Method for determining whether a biological sample originates from liver cancer tissue

Abstract

The present invention relates to a method for determining whether a biological sample of unknown origin is derived from liver cancer tissue; and a composition containing a liver cancer tissue-specific DNA methylation marker for implementing the method. The liver cancer tissue-specific DNA methylation marker exhibits low-level methylation in normal liver tissue and other tissues, and exhibits high-level methylation only in liver cancer tissue, and therefore can be used to very accurately determine whether a biological sample is derived from liver cancer tissue.

Description

Technical Field

[0001] The present invention relates to a method for determining whether a biological sample of unknown origin is derived from liver cancer tissue, and a composition containing liver cancer tissue-specific DNA methylation markers for performing the method. Background Art

[0002] Cells in the human body share the same genetic information, yet each cell's function and appearance are remarkably diverse. This is because specific genes are expressed in each cell, resulting in distinct cell differentiation processes and distinct cellular phenotypes. Several factors, such as DNA methylation, histone modifications, and tissue-specific transcription factors, are involved in the expression of these specific genes. Specifically, DNA methylation, particularly methylation at CpG sites, is an essential element of cell-specific gene expression, and each cell or tissue is known to have a unique DNA methylation signature. Therefore, using DNA methylation signatures, the origin of a cell or tissue can be easily identified.

[0003] Identifying the origin of cells or tissues can enable early diagnosis of diseases and improve the accuracy of diagnosis. For example, circulating cell-free DNA (cfDNA) exists in the blood circulating in the living body, which may contain circulating tumor DNA (ctDNA) shed from tumor cells. Even if the presence of ctDNA is detected by liquid biopsy, in order to accurately diagnose cancer, the tissue of ctDNA origin must be additionally confirmed, and in this process, the diagnosis of cancer will be delayed. However, if the tissue of cancer origin can be identified at the same time as the corresponding ctDNA is detected, early diagnosis of cancer is possible, and there is an increasing need for a method to identify the origin of biological samples for early diagnosis of other diseases as well as cancer.

[0004] The present inventors studied a method for determining the origin of tissues and cells of unknown origin based on DNA methylation data, and as a result, identified a marker with a high methylation level specifically for liver cancer tissue, thereby completing the present invention. Summary of the Invention

[0005] Technical issues

[0006] An object of the present invention is to provide a method for determining whether a biological sample of unknown origin is derived from liver cancer tissue, a method for confirming that DNA in a biological sample is derived from liver cancer tissue, and a composition for implementing these methods.

[0007] Technical Solution

[0008] To achieve the above objectives, one aspect of the present invention provides a method for determining whether a biological sample is derived from liver cancer tissue, comprising the following steps:

[0009] (a) isolating DNA from a biological sample isolated from a subject; and

[0010] (b) The methylation level of the CpG site of the sequence represented by SEQ ID NO: 1 in the isolated DNA was measured.

[0011] In one embodiment of the present invention, step (b) can further measure the methylation level of the CpG sites of the sequence represented by SEQ ID NO: 2 in the isolated DNA. For example, the method for determining whether a biological sample of the present invention is derived from liver cancer tissue can measure the methylation level of the CpG sites of the sequence represented by SEQ ID NO: 1 alone, or the methylation level of the CpG sites of the sequences represented by SEQ ID NO: 1 and 2 in step (b).

[0012] In a specific embodiment of the present invention, the subject may be a human, and the biological sample includes, but is not limited to, tissues, tissue fragments, cells, cell fragments, blood, plasma, body fluids, feces, and urine isolated from the subject. Tissues, tissue fragments, cells, and cell fragments may be isolated from blood, plasma, body fluids, urine, and the like collected from the subject. Furthermore, the DNA may be DNA isolated from tissues, cells, and the like, or it may be cell-free DNA (cfDNA) floating in blood, plasma, body fluids, and the like, or circulating tumor DNA (ctDNA) shed from tumor cells.

[0013] The term "methylation" used in this specification refers to the binding of a methyl group (-CH3) to a base constituting DNA, and preferably refers to methylation occurring on cytosine at a specific CpG site in specific DNA.

[0014] The term "methylation level" used in this specification refers to the quantitative evaluation of the methylation status of CpG sites present in a specific DNA sequence. The methylation status refers to the presence or absence of 5-methyl-cytosine at one or more CpG sites in a DNA sequence.

[0015] The term "CpG site" as used herein refers to a sequence where cytosine (C) and guanine (G) are linked by a phosphate group and can be found in DNA sequences including promoter regions, protein-coding regions (open reading frames, ORFs), and terminator regions. Methylation of CpG sites is known to be involved in maintaining genomic stability and regulating gene expression.

[0016] The present inventors attempted to discover liver cancer tissue-specific methylation markers and discovered the cg13204512 (SEQ ID NO: 1) and cg00108164 (SEQ ID NO: 2) markers, which have high methylation levels in liver cancer tissue samples and low methylation levels in other tissues, including normal liver tissue and blood. The target methylation sites for both markers are CpG sites located at nucleotide position 61 in the sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively. However, since methylation may occur even at CpG sites other than the target methylation site, the present invention measures the methylation levels of all CpG sites present in the sequences represented by SEQ ID NO: 1 and 2, including the target CpG site.

[0017] As used herein, the term "liver cancer tissue-specific methylation marker" refers to a methylation marker that exhibits a specific methylation level only in DNA from liver cancer tissue, compared to the methylation level of DNA isolated from normal liver tissue and other tissues. In the present invention, a "liver cancer tissue-specific methylation marker" refers to a marker that exhibits a high methylation level only in DNA from liver cancer tissue, compared to the methylation level of DNA isolated from normal liver tissue and other tissues.

[0018] In a specific embodiment of the present invention, (b) can be implemented by a method selected from the group consisting of PCR, methylation-specific PCR, real-time methylation-specific PCR, MethyLight PCR, MehtyLight digital PCR, EpiTYPER, PCR using methylated DNA-specific binding protein, quantitative PCR, DNA chip, molecular beacon, MS-HRM (methylation-sensitive high-resolution melting), asymmetric PCR, asymmetric PCR MS-HRMA (asymmetric PCR methylation-sensitive high-resolution melting analysis), recombinase polymerase amplification, LAMP (loop-mediated isothermal amplification), Eclipse probe, next-generation sequencing panel (NGS panel), pyrosequencing and disulfide sequencing.

[0019] For example, methylation levels can be identified by microarrays, which can use probes immobilized on a solid surface. The probes can comprise a sequence complementary to a 10 to 100 consecutive nucleotide sequence comprising a CpG site.

[0020] In a specific embodiment of the present invention, the method may further comprise, after (b), (c) comparing the methylation level with the methylation level of a control group.

[0021] In the present invention, a control group refers to DNA isolated from a biological sample whose tissue of origin is known. All tissues in the living body, including normal liver tissue and liver cancer tissue, can be used. For example, if the methylation level of DNA isolated from the biological sample is similar to that of DNA isolated from the control group, the biological sample can be determined to be derived from liver cancer tissue. If the methylation level of DNA isolated from the biological sample is higher than that of the control group, the biological sample can be determined to be derived from liver cancer tissue.

[0022] Another aspect of the present invention provides a composition for determining whether a biological sample is derived from liver cancer tissue, comprising a reagent capable of measuring the methylation level of the sequence represented by SEQ ID NO: 1.

[0023] In a specific embodiment of the present invention, there may be one or more CpG sites, including the CpG site located at nucleotide position 61 in the sequences shown by SEQ ID NO: 1 and SEQ ID NO: 2.

[0024] In the present invention, the composition may further comprise a reagent capable of detecting the methylation level of the sequence represented by SEQ ID NO: 2. The reagent capable of measuring the methylation level may be a primer, a probe, or an antisense nucleic acid that binds to the CpG sites of the sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2, and the primer, probe, or antisense nucleic acid may be used as a hybridizable array element and may be immobilized on a substrate.

[0025] Substrate is suitable solid or semisolid support, for example, it can comprise film, filter, chip, slide, wafer, fiber, magnetic bead or non-magnetic bead, gel, tubing, plate, polymer, microparticle and capillary.But hybridization array element can be fixed on substrate by chemical binding method, covalent binding method such as UV or linking agent (for example: ethylene glycol oligomer and diamine).

[0026] In one embodiment of the present invention, DNA isolated from a biological sample (sample DNA) can be hybridized with the array elements when applied to a hybridizable array. Hybridization conditions can be modified in various ways according to techniques known in the art, and hybridization levels can be detected and analyzed in various ways. Furthermore, to provide a signal that allows confirmation of hybridization, the sample DNA and / or primers, probes, or antisense nucleic acids can be labeled and linked to oligonucleotides.

[0027] Labels may include, but are not limited to, fluorophores (e.g., fluorescein, phycoerythrin, rhodamine, lissamine, Cy3, and Cy5 (Pharmacia)), chromophores, chemiluminescent groups, magnetic particles, radioisotopes (P32 and S35), enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, enzyme substrates, heavy metals (e.g., gold), antibodies, streptavidin, biotin, digoxigenin, and haptens with specific binding partners (e.g., chelating groups).

[0028] In the present invention, hybridization of primers, probes, or antisense nucleic acids with sample DNA depends on various factors, such as reaction temperature, hybridization and washing time, buffer composition and its pH and ionic strength, nucleotide length, nucleotide sequence, the number of GC sequences, etc. Detailed conditions for hybridization can be found in Joseph Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and MLM Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc. NY (1999).

[0029] After the hybridization reaction, the hybridization signal generated by the hybridization reaction can be detected. For example, when the probe is labeled with an enzyme, hybridization can be confirmed by reacting the substrate of the enzyme with the hybridization reaction product.

[0030] As enzymes and substrates, peroxidase (e.g., horseradish peroxidase) and chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and catechol), TMB (tetramethylbenzidine), ABTS (2,2'-azabis[3-ethylbenzothiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine can be used; alkaline phosphatase and bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium chloride (NBT), naphthol-AS-B1-phosphate and ECF substrate; glucose oxidase and t-NBT (nitro blue tetrazolium chloride) and m-PMS (phenazine methylsulfate).

[0031] Another aspect of the present invention provides a composition for diagnosing liver cancer, comprising a reagent capable of measuring the methylation level of the sequence represented by SEQ ID NO: 1.

[0032] In the present invention, the composition for diagnosing liver cancer may further include a reagent capable of measuring the methylation level of the sequence represented by SEQ ID NO: 2, wherein the reagent capable of measuring the methylation level includes a primer, probe, or antisense nucleic acid that binds to the CpG site of the sequence consisting of SEQ ID NO: 1 and SEQ ID NO: 2. The sequence represented by SEQ ID NO: 1 or 2 may be genomic DNA, or may be a sequence in which unmethylated cytosine is converted to uracil by bisulfite treatment.

[0033] In the present invention, DNA derived from liver cancer tissue can be detected by isolating DNA from a biological sample isolated from a subject and measuring the methylation level of a liver cancer tissue-specific marker. Consequently, liver cancer can be diagnosed by confirming the presence of DNA derived from liver cancer tissue. Furthermore, the composition for diagnosing liver cancer of the present invention is characterized in that liver cancer can be diagnosed using biological samples from various sources.

[0034] In the present invention, biological samples include tissues, tissue fragments, cells, cell fragments, blood, plasma, body fluids, feces and urine separated from a subject, but are not limited thereto.

[0035] Beneficial effects

[0036] The method for determining whether a biological sample is derived from liver cancer tissue and the composition for implementing the method use a liver cancer tissue-specific DNA methylation marker, and the liver cancer tissue-specific marker has a low methylation level in normal liver tissue and other tissues, and has a high methylation level only in liver cancer tissue, so it can very accurately determine whether the biological sample is derived from liver tissue.

[0037] BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The process of discovering liver cancer tissue-specific markers is schematically shown.

[0039] Figure 2 Shown is a variable importance map (VarImp map) of discovered liver cancer tissue-specific markers.

[0040] Figure 3 Validation results of the performance of the finally selected cg13204512 and cg00108164 markers are shown.

[0041] Figure 4 The results of the confirmation of the methylation levels of the finally selected cg13204512 and cg00108164 markers in normal liver tissue (LIHC_N), liver cancer tissue (LIHC_T), and pan-tumor tissue excluding liver (Pan_T) are shown.

[0042] Figure 5 Shown are the results of confirming the methylation levels of the cg13204512 marker in various normal tissues (A) and cancer tissues (B).

[0043] Figure 6 Shown are the results of confirming the methylation levels of the cg00108164 marker in various normal tissues (A) and cancer tissues (B).

[0044] Figure 7 Validation results of methylation levels of cg13204512 and cg00108164 markers in major normal and cancer tissues are shown: bladder (BL), breast (BR), cervix (CE), colon (CO), esophagus (ES), glioblastoma (GB), head and neck (HN), kidney (KI), liver (LI), lung (LU), pancreas (PA), paraganglioma (PC), prostate (PR), rectum (RE), sarcoma (SA), skin (SK), stomach (ST), thymus (TH), uterus (UC), and blood (B). DETAILED DESCRIPTION

[0045] Hereinafter, the present invention will be described in more detail through one or more specific examples. However, these embodiments are intended to illustrate one or more specific embodiments, but the scope of the present invention is not limited by these embodiments.

[0046] Example 1: Discovery of liver cancer tissue-specific methylation markers

[0047] DNA methylation data for various cancers were downloaded from The Cancer Genome Atlas (hereinafter referred to as TCGA). From the data downloaded from TCGA, methylated CpG sites were selected from liver cancer tissue samples (n=379), while unmethylated CpG sites were selected from cancer samples of other tissues (n=7260). The standard was to classify samples as methylated if 50% or more were methylated, and as unmethylated if 90% or more were unmethylated.

[0048] Then, we further selected non-methylated CpG sites in 90% of cancer samples (pan-tumor) except liver cancer, and CpG sites with 30% or more different methylation levels in normal liver tissues and liver cancer tissues.

[0049] exist Figure 1 , the process of discovering the liver cancer tissue-specific marker of the present invention is schematically shown.

[0050] Example 2: Verification of the performance of liver cancer tissue-specific methylation markers

[0051] The variable importance map (VarImp map) of the liver cancer tissue-specific markers found in Example 1 above was produced by the random forest method, as shown in FIG. Figure 2 As shown. Figure 2 In the figure, the Mean Decrease in Accuracy (X-axis) indicates how well each marker improves the accuracy of the HCC / normal liver tissue classification, while the Mean Decrease in Gini Index (Y-axis) indicates how well each marker improves the impurity in the HCC / normal liver tissue / other tissue classification. In other words, a larger value means that the marker can clearly distinguish HCC tissue from normal liver tissue and other tissues.

[0052] Next, the liver cancer tissue-specific marker was tested by randomly dividing the methylation data from the cancer tissue used in Example 1 into training data and validation data. The results, as shown in Table 1 below, confirm that the discovered marker distinguishes liver tissue from other tissues with high efficiency and accuracy. The performance of the liver tissue-specific marker showed an accuracy of 0.9915, a sensitivity of 0.8816, a specificity of 0.9973, and an area under the curve (AUC) of 0.9668.

[0053] Table 1

[0054]

[0055] Then, we further verified Figure 1 The performance of two markers (cg13204512, cg00108164) with high importance in the variable importance map of . Figure 2 As shown, the area under the curve (AUC) of the two markers was 0.9725, confirming the good performance of distinguishing liver cancer tissue from other tissues.

[0056] Based on the above results, cg13204512 and cg00108164 markers were finally selected as liver cancer tissue-specific markers. The information and sequences of cg13204512 and cg00108164 markers are described in Tables 2 and 3 below.

[0057] Table 2

[0058]

[0059] Table 3

[0060]

[0061] - In the table, [CG] refers to the target methylation site for each probe.

[0062] Example 3: Validation of the finally selected liver cancer tissue-specific markers

[0063] The methylation levels of the finally selected cg13204512 and cg00108164 markers in liver cancer tissues, normal liver tissues, and other tissues were determined. Figure 4 As shown in Figures A and B, it can be seen that these two markers have low methylation levels in normal liver tissue (LIHC_N) and pan-tumor tissue (Pan_T) excluding the liver, and have high methylation levels in liver cancer tissue (LIHC_T).

[0064] exist Figures 5 to 7 , the methylation levels of the cg13204512 and cg00108164 markers confirmed in liver tissue and other tissues are shown.

[0065] From the current results, it can be seen that the cg13204512 and cg00108164 markers have high methylation levels in liver cancer tissues and low methylation levels in normal liver tissues and other tissues. Therefore, these two markers can be used as liver cancer tissue-specific markers.

Claims

1. Use of a composition in preparing a kit for determining whether a biological sample originates from liver cancer tissue, the composition comprising a reagent capable of measuring the methylation level of a CpG site located at nucleotides 61-62 in a sequence shown in SEQ ID NO: 1 and a reagent capable of measuring the methylation level of a CpG site located at nucleotides 61-62 in a sequence shown in SEQ ID NO:

2.

2. The use according to claim 1, wherein The biological sample is selected from the group consisting of tissues, cells, body fluids and feces isolated from a subject.

3. The use according to claim 1, wherein The biological sample is selected from the group consisting of tissue fragments, cell fragments, blood and urine isolated from a subject.

4. The use according to claim 1, wherein The biological sample is plasma isolated from a subject.

5. The use according to claim 1, wherein Measuring the methylation level is performed by a method selected from the group consisting of PCR, DNA chip, molecular beacon, MS-HRM (methylation-sensitive high-resolution melting), recombinase polymerase amplification, LAMP amplification (loop-mediated isothermal amplification), Eclipse probe, next generation sequencing panel (NGS panel), pyrosequencing and disulfide sequencing.

6. The use according to claim 1, wherein Measuring the methylation level is performed by a method selected from the group consisting of methylation-specific PCR, EpiTYPER, quantitative PCR, and asymmetric PCR.

7. The use according to claim 1, wherein Measuring the methylation level is performed by a method selected from the group consisting of real-time methylation-specific PCR, MethyLight PCR, and asymmetric PCR MS-HRMA (asymmetric PCR methylation-sensitive high-resolution melting analysis).

8. The use according to claim 1, wherein MethyLight digital PCR was used to measure methylation levels.

9. A composition for determining whether a biological sample is derived from liver cancer tissue, comprising a reagent capable of measuring the methylation level of a CpG site located at nucleotides 61-62 in the sequence shown in SEQ ID NO: 1 and a reagent capable of measuring the methylation level of a CpG site located at nucleotides 61-62 in the sequence shown in SEQ ID NO:

2.

10. The composition for determining whether a biological sample is derived from liver cancer tissue according to claim 9, wherein: The reagent capable of measuring the methylation level is a primer, a probe or an antisense nucleic acid.

11. A composition for diagnosing liver cancer, comprising a reagent capable of measuring the methylation level of a CpG site located at nucleotides 61-62 in the sequence shown in SEQ ID NO: 1 and a reagent capable of measuring the methylation level of a CpG site located at nucleotides 61-62 in the sequence shown in SEQ ID NO:

2.

12. The composition for diagnosing liver cancer according to claim 11, wherein The reagent is a primer, a probe or an antisense nucleic acid.

13. Use of a composition in the preparation of a kit for diagnosing liver cancer, the composition comprising a reagent capable of measuring the methylation level of a CpG site located at nucleotides 61-62 in the sequence shown in SEQ ID NO: 1 and a reagent capable of measuring the methylation level of a CpG site located at nucleotides 61-62 in the sequence shown in SEQ ID NO:

2.

14. The use according to claim 13, wherein The reagent is a primer, a probe or an antisense nucleic acid.

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