embodiments, the set of nucleic acids each has a sequence from a subject without HCC. Related system embodiments include the described set of nucleic acids and a database of nucleic acid sequences related to the set of nucleic acids. Some embodiments further include bisulfite reagents. Also, some embodiments further include nucleic acid sequencers.

[0045] In certain embodiments, a method for detecting HCC in a sample (e.g., a plasma sample) collected from a subject is provided, which comprises: a) collecting a sample containing DNA from the subject; b) treating the collected DNA with a reagent that selectively modifies non-methylated cytosine residues in the collected DNA to generate modified residues but does not modify methylated cytosine residues; c) measuring the methylation level of one or more DNA methylation markers in the DNA that has undergone the treatment of step b), wherein the one or more DNA methylation markers include bases of differentially methylated regions (DMRs) shown by DMR1-400 (from Tables 1 and 4); d) comparing the measured methylation level of the one or more DNA methylation markers with a methylation level reference of the one or more DNA methylation markers for subjects without HCC; and e) identifying that the subject has HCC if a difference exists.

[0046] In some embodiments, measuring an increase in methylation in one or more DNA methylation markers includes measuring a change in methylation within a region selected from the group consisting of CpG islands and CpG island shores.

[0047] In some embodiments, the measurement of increased methylation within the CpG island or CpG shore includes increased methylation within the coding or regulatory region of the DNA methylation marker.

[0048] In some embodiments, measuring the methylation level of one or more DNA methylation markers in the DNA that has undergone the treatment of step b) includes measuring the methylation score and / or the frequency of methylation of the one or more DNA methylation markers. In some embodiments, the treatment of step b) is achieved by bisulfite modification of the collected DNA.

[0049] In some embodiments, measuring the methylation level of one or more DNA methylation markers in the DNA that has undergone the treatment of step b) is achieved by a technique selected from the group consisting of methylation-specific PCR, quantitative methylation-specific PCR, methylation-sensitive DNA restriction enzyme analysis, quantitative bisulfite pyrosequencing, and bisulfite genomic sequencing PCR.

[0050] Further embodiments will be apparent to those skilled in the art based on the disclosure contained herein.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0052] Detailed Description Provided herein is a technique for screening hepatocellular carcinoma, and specifically, but not limited to, methods, compositions, and related uses for detecting the presence of hepatocellular carcinoma.

[0053] When the technology is described in this specification, the headings of the sections used are for purposes of organization only and are in no way to be construed as limiting the subject matter.

[0054] In this detailed description of various embodiments, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the embodiments of the present disclosure. However, it will be understood by those skilled in the art that these various embodiments may be practiced without these specific details. In other instances, structures and devices are shown in block diagram form. Further, it will be readily understood by those skilled in the art that the methods presented and the specific order of execution thereof are illustrative, and that these orders may be changed and still remain within the spirit and scope of the various embodiments disclosed herein.

[0055] All documents and materials of the same kind cited in this application, namely, but not limited to, patents, patent applications, articles, books, theses, and Internet web pages, etc., are hereby expressly incorporated by reference in their entirety for all purposes. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the various embodiments described herein belong. In the event that the definition of a term in an incorporated reference is deemed to differ from the definition provided in this disclosure, the definition provided in this disclosure shall control.

[0056] Definitions To facilitate understanding of the present technology, some terms and expressions are defined below. Further definitions are set forth throughout the detailed description.

[0057] Throughout this specification and the claims, the following terms take the meanings that are explicitly associated with them in this specification, unless the context clearly indicates otherwise. As used herein, the phrase "in one embodiment" does not necessarily refer to the same embodiment, but it may. Further, as used herein, the phrase "in another embodiment" does not necessarily refer to a different embodiment, but it may. Thus, as described below, the various embodiments of the present invention can be readily combined without departing from the scope or spirit of the present invention.

[0058] Further, as used herein, the term "or" is an inclusive "or" operator and is equivalent to the term "and / or" unless the context clearly indicates otherwise. The term "based on" is not exclusive and permits being based on additional factors not recited unless the context clearly indicates otherwise. Further, throughout this specification, the meanings of "a", "an", and "the" include plural references. The meaning of "in" includes "in" and "on".

[0059] 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. Examples of "nucleic acids" include, without limitation, single-stranded and double-stranded nucleic acids. The term "nucleic acid" as used herein also includes the above-described DNA containing one or more modified bases. Thus, DNA having a modified backbone for stability or other reasons is a "nucleic acid". The term "nucleic acid" as used herein encompasses such chemically, enzymatically, or metabolically modified forms of nucleic acids, as well as the chemical forms of DNA characteristic of viruses and cells, including, for example, simple and complex cells.

[0060] The terms "oligonucleotide", "polynucleotide", "nucleotide", or "nucleic acid" refer to a molecule having two or more, preferably more than three, and usually more than ten deoxyribonucleotides or ribonucleotides. The exact size is determined by many factors and similarly by the ultimate function or use of the oligonucleotide. The oligonucleotide can be produced by any method including chemical synthesis, DNA replication, reverse transcription, or combinations thereof. Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides of RNA are uracil, adenine, cytosine, and guanine.

[0061] As used herein, the terms "locus" or "region" of a nucleic acid refer to a small region of the nucleic acid, such as a gene, a single base, a CpG island, etc. on a chromosome.

[0062] The terms "complementary" and "complementarity" refer to nucleotides (e.g., a single nucleotide) or polynucleotides (e.g., a sequence of nucleotides) that are linked by base pairing rules. For example, the sequence 5'-A-G-T-3' is complementary to the sequence 3'-T-C-A-5'. Complementarity may be "partial" where only some of the bases of the nucleic acid conform to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands results in the efficiency and strength of hybridization between the nucleic acid strands. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids.

[0063] The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that contains the coding sequence necessary for the production of RNA, or for the production of a polypeptide or its precursor. A functional polypeptide can be encoded by the full-length coding sequence or, as long as the desired activity or functional property of the polypeptide (e.g., enzymatic activity, ligand binding, signal transduction, etc.) is retained, by any portion of the coding sequence. When used with respect to a gene, the term "portion" refers to a fragment of that gene. The fragment can range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, "nucleotides containing at least a portion of a gene" can include either a fragment of the gene or the entire gene.

[0064] The term "gene" also encompasses the coding region of a structural gene such that the gene corresponds to the length of the full-length mRNA (e.g., including the coding sequence, regulatory sequences, structural sequences, and other sequences), and includes sequences located adjacent to the coding regions at both the 5' and 3' termini, for example, within about 1 kb of either terminus. The sequence located 5' to the coding region and present in the mRNA is called the 5' non-translated or untranslated sequence. The sequence located 3' or downstream of the coding region and present in the mRNA is called the 3' non-translated or 3' untranslated sequence. The term "gene" encompasses both cDNA and genomic forms of the gene. In some organisms (e.g., eukaryotes), the genomic form or clone of a gene contains a coding region interrupted by non-coding sequences called "introns" or "intervening regions" or "intervening sequences". Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA), and introns can contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nucleus or primary transcript, and thus are not present in the messenger RNA (mRNA) transcript. The mRNA functions to specify the amino acid sequence or order of the nascent polypeptide during translation.

[0065] In addition to containing introns, genomic form genes can also include sequences located at both the 5' and 3' ends of the sequences present in the RNA transcript. These sequences are called "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the untranslated sequences present in the mRNA transcript). The 5' flanking region can include regulatory sequences such as promoters and enhancers that control or affect the transcription of the gene. The 3' flanking region can include sequences that direct the termination of transcription, post-transcriptional cleavage, and polyadenylation.

[0066] The term "allele" refers to genetic diversity, which includes, but is not limited to, variants and mutants, polymorphic loci, and single nucleotide polymorphic loci, frameshifts, and splice variants. Alleles can occur naturally within a population or can occur during the lifetime of any particular individual in the population.

[0067] Thus, the terms "variant" and "mutant" when used with respect to a nucleotide sequence refer to a nucleic acid sequence that differs from another, usually related, nucleotide acid sequence by one or more nucleotides. "Diversity" is the difference between two different nucleotide sequences, usually with one of the sequences being a reference sequence.

[0068] "Amplification" is a special case of nucleic acid replication that involves template specificity. This is contrasted with non-specific template replication (e.g., replication that is dependent on a template but not on a specific template). Template specificity is here distinguished from the fidelity of replication (e.g., the synthesis of the appropriate polynucleotide sequence) and nucleotide (ribo or deoxyribo) specificity. Template specificity is often described in terms of "target" specificity. Target sequences are "targets" in the sense that they are required to be selected from other nucleic acids. Amplification techniques are mainly designed for this selection.

[0069] The amplification of nucleic acids generally refers to the generation 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, 10 to 100 copies of a polynucleotide molecule, which may or may not be exactly the same), and the amplification product or amplicon is generally detectable. The amplification of polynucleotides involves various chemical and enzymatic processes. The generation of multiple DNA copies from one or several copies of a target or template DNA molecule in the process of polymerase chain reaction (PCR) or ligase chain reaction (LCR, see, e.g., U.S. Patent No. 5,494,810) is a certain type of amplification. Further types of amplification include, but are not limited to, allele-specific PCR (see, e.g., U.S. Patent No. 5,639,611), assembly PCR (see, e.g., U.S. Patent No. 5,965,408), helicase-dependent amplification (see, e.g., U.S. Patent No. 7,662,594), hot start PCR (see, e.g., U.S. Patents Nos. 5,773,258 and 5,338,671), sequence-specific PCR, inverse PCR (see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186), ligation-mediated PCR (see, e.g., Guilfoyle, R., et al., Nucleic Acids Research, 25:1854-1858 (1997), U.S. Patent No. 5,508,169), methylation-specific PCR (see, e.g., Herman, et al., (1996) PNAS 93(13)9821-9826), miniprimer PCR, multiplex ligation-dependent probe amplification (see, e.g., Schouten, et al., (2002) Nucleic Acids Research 30(12):e57), 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., (see (2008) BMC Genetics 9:80), nested PCR, overlap extension PCR (see, for example, Higuchi, et al., (1988) Nucleic Acids Research 16(15) 7351-7367), real-time PCR (see, for example, Higuchi, et al., (1992) Biotechnology 10:413-417, Higuchi, et al., (1993) Biotechnology 11:1026-1030), reverse transcription PCR (see, for example, Bustin, S.A. (2000) J. Molecular Endocrinology 25:169-193), solid-phase PCR, thermal asymmetric interlaced PCR, and touchdown PCR (see, for example, 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). Amplification of polynucleotides can also be achieved using digital PCR (see, for example, 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, US Patent Application Publication No. 20070202525).

[0070] The term "polymerase chain reaction" ("PCR") refers to the methods of U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188 to K.B. Mullis, which describe methods for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This method of amplifying a target sequence consists of introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, and then performing thermal cycles in the correct order in the presence of a DNA polymerase. These two primers are complementary to their respective strands of the double-stranded target sequence. To cause amplification, the mixture is denatured and then the primers are annealed to their complementary sequences within the target molecule. After annealing, these primers are extended with a polymerase to form new pairs of complementary strands. The steps of denaturation, primer annealing, and polymerase extension are repeated a number of times (i.e., denaturation, annealing, and extension constitute one "cycle" and there can be a number of "cycles"), and an amplified segment of the desired target sequence at a high concentration can be obtained. 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 thus this length is a controllable parameter. From the perspective of the repetition of this process, this method is called the "polymerase chain reaction" ("PCR"). Since the desired amplified segments of the target sequence become the dominant sequences (in terms of concentration) in the mixture, they are said to be "amplified by PCR" and are "PCR products" or "amplicons".

[0071] Template specificity is achieved by enzyme selection in most amplification techniques. Amplification enzymes are enzymes that process only specific sequences of nucleic acids in a heterogeneous mixture of nucleic acids under the conditions in which they are used. For example, in the case of Qβ replicase, MDV-1 RNA is the specific template for the replicase (Kacian et al., Proc. Natl. Acad. Sci. USA, 69:3038

[1972] ). Other nucleic acids are not replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has strict specificity for its own promoter (Chamberlin et al, Nature, 228:227

[1970] ). In the case of T4 DNA ligase, the enzyme does not ligate two oligonucleotides or polynucleotides where there is a mismatch at the ligation junction between the oligonucleotide or polynucleotide substrate and the template (Wu and Wallace (1989) Genomics 4:560). Finally, thermostable template-dependent DNA polymerases (e.g., Taq and Pfu DNA polymerases) are found to exhibit high specificity for sequences surrounded and thus defined by primers due to their ability to function at high temperatures. High temperatures favor the hybridization of primers to target sequences and result in thermodynamic conditions that are unfavorable for hybridization to non-target sequences (H.A. Erlich (ed.), PCR Technology, Stockton Press

[1989] ).

[0072] As used herein, the term "nucleic acid detection assay" refers to any method for determining the nucleotide composition of a target nucleic acid. Nucleic acid detection assays include, but are not limited to, DNA sequencing, probe hybridization, structure-specific cleavage assays (e.g., INVADER assay, Hologic, Inc.) (e.g., U.S. Pat. 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 US2009 / 0253142), enzyme mismatch cleavage assays (e.g., Variagenics, U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770), polymerase chain reaction, branched hybridization assays (e.g., Chiron, U.S. Pat. Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802), rolling circle replication (e.g., U.S. Pat. Nos. 6,210,884, 6,183,960, and 6,235,502), NASBA (e.g., U.S. Pat. No. 5,409,818), molecular labeling techniques (e.g., U.S. Pat. No. 6,150,097), E sensor technology (Motorola, U.S. Pat. Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573), cycling probe technology (e.g., U.S. Pat. Nos. 5,403,711, 5,011,769, and 5,660,988), Dade 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), ligase chain reaction (e.g., Barnay Proc. Natl. Acad. Sci USA 88, 189-93 (1991)), and sandwich hybridization assays (e.g., U.S. Pat. No. 5,288,609).

[0073] The term "amplifiable nucleic acid" refers to a nucleic acid that can be amplified by any amplification method. An "amplifiable nucleic acid" is typically intended to include a "sample template".

[0074] The term "sample template" refers to a nucleic acid derived from a sample that is analyzed for the presence of a "target" (defined below). In contrast, a "background template" is used with respect to nucleic acids other than the sample template, which may or may not be present in the sample. A background template is most often inadvertent. It may be the result of carryover or may be due to the presence of nucleic acid contaminants that are required to be purified from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.

[0075] The term "primer" refers to an oligonucleotide that can act as an initiation point for synthesis of an extension product of a primer complementary to a nucleic acid strand when placed under conditions (e.g., in the presence of nucleotides and an inducer, such as DNA polymerase, and at an appropriate temperature and pH) that induce synthesis of the extension product of the primer, whether it occurs naturally as seen in a purified restriction digest or is synthetically produced. A primer is preferably single-stranded for maximum efficiency in amplification, but alternatively may be double-stranded. In the double-stranded case, the primer is first treated to separate the strands and then used for preparation of the extension product. Preferably, a primer is an oligodeoxyribonucleotide. A primer must be of sufficient length to initiate synthesis of an extension product in the presence of an inducer. The exact length of a primer depends on many factors, including temperature, the source of the primer, and the use of the method.

[0076] The term "probe" refers to an oligonucleotide (e.g., a nucleotide sequence) that can hybridize to another oligonucleotide of interest, whether it occurs naturally as found in a purified restriction digest or is produced synthetically, recombinantly, or by PCR amplification. The probe may be single-stranded or double-stranded. Probes are useful for the detection, identification, and isolation of specific gene sequences (e.g., "capture probes"). Any probe used in the present invention may, in some embodiments, be labeled with any "reporter molecule" so as to be detectable by any detection system, including but not limited to enzyme (e.g., ELISA and enzyme-based histochemical assays), fluorescence, radioactivity, and luminescence systems. The present invention is not intended to be limited to any particular detection system or label.

[0077] As used herein, "methylation" refers to cytosine methylation at the C5 or N4 position of cytosine, N6 of adenine, or methylation of other types of nucleic acids. Since conventional in vitro DNA amplification methods do not retain the methylation pattern of the amplification template, in vitro amplified DNA is usually not methylated. However, "unmethylated DNA" or "methylated DNA" may also refer to amplified DNA whose original template is respectively unmethylated or methylated.

[0078] Accordingly, as used herein, "methylated nucleotide" or "methylated nucleotide base" refers to the presence of a methyl moiety on a nucleotide base that is not present in the typical nucleotide bases where such methyl moiety is widely recognized. For example, cytosine does not contain a methyl moiety in its pyrimidine ring, while 5-methylcytosine contains a methyl moiety at the 5-position of its pyrimidine ring. Thus, cytosine is not a methylated nucleotide, while 5-methylcytosine is a methylated nucleotide. In another example, thymine contains a methyl moiety at the 5-position of its pyrimidine ring, but for the purposes of this specification, since thymine is a typical nucleotide base of DNA, when present in DNA, thymine is not considered a methylated nucleotide.

[0079] As used herein, the term "methylated nucleic acid molecule" refers to a nucleic acid molecule that contains one or more methylated nucleotides.

[0080] 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 the nucleic acid molecule. For example, a nucleic acid molecule containing methylated cytosine is considered to be methylated (e.g., the methylation state of the nucleic acid molecule is methylation). A nucleic acid molecule that does not contain any methylated nucleotides is considered to be unmethylated.

[0081] The methylation state of a particular nucleic acid sequence (e.g., a gene marker or DNA region described herein) may indicate the methylation state of all bases of the sequence, or may indicate the methylation state of a subset of bases (e.g., one or more cytosines) within the sequence, or may indicate information regarding the density of local methylation within the sequence, with or without providing the exact position information within the sequence where methylation occurs.

[0082] The methylation state of a nucleotide locus of a nucleic acid molecule refers to the presence or absence of a methylated nucleotide at a particular locus of the nucleic acid molecule. For example, the methylation state of cytosine at the 7th nucleotide of a nucleic acid molecule is methylation if the nucleotide present at the 7th nucleotide of the nucleic acid molecule is 5-methylcytosine. Similarly, the methylation state of cytosine at the 7th nucleotide of a nucleic acid molecule is unmethylation if the nucleotide present at the 7th nucleotide of the nucleic acid molecule is cytosine (not 5-methylcytosine).

[0083] The methylation state can optionally be represented or indicated by a "methylation value" (e.g., representing the frequency, proportion, ratio, percentage, etc. of methylation). The methylation value can be generated, for example, by quantifying the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, by comparing amplification profiles after bisulfite reaction, or by comparing the sequences of bisulfite-treated and untreated nucleic acids. Thus, a value, such as a methylation value, represents the methylation state and can thus be used as a numerical indicator of the methylation state across multiple copies of a locus. This is particularly useful when it is desirable to compare the methylation state of a sequence in a sample to a threshold or reference value.

[0084] As used herein, "methylation frequency" or "methylation percentage (%)" refers to the number of instances in which a molecule or locus is methylated relative to the number of instances in which the molecule or locus is unmethylated.

[0085] Thus, the methylation state represents the methylation state of nucleic acids (e.g., genomic sequences). Further, the methylation state refers to the characteristics of nucleic acid segments at specific genomic loci associated with methylation. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues in this DNA sequence are methylated, the position(s) of the methylated C residue(s), the frequency or percentage of methylated C across any specific region of the nucleic acid, and differences in methylation of alleles, for example, due to differences in the origin of the alleles. 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 specific region of the nucleic acids in a biological sample. For example, when cytosine (C) residue(s) within a nucleic acid sequence are methylated, it can be referred to as "hypermethylated" or "increased methylation", while when cytosine (C) residue(s) within a DNA sequence are not methylated, it can be referred to as "hypomethylated" or "decreased methylation". Similarly, when cytosine (C) residue(s) within a nucleic acid sequence are methylated compared to another nucleic acid sequence (e.g., from a different region or a different individual, etc.), that sequence is considered hypermethylated or increased in methylation compared to the other nucleic acid sequence. Alternatively, when cytosine (C) residue(s) within a DNA sequence are not methylated compared to another nucleic acid sequence (e.g., from a different region or a different individual, etc.), that sequence is considered hypomethylated or decreased in methylation compared to the other nucleic acid sequence. Further, the term "methylation pattern" as used herein refers to the collective sites of methylated and unmethylated nucleotides across a region of a nucleic acid. Two nucleic acids can have the same or similar frequency or percentage of methylation but different methylation patterns if the number of methylated and unmethylated nucleotides is the same or similar across that region, but the positions of the methylated and unmethylated nucleotides are different.An array is said to have "differential methylation", or "methylation differences", or "different methylation states" if the degree of methylation is different (e.g., one has increased or decreased methylation relative to the other), the frequency is different, or the pattern is different. The term "differential methylation" refers to the difference in the level or pattern of nucleic acid methylation in a cancer-positive sample compared to that in a cancer-negative sample. This may also refer to the difference in level or pattern between patients in whom cancer has recurred after surgery and those in whom it has not. Differential methylation and specific levels or patterns of DNA methylation are prognostic and predictive biomarkers, for example, when the correct cut-off or predictive properties are defined.

[0086] The frequency of methylation states can be used to describe a population of individuals or samples from a single individual. For example, a nucleotide locus having a methylation state frequency of 50% means that 50% of the cases are methylated and 50% of the cases are unmethylated. Using such a frequency, for example, a nucleotide locus or nucleic acid region can be shown to be methylated to what extent in a population of individuals or a group 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 frequency of the methylation state of the first population or pool is different from the frequency of the methylation state of the second population or pool. Using such a frequency, for example, a nucleotide locus or nucleic acid region can also be shown to be methylated to what extent in a single individual. For example, using such a frequency, the degree to which a cell population from a tissue sample is methylated or not methylated at a nucleotide locus or nucleic acid region can be shown.

[0087] As used herein, "nucleotide locus" refers to the position of a nucleotide in a nucleic acid molecule. A nucleotide locus of a methylated nucleotide refers to the position of the methylated nucleotide in a nucleic acid molecule.

[0088] Typically, methylation of human DNA occurs in a dinucleotide sequence that includes adjacent guanine and cytosine, where the cytosine is located 5' to the guanine (also called a CpG dinucleotide sequence). Most cytosines within CpG dinucleotides are methylated in the human genome, but remain partially unmethylated in specific genomic regions of high CpG dinucleotides known as CpG islands (see, e.g., Antequera et al. (1990) Cell 62:503-514).

[0089] As used herein, the term "CpG island" refers to a high G:C region of genomic DNA that contains an increased number of CpG dinucleotides relative to the genomic DNA as a whole. CpG islands can be at least 100, 200, or more base pairs in length, where the G:C content of the region is at least 50%, and the ratio of the observed CpG frequency to the predicted frequency is 0.6. In some cases, CpG islands can be at least 500 base pairs in length, where the G:C content of the region is at least 55%, and the ratio of the observed CpG frequency to the predicted frequency is 0.65. The observed CpG frequency relative to the predicted frequency can be calculated according to the method described by Gardiner-Garden et al (1987) J. Mol. Biol. 196:261-281. For example, the observed CpG frequency relative to the predicted 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 predicted 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. The methylation status is usually measured in the promoter region of CpG islands, for example. However, it will be appreciated that other sequences in the human genome, such as CpA and CpT, are prone to DNA methylation (see, for example, Ramsahoye (2000) Proc. Natl. Acad. Sci. USA 97:5237-5242, Salmon and Kaye (1970) Biochim. Biophys. Acta. 204:340-351, Grafstrom (1985) Nucleic Acids Res. 13:2827-2842, Nyce (1986) Nucleic Acids Res. 14:4353-4367, Woodcock (1987) Biochem. Biophys. Res. Commun. 145:888-894).

[0090] As used herein, a reagent that modifies nucleotides of a nucleic acid molecule according to the methylation state of the nucleic acid molecule, i.e., a methylation-specific reagent, refers to a compound, 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. A method of treating a nucleic acid molecule with such a reagent may include contacting the nucleic acid molecule with the reagent and, if desired, combining with additional steps to achieve a desired change in the nucleotide sequence. Such a change in the nucleotide sequence of the nucleic acid molecule can result in a nucleic acid molecule in which each methylated nucleotide is modified to a different nucleotide. Such a change in the nucleotide sequence of the nucleic acid can result in a nucleic acid molecule in which each unmethylated nucleotide is modified to a different nucleotide. Such a change in the nucleotide sequence of the nucleic acid can result in a nucleic acid molecule in which each selected unmethylated nucleotide (e.g., each unmethylated cytosine) is modified to a different nucleotide. Use of such a reagent to alter the nucleotide sequence of a nucleic acid can result in a nucleic acid molecule in which each nucleotide that is a methylated nucleotide (e.g., each methylated cytosine) is modified to a different nucleotide. As used herein, use of a reagent that modifies a selected nucleotide refers to a reagent that modifies one of the four nucleotides (C, G, T, and A for DNA and C, G, U, and A for RNA) that normally occur in a nucleic acid molecule, and thus the reagent modifies that one nucleotide without modifying the other three nucleotides. In one exemplary embodiment, such a reagent modifies a selected unmethylated nucleotide to produce a different nucleotide. In another exemplary embodiment, such a reagent can deaminate an unmethylated cytosine nucleotide. An exemplary reagent is bisulfite.

[0091] As used herein, the term "bisulfite reagent" refers, in some embodiments, to a reagent that includes bisulfite, disulfite, hydrogen sulfite, or combinations thereof for differentiating methylated and unmethylated cytidines, e.g., in a CpG dinucleotide sequence.

[0092] The term "methylation assay" refers to any assay for measuring the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence.

[0093] The term "MS AP-PCR" (methylation-sensitive arbitrarily primed polymerase chain reaction) enables a global scan of the genome using high-CG primers and focuses on regions most likely to contain CpG dinucleotides, and refers to a technique recognized in the art as described in Gonzalgo et al. (1997) Cancer Research 57:594-599.

[0094] The term "MethyLight™" refers to a fluorescence-based real-time PCR technique recognized in the art as described in Eads et al. (1999) Cancer Res. 59:2302-2306.

[0095] The term "HeavyMethyl™" refers to an assay in which methylation-specific blocking probes (also referred to herein as blockers) that cover the CpG moieties between or covered by amplification primers enable methylation-specific selective amplification of a nucleic acid sample.

[0096] The term "HeavyMethyl™ MethyLight™ assay" refers to the HeavyMethyl™ MethyLight™ assay, which is a variant of the MethyLight™ assay, where the MethyLight™ assay is combined with methylation-specific blocking probes that cover the CpG positions between the amplification primers.

[0097] The term "Ms-SNuPE" (methylation-sensitive single nucleotide primer extension) refers to an assay recognized in the art as described in Gonzalgo & Jones (1997) Nucleic Acids Res. 25:2529-2531.

[0098] The term "MSP" (methylation-specific PCR) refers to a methylation assay recognized in the art as described in Herman et al. (1996) Proc. Natl. Acad. Sci. USA 93:9821-9826, and U.S. Patent No. 5,786,146.

[0099] The term "COBRA" (combined bisulfite restriction analysis) refers to a methylation assay recognized in the art as described in Xiong & Laird (1997) Nucleic Acids Res. 25:2532-2534.

[0100] The term "MCA" (methylated CpG island amplification) refers to a methylation assay as described in Toyota et al. (1999) Cancer Res. 59:2307-12, and WO00 / 26401A1.

[0101] As used herein, "selected nucleotide" refers to one of the four nucleotides (C, G, T, and A in DNA and C, G, U, and A in RNA) that normally occur in a nucleic acid molecule, and may include methylated derivatives of the normally occurring nucleotides (e.g., if C is the selected nucleotide, both methylated and non-methylated C are included in the meaning of the selected nucleotide). On the other hand, 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.

[0102] The term "methylation-specific restriction enzyme" or "methylation-sensitive restriction enzyme" refers to an enzyme that selectively digests nucleic acids depending on the methylation state of its recognition site. In the case of a restriction enzyme that specifically cleaves when the recognition site is not methylated or is hemimethylated, cleavage does not occur or occurs with a significantly reduced efficiency when the recognition site is methylated. In the case of a restriction enzyme that specifically cleaves when the recognition site is methylated, cleavage does not occur or occurs with a significantly reduced efficiency when the recognition site is not methylated. Preferred are methylation-specific restriction enzymes whose recognition sequence contains a CG dinucleotide (for example, a recognition sequence such as CGCG or CCCGGG). Even more preferred for some embodiments are restriction enzymes that do not cleave when the cytosine of this dinucleotide is methylated at carbon atom C5.

[0103] As used herein, "different nucleotide" refers to a nucleotide that is chemically different from a selected nucleotide, and thus, typically, the different nucleotide has Watson-Crick base pairing properties different from those of the selected nucleotide, such that the normally occurring nucleotide complementary to the selected nucleotide is not the same as the normally occurring nucleotide complementary to the different nucleotide. For example, if C is the selected nucleotide, U or T may be the different nucleotide, as exemplified by the complementarity of C and G and the complementarity of U or T and A. As used herein, a nucleotide complementary to a selected nucleotide or a nucleotide complementary to a different nucleotide refers to a nucleotide that base pairs with the selected nucleotide or different nucleotide with a higher affinity than base pairing of the complementary nucleotide with three of the four normally occurring nucleotides under high stringency conditions. Examples of complementarity are the Watson-Crick base pairs of DNA (e.g., A-T and C-G) and RNA (e.g., A-U and C-G). Thus, for example, G base pairs with C with a higher affinity than G base pairs with G, A, or T under high stringency conditions, and thus, if C is the selected nucleotide, G is the nucleotide complementary to the selected nucleotide.

[0104] As used herein, "sensitivity" of a given marker refers to the percentage of samples that report a DNA methylation value above a threshold that distinguishes between neoplastic and non-neoplastic samples. In some embodiments, positive is defined as a histologically confirmed neoplasm that reports a DNA methylation value above a threshold (e.g., in the range associated with the disease), and false negative is defined as a histologically confirmed neoplasm that reports a DNA methylation value below the threshold (e.g., in the range associated with no disease). The value of sensitivity thus reflects the probability that a DNA methylation measurement of a given marker from a known lesion sample is 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 its clinical symptoms when applied to a subject with clinical symptoms.

[0105] As used herein, the "specificity" of a given marker 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 range associated with no disease) 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 range associated with no disease). The specificity value thus reflects the probability that the DNA methylation measurement of a given marker from a known non-neoplastic sample is within the range of the non-disease associated measurement. 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 a patient without that clinical condition.

[0106] The term "AUC" used herein is an abbreviation for "area under the curve". In particular, it refers to the area under the receiver operating characteristic (ROC) curve. The ROC curve is a plot of the true positive rate against the false positive rate for different possible cut points of a diagnostic test. It shows the trade-off between sensitivity and specificity depending on the cut point selected (any increase in sensitivity is accompanied by a decrease in specificity). The area under the ROC curve (AUC) is a measure of the accuracy of a diagnostic test (the larger the area, the better. The optimum is 1. A random test has a ROC curve on the diagonal with an area of ​​0.5. For reference, see J.P.Egan. (1975) Signal Detection Theory and ROC Analysis, Academic Press, New York).

[0107] The term "tumor" as used herein refers to "an abnormal mass of tissue whose growth exceeds and is out of step with that of normal tissue." See, e.g., Willis RA, "The Spread of Tumors in the Human Body," London, Butterworth & Co, 1952.

[0108] As used herein, the term "adenoma" refers to a benign tumor of glandular origin. These tumors are bipotential and may progress over time to become malignant.

[0109] The terms "precancerous condition" or "preneoplastic" and their equivalents refer to any cell proliferative disorder that is undergoing malignant transformation.

[0110] A "site" or "region" of a tumor, adenoma, cancer, etc. is a tissue, organ, cell type, anatomical region, body part, etc. within the body of a subject in which the tumor, adenoma, cancer, etc. is located.

[0111] The application of a "diagnostic" test as used herein includes detecting or identifying the condition or state of a subject, determining the likelihood that a subject has a given disease or condition, determining the likelihood that a subject having a disease or condition will respond to treatment, determining the prognosis (or its estimated progression or regression) of a subject having a disease or condition, and determining the effectiveness of treatment for a subject having a disease or condition. For example, a certain diagnosis can be used to detect the presence or likelihood of a subject having a tumor, or the likelihood that such a subject will favorably respond to a compound (e.g., a pharmaceutical, e.g., a drug) or other treatment.

[0112] As used herein, the term "marker" refers to a substance (e.g., a nucleic acid or a region of a nucleic acid) that can diagnose a disorder (e.g., a non-cancerous disorder) (e.g., a cancerous disorder) by distinguishing normal cells from disorder-related cells (e.g., non-cancerous cells associated with the disorder) (e.g., cancerous cells associated with the disorder), for example based on their methylation status.

[0113] As used in reference to nucleic acids, the term "isolated" when used in reference to "isolated oligonucleotides" refers to a nucleic acid sequence that has been identified and separated from at least one nucleic acid contaminating substance with which it is ordinarily associated in its natural source. An isolated nucleic acid exists in a form or context 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 exist in nature. Examples of non-isolated nucleic acids include a given DNA sequence (e.g., a gene) found in a host cell's chromosome in proximity to an adjacent gene, an RNA sequence, e.g., a particular mRNA encoding a particular protein that exists in a cell as a mixture with many other mRNAs encoding a number of proteins. However, an isolated nucleic acid encoding a particular protein includes, for example, such nucleic acid within a cell that normally expresses the protein, where the nucleic acid is at a chromosomal location different from that of the natural cell or has a nucleic acid sequence arranged laterally that is different from that found in nature. The isolated nucleic acid or oligonucleotide may exist in single-stranded form or in double-stranded form. When an isolated nucleic acid or oligonucleotide is utilized to express a protein, the oligonucleotide includes at least the sense strand or coding strand (i.e., the oligonucleotide can be single-stranded), but may also include both the sense strand and the antisense strand (i.e., the oligonucleotide can be double-stranded). An isolated nucleic acid can be combined with other nucleic acids or molecules after isolation from its natural or normal environment. For example, an isolated nucleic acid may be included in a host cell in which it is placed, for example, for heterologous expression.

[0114] The term "purified" refers to any molecule of a nucleic acid or amino acid sequence that has been removed from its natural environment, isolated, or separated. An "isolated nucleic acid sequence" can thus be a purified nucleic acid sequence. A "substantially purified" molecule contains less than 60%, preferably less than 75%, and more preferably less than 90% of other components that are naturally associated with it. The terms "purified" or "purifying" as used herein also refer to the removal of contaminants from a sample. Removal of contaminating proteins increases the proportion of the polypeptide or nucleic acid of interest in the sample. In another example, recombinant polypeptides are expressed in plant, bacterial, yeast, or mammalian host cells, and these polypeptides are purified by removal of host cell proteins, thereby increasing the proportion of the recombinant polypeptide in the sample.

[0115] The term "composition comprising" a given polynucleotide sequence or polypeptide broadly refers to any composition that contains the given polynucleotide sequence or polypeptide. The composition can include an aqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS), and other components (e.g., Denhardt's solution, milk powder, salmon sperm DNA, etc.).

[0116] The term "sample" is used in its broadest sense. In one sense, it can refer to animal cells or tissues. In another sense, it means any source, as well as specimens or cultures derived from biological and environmental samples. Biological samples can be taken from plants or animals (including humans) and include fluids, solids, tissues, and gases. Environmental samples include environmental substances such as surface materials, soil, water, and industrial samples. These examples are not to be construed as limiting the types of samples applicable to the present invention.

[0117] As used herein, the term "remote sample," as used in some contexts, relates to a sample that is indirectly obtained from a site that is not the cell, tissue, or organ source of the sample. For example, when a pancreatic-derived sample substance is evaluated in a fecal sample (e.g., not from a sample taken directly from the pancreas), this sample is a remote sample.

[0118] As used herein, the terms "patient" or "subject" refer to an organism that is subjected to the various tests provided by the 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.

[0119] As used herein, the term "kit" refers to any delivery system for delivering a substance. In the context of a reaction assay, such a delivery system includes a system that enables the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in a suitable container) and / or support materials (e.g., buffers, instructions for performing the assay, etc.) from one location to another. For example, a kit comprises one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or support materials. As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separate containers, each containing a small portion of the total kit components. These containers can be supplied together or separately to the intended recipient. For example, the first container may contain an enzyme for use in an assay, while the second container contains oligonucleotides. The term "fragmented kit" is intended to, but not limited to, encompass kits containing analyte-specific reagents (ASRs) regulated by section 520(e) of the Federal Food, Drug, and Cosmetic Act. In fact, any delivery system that includes two or more separate containers, each containing a small portion of the total kit components, is included in the term "fragmented kit". In contrast, a "mixed kit" refers to a delivery system that includes all of the components of a reaction assay in a single container (e.g., a single box containing each of the desired components). The term "kit" includes both fragmented kits and mixed kits.

[0120] Embodiments of the technology Provided herein are technologies for screening (e.g., detecting) HCC, and specifically, but not limited to, methods, compositions, and related uses for detecting the presence of HCC in a subject.

[0121] A marker and / or panel of markers has been confirmed to be able to detect HCC (see Examples I, II, and III) (e.g., ACP1, BDH1, Chr12.133, CLEC11A, DAB2IP, DBNL, EMX1, EFNB2, HOXA1, LRRC4, SPINT2, TSPYL5, CCNJ_3707, CCNJ_3124, PFKP, SCRN1, and ECE1) (e.g., chromosomal regions with the annotations shown in Tables 1 and 4).

[0122] Although the disclosure herein refers to certain exemplary embodiments, it should be understood that these embodiments are presented by way of example and not limitation.

[0123] The method includes measuring the methylation status of at least one methylated marker in a biological sample isolated from a subject, wherein a change in the methylation status of the marker indicates the presence or class of HCC. Certain embodiments include differential methylation regions (DMRs, e.g., DMR1-400 (from Tables 1 and 4)) and relate to markers used for the diagnosis (e.g., screening) of HCC.

[0124] In addition to embodiments where the methylation analysis of at least one marker, marker region, or marker base containing the DMRs (e.g., DMR1-400) provided herein and described in Tables 1 and 3 is analyzed, the technique further provides a panel of markers including at least one marker, marker region, or marker base containing a DMR useful for the detection of HCC in a subject.

[0125] Some embodiments of the technique are based on the analysis of the CpG methylation status of at least one marker, marker region, or marker base containing a DMR.

[0126] In some embodiments, the present technology provides for the use of bisulfite technology in combination with one or more methylation assays for measuring the methylation status of CpG dinucleotide sequences within at least one marker that includes DMRs (e.g., those provided in Tables 1 and 4 (e.g., DMR1 - 400)). Genomic CpG dinucleotides may or may not be methylated (alternatively known as up - and down - methylation, respectively). However, the methods of the present invention are suitable for analysis within the background of a heterogeneous biological sample, such as a low - concentration tumor cell or a biological substance derived therefrom, in a remote sample (e.g., blood, organ eluate, or feces). Thus, when analyzing the methylation status of CpG positions within such a sample, a quantitative assay may be used to measure the level (e.g., percentage, ratio, proportion, rate, or degree) of methylation at a particular CpG position.

[0127] According to the present technology, measurement of the methylation status of CpG dinucleotide sequences in markers that include DMRs is useful in both the diagnosis and characterization of HCC in a subject.

[0128] Combination of markers In some embodiments, the technology relates to evaluating the methylation status of a combination of markers that includes two or more DMRs from Table 1 and / or 4 (e.g., two or more DMRs from DMR numbers 1 - 400). In some embodiments, evaluating the methylation status of two or more markers increases the specificity and / or sensitivity of screening or diagnosis for identifying the presence of HCC in a subject.

[0129] Various cancers are predicted by various combinations of markers. For example, they are identified by statistical techniques related to the specificity and sensitivity of prediction. The technology provides methods for identifying combinations of predictions and effective combinations of predictions for several cancers.

[0130] For example, a marker and / or a panel of markers has been determined to be capable of detecting HCC (see Examples I, II, and III) (e.g., ACP1, BDH1, Chr12.133, CLEC11A, DAB2IP, DBNL, EMX1, EFNB2, HOXA1, LRRC4, SPINT2, TSPYL5, CCNJ_3707, CCNJ_3124, PFKP, SCRN1, and ECE1) (e.g., chromosomal regions having the annotations shown in Tables 1 and 4).

[0131] Assay method for methylation status Regarding the presence of 5-methylcytosine, the method most frequently used for analyzing nucleic acids is based on the bisulfite method described by Frommer et al. (Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89:1827-31) for detecting 5-methylcytosine in DNA or a modification thereof. This bisulfite method for mapping 5-methylcytosine is based on the observation that cytosine, rather than 5-methylcytosine, reacts with hydrogen sulfite ions (also known as bisulfite). This reaction is typically carried out according to the following steps. First, cytosine reacts with hydrogen sulfite to form sulfonated cytosine. Next, sulfonated uracil is obtained by spontaneous deamination of this sulfonated reaction intermediate. Finally, this sulfonated uracil is desulfonated under alkaline conditions to yield uracil. Since uracil forms a base pair with adenine (and thus behaves like thymine), while 5-methylcytosine base pairs with guanine (and thus behaves like cytosine), detection is possible. This enables the discrimination between methylated and non-methylated cytosine, for example, by bisulfite genomic sequencing (Grigg G, & Clark S, Bioessays (1994) 16:431-36, Grigg G, DNA Seq. (1996) 6:189-98) or, for example, by methylation-specific PCR (MSP) disclosed in U.S. Patent No. 5,786,146.

[0132] Some prior art methods involve encapsulating the DNA to be analyzed in an agarose matrix, thereby preventing DNA diffusion and regeneration (bisulfite only reacts with single-stranded DNA) and replacing the precipitation and purification steps with high-speed dialysis (Olek A, et al. (1996) “A modified and improved method for bisulfite based cytosine methylation analysis” Nucleic Acids Res. 24:5064-6). Thus, it is possible to analyze the methylation state of individual cells, explaining the utility and sensitivity of this method. A review of conventional methods for detecting 5-methylcytosine is provided in Rein, T., et al. (1998) Nucleic Acids Res. 26:2255.

[0133] The bisulfite technique typically involves, following bisulfite treatment, amplifying a short, specific fragment of a known nucleic acid and then analyzing the product either by sequencing (Olek & Walter (1997) Nat. Genet. 17:275-6) or by analyzing individual cytosine positions by primer extension reactions (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 the bisulfite technique for detecting methylation of individual genes has been described (Grigg & Clark (1994) Bioessays 16:431-6, Zeschnigk et al. (1997) Hum Mol Genet. 6:387-95, Feil et al. (1994) Nucleic Acids Res. 22:695, Martin et al. (1995) Gene 157:261-4, WO9746705, WO9515373).

[0134] Various methylation assay techniques are known in the art and can be used in combination with bisulfite treatment according to the technology of the present invention. These assays enable the measurement of the methylation status of one or more CpG dinucleotides (e.g., CpG islands) within a nucleic acid sequence. Such assays include, among other techniques, sequencing of bisulfite-treated nucleic acids, PCR (for sequence-specific amplification), Southern blot analysis, and the use of methylation-sensitive restriction enzymes.

[0135] 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, digestion of PCR products amplified from bisulfite-converted DNA with restriction enzymes has found use in the evaluation of methylation status, as described, for example, in 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).

[0136] COBRA (trademark) analysis is a quantitative methylation assay useful for measuring DNA methylation levels at specific loci in small amounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997). Briefly, digestion with restriction enzymes 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 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 a specific labeled hybridization probe. The methylation level in the original DNA sample is linearly and quantitatively represented over a wide range of DNA methylation levels by the relative amounts of digested and undigested PCR products. Furthermore, this technique can be reliably applied to DNA derived from microdissected paraffin-embedded tissue samples.

[0137] Typical reagents for COBRA (trademark) analysis (e.g., those that can be found in a typical COBRA (trademark)-based kit) can 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, a kinase labeling kit for oligonucleotide probes, and labeled nucleotides. Furthermore, bisulfite conversion reagents can include DNA denaturation buffers, sulfonation buffers, DNA recovery reagents or kits (e.g., precipitation, ultrafiltration, affinity columns), desulfonation buffers, and DNA recovery components.

[0138] Preferably, assays such as "MethyLight (trademark)" (fluorescent-based real-time PCR technology) (Eads et al., Cancer Res. 59:2302-2306, 1999), Ms-SNuPE (trademark) (methylation-sensitive single nucleotide primer extension) reaction (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 methylation CpG island amplification ("MCA", Toyota et al., Cancer Res. 59:2307-12, 1999) are used alone or in combination with one or more of these methods.

[0139] The "HeavyMethyl (trademark)" assay, i.e., technology, is a quantitative method for assessing methylation differences based on methylation-specific amplification of bisulfite-treated DNA. Methylation-specific blocking probes ("blockers") that cover CpG positions between or covered by these amplification primers enable methylation-specific selective amplification of nucleic acid samples.

[0140] The term "HeavyMethyl (trademark) MethyLight (trademark)" assay refers to the HeavyMethyl (trademark) MethyLight (trademark) assay, which is a variant of the MethyLight (trademark) assay, where the MethyLight (trademark) assay is combined with methylation-specific blocking probes that cover CpG positions between amplification primers. This HeavyMethyl (trademark) assay may also be used in combination with methylation-specific amplification primers.

[0141] Examples of common reagents for HeavyMethyl (trademark) analysis (e.g., those that can be found in a normal MethyLight (trademark) - based kit) include, but are not limited to, PCR primers, blocking oligonucleotides, optimized PCR buffers and deoxynucleotides, and Taq polymerase for specific loci (e.g., specific genes, markers, DMRs, gene regions, marker regions, bisulfite - treated DNA sequences, CpG islands, or bisulfite - treated DNA sequences or CpG islands, etc.).

[0142] MSP (methylation - specific PCR) enables the assessment of the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation - sensitive restriction enzymes (Herman et al. Proc. Natl. Acad. Sci. USA 93:9821 - 9826, 1996; U.S. Patent No. 5,786,146). Briefly, DNA is modified with sodium bisulfite, which converts unmethylated cytosine, but not methylated cytosine, to uracil, and the product is subsequently amplified with primers specific for methylated DNA as opposed to unmethylated. MSP requires only small amounts of DNA, is sensitive to 0.1% methylated alleles at a given CpG island locus, and can be performed on DNA extracted from paraffin - embedded samples. Examples of common reagents for MSP analysis (e.g., those that can be found in a normal MSP - based kit) include, but are not limited to, methylation - and unmethylation - specific PCR primers, optimized PCR buffers and deoxynucleotides, and specific probes for specific loci (e.g., specific genes, markers, DMRs, gene regions, marker regions, bisulfite - treated DNA sequences, CpG islands, etc.).

[0143] The MethyLight™ assay is a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (e.g., TaqMan®) that does not require further manipulation after the PCR step (Eads et al., Cancer Res. 59:2302-2306, 1999). Briefly, the MethyLight™ process begins with a pooled sample of genomic DNA and converts this by sodium bisulfite reaction according to standard procedures into a pooled mixture of methylation-dependent sequence differences (in this bisulfite process, unmethylated cytosine residues are converted to uracil). Subsequently, for example, fluorescence-based PCR is performed in a "biased" reaction using PCR primers that overlap known CpG dinucleotides. Sequence discrimination occurs at both the level of the amplification step and the fluorescence detection step.

[0144] The MethyLight™ assay is used as a quantitative test for methylation patterns in nucleic acids, e.g., genomic DNA samples, and sequence discrimination occurs at the level of probe hybridization. In the quantitative form, the PCR reaction gives methylation-specific amplification in the presence of a fluorescent probe that overlaps a specific putative methylation site. An unbiased control for the amount of input DNA is provided by a reaction in which neither the primers nor the probe overlaps any CpG dinucleotide. Alternatively, a qualitative test of genomic methylation is achieved by probing a biased PCR pool with a control oligonucleotide that does not cover known methylation sites (e.g., the fluorescence-based HeavyMethyl™ and MSP techniques) or with either of the oligonucleotides that cover potential methylation sites.

[0145] The MethyLight™ procedure is used with any suitable probe (e.g., a "TaqMan®" probe, a 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 a TaqMan® probe and, e.g., MSP primers, and / or HeavyMethyl blocker oligonucleotides and TaqMan® probe. The TaqMan® probe is doubly labeled with fluorescent "reporter" and "quencher" molecules and is designed to be specific for regions of relatively high GC content, so that it melts at a temperature approximately 10° C. higher than the forward or reverse primers in the PCR cycle. This allows the TaqMan® probe to remain fully hybridized during the annealing / extension step of PCR. As Taq polymerase enzymatically synthesizes new strands during PCR, the ultimately annealed TaqMan® probe is reached. The 5' to 3' endonuclease activity of Taq polymerase then digests and removes the TaqMan® probe, releasing the fluorescent reporter molecule, and a real-time fluorescence detection system is used for quantitative detection of its signal, now with the quencher removed.

[0146] Typical reagents for MethyLight™ analysis (e.g., those that may be found in a typical MethyLight™-based kit) can 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.), TaqMan® or Lightcycler® probes, optimized PCR buffers and deoxynucleotides, and Taq polymerase.

[0147] The QM (trademark) (Quantitative Methylation) assay is an alternative quantitative test for methylation patterns in genomic DNA samples, and sequence discrimination occurs at the level of probe hybridization. In this quantitative type, the PCR reaction gives unbiased amplification in the presence of a fluorescent probe overlapping a specific putative methylation site. An unbiased control for the amount of DNA input is provided by a reaction where neither the primers nor the probe overlap any CpG dinucleotide. Alternatively, qualitative tests of genomic methylation are achieved by probing a biased PCR pool with control oligonucleotides that do not cover known methylation sites (fluorescent-based HeavyMethyl (trademark) and MSP techniques), or with oligonucleotides that cover potential methylation sites.

[0148] The QM (trademark) process can be used in an amplification process with any suitable probe, such as a "TaqMan (registered trademark)" probe, a Lightcycler (registered trademark) probe, etc. For example, double-stranded genomic DNA is treated with sodium bisulfite and subjected to unbiased primers and TaqMan (registered trademark) probes. The TaqMan (registered trademark) probe is doubly labeled with fluorescent "reporter" and "quencher" molecules and is designed to be specific for regions of relatively high GC content, so that it melts at a temperature approximately 10 °C higher than the forward or reverse primers in the PCR cycle. This allows the TaqMan (registered trademark) probe to remain fully hybridized during the annealing / extension step of PCR. As Taq polymerase enzymatically synthesizes new strands during the PCR process, it eventually reaches the finally annealed TaqMan (registered trademark) probe. The 5' to 3' endonuclease activity of Taq polymerase then digests and removes the TaqMan (registered trademark) probe, releasing the fluorescent reporter molecule, and a real-time fluorescence detection system is used for quantitative detection of its signal with the quencher removed here. Ordinary reagents for QM (trademark) analysis (such as those that can be found in ordinary QM (trademark)-based kits) can include, but are not limited to, PCR primers for specific loci (such as specific genes, markers, DMRs, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.), TaqMan (registered trademark) or Lightcycler (trademark) probes, optimized PCR buffers and deoxynucleotides, and Taq polymerase.

[0149] The Ms-SNuPE (trademark) technique is a quantitative method for assessing differences in methylation at specific CpG sites based on bisulfite treatment of DNA followed by primer extension of single nucleotides (Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997). Briefly, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence is then carried out using PCR primers specific for bisulfite-converted DNA, and the resulting product is isolated and used as a template for methylation analysis at the CpG site of interest. Small amounts of DNA (e.g., microdissected pathological sections) can be analyzed, avoiding the use of restriction enzymes for measuring the methylation status at CpG sites.

[0150] Typical reagents for Ms-SNuPE (trademark) analysis (e.g., those that can be found in a typical Ms-SNuPE (trademark)-based kit) include, but are not limited to, PCR primers for specific loci (e.g., specific genes, markers, DMRs, gene regions, marker regions, bisulfite-treated DNA sequences, CpG islands, etc.), optimized PCR buffers and deoxynucleotides, gel extraction kits, positive control primers, Ms-SNuPE (trademark) primers for specific genes, reaction buffers (for Ms-SNuPE reactions), and labeled nucleotides. Further, 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.

[0151] Reduced Representation Bisulfite Sequencing (RRBS) begins with bisulfite treatment of nucleic acids to convert all unmethylated cytosines to uracil, followed by digestion with a restriction enzyme (e.g., an enzyme that recognizes sites containing CG sequences such as MspI) and complete sequencing of the fragments after ligation to adapter ligands. By selecting the restriction enzyme, fragments for CpG-dense regions are enriched and the number of unnecessary sequences that can be located at multiple genetic positions during the analysis process is reduced. Thus, RRBS reduces the complexity of nucleic acid samples 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 digestion with a restriction enzyme contain DNA methylation information regarding at least one CpG dinucleotide. Thus, RRBS enriches samples for promoters, CpG islands, and other genomic features that have a high frequency of restriction enzyme cleavage sites within those regions and, in turn, provides an assay for evaluating the methylation status of one or more genomic loci.

[0152] A typical protocol for RRBS includes steps of digesting the nucleic acid sample with a restriction enzyme such as MspI, filling in overhangs and A-tailing, ligating an adapter, bisulfite conversion, and PCR. See, for example, 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.

[0153] In some embodiments, the methylation status is evaluated using a quantitative allele-specific real-time target and signal amplification (QuARTS) assay. Three reactions are carried out sequentially in each QuARTS assay, including amplification in the primary reaction (Reaction 1) and target probe cleavage (Reaction 2), and FRET cleavage and fluorescence signal generation in the secondary reaction (Reaction 3). When the target nucleic acid is amplified with specific primers, a specific detection probe with a flap sequence loosely binds to the amplicon. The presence of a specific invasive oligonucleotide at the target binding site causes a cleavage, releasing the flap sequence by 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 invasive oligonucleotide for the FRET cassette, resulting in cleavage between the fluorophore and quencher of the FRET cassette, which generates a fluorescence signal. This cleavage reaction cleaves multiple probes per target, and thus releases multiple fluorophores per flap, providing exponential signal amplification. QuARTS can adequately detect multiple targets in a single reaction by using different dyes with the FRET cassette. For example, see Zou et al. (2010) “Sensitive quantification of methylated markers with a novel methylation specific technology” Clin Chem 56:A199, U.S. Patent Application Nos. 12 / 946,737, 12 / 946,745, 12 / 946,752, and 61 / 548,639.

[0154] The term "bisulfite reagent" refers to a reagent containing bisulfite, disulfite, hydrogen sulfite, or a combination thereof, and as disclosed herein, is useful for distinguishing between methylated and non-methylated CpG dinucleotide sequences. The treatment method is known in the art (for example, PCT / EP2004 / 011715). The bisulfite treatment is preferably 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, for example, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid or trihydroxybenzoic acid and its derivatives, for example, gallic acid (see PCT / EP2004 / 011715). This bisulfite conversion is preferably carried out at a reaction temperature of 30°C to 70°C, whereby the temperature rises above 85°C for a short time during the reaction (see PCT / EP2004 / 011715). The DNA treated with this bisulfite is preferably purified before quantification. This may be carried out by any method known in the art, for example, but not limited to, by ultrafiltration, for example, using a Microcon™ column (manufactured by Millipore™). This purification is carried out according to the modified manufacturer's protocol (see, for example, PCT / EP2004 / 011715).

[0155] In some embodiments, the fragments of the treated DNA are amplified using a set of primer oligonucleotides according to the present invention (see, for example, Table 2) and an amplification enzyme. The 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 usually have a length of 100 to 2000 base pairs.

[0156] In another embodiment of the method, the methylation status of CpG positions within or near a marker that includes DMR (e.g., DMR1-400, Tables 1 and 4) can be detected by use of methylation-specific primer oligonucleotides. This technique (MSP) is described in Herman U.S. Patent No. 6,265,171. Use of methylation status-specific primers for amplification of bisulfite-treated DNA allows discrimination between methylated and unmethylated nucleic acids. An MSP primer pair includes at least one primer that hybridizes to a bisulfite-treated CpG dinucleotide. Thus, the sequence of the primer includes at least one CpG dinucleotide. An MSP primer specific for unmethylated DNA includes a "T" at the position of the C position of the CpG.

[0157] The fragment obtained by amplification can carry a directly or indirectly detectable label. In some embodiments, these labels are fluorescent labels, radionuclides, or separable molecular fragments having a typical mass detectable by a mass spectrometer. Where the label is a mass label, some embodiments assume that the labeled amplicon has a single positive or negative net charge, allowing for better detectability by a mass spectrometer. This detection can be carried out and visualized, for example, by matrix-assisted laser desorption / ionization mass spectrometry (MALDI) or using electrospray ionization mass spectrometry (ESI).

[0158] Methods for isolating DNA suitable for these assay techniques are known in the art. In particular, some embodiments include the isolation of nucleic acids described in U.S. Patent Application No. 13 / 470,251 ("Isolation of Nucleic Acids").

[0159] Method In some embodiments, a technique that includes the following steps, i.e., a method, is provided. 1) Contacting a nucleic acid collected from a subject (e.g., genomic DNA, e.g., isolated from a body fluid such as a blood sample (e.g., a plasma sample), a fecal sample, or a tissue sample) with at least one reagent or a series of reagents that distinguish methylated and non-methylated CpG dinucleotides within at least one marker containing a DMR (e.g., DMR1-400 (from Tables 1 and 4)). 2) Detecting the absence of HCC (e.g., provided with a sensitivity of 80% or more and a specificity of 80% or more).

[0160] In some embodiments, a technique, i.e., a method, is provided that includes the following steps. 1) Contacting a nucleic acid collected from a subject (e.g., genomic DNA, e.g., isolated from a body fluid such as a blood sample (e.g., a plasma sample), a fecal sample, or a tissue sample) with at least one reagent or a series of reagents that distinguish methylated and non-methylated CpG dinucleotides within at least one marker containing a DMR (e.g., DMR1-400 (from Tables 1 and 4)). 2) Classifying HCC (e.g., provided with a sensitivity of 80% or more and a specificity of 80% or more). Preferably, the sensitivity is about 70% to about 100%, or about 80% to about 90%, or about 80% to about 85%. Preferably, the specificity is about 70% to about 100%, or about 80% to about 90%, or about 80% to about 85%.

[0161] Genomic DNA can be isolated by any method, including the use of commercially available kits. Briefly, if the DNA of interest is encapsulated within a cell membrane, the biological sample needs to be disrupted and lysed by enzymatic, chemical, or mechanical means. Proteins and other contaminating substances 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 various methods, including salting out, organic extraction, or binding the DNA to a solid support. The choice of method is influenced by several factors, including time, cost, and the required amount of DNA. All clinical sample types containing neoplastic or preneoplastic substances, such as cell lines, histological slides, biopsies, paraffin-embedded tissues, body fluids, feces, colon eluates, urine, plasma, serum, whole blood, isolated blood cells, cells isolated from blood, and combinations thereof, are suitable for use in the methods of the present invention.

[0162] The technique is 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 fecal samples, or from blood, or from plasma samples, using, for example, direct gene capture or related methods detailed in U.S. Patent Application No. 61 / 485386.

[0163] The genomic DNA sample is then treated with at least one reagent, or a series of reagents, that distinguish methylated and non-methylated CpG dinucleotides within at least one marker, including, for example, DMRs (e.g., DMR1-400, as shown by Tables 1 and 4).

[0164] 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 with respect to hybridization behavior. However, in some embodiments, the reagent can be a methylation-sensitive restriction enzyme.

[0165] In some embodiments, the genomic DNA sample is processed 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 with respect to hybridization behavior. In some embodiments, this processing is performed using bisulfite (hydrogen sulfite, disulfite), followed by alkaline hydrolysis.

[0166] The processed nucleic acid is then analyzed to determine the methylation status of a target gene sequence (DMR, for example, at least one gene, genomic sequence, or nucleotide from a marker comprising at least one DMR selected from DMR1 - 400, for example, those shown in Tables 1 and 4). The analysis method can be selected from those known in the art including those described herein, for example, QuARTS and MSP described herein.

[0167] The technique relates to the analysis of any sample associated with HCC. For example, in some embodiments, the sample includes a plasma sample derived from a patient. In some embodiments, the sample includes tissue and / or body fluid collected from a patient. In some embodiments, the sample includes liver tissue. In some embodiments, the tissue includes secretions. In some embodiments, the sample includes blood, plasma, and / or serum. In some embodiments, the subject is human. These samples may include cells, tissues, and / or secretions from the upper digestive tract, the lower digestive tract, or both the upper and lower digestive tracts. The sample may include cells, secretions, or tissues from the liver, bile duct, pancreas, stomach, colon, rectum, esophagus, small intestine, appendix, duodenum, polyp, gallbladder, anus, and / or peritoneum. In some embodiments, the sample includes cytosol, ascites, urine, feces, pancreatic juice, fluid obtained during endoscopy, blood, mucus, or saliva. In some embodiments, the sample is a fecal sample.

[0168] Such samples can be obtained by various means known in the art, for example, obvious to those skilled in the art. For example, urine and fecal samples are readily available, while blood, ascites, serum, or pancreatic fluid samples can be obtained parenterally, for example, by using a needle and syringe. Cell-free or substantially cell-free samples can be obtained by subjecting the sample to various techniques known in the art, including but not limited to centrifugation and filtration. It is generally preferred to obtain samples without using invasive techniques, although in some cases it may be preferred to obtain samples such as tissue homogenates, tissue sections, and biopsy specimens.

[0169] In some embodiments of the technology, a method for diagnosing HCC in a subject is provided. As used herein, the terms "diagnosing" and "diagnosis" refer to a method by which one of ordinary skill in the art can infer and even determine whether a subject is suffering from a given disease or condition, or whether the subject may develop a given disease or condition in the future. One of ordinary skill in the art often diagnoses based on one or more diagnostic indicators, for example, a biomarker (e.g., a DMR disclosed herein) whose methylation status indicates the presence, severity, or absence of the condition.

[0170] Along with diagnosis, the clinical prognosis of cancer (e.g., for HCC) is related to determining the malignancy of the cancer and the likelihood of tumor recurrence to plan the most effective treatment. If a more accurate prognostic diagnosis can be made, or even the potential risk of cancer development can be evaluated, an appropriate treatment, and in some cases a less aggressive treatment, can be selected for the patient. Evaluation of cancer biomarkers (e.g., measurement of methylation status) is useful for separating subjects with a good prognosis and / or a low risk of cancer development who do not require treatment or require limited treatment from subjects with a higher likelihood of cancer development or cancer recurrence for whom more intensive treatment is effective.

[0171] As such, as used herein, "performing a diagnosis" or "diagnosing" further includes determining the risk of developing cancer or determining a prognosis, whereby, based on the measured value of a diagnostic biomarker (e.g., DMR) disclosed herein, the clinical outcome is predicted (regardless of the presence or absence of medical treatment), an appropriate treatment is selected (or whether the treatment is effective), or the current treatment is managed and potentially changed. Further, in some embodiments of the subject matter of the present disclosure, multiple measurements of a biomarker over time can be made to facilitate diagnosis and / or prognosis. The temporal change of the biomarker can be used to predict the clinical outcome, monitor the progression of HCC, and / or observe the effectiveness of an appropriate treatment for cancer. In such embodiments, for example, during the course of an effective treatment, a temporal change in the methylation state of one or more biomarkers (e.g., DMR) disclosed herein in a biological sample (and potentially one or more additional biomarkers if observed) can be expected.

[0172] The subject matter of the present disclosure further provides, in some embodiments, a method of determining whether to initiate or continue the prevention or treatment of HCC in a subject. In some embodiments, the method comprises providing a series of biological samples from the subject over a period of time, analyzing the series of biological samples to measure 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 biomarker over the period are used to predict the risk of developing HCC, predict the clinical outcome, determine whether to initiate or continue the prevention or treatment of the cancer, and determine whether the current treatment is effectively treating HCC. For example, a first time point can be selected prior to the start of treatment, and a second time point can be selected at a time after the start of the treatment. The methylation status can be measured in each of the samples taken from different time points, and qualitative and / or quantitative differences are noted. Changes in the methylation status of the biomarker levels from different samples can correlate with the risk of a disorder (e.g., the risk of HCC), prognosis, determination of treatment efficacy, and / or progression in the subject.

[0173] In preferred embodiments, the methods and compositions of the invention are for treatment or diagnosis at an early stage of the disease, e.g., before the symptoms of the disease appear. In some embodiments, the methods and compositions of the invention are for treatment or diagnosis of a disease at a certain clinical stage.

[0174] As described above, in some embodiments, multiple measurements of one or more diagnostic or prognostic biomarkers can be taken, and the time-course of the markers can be used to make a diagnosis or prognosis. For example, a diagnostic marker can be measured at a first time point and again at a second time point. In such embodiments, an increase in the marker from the first time point to the second time point can be diagnostic of a particular type or severity of disorder, or a given prognosis. Similarly, a decrease in the marker from the first time point to the second time point can be indicative of a particular type or severity of disorder, or a given prognosis. Additionally, the degree of change of one or more markers can be related to the severity of the disorder and future adverse events. Those skilled in the art will understand that in certain embodiments, comparative measurements can be made for the same biomarker at multiple time points, while in other embodiments, a given biomarker can be measured at one time point and a second biomarker can be measured at a second time point, and comparison of these markers can provide diagnostic information.

[0175] As used herein, the phrase "make a prognosis" refers to a method by which one of ordinary skill in the art can predict the course or outcome of a condition in a subject. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, nor does it refer to the likelihood that a given course or outcome will in fact occur as expected based on the methylation status of a biomarker (e.g., a DMR). Instead, one of ordinary skill in the art will understand that the term "prognosis" refers to an increased probability that a particular course or outcome will occur, i.e., that a particular course or outcome is more likely to occur in a subject exhibiting a given condition as compared to an individual not exhibiting the condition. For example, in an individual not exhibiting the condition (e.g., having the normal methylation status of one or more DMRs), the likelihood of a given outcome may be extremely low.

[0176] In some embodiments, a statistical analysis correlates a prognostic indicator with a tendency towards an adverse outcome. For example, in some embodiments, a methylation state that is different from that of a normal control sample from a patient without a disorder, as measured at a statistically significant level, may suggest that a subject is more likely to develop a disorder than a subject having a level more similar to the methylation state in the control sample. Further, a change in methylation state from a baseline (e.g., “normal”) level may reflect the prognosis of a subject, and the degree of change in methylation state 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 a p-value (see, for example, Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983). Exemplary confidence intervals for the subject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9%, and 99.99%, and exemplary p-values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

[0177] In other embodiments, a threshold for the degree of change in the methylation state of a prognostic or diagnostic biomarker (e.g., DMR) disclosed herein can be defined, and the degree of change in the methylation state of the biomarker in a biological sample is simply compared to the threshold for the degree of change in the methylation state. Preferred threshold changes in the methylation state of the biomarker 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 still other embodiments, a “nomogram” can be defined, whereby the methylation state of a prognostic or diagnostic indicator (biomarker or combination of biomarkers) is directly related to the associated tendency towards a given outcome. One of ordinary skill in the art is familiar with the use of such nomograms for relating two numerical values, understanding that the uncertainty in this measurement is the same as the uncertainty in the marker concentration, since the measured value of an individual sample, rather than the mean of the population, is being referenced.

[0178] In some embodiments, a control sample is analyzed simultaneously with the biological sample so that the results obtained from the biological sample can be compared with the results obtained from the control sample. Further, it is contemplated that a standard curve against which the results of an assay on a biological sample can be compared may be provided. Such a standard curve depicts the methylation state of a biomarker as a function of assay units, for example, fluorescence signal intensity if a fluorescent label is used. Using samples taken from multiple donors, the standard curve can be provided not only with respect to the control methylation state of one or more biomarkers in normal tissue, but also with respect to the "at risk" levels of one or more biomarkers in tissue taken from donors with metaplasia or a disorder (e.g., HCC). In certain embodiments of the method, a subject is identified as having HCC when an abnormal methylation state of one or more of the DMRs provided herein is identified in a biological sample from the subject. In other embodiments of the method, detection of an abnormal methylation state of one or more such biomarkers in a biological sample from the subject results in the subject being identified as having HCC.

[0179] Analysis of the markers may be performed separately or simultaneously from additional markers within one test sample. For example, several markers can be integrated into one test for efficient processing of diverse samples and potentially to increase diagnostic and / or prognostic accuracy. Further, those skilled in the art will recognize the value of examining multiple samples (e.g., at successive time points) from the same subject. Such testing of successive samples can enable identification of changes in the methylation state of the markers over time. Not only changes in the methylation state, but also the absence of changes in the methylation state can provide useful information regarding the disease state. This includes, but is not limited to, identification of the subject's outcome, including the approximate time since the event, the presence and amount of salvageable tissue, the appropriateness of drug therapy, the effectiveness of various treatments, and the risk of future events.

[0180] The analysis of biomarkers can be performed in various physical formats. For example, the use of microtiter plates or automation can be used to facilitate the processing of a large number of test samples. Alternatively, a single-sample format can be developed to facilitate timely emergency treatment and diagnosis, for example, during external transportation or in the setting of an emergency treatment room.

[0181] In some embodiments, the subject is diagnosed with HCC if there is a measurable difference in the methylation state of at least one biomarker in the sample when compared to the methylation state of a control. Conversely, if no change in the methylation state is identified in the biological sample, the subject may be identified as not having HCC, having no risk of HCC, or having a low risk of HCC. In this regard, subjects with HCC or at risk thereof can be distinguished from subjects with low to substantially no risk of HCC or its risk. Those subjects at risk of developing HCC can be scheduled for more intensive and / or regular screening.

[0182] As described above, depending on the embodiment of the method of the present technique, detecting a change in the methylation state of one or more biomarkers can be either a qualitative measurement or a quantitative measurement. Thus, the step of diagnosing that a subject has HCC or has a risk of developing HCC involves making a measurement of a particular threshold, 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 method, the control methylation state is any detectable methylation state of the biomarker. In other embodiments of the method where control samples are tested simultaneously with the biological samples, the predetermined methylation state is the methylation state of the control sample. In other embodiments of the method, the predetermined methylation state is based on and / or specified by a standard curve. In other embodiments of the method, the predetermined methylation state is a specific state or range of states. Thus, the predetermined methylation state can be selected, in part, within an acceptable range that would be apparent to one of ordinary skill in the art based on the embodiment of the method being implemented and the desired level of specificity, etc.

[0183] Furthermore, with respect to the diagnostic method, a preferred subject is a vertebrate subject. A preferred vertebrate is a warm-blooded animal, and a preferred warm-blooded vertebrate is a mammal. A preferred mammal is most preferably a human. As used herein, the term "subject" includes both human and animal subjects. Accordingly, therapeutic uses in animals are provided herein. Accordingly, the present technology provides for the diagnosis of not only mammals such as humans, but also important animals at risk of extinction, such as the Siberian tiger, economically important animals, such as animals raised for human consumption, and / or socially important animals for 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, pigs (including pig, hog, and swine including wild boar), ruminants and / or ungulates such as beef cattle, bulls, sheep, giraffes, deer, goats, bison, and camels, and horses. Accordingly, the present technology also provides for the diagnosis and treatment of domesticated pigs, ruminants, ungulates, horses (including racehorses), etc., without being limited thereto. The subject matter of the present disclosure further includes a system for diagnosing HCC in a subject. The system can be provided, for example, as a commercially available kit for use in screening for the risk of such a disorder in a subject from whom a biological sample has been taken. An exemplary system provided in accordance with the present technology includes assessing the methylation status of the DMRs shown in Table 1 and / or 4.

Example

[0184] Example I. This example describes the identification of 311 differential methylation regions (DMRs) for distinguishing DNA samples of HCC from DNA derived from normal controls (e.g., non-HCC individuals with or without cirrhosis).

[0185] The experiment was conducted in four steps.

[0186] First, the discovery of DNA methylation markers was performed using RRBS on DNA extracted from frozen tumor HCC tissues (with and without cirrhosis) and frozen normal liver tissues (with and without cirrhosis), as well as buffy coat samples from healthy volunteers. Discriminatory differentially methylated regions (DMRs) were identified using stringent filtering criteria, re-assayed in either the same or an enlarged sample, and the reproducibility of the results was ensured using real-time methylation-specific quantitative PCR assay (qMSP) (technical validation).

[0187] Second, candidate markers were selected by further ranking the criteria for biological validation by blinded qMSP assay on DNA extracted from independent archived cases and control tissues.

[0188] Third, the sequencing results for these candidate markers were compared across the pan-GI RRBS sequencing set and the level of methylation specificity was measured.

[0189] Fourth, another decision model was applied to select a small set of HCC markers that would perform best in a blood-based environment where most of the DNA is of non-liver origin. The selected markers were then examined blindly in independent plasma samples to evaluate HCC detection in a clinical medium.

[0190] These four steps are summarized in Figure 1.

[0191] Results Unbiased genome-wide methylation profiling was performed on DNA extracted from 18 HCCs and 35 controls (9 cirrhotic and 26 normal liver) tissues. Through tissue validation, the best DMRs were confirmed using methylation-specific PCR on DNA extracted from independent tissues from 75 HCCs and 29 controls (16 cirrhotic and 13 normal liver). A blinded quantitative allele-specific real-time target and signal amplification assay targeting the top DMRs was then performed on plasma DNA from an independent set of patients including 21 HCC cases (9 BCLC [Barcelona Clinic Liver Cancer staging] stage A, 6 stage B, 6 stage C) and 33 cirrhotic controls. Recursive partitioning decision analysis was used to identify the best combination of DMRs. In the initial profiling, 311 DMRs with an AUC greater than 0.75 were identified. After biological validation, the top 12 DMRs (ACP1, BDH1, Chr12.133, CLEC11A, DAB2IP, DBNL, EMX1, EFNB2, HOXA1, LRRC4, SPINT2, TSPYL5, CCNJ_3707, CCNJ_3124, PFKP, SCRN1, and ECE1) were selected and advanced to plasma testing. EMX1, the most discriminative marker in plasma, had an AUC of 0.89 alone. A combination of three complementary markers (EMX1, LRRC4, and BDH1) identified 20 / 21 HCCs and 32 / 33 controls in plasma, with one HCC having low levels of BDH1 and one control having elevated LRRC4. This panel had 95% sensitivity (95% CI, 74% - 100%) for HCC with 97% specificity (95% CI, 82% - 100%) and achieved an AUC of 0.98 (see Figure 2). Information on the area under the receiver operating characteristic curve for ACP1, Chr12.133, CLEC11A, DAB2IP, DBNL, EMX1, HOXA1, LRRC4, SPINT2, and TSPYL5 from this biological validation stage is shown in Figures 3A - I.

[0192] Figures 4A - CC provide box - and - whisker plots (logarithmic scale) of 27 gastric cancer markers (29 with fit - point analysis) from the biological tissue verification data. The samples are arranged with normal liver at the left end, followed by HCC without cirrhosis, HCC with cirrhosis, and cirrhotic control (inflammatory). The vertical axis is the degree of methylation (normalized against the β - actin chain).

[0193] Figure 5 shows the performance of 27 HCC cancer markers in 75 HCC tissue samples and 29 controls (16 cirrhotic, 13 normal liver) in a 95% normal - specificity matrix format. The markers are listed vertically and the samples are listed horizontally. The samples are arranged with normal liver (NI) at the left end, followed by HCC without cirrhosis (HN), HCC with cirrhosis (HC), and cirrhotic control (In). Positive hits are light - gray and misses are dark - gray. This plot allows for complementary evaluation of the markers. Note: Two markers, namely, TBX15 and EGR2, were analyzed a second time using the fit - point method for the qMSP data and are included here.

[0194] Table 1 shows information on DMRs for distinguishing HCC from normal controls.

[0195] Table 2 shows primer information regarding the DMRs selected from Table 1.

[0196] Table 3 provides information on the AUC and ratio of specific DMRs in the comparison of HCC and normal liver. This ratio is the ratio of the methylation degree of the cases to that of the controls.

[0197]

Table 1

[0198]

Table 2

[0199]

Table 3

[0200] Such experiments also led to the identification of 89 liver epithelial DMRs that are methylated in liver (cancer and normal) but not in normal leukocyte DNA samples.

[0201] Table 4 shows information on liver epithelial DMRs that are methylated in liver (cancer and normal) but not in normal leukocyte DNA samples.

[0202]

Table 4

[0203] Test Subjects and Samples This study was approved by the Mayo Clinic Institutional Review Board (Rochester, MN). Fresh frozen (FF) tissue, plasma, and buffy coat samples were provided by an IRB-approved patient biobank. Tumor tissue sections were reviewed by a specialized GI pathologist to confirm the diagnosis and estimate the tumor cellularity. Sections were then macrodissected. Genomic DNA was purified using the QiaAmp Mini kit (Qiagen, Valencia CA), followed by repurification with the AMPure XP kit (Beckman Coulter, Brea CA).

[0204] Preparation of Reduced Representation Bisulfite Sequencing libraries The array determination library was prepared using a modified version of the previously published method. Genomic DNA (300 ng) was digested overnight with 10 U of MspI. All enzymes used in subsequent steps were provided by New England Biolabs (NEB) unless otherwise specified. Fragments were end-repaired and A-tailed using 5 U of Klenow fragment (3'-5' exo-), and ligated overnight to TruSeq adapters (Illumina) containing barcode sequences and ubiquitously methylated cytosine. Binding efficiency and fragment quality were evaluated using SYBR Green qPCR (LightCycler480 - Roche). Samples were bisulfite-treated and purified (twice) using the modified EpiTect protocol (Qiagen), followed by a final AMPure XP clean-up. qPCR was used to determine the optimal PCR cycles for library enrichment. The following conditions were used for enrichment PCR. Each 50 μL reaction contained 5 μL of 10x buffer, 1.25 μL of 10 mM each deoxyribonucleotide triphosphate (dNTP), 5 μL of primer cocktail (approx. 5 μM), 15 μL of sample, 1 μL of PfuTurbo Cx hot start, and 22.75 μL of water. The temperature and time were 95°C - 5 min, 98°C - 30 s, 12 - 16 cycles of 98°C - 10 s, 65°C - 30 s, 72°C - 30 s, 72°C - 5 min, and hold at 4°C, respectively. Samples were quantified using the PicoGreen assay (Molecular Probes), pooled into a randomized quadruplicate library, and tested for size verification on a Bioanalyzer 2100 (Agilent). An additional round of AMPure XP purification / size selection was performed at empirically determined buffer concentrations to minimize adapter dimer contamination and remove inserts larger than 350 bp. Final library evaluation was achieved by qPCR using PhiX control standards (Illumina) and adapter-specific primers.

[0205] Ultraparallel sequencing and bioinformatics Samples were loaded into the flow cell according to a randomized lane assignment, and additional lanes were reserved for internal assay controls. Sequencing was performed on an Illumina HiSeq 2000 by the Next Generation Sequencing Core at the Mayo Clinic Medical Genome Facility. Reads were unidirectional for 101 cycles. Each lane of the flow cell generated between 100 million and 120 million reads, sufficient for a median coverage of 30 - 50x sequence determination depth for the sequences to be aligned. Standard Illumina pipeline software called bases and generated reads in fastq format. SAAP-RRBS (Streamlined Analysis and Annotation Pipeline for reduced representation bisulfite sequencing) was used for sequence read evaluation and cleanup, alignment to the reference genome, extraction of methylation status, and reporting and annotation of CpGs. CpGs with low coverage (≤10) were excluded. Tertiary analysis consisted of removing CpGs with no information or low sample coverage, and identifying methylated CpG regions with low background and high density clusters within a sliding 100bp window. The read-depth criteria were based on the desired power to detect a 10% difference in methylation % between cases and controls. Statistical significance was determined by logistic regression of methylation percentage for each DMR based on read count. To account for different read depths between individual subjects, an over-dispersed logistic regression model was used, and the dispersion parameter was estimated using the Pearson chi-squared statistic of the residuals from the approximate model. DMRs ranked according to the significance level were further examined when the methylation % in the control group was ≤1% and ≥10% in cancer. This yielded hundreds of potential candidates in most organ sites. Additional filters used were the area under the receiver operating characteristic curve, the signal-to-background methylation % ratio, and positive sample-to-sample co-methylation of CpGs across the entire DMR (and its absence in controls).

[0206] Technical and Biotissue Validation Methylation-specific PCR (MSP) assays were developed for 30 of the most promising DMRs from the liver discovery dataset as judged by the above criteria. Primers were designed either by software (Methprimer - University of California, San Francisco CA, MSP Primer - Johns Hopkins University, Baltimore, MD) or manually. Assays were rigorously tested and optimized by SYBR Green qPCR against bisulfite-converted (methylated and unmethylated genomic DNA), unconverted, and no-template controls. Assays that cross-reacted with negative controls were either redesigned or discarded. Additionally, melt curve analysis was performed to confirm that specific amplification was occurring. At the technical validation stage, the same samples used for RRBS discovery were retested by qMSP. A β-actin assay designed to not see methylation was used as the denominator representing total DNA copies. These data were analyzed by logistic regression, and the AUC and signal-to-background results were compared to discovery values. Markers with slightly less than half did not work well and were excluded. The remaining (N = 16) were examined by qMSP in an expanded set of 104 independent tissue samples. Additionally, the experiment included 11 methylated cancer markers. These were identified and validated in previous sequencing studies in other GI cancers (colon, esophagus, pancreas, bile duct) and are strong multi-organ cancer markers. The outcome measures were AUC and ratios (Table 3). Box-and-whisker plots and complementary matrices for the assayed markers are depicted in Figures 4 and 5, respectively.

[0207] Validation in all organs To evaluate how the best methylation markers function outside the liver, in experiments, a comparative CpG methylation % matrix was constructed using sequencing reads for validation DMRs across HCC samples as well as other major GI cancers that had been previously sequenced, namely, colon, pancreatic, esophageal, and gastric cancers. The final marker panel was selected based on 1) overall performance in the biological tissue validation stage and 2) the site-specific characteristics of the markers across other cancers and tested in plasma. To best detect HCC in blood, a robust 12-marker panel that would show both universal and liver-specific cancer signals was selected on the premise of an excess of non-HCC DNA. Ten of these markers were from tissue validation, and the two additional markers, EFNB2 and BDH1, which show abnormal liver site specificity, were designed and used directly from RRBS data without subsequent tissue validation.

[0208] Verification in Plasma Plasma DNA was extracted from 2 mL fractions by an automated silica bead method developed at Exact Sciences.

[0209] [Table 5]

[0210] This DNA was then bisulfite-converted and purified using an in-house method outlined below.

[0211] [Table 6]

[0212] Samples were run on a real-time PCR instrument (Roche LC480) in the QuARTs format (see U.S. Patent No. 8,361,720) using primers and probes made from the DMR sequence (see Table 2), GoTaq DNA polymerase (Promega), Cleavase II (Hologic), and a fluorescence resonance energy transfer reporter cassette (FRET) (Biosearch Technologies) containing FAM, HEX, and Quasar 670 dyes.

[0213] Figure 6 shows the oligonucleotide sequences of the FRET cassettes used for the detection of methylated DNA signatures by the QuARTs (Quantitative Allele-Specific Real-Time Target and Signal Amplification) assay. Each FRET sequence contains fluorophores and quenchers that can be multiplexed together in three separate assays.

[0214] Plasmids containing the marker sequence of interest were obtained from Genscript, diluted with 1× QuARTs reagent, and set to 1 copy per reaction with a set concentration of 15 ul. This reaction mixture was dispensed into each of the 384 wells, 45 cycles were repeated on the LightCycler, and data were collected. Wells were given a call of either containing or not containing the sample. The Poisson probability variable was set to 1, and the value of the average success rate was input by trial and error and used to calculate the cumulative probability for that value. When the cumulative probability was equal to the percentage of wells with a signal, the correct average success rate, in this case the copy number, was found. These plasmids were diluted and used as assay standards.

[0215] QuARTs-X (see U.S. Provisional Patent No. 62 / 249,097) is performed by first creating a pre-amplification plate of the sample using primers for up to 12 targets that undergo 11 cycles of amplification. This product is then diluted 1:9 and used as a template for subsequent QuARTs reactions that contain only 3 targets in a triple reaction. The standard used to calculate the number of strands does not go through this pre-amplification. By pre-amplifying the sample instead of the standard, the sensitivity of this assay is increased.

[0216] The results were analyzed by recursive partitioning (rPart). Using logistic regression to integrate multiple methylation markers into a single risk score is a standard approach. However, it is difficult to discover and / or model higher-order interactions among markers within a logistic model. If such effects exist, this limits the predictive ability of our panel of markers. Recursive partitioning tree (rPart) is a decision tree approach that can discover higher-order interactions among markers in a way that maximizes the prediction accuracy of the marker panel. In rPart, the sensitivity and specificity of HCC blood samples for the combination of the top three markers (EMX1, BDH1, LRRC4) were 97% and 95% respectively (Figure 7A). Another combination of three markers was modeled. That is, Option number 1 (EMX1, DAB2IP, TSPYL5): Specificity = 100% Sensitivity = 90% (Figure 7B) Option number 2 (EMX1, HOXA1, ACP1): Specificity = 88% Sensitivity = 100% (Figure 7C) Option number 3 (EMX1, EFNB2, SPINT2): Specificity = 100% Sensitivity = 90% (Figure 7D) EMX1, an excellent single marker in plasma, had an AUC of 0.89 with a specificity of 100% and a sensitivity of 77%. The signal for EMX1 showed a higher beta-actin normalized signal as the stage increased (Figure 8).

[0217] Example II. Tissue samples (HCC75, cirrhosis 20, normal 30) were prepared with primers and probes made from the DMR sequences (see Table 5), GoTaq DNA polymerase (Promega), Cleavase 2.0 (Hologic), and FAM, HEX, and Quasar Run on a real-time PCR instrument (Roche LC480) in QuARTs format (see U.S. Patent No. 8,361,720) using a fluorescence resonance energy transfer reporter cassette (FRET) containing 670 pigment (Biosearch Technologies). Table 6 shows the ability of each marker to distinguish HCC from cirrhosis and normal with 100% sensitivity.

[0218]

Table 7

[0219]

Table 8

[0220] Example III. The main objective was to determine a panel of markers for predicting hepatocellular carcinoma (HCC). Plasma from 244 subjects (95 with hepatocellular carcinoma and 149 controls) was adjusted to 2 mL and extracted. The 149 controls consisted of 51 cirrhotic patients and 98 normal patients.

[0221] Figure 9 shows the relative importance of each of the methylation markers examined in this analysis. The cross-validation estimates of sensitivity and specificity for the entire panel of markers were 75% and 96%, respectively.

[0222] With a specificity of 88.6% for controls compared to normal, the following panel of markers (Chr12.133, CLEC11A, EMX1, HOXA1, CCNJ_3707) had a sensitivity of 85.3% for HCC (Table 7).

[0223]

Table 9

[0224] The same panel (Chr12.133, CLEC11A, EMX1, HOXA1, CCNJ_3707) had the following breakdown of specificities for normal and cirrhotic (control group) patients (Table 8).

[0225]

Table 10

[0226] An exemplary procedure for isolating DNA from a 4 mL plasma sample is carried out as follows. · Add 300 μL of proteinase K (20 mg / mL) to 2 mL of the plasma sample and mix. If the sample is plasma less than 2 mL, add 10 mM Tris-HCl, 0.1 mM EDTA solution to adjust to 2 mL. · Add 6 mL of plasma lysis buffer 1 to the plasma and mix at room temperature.

[0227] The plasma lysis buffer is - 4.3 M guanidine thiocyanate - 10% IGEPAL CA-630 (octylphenoxypoly(ethyleneoxy)ethanol, branched) (5.3 g of IGEPAL CA-630 is mixed with 45 mL of 4.8 M guanidine thiocyanate) · Add 200 μL of magnetic silica-binding beads [16 μg of beads / μL] and mix again. · Add 7 mL of lysis buffer 2 to the tube.

[0228] Plasma lysis buffer 2 is prepared by mixing 60% of lysis buffer 1 with 40% isopropanol. · Mix the sample with lysis buffer 2 for 60 minutes. · Place the tube on a magnet and collect the beads for 10 minutes. Aspirate and discard the supernatant. · Add 1000 μL of wash buffer (10 mM Tris HCl, 80% EtOH) to the beads and incubate at 30 °C for 3 minutes with shaking. · Place the tube on a magnet and collect the beads. Aspirate and discard the supernatant. · Add 500 μL of washing buffer to the beads and incubate at 30 °C for 3 minutes with shaking. · Place the tube on a magnet to collect the beads. Aspirate and discard the supernatant. · Add 250 μL of washing buffer, incubate at 30 °C for 3 minutes with shaking. · Place the tube on a magnet to collect the beads. Aspirate and discard the remaining buffer. · Add 250 μL of washing buffer, incubate at 30 °C for 3 minutes with shaking. · Place the tube on a magnet to collect the beads. Aspirate and discard the remaining buffer. · Dry the beads at 70 °C for 15 minutes with shaking. · Add 125 μL of elution buffer (10 mM Tris HCl, pH 8.0, 0.1 mM EDTA) to the beads and incubate at 65 °C for 25 minutes with shaking. · Place the tube on a magnet to collect the beads for 10 minutes. · Aspirate and transfer the supernatant containing DNA to a new container or tube. Bisulfite conversion I. Sulfonation of DNA using ammonium bisulfite 1. Combine in each tube 64 μL of DNA, 7 μL of 1N NaOH, and 9 μL of carrier solution containing 0.2 mg / mL of BSA and 0.25 mg / mL of fish DNA. 2. Incubate at 42 °C for 20 minutes. 3. Add 120 μL of 45% ammonium bisulfite and incubate at 66 °C for 75 minutes. 4. Incubate at 4 °C for 10 minutes. II. Desulfonation using magnetic beads Materials Magnetic beads (Promega MagneSil Paramagnetic Particles, Promega catalog number AS1050, 16 μg / μL). Binding buffer: 6.5 - 7 M guanidine hydrochloride. Post-conversion washing buffer: 80% ethanol containing 10 mM Tris HCl (pH 8.0). Buffers for desulfonation: 70% isopropyl alcohol and 0.1N NaOH were selected as the buffers for desulfonation.

[0229] Mix the samples using any device or technique suitable for mixing or incubating the samples essentially at the temperatures and mixing rates described below. For example, a Thermomixer (Eppendorf) can be used for mixing or incubating the samples. An exemplary desulfonation is as follows. 1. Vortex the bottle for 1 minute to thoroughly mix the bead stock. 2. Aliquot 50 μL of the beads into a 2.0 mL tube (e.g., manufactured by USA Scientific). 3. Add 750 μL of the binding buffer to the beads. 4. Add 150 μL of the sulfonated DNA from Step I. 5. Mix (e.g., at 1000 RPM at 30 °C for 30 minutes). 6. Place the tube on a magnet stand and let it stand for 5 minutes. While the tube is on the stand, remove and discard the supernatant. 7. Add 1,000 μL of the washing buffer. Mix (e.g., at 1000 RPM at 30 °C for 3 minutes). 8. Place the tube on a magnet stand and let it stand for 5 minutes. While the tube is on the stand, remove and discard the supernatant. 9. Add 250 μL of the washing buffer. Mix (e.g., at 1000 RPM at 30 °C for 3 minutes). 10. Place the tube on a magnetic rack and after 1 minute, remove and discard the supernatant. 11. Add 200 μL of the desulfonation buffer. Mix (e.g., at 1000 RPM at 30 °C for 5 minutes). 12. Place the tube on a magnetic rack and after 1 minute, remove and discard the supernatant. 13. Add 250 μL of the washing buffer. Mix (e.g., at 1000 RPM at 30 °C for 3 minutes). 14. Place the tube on a magnetic rack and after 1 minute, remove and discard the supernatant. Add 15.250 μL of washing buffer to the tube. Mix (e.g., at 1000 RPM at 30 °C for 3 minutes). 16. Place the tube on the magnetic rack and after 1 minute, remove and discard the supernatant. 17. Incubate all tubes at 30 °C with the lids open for 15 minutes. 18. Remove the tube from the magnetic rack and add 70 μL of elution buffer directly to the beads. 19. Incubate the beads with the elution buffer (e.g., at 1000 RPM at 40 °C for 45 minutes). 20. Place the tube on the magnetic rack for about 1 minute and remove and store the supernatant.

[0230] The converted DNA is then used for pre-amplification and / or flap endonuclease assay.

[0231] All publications and patents mentioned in the above specification are hereby incorporated by reference in their entirety for all purposes. Various modifications and variations of the described technology's compositions, methods, and uses will be apparent to those skilled in the art without departing from the scope and spirit of the described technology. Although the technology has been described in relation to specific exemplary embodiments, it should be understood that the invention as claimed should not be limited more than necessary to such specific embodiments. Indeed, various modifications of the methods described for practicing the invention that are apparent to those skilled in the art of pharmacology, biochemistry, medical science, or related fields are intended to be within the scope of the following claims.

Claims

**Claim 1**: A method for detecting the methylation status of a gene in a sample, comprising: processing the sample taken from a human subject with a reagent that modifies DNA by a methylation-specific method, and measuring the methylation level of at least one differentially methylated region (DMR) by amplifying the at least one differentially methylated region (DMR) using a primer set, wherein the at least one DMR is derived from EMX1, and when the methylation level of the at least one DMR measured in the sample taken from the human subject is increased compared to the methylation level of the DMR measured in a control sample without HCC, the sample is used as an indicator for determining that the human subject is a human subject with or suspected of having HCC. **Claim 2** The method further comprises measuring the methylation level of at least one additional DMR derived from a gene selected from Chr12.133 and / or CLEC11A, and the methylation level of the at least one additional DMR from Chr12.133 and / or CLEC11A is used as an indicator for determining that the human subject is a human subject with or suspected of having hepatocellular carcinoma (HCC). The method according to claim 1. **Claim 3** Measuring the methylation level of the at least one DMR comprises measuring the presence or absence of methylation at one or more CpG positions. The method according to claim 1. **Claim 4** The one or more CpG positions are present in the coding region, non-coding region, and / or regulatory region of the gene. The method according to claim 3. **Claim 5** Measuring the methylation level of the at least one DMR comprises measuring the methylation frequency. The method according to claim 1. **Claim 6** Measuring the methylation level of the at least one DMR comprises measuring the methylation pattern. The method according to claim 1. **Claim 7** The reagent for modifying DNA by a methylation-specific method comprises one or more of a methylation-sensitive restriction enzyme, a methylation-dependent restriction enzyme, and / or a bisulfite reagent. The method according to claim 1. **Claim 8** Measuring the methylation level of the at least one DMR includes performing at least one of 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, according to the method of claim 1.

9. The method according to claim 1, wherein the at least one DMR has an increased methylation percentage or a high methylation rate as compared to a control sample without HCC.

10. The method according to claim 1, wherein the sample is a blood sample, a fecal sample, or a tissue sample.

11. The method according to claim 10, wherein the blood sample is a plasma sample, a serum sample, or a whole blood sample.

12. The method according to claim 10, wherein the tissue sample is a gastric tissue sample, a pancreatic tissue sample, a liver tissue sample, or a colorectal tissue sample.

13. The method according to claim 2, wherein the at least one DMR includes Chr12.

133.

14. The method according to claim 2, wherein the at least one DMR further includes CLEC11A.

15. The method according to claim 2, wherein the at least one DMR further includes Chr12.133 and CLEC11A.

16. The method according to claim 1 further includes measuring the methylation level of at least one DMR derived from a reference gene.

17. The primer set specific to EMX1 includes SEQ ID NO: 16 and 17, or SEQ ID NO: 91 and 92; The primer set specific to Chr12.133 includes SEQ ID NO: 25 and 26, or SEQ ID NO: 49 and 50; and The primer set specific to CLEC11A includes SEQ ID NO: 7 and 8, or SEQ ID NO: 52 and 53, according to the method of claim 2.

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