Method for determining upper tract urothelial carcinoma

Abstract

Provided is a method for diagnosing upper tract urothelial carcinoma which is minimally invasive and highly reliable. A method for identifying cells or tissue having upper tract urothelial carcinoma, said method involving: the detection of the DNA methylation level at one or more of the prescribed CpG sites in genomic DNA derived from upper tract urothelial cells or tissue containing the same; and the identification of whether or not upper tract urothelial carcinoma is present in said cells or tissue containing the same on the basis of the detected DNA methylation level.

Description

[Technical field]

[0001] The present invention relates to a method for determining upper tract urothelial carcinoma. [Background technology]

[0002] Urothelial carcinoma (UC) is a cancer occurring in the upper urinary tract (i.e., the renal pelvis and ureter) and bladder, and is the second most common urological malignant tumor after prostate cancer. The most common type of UC is urinary bladder cancer (BUC). On the other hand, upper urinary tract urothelial carcinoma (UTUC) is relatively rare, but generally has a poor prognosis.

[0003] The first symptom of UC patients is usually gross or non-gross hematuria. Patients with hematuria usually undergo spontaneous urine cytology, and those with a positive result undergo further cystoscopy. UTUC is suspected if no tumor is identified in the bladder.

[0004] Traditionally, ureteral catheterization has been performed as a diagnostic method for UTUC for retrograde ureteral pyelography, ureteroscopy, and selective urine cytology sampling. However, ureteral catheterization is highly invasive because a catheter is inserted into the narrow bladder, and sometimes induces complications including urinary tract infection and ureteral damage / dissection. In addition, selective urine cytology does not always provide sufficient sensitivity and specificity due to the destruction and degeneration of exfoliated cancer cells. For this reason, ureteral catheterization has tended to be avoided in recent years. In addition, MRI is non-invasive, but has low resolution and cannot adequately depict the renal calyx. In recent years, CT-urography has been recommended as a diagnostic method for UTUC. However, CT-urography has problems such as a higher radiation dose than retrograde pyelography, and is contraindicated in cases of contrast allergy or reduced renal function. In addition, imaging diagnostics such as CT-urography and MRI cannot detect flat intraepithelial carcinoma without thickening of the urothelium. It is desirable to develop a minimally invasive and reliable method for diagnosing UTUC.

[0005] In recent years, it has become clear that abnormalities in DNA methylation are deeply involved in carcinogenesis, and this has attracted attention. Aberrant DNA methylation in CpG islands in the promoter regions of some genes is known to be a characteristic epigenetic abnormality in cancer cells. CpG islands are regions in which the two-base sequence cytosine (C)-guanine (G) via a phosphodiester bond (p) appears frequently. Abnormal DNA methylation in CpG islands is involved in carcinogenesis through inactivation of tumor suppressor genes.

[0006] Regarding urothelial carcinoma, studies by the inventors' group and other groups have reported that noncancerous urothelium and UC show different DNA methylation profiles (Non-Patent Documents 1 to 6), and have also suggested that changes in DNA methylation can be used to diagnose UC (Non-Patent Document 7). However, although these previous reports suggest the possibility of risk assessment for UC based on DNA methylation abnormalities, they do not suggest that a diagnostic method for upper tract urothelial carcinoma can be established based on DNA methylation abnormalities to replace conventional ureteral catheterization and CT-urography.

[0007] On the other hand, differences in DNA methylation profiles and gene mutations between UTUC and BUC have been reported recently (Non-Patent Documents 8 to 9). At least part of the differences in DNA methylation profiles between UTUC and BUC may be due to differences in their gene mutations.

[0008] As a method for analyzing methylated DNA, a method using the bisulfite method has already been established and is widely used. As a method for analyzing methylated DNA based on the bisulfite method, the Methylation-Specific PCR (MSP) method, the Combined Bisulfite Restriction Analysis (COBRA) method, and the BAC array-based methylated CpG island amplification method (BAMCA method) are often used. Patent Document 1 discloses a method for detecting a risk of poor prognosis of renal cell carcinoma by detecting the methylation level at the CpG site of a specific gene by a bead array method, mass spectrometry (MassARRAY method), pyrosequencing, methylation-sensitive high-resolution melting curve analysis, quantitative PCR, direct sequencing of bisulfite-treated products, the COBRA method, or the like. Patent Documents 2 and 3 disclose a method for determining the prognosis of cancer by detecting the methylation level at the CpG site of a specific gene based on the difference in retention time by ion exchange chromatography. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2013 / 168644 [Patent Document 2] International Publication No. 2015 / 129916 [Patent Document 3] International Publication No. 2017 / 038983 [Non-patent literature]

[0010] [Non-Patent Document 1] Carcinogenesis, 2011, 32:462-9 [Non-Patent Document 2] Cancer Sci, 2010, 101:231-40 [Non-Patent Document 3] J Urology, 2005, 173:243-6 [Non-Patent Document 4] Curr Urol Rep, 2018, 19:102 [Non-Patent Document 5] Epigenomics, 2016, 8:1415-1428 [Non-Patent Document 6] Nature, 2014, 507:315-22 [Non-Patent Document 7] Carcinogenesis, 2019, bgz112 [Non-Patent Document 8] Int J Mol Sci, 2019, 20:E2657 [Non-Patent Document 9] Eur Urol, 2017, 72:641-649 Summary of the Invention [Problem to be solved by the invention]

[0011] A minimally invasive and reliable method for diagnosing upper tract urothelial carcinoma (UTUC) is desired. The present invention provides a method for diagnosing UTUC based on DNA methylation levels. [Means for solving the problem]

[0012] We identified CpGs that exhibited different DNA methylation profiles between upper tract urothelial carcinoma and non-cancerous upper tract urothelium, and further found that UTUC and bladder cancer (BUC) could be distinguished based on the DNA methylation profiles of these CpGs.

[0013] Thus, the present invention provides the following: [1] A method for determining whether a cell or tissue has upper tract urothelial carcinoma, comprising: positions 158,799,748, 158,799,765, 158,799,775, 158,799,777, and 158,799,789 on chromosome 7; positions 111,813,685, 111,813,690, and 111,813,698 on chromosome 1; positions 240,375,464 on chromosome 1; positions 28,956,374, 28,956,381, 28,956,384, and 28,956,387 on chromosome 6; positions 91,182,528 and 91,182,529 on chromosome 1 in genomic DNA derived from upper urothelial cells or tissues containing same detecting the DNA methylation level of at least one CpG site selected from the group consisting of: positions 2,534 on chromosome 1; positions 119,535,921, 119,535,925, 119,535,928, and 119,535,937 on chromosome 1; positions 4,850,072, 4,850,075, and 4,850,090 on chromosome 7; positions 31,846,849, 31,846,844, 31,846,839, and 31,846,833 on chromosome 11; and positions 106,187,192 and 106,187,210 on chromosome 14; determining whether the cell or tissue has upper tract urothelial carcinoma based on the detected DNA methylation level; The method includes: [2] The method described in [1], wherein the detection of the DNA methylation level comprises detecting the DNA methylation level of the at least one CpG site using bisulfite-treated genomic DNA. [3] The method described in [2], wherein the detection of the DNA methylation level is performed using pyrosequencing, mass spectrometry, bead array or ion exchange chromatography. [4] The method according to any one of [1] to [3], wherein the genomic DNA is DNA derived from upper urothelial cells contained in urine. [5] Positions 158,799,748, 158,799,765, 158,799,775, 158,799,777, and 158,799,789 on chromosome 7, which have a chain length of at least 12 bases and have been subjected to bisulfite treatment; positions 111,813,685, 111,813,690, and 111,813,698 on chromosome 1; position 240,375,464 on chromosome 1; positions 28,956,374, 28,956,381, 28,956,384, and 28,956,387 on chromosome 6; and positions 91,182,528 and 91,182,534 on chromosome 1. a primer or probe for determining upper tract urothelial carcinoma, which hybridizes to at least one CpG site selected from the group consisting of: positions 119,535,921, 119,535,925, 119,535,928, and 119,535,937 on chromosome 1; positions 4,850,072, 4,850,075, and 4,850,090 on chromosome 7; positions 31,846,849, 31,846,844, 31,846,839, and 31,846,833 on chromosome 11; and positions 106,187,192 and 106,187,210 on chromosome 14. [6] The primer or probe according to [5], which hybridizes to a bisulfite-treated product of DNA comprising any one of the nucleotide sequences of SEQ ID NOs: 31 to 40. [7] The primer or probe according to [5] or [6], which is selected from the group consisting of a polynucleotide having a nucleotide sequence of any one of SEQ ID NOs: 1 to 30 and its complementary strand. [8] A primer set consisting of a combination of polynucleotides of SEQ ID NOs: 1 and 2, or a combination of complementary strands thereof; A primer set consisting of a combination of polynucleotides of SEQ ID NOs: 3 and 4, or a combination of complementary strands thereof; A primer set consisting of a combination of polynucleotides of SEQ ID NOs: 5 and 6, or a combination of complementary strands thereof; A primer set consisting of a combination of polynucleotides of SEQ ID NOs: 7 and 8, or a combination of complementary strands thereof; A primer set consisting of a combination of polynucleotides of SEQ ID NOs: 9 and 10, or a combination of complementary strands thereof; A primer set consisting of a combination of polynucleotides of SEQ ID NOs: 11 and 12, or a combination of complementary strands thereof; A primer set consisting of a combination of polynucleotides of SEQ ID NOs: 13 and 14, or a combination of complementary strands thereof; A primer set consisting of a combination of polynucleotides of SEQ ID NOs: 15 and 16, or a combination of complementary strands thereof; A primer set consisting of a combination of polynucleotides of SEQ ID NOs: 17 and 18, or a combination of complementary strands thereof; or A primer set consisting of a combination of polynucleotides of SEQ ID NOs: 19 and 20, or a combination of complementary strands thereof; The primer or probe according to [7], [9] The primer or probe according to [7], which is a probe consisting of a polynucleotide having a nucleotide sequence of any one of SEQ ID NOs: 21 to 30 or a complementary strand thereof.

[10] A method for determining upper tract urothelial carcinoma in a subject, comprising: positions 158,799,748, 158,799,765, 158,799,775, 158,799,777, and 158,799,789 on chromosome 7; positions 111,813,685, 111,813,690, and 111,813,698 on chromosome 1; positions 240,375,464 on chromosome 1; positions 28,956,374, 28,956,381, 28,956,384, and 28,956,387 on chromosome 6; positions 91,182,528 and 91,182,529 on chromosome 1 in genomic DNA derived from upper urothelial cells or tissues containing same of the subject; detecting the DNA methylation level of at least one CpG site selected from the group consisting of: positions 82,534 on chromosome 1; positions 119,535,921, 119,535,925, 119,535,928, and 119,535,937 on chromosome 1; positions 4,850,072, 4,850,075, and 4,850,090 on chromosome 7; positions 31,846,849, 31,846,844, 31,846,839, and 31,846,833 on chromosome 11; and positions 106,187,192 and 106,187,210 on chromosome 14; and determining whether the subject has upper tract urothelial carcinoma based on the detected DNA methylation level; The method includes:

[11] The method described in

[10] , wherein the detection of the DNA methylation level comprises detecting the DNA methylation level of the at least one CpG site using bisulfite-treated genomic DNA.

[12] The method described in

[11] , wherein the detection of the DNA methylation level is performed using pyrosequencing, mass spectrometry, bead array or ion exchange chromatography.

[13] The method according to any one of

[10] to

[12] , wherein the genomic DNA is DNA derived from upper urothelial cells contained in urine. Effect of the Invention

[0014] According to the present invention, tissues having UTUC can be determined minimally invasively with high sensitivity and specificity. The present invention enables accurate diagnosis of UTUC while reducing the physical burden on patients during UTUC testing. Furthermore, the present invention enables UTUC testing in patients for whom conventional ureteral catheterization or CT-urography is difficult to apply, thereby contributing to improving the survival rate of UTUC patients. [Brief description of the drawings]

[0015] [Figure 1] Principal component analysis of Infinium assay results for 473,228 CpG sites. Circles: normal upper tract urothelial tissue samples (C) (n=26), triangles: non-cancerous upper tract urothelial tissue samples from UTUC patients (N) (n=31), crosses: UTUC tissue samples (UTUC) (n=31). [Diagram 2] Unsupervised hierarchical clustering using Infinium assay results for C, N and UTUC samples. [Diagram 3] Quantitative DNA methylation levels of 10 CpG sites by pyrosequencing. CpG sites are represented by their probe IDs in the Infinium assay. [Figure 4] ROC curves of sensitivity and specificity for distinguishing UTUC samples (n = 30) from N samples (n = 31) for 37 markers. [Diagram 5] Continued from Figure 4. [Figure 6] Example chromatogram of DNA from a urine sample of a UTUC patient, along with chromatograms of a negative control (unmethylated DNA) and a positive control (methylated DNA). [Figure 7] The original peaks in the chromatogram obtained from the urine sample of the UTUC patient shown in Figure 6 and peaks estimated to be their constituent components are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] As used herein, the term "upper urinary tract" refers to the upper region of the urinary tract extending from the renal pelvis to the urethra, i.e., the renal pelvis and ureter, and does not include the more distal urinary tract, i.e., the bladder and urethra. As used herein, the term "upper urinary tract urothelial carcinoma (UTUC)" refers to cancer occurring in the epithelial cells of the upper urinary tract, and does not include cancer occurring in the urinary tract distal to the ureter, such as urinary bladder urothelial carcinoma (BUC). As used herein, the term "upper urinary tract urothelial carcinoma" includes primary cancer and cancer resulting from metastasis of primary upper urinary tract cancer within the upper urinary tract.

[0017] As used herein, the term "subject" includes humans, for example, those in need of assessment of UTUC, and more specific examples include medical examination recipients and patients suspected of having UTUC.

[0018] As used herein, the term "CpG site" refers to a site in DNA where a phosphodiester bond (p) is formed between cytosine (C) and guanine (G). A region where CpG sites appear frequently is called a CpG island. CpG sites contained in a CpG island often cause DNA methylation abnormalities associated with cancer. As used herein, unless otherwise defined, the term "CpG site" preferably refers to a CpG site contained in a CpG island, and more preferably refers to a CpG site present in the vicinity of a gene.

[0019] As used herein, "DNA methylation" refers to a state in which the 5th carbon of cytosine in DNA is methylated. As used herein, the "DNA methylation level" of a CpG site refers to the proportion of DNA methylated at the CpG site. As used herein, high and low DNA methylation levels refer to high and low proportions of DNA methylation, respectively.

[0020] As used herein, "at least 90% identity" with respect to amino acid sequences and nucleotide sequences means 90% or more identity, preferably 93% or more identity, more preferably 95% or more identity, even more preferably 96% or more identity, even more preferably 97% or more identity, even more preferably 98% or more identity, and even more preferably 99% or more identity.

[0021] As shown in the Examples below, the present inventors have demonstrated that the DNA methylation levels at CpG sites located at specific loci on chromosomes are different between tissues without UTUC derived from non-UTUC or UTUC patients and tissues with UTUC derived from UTUC patients. The DNA methylation levels at these CpG sites also differed between UTUC and BUC. Therefore, the DNA methylation levels at these CpG sites can be used as markers for determining UTUC.

[0022] CpG sites that can be used for UTUC determination in the present invention are shown in Table 1. In this specification, the chromosomal positions of CpG sites are expressed based on the positions on the NCBI database Genome Build 37, which is the reference human genome sequence.

[0023] [Table 1]

[0024] In the present invention, the DNA methylation levels of the CpG sites shown in Table 1 may be used as markers for determining UTUC.

[0025] The CpG sites shown in Table 1 are classified into marker groups 1 to 9. Each marker group is composed of CpG sites adjacent to each other, and these adjacent CpG sites are collectively subjected to DNA methylation or non-methylation associated with UTUC. Therefore, in the present invention, a statistical value of the DNA methylation level of the CpG sites included in a marker group (for example, but not limited to, a total value, an average value, etc.) can also be used as a marker for determining UTUC.

[0026] Therefore, the present invention provides a method for determining whether a cell or tissue has UTUC, comprising the steps of: Detecting the DNA methylation level of at least one CpG site selected from the group consisting of the CpG sites shown in Table 1 in genomic DNA derived from upper urothelial cells of a subject or a tissue containing the same; and determining whether the cell or tissue has UTUC based on the detected DNA methylation level; The present invention provides a method comprising the steps of: The present invention also provides a method for determining UTUC in a subject, comprising the steps of: Detecting the DNA methylation level of at least one CpG site selected from the group consisting of the CpG sites shown in Table 1 in genomic DNA derived from upper urothelial cells of a subject or a tissue containing the same; and determining whether the subject has UTUC based on the detected DNA methylation level; The present invention provides a method comprising the steps of: The present invention also provides a method for obtaining data used to determine whether a subject has UTUC, or data used to determine whether a cell or tissue has UTUC, comprising the steps of: Detecting the DNA methylation level of at least one CpG site selected from the group consisting of the CpG sites shown in Table 1 in genomic DNA derived from upper urothelial cells of a subject or a tissue containing the same; and Obtaining data for determining whether the subject has UTUC or not, or data for determining whether the cell or tissue has UTUC or not, based on the detected DNA methylation level; The present invention provides a method comprising the steps of:

[0027] In the above-mentioned method of the present invention, it is sufficient to detect the DNA methylation level of any one of the CpG sites shown in Table 1, but the DNA methylation levels of any two or more CpG sites may be detected. When detecting the DNA methylation levels of two or more CpG sites, a combination of CpG sites belonging to the same marker group shown in Table 1 may be used, or a combination of CpG sites belonging to different marker groups may be used. Alternatively, a statistical value of the DNA methylation level of a CpG site included in any of the marker groups shown in Table 1 (for example, including but not limited to a total value, an average value, etc.) may be detected. The statistical values ​​of the DNA methylation level derived from different marker groups may be used in combination. One or more statistical values ​​of the DNA methylation level may be used in combination with the DNA methylation level of a CpG site belonging to a marker group different from them.

[0028] The genomic DNA derived from upper urothelial cells or tissues containing the same used in the method of the present invention includes genomic DNA prepared from upper urothelial cells or tissues containing the same, and DNA derived from upper urothelial cells in urine. Examples of the urothelial cells or tissues containing the same include fresh upper urothelial tissues collected in surgery or biopsy, frozen upper urothelial tissues collected and frozen, upper urothelial tissues collected and formalin-fixed and paraffin-embedded, and upper urothelial cells concentrated from urine. Among these, frozen upper urothelial tissues are preferred from the viewpoint of suppressing the degradation of genomic DNA in the tissue and enabling more efficient detection of DNA methylation levels. On the other hand, DNA derived from upper urothelial cells contained in excreted urine is preferred from the viewpoint of non-invasiveness and ease of collection.

[0029] The method for preparing sample DNA from the above-mentioned upper urinary tract epithelial cells or tissues containing them is not particularly limited, and any known method can be appropriately selected and used. Known methods for preparing DNA include the phenol-chloroform method, or a DNA extraction method using a commercially available DNA extraction kit, such as QIAamp DNA Mini kit (Qiagen), Clean Columns (NexTec), AquaPure (Bio-Rad), ZR Plant / Seed DNA Kit (Zymo Research), prepGEM (ZyGEM), BuccalQuick (TrimGen), etc. Urinary ur ...

[0030] Preferably, the prepared genomic DNA is bisulfite-treated. The method for bisulfite treatment of DNA is not particularly limited, and any known method can be appropriately selected and used. Known methods for bisulfite treatment include, for example, EZ DNA Methylation-Gold TM Examples of methods include methods using commercially available kits such as the CpG Bisulfite Kit (Zymo Research), EpiTect Bisulfite Kit (Qiagen), MethylEasy (Human Genetic Signatures Pty), Cells-to-CpG Bisulfite Conversion Kit (Applied Biosystems), and CpGenome Turbo Bisulfite Modification Kit (MERCK MILLIPORE).

[0031] Furthermore, the bisulfite-treated DNA may be amplified. The amplification method is not particularly limited, but PCR is preferably used. The amplification method and conditions can be appropriately selected from known methods and conditions depending on the sequence, length, amount, etc. of the DNA to be amplified.

[0032] When the bisulfite-treated DNA is amplified by PCR or the like, DNA containing at least one CpG site shown in Table 1 may be amplified. Preferred examples of the region to be amplified include one or more selected from DNA regions containing any of the nucleotide sequences of SEQ ID NOs: 31 to 40. The chain length of the PCR amplified product can be appropriately selected in consideration of factors such as shortening the PCR amplification time, shortening the methylation level detection time, and the accuracy of methylation level detection. For example, the chain length of the PCR amplified product is preferably 500 bp or less, more preferably 300 bp or less, and even more preferably 100 bp or less, while the lower limit is 30 to 40 bp, which is the chain length of the PCR amplified product when a primer of about 15 mer that avoids non-specific hybridization in PCR is used. Alternatively, it is preferable to design primers so that the content of the CpG site shown in Table 1 in the PCR amplified product is rich. For example, the cytosine of the CpG site is preferably contained in 2% or more, more preferably 5% or more, relative to the chain length of the PCR amplified product.

[0033] In the method of the present invention, the method for detecting the DNA methylation level at a CpG site may be any method capable of quantifying the DNA methylation level at a given CpG site, and may be appropriately selected from known methods, such as the following methods 1 to 8.

[0034] The first method is a method for quantifying DNA methylation at a CpG site by utilizing a single-base extension reaction using a probe constructed to have a base complementary to methylated cytosine or non-methylated cytosine at the 3' end. For example, the first method is a method based on the following principle. First, the genomic DNA is treated with bisulfite. Note that this bisulfite treatment converts non-methylated cytosine residues to uracil, but does not convert methylated cytosine residues (see Clark SJ et al., Nucleic Acids Res, 1994, vol. 22, pp. 2990-7). Next, using the bisulfite-treated genomic DNA as a template, whole genome amplification is performed, and the DNA is enzymatically fragmented (usually about 300-600 bp) and dissociated into single strands.

[0035] In the first method, a probe is prepared that hybridizes to genomic DNA converted by bisulfite treatment, and the base at the 3' end of the probe is a base complementary to the cytosine at the CpG site. That is, if the CpG site is methylated, the base at the 3' end of the probe is guanine, and if the CpG site is not methylated, the base at the 3' end of the probe is adenine.

[0036] Then, the single-stranded DNA fragment is hybridized with two kinds of probes that are different only in the base at the 3' end that is complementary to the CpG site, and a single-base extension reaction is carried out in the presence of a fluorescently labeled base. As a result, when the CpG site of the single-stranded fragment is methylated, a fluorescently labeled base is incorporated by a single-base extension reaction into a probe (methylation detection probe) whose 3'-end base is guanine, but a fluorescently labeled base is not incorporated into a probe (non-methylation detection probe) whose 3'-end base is adenine due to a mismatch of the base at the 3' end. On the other hand, when the CpG site of the single-stranded fragment is not methylated, a fluorescently labeled base is incorporated into the non-methylation detection probe, but a fluorescently labeled base is not incorporated into the methylation detection probe. Therefore, the DNA methylation level can be calculated from the intensity of the fluorescence emitted by the methylation detection probe and / or the non-methylation detection probe.

[0037] In another embodiment of the first method, instead of the methylation detection probe and the unmethylation detection probe, a probe that hybridizes with genomic DNA converted by bisulfite treatment and has a base at the 3' end that is complementary to the guanine at the CpG site may be used. Then, the probe is hybridized with the single-stranded DNA fragment, and a single-base extension reaction is performed in the presence of guanine labeled with a fluorescent substance and / or adenine labeled with a fluorescent dye different from the fluorescent substance. As a result, when the CpG site is methylated, fluorescently labeled guanine is incorporated into the probe, while when the CpG site is not methylated, fluorescently labeled adenine is incorporated into the probe, so that the DNA methylation level can be calculated from the intensity of fluorescence emitted by each fluorescent substance incorporated into the probe.

[0038] A preferred example of the first technique is the bead array method (eg, the Infinium assay provided by Illumina, Inc.).

[0039] The second method is a method for quantifying methylated DNA by mass spectrometry. For example, the second method is based on the following principle. First, the genomic DNA is treated with bisulfite. Then, the bisulfite-treated genomic DNA is used as a template to amplify DNA containing at least one CpG site with a primer to which a T7 promoter has been added. Next, the amplified DNA is transcribed into RNA, and a base-specific cleavage reaction is carried out with RNase. Then, the cleavage reaction product is subjected to a mass spectrometer to measure the mass.

[0040] The mass derived from the methylated cytosine residue (mass of cytosine) obtained by mass measurement is then compared with the mass derived from the unmethylated cytosine residue (mass of uracil) to calculate the DNA methylation level at the CpG site.

[0041] A preferred example of the second technique is a DNA methylation analysis method using a mass spectrometer (eg, MassARRAY (registered trademark), see Jurinke C et al., Mutat Res, 2005, vol. 573, pp. 83-95).

[0042] In MassARRAY (registered trademark), DNA containing a CpG site after bisulfite treatment is amplified, transcribed into RNA, and then specifically cleaved at the uracil site by RNAase. This generates RNA fragments of different lengths depending on the presence or absence of DNA methylation. By subjecting the obtained RNA fragments to mass spectrometry, fragments with methylated CpG sites and non-methylated fragments are separated and detected according to the difference in molecular weight. Primers for amplifying DNA containing the CpG site can be designed using EpiDesigner (manufactured by SEQUENOM, primer design software for MassARRAY) or the like. For mass spectrometry of RNA fragments, MALDI-TOF MAS (e.g., MassARRAY Analyzer 4, manufactured by SEQUENOM) capable of detecting the difference in mass of a single base is used. The methylation level of DNA is calculated from the ratio of the mass of RNA fragments derived from methylated DNA to that of RNA fragments derived from non-methylated DNA.

[0043] The third method is based on the following principle. First, the genomic DNA is treated with bisulfite. The bisulfite treatment converts unmethylated cytosine residues to uracil, but in the following extension reaction (sequence reaction), uracil is represented as thymine. Next, the bisulfite-treated genomic DNA is used as a template to amplify DNA containing at least one CpG site. The amplified DNA is dissociated into single strands. Next, only one strand of the dissociated single-stranded DNA is separated. Then, an extension reaction is performed one base at a time from the vicinity of the base of the CpG site, and the pyrophosphate generated during this is enzymatically made to emit light, and the intensity of the light emission is measured. The intensity of the light emission derived from the methylated cytosine residue obtained in this way (light emission intensity of cytosine) is compared with the intensity of the light emission derived from the unmethylated cytosine residue (light emission intensity of thymine), and the DNA methylation level (%) at the CpG site is calculated, for example, by the following formula. DNA methylation level (%) = cytosine emission intensity × 100 / (cytosine emission intensity + thymine emission intensity).

[0044] An example of the third technique is the Pyrosequencing method (registered trademark) (see Anal. Biochem. (2000) 10:103-110).

[0045] The fourth method is based on the following principle. First, the genomic DNA is treated with bisulfite. Next, in a reaction system containing an intercalator that emits fluorescence when inserted between DNA double strands, the bisulfite-treated genomic DNA is used as a template to amplify a nucleotide containing at least one CpG site. Next, the temperature of the reaction system is changed, and the fluctuation in the intensity of the fluorescence emitted by the intercalator is detected. The melting curve of the nucleotide containing at least one CpG site is compared with the melting curve of the amplification product using methylated and unmethylated control samples as templates, and the DNA methylation level at the CpG site is calculated.

[0046] An example of the fourth technique is methylation-sensitive high resolution melting (MS-HRM; see Wojdacz TK et al., Nat Protoc, 2008, vol. 3, pp. 1903-8).

[0047] The fifth method is based on the following principle. First, the genomic DNA is treated with bisulfite. Next, a primer set capable of amplifying the CpG site when it is methylated and a primer set capable of amplifying the CpG site when it is not methylated are prepared. Then, the bisulfite-treated genomic DNA is used as a template to amplify nucleotides containing at least one CpG site using these primer sets. Then, the amount of the obtained amplification product, i.e., the amount of the amplification product specific to the methylated CpG site and the amount of the amplification product specific to the unmethylated CpG site, is compared to calculate the DNA methylation level at the CpG site.

[0048] Further, in another embodiment of the fifth method, first, the genomic DNA is subjected to a bisulfite treatment. Next, an oligonucleotide probe having a nucleotide capable of hybridizing when the CpG site is methylated and labeled with a reporter fluorescent dye and a quencher fluorescent dye is prepared. Also, an oligonucleotide probe having a nucleotide capable of hybridizing when the CpG site is not methylated and labeled with a reporter fluorescent color different from the reporter fluorescent dye and a quencher fluorescent dye is prepared. Then, the oligonucleotide probe is hybridized to the bisulfite-treated genomic DNA, and further, the nucleotides including the CpG site are amplified using the genomic DNA hybridized with the oligonucleotide probe as a template. Then, the fluorescence emitted by the reporter fluorescent dye is detected due to the decomposition of the oligonucleotide probe accompanying the amplification. The intensity of fluorescence emitted by the thus detected methylated cytosine CpG site-specific reporter fluorescent dye is compared with the intensity of fluorescence emitted by the unmethylated cytosine CpG site-specific reporter fluorescent dye to calculate the DNA methylation level at the CpG site.

[0049] The fifth technique includes, for example, a methylation-specific quantitative PCR method (methylation-specific polymerase chain reaction (MS-PCR) using real-time quantitative PCR) such as the MethyLight method using TaqMan probe (registered trademark).

[0050] The sixth method is based on the following principle. First, the genomic DNA is treated with bisulfite. Next, a direct sequencing reaction is performed using the bisulfite-converted nucleotides containing the CpG site as a template. Then, the DNA methylation level at the CpG site is calculated by comparing the fluorescence intensity based on the determined base sequence, i.e., the fluorescence intensity derived from the methylated cytosine residue (fluorescence intensity of cytosine) with the fluorescence intensity derived from the unmethylated cytosine residue (fluorescence intensity of thymine).

[0051] Furthermore, in another embodiment of the sixth method, the genomic DNA is first subjected to a bisulfite treatment. Then, the bisulfite-converted nucleotides containing the CpG site are cloned by PCR or the like. The base sequences of the resulting multiple cloning products are determined, and the number of cloning products having a base sequence specific to a methylated cytosine CpG site is compared with the number of cloning products having a base sequence specific to an unmethylated cytosine CpG site to calculate the DNA methylation level at the CpG site.

[0052] The sixth technique includes, for example, bisulfite direct sequencing and bisulfite cloning sequencing (see Kristensen LS et al., Clin Chem, 2009, vol. 55, pp. 1471-83).

[0053] The seventh method is based on the following principle. First, the genomic DNA is treated with bisulfite. Then, the region containing the CpG site is amplified by PCR using the bisulfite-converted nucleotides containing the CpG site as a template. Next, the amplified DNA fragment is treated with a restriction enzyme that recognizes a portion where the sequence differs between when the CpG site is methylated and when it is not. Then, the DNA methylation level of the CpG site can be calculated by quantitatively analyzing the band densities of the restriction enzyme fragments derived from the methylated CpG site and the restriction enzyme fragments derived from the unmethylated CpG site, which are fractionated by electrophoresis.

[0054] The seventh technique is, for example, COBRA (combined analysis using bisulfite and restriction enzymes).

[0055] The eighth method is a method using ion exchange chromatography. For example, the eighth method is a method based on the following principle. First, the genomic DNA is treated with bisulfite. Then, the DNA is fragmented to obtain DNA fragments containing CpG sites. Using the obtained DNA fragments as templates, the regions containing the CpG sites are amplified by PCR or the like. Next, the amplified DNA fragments are subjected to ion exchange chromatography to separate DNA in which the CpG sites are methylated from DNA in which the CpG sites are not methylated.

[0056] In the eighth method, the length of the PCR amplification product can be appropriately selected taking into consideration factors such as shortening the PCR amplification time, shortening the analysis time in ion exchange chromatography, and maintaining separation performance. For example, the length of the PCR amplification product is preferably 500 bp or less, more preferably 300 bp or less, and even more preferably 100 bp or less, while the lower limit is 30 to 40 bp, which is the length of the PCR amplification product when using a primer of about 15 mer that can avoid non-specific hybridization in PCR. On the other hand, it is preferable to design the primer so that the content of CpG sites in the PCR amplification product is rich. For example, the cytosine of the CpG site is preferably contained in 2% or more, more preferably 5% or more, relative to the length of the PCR amplification product.

[0057] In the eighth method, the unmethylated cytosine residue is converted to uracil by bisulfite treatment of genomic DNA, and then further converted to thymine by PCR. On the other hand, the methylated cytosine residue remains as cytosine even after bisulfite treatment and PCR. Due to this difference in base, a fragment containing methylated cytosine (methylated fragment) and an unmethylated fragment are detected as separate peaks with different retention times in ion exchange chromatography. That is, the methylated fragment is detected as a peak with a shorter retention time than the unmethylated fragment. Therefore, it is possible to determine whether or not the DNA at the CpG site is methylated based on the retention time of the peak in ion exchange chromatography. Furthermore, when the DNA fragment subjected to ion exchange chromatography contains multiple CpG sites, the more CpG sites are methylated, the shorter the retention time of the peak. Therefore, the DNA methylation level of the CpG site can be calculated based on the retention time of the peak. Alternatively, it is also possible to calculate the abundance or abundance ratio of the methylated fragment and the unmethylated fragment based on the area or height of the peak.

[0058] Preferably, the DNA methylation level of the CpG site is determined by comparing with a sample (control) having a known DNA methylation level, or by using a calibration curve prepared in advance using a sample having a known DNA methylation level. Alternatively, a retention time (also referred to as a reference retention time in this specification) that is a reference for separating the retention time of the peak of a methylated fragment in which the CpG site is highly methylated from the retention time of the peak of a fragment with a low methylation level is determined in advance using a sample having a known DNA methylation level. For example, a fragment detected at a retention time earlier than the reference retention time is determined to be highly methylated DNA.

[0059] The ion exchange chromatography carried out in the eighth method is preferably anion exchange chromatography. The column packing material is not particularly limited as long as it is a base particle having a strong cationic group on the surface, but the base particle having both a strong cationic group and a weak cationic group on the packing surface as shown in International Publication No. 2012 / 108516 is preferred. More preferably, the base particle is a base particle containing a coated polymer particle in which a layer of a hydrophilic polymer having a strong cationic group (preferably a quaternary ammonium salt) is copolymerized on the surface of a hydrophobic crosslinked polymer particle, and a weak cationic group (preferably a tertiary amino group) introduced on the surface of the coated polymer particle. The column temperature during the chromatographic analysis is preferably 30°C or higher and less than 90°C.

[0060] The above are examples of techniques that can be suitably used as a "technique for detecting DNA methylation level" in the present invention, but the techniques are not limited thereto. In the first to eighth techniques, as described above, the genomic DNA prepared from upper urothelial cells or tissues is further subjected to bisulfite treatment. Therefore, the genomic DNA used for detecting the DNA methylation level of CpG sites in the method of the present invention is preferably bisulfite-treated genomic DNA derived from upper urothelial cells or tissues containing the same.

[0061] In the method of the present invention, the presence or absence of UTUC in a test upper urothelial cell or a tissue containing the same, or the presence or absence of UTUC in a subject, is determined based on the detected DNA methylation level at the CpG site. A person skilled in the art can appropriately set a specific indicator for the determination in accordance with the method for detecting DNA methylation level.

[0062] An embodiment of the procedure for determining the presence or absence of UTUC is described below. In the first embodiment, first, for each DNA methylation level detection method, a receiver operating characteristic (ROC) analysis is performed for each CpG site to obtain sensitivity (positive rate) and specificity (negative rate), and the DNA methylation level at which the sum of sensitivity and specificity is maximized is set as an index (cutoff value).

[0063] In the first embodiment, for CpG sites whose methylation level is increased by carcinogenesis of upper urothelial tissue (e.g., CpG sites included in marker groups 1, 2, 4, 8, and 9 shown in Table 1), if the DNA methylation level detected by the method of the present invention is higher than the cutoff value, the DNA methylation level is determined to exceed the diagnostic threshold, and the test cell or tissue, or the subject is determined to have UTUC, but if the detected methylation level is equal to or lower than the cutoff value, the test cell or tissue, or the subject is determined to not have UTUC. On the other hand, for CpG sites whose methylation level is decreased by carcinogenesis of epithelial urothelial tissue (e.g., CpG sites included in marker groups 3, 5, 6, and 7 shown in Table 1), if the DNA methylation level detected by the method of the present invention is lower than the cutoff value, the DNA methylation level is determined to exceed the diagnostic threshold, and the test cell or tissue, or the subject is determined to have UTUC, but if the detected methylation level is equal to or higher than the cutoff value, the test cell or tissue, or the subject is determined to not have UTUC.

[0064] In the first embodiment, when the methylation levels of multiple CpG sites are detected, the number or percentage of CpG sites whose DNA methylation levels exceed the diagnostic threshold can be used as an index for UTUC. For example, when the methylation levels of all the examined CpG sites exceed the diagnostic threshold, the test cell or tissue, or the subject can be determined to have UTUC. Alternatively, when the methylation levels of a certain percentage or more of the examined CpG sites exceed the diagnostic threshold, the test cell or tissue, or the subject can be determined to have UTUC. Alternatively, when the methylation levels of a certain number or more of the CpG sites exceed the diagnostic threshold, the test cell or tissue, or the subject can be determined to have UTUC. On the other hand, the test cell or tissue, or the subject that does not meet these criteria can be determined not to have UTUC.

[0065] In a second embodiment of the procedure for determining UTUC, the retention time of a peak obtained in ion exchange chromatography analysis (method 8 above) of DNA (sample) containing a target CpG site from a test cell or tissue, or a subject, is compared with the retention time for DNA containing the unmethylated target CpG site (negative control) or DNA containing the methylated target CpG site (positive control) to determine the methylation level of the CpG site or to determine the presence or absence of UTUC.

[0066] In the second embodiment, for CpG sites whose methylation level is increased by carcinogenesis of upper urothelial tissue (e.g., CpG sites included in marker groups 1, 2, 4, 8, and 9 shown in Table 1), if a peak with a retention time shorter than that of the negative control is detected from the sample, the sample is determined to be methylated, or the test cell or tissue, or the subject is determined to have UTUC. Alternatively, if a peak with a retention time equivalent to that of the positive control is detected from the sample, the sample is determined to be methylated, or the test cell or tissue, or the subject is determined to have UTUC. On the other hand, for CpG sites whose methylation level is reduced by carcinogenesis of upper urothelial tissue (e.g., CpG sites included in marker groups 3, 5, 6, and 7 shown in Table 1), if a peak with a retention time longer than that of the positive control is detected from the sample, the sample is determined to be unmethylated, or the test cell or tissue, or the subject is determined to have UTUC. Alternatively, if a peak with a retention time equivalent to that of the negative control is detected from the sample, the sample is determined to be unmethylated, or the test cell or tissue, or the subject is determined to have UTUC.

[0067] When the genomic DNA to be examined for the methylation level of the target CpG site is urinary DNA, it may contain DNA derived from UTUC cells as well as DNA derived from cells of different lineages such as non-cancerous epithelial cells and blood cells. Even in such a case, the presence or absence of DNA derived from UTUC cells can be detected by the above-mentioned method 8. That is, when DNA derived from a plurality of different cells is mixed in the sample DNA, the peak of the sample DNA obtained by the chromatography analysis by the eighth method will show a bimodal, shouldered or broad peak shape (see, for example, FIG. 6 described later). Such an original peak can be separated into peaks derived from different cell lineages, i.e., peaks with different DNA methylation levels. For example, fitting can be performed on the assumption that there are multiple normal distribution shape peaks for the original peak, and peaks contained in the original peak can be estimated. Next, for each estimated peak, the methylation level of the target CpG site is calculated based on its retention time. For example, the methylation level of each estimated peak can be calculated using a calibration curve created based on the retention time of DNA with a known methylation level. By comparing the methylation level of the estimated peak with a cutoff value in the same procedure as in the first embodiment described above, it can be determined whether the urine-derived DNA contains UTUC cell-derived DNA or whether the subject has UTUC.

[0068] Thus, according to the present invention, UTUC can be determined with a less invasive and simpler procedure than conventional UTUC examination methods such as ureteral catheterization and CT-urography. The present invention enables UTUC diagnosis with high sensitivity and specificity while reducing the physical burden on the patient during UTUC examination. The present invention also enables UTUC examination in patients for whom conventional ureteral catheterization and CT-urography are difficult to apply, such as patients suspected of urethral damage, patients allergic to contrast media, and patients with reduced renal function.

[0069] Furthermore, the present invention provides a method for treating UTUC, comprising treating a subject diagnosed with UTUC by the method of the present invention. Treatment methods include, but are not limited to, surgery, chemotherapy using drugs or radiation, etc.

[0070] The present invention further provides a primer or probe for determining the presence or absence of UTUC in upper urothelial cells, tissues containing the same, or subjects, which has a chain length of at least 12 bases and hybridizes to at least one CpG site selected from the group consisting of the CpG sites shown in Table 1 after bisulfite treatment.

[0071] An example of the primer or probe of the present invention is a primer or probe that hybridizes to bisulfite-treated DNA containing at least one CpG site shown in Table 1. Another example of the primer of the present invention is a primer that amplifies bisulfite-treated DNA containing at least one CpG site shown in Table 1. Preferred examples of DNA containing at least one CpG site shown in Table 1 include DNA containing any of the nucleotide sequences of SEQ ID NOs: 31 to 40.

[0072] A preferred example of the primer or probe of the present invention is a primer or probe that hybridizes to DNA containing at least one bisulfite-treated CpG site shown in Table 1 and has a base complementary to methylated cytosine or unmethylated cytosine at its 3' end (e.g., a primer or probe that can be used in the first technique described above).

[0073] Another preferred example of the primer or probe of the present invention is a primer (sequencing primer) that can carry out an extension reaction of one base at a time from the vicinity of a base of a bisulfite-treated DNA fragment containing at least one CpG site shown in Table 1 (for example, a primer or probe that can be used in the above-mentioned third method). Another preferred example is a probe that hybridizes to a nucleotide containing a CpG site in a bisulfite-treated DNA fragment containing at least one CpG site shown in Table 1 (for example, a primer or probe that can be used in the above-mentioned fourth method).

[0074] Another preferred example of the primer or probe of the present invention is a primer set capable of specifically amplifying a region containing a methylated or unmethylated CpG site in a DNA fragment containing at least one CpG site shown in Table 1 that has been bisulfite-treated (for example, a primer set that can be used for PCR amplification in the above-mentioned fifth or eighth method).

[0075] The length of the primer or probe of the present invention may be at least 12 bases, but is preferably at least 15 bases, more preferably at least 20 bases, and even more preferably 15 to 40 bases. The primer or probe of the present invention may be labeled (e.g., fluorescently labeled). The primer or probe of the present invention is preferably a primer or probe that can be used in any of the first to eighth techniques. The primer of the present invention is preferably a PCR primer.

[0076] Preferred examples of the primer or probe of the present invention include polynucleotides consisting of the nucleotide sequences of SEQ ID NOs: 1 to 30 and their complementary strands. Other preferred examples include polynucleotides consisting of nucleotide sequences having at least 90% sequence identity with SEQ ID NOs: 1 to 30 and their complementary strands. More preferred examples include a primer set consisting of a combination of polynucleotides of SEQ ID NO: 1 and 2 or a combination of their complementary strands; a primer set consisting of a combination of polynucleotides of SEQ ID NO: 3 and 4 or a combination of their complementary strands; a primer set consisting of a combination of polynucleotides of SEQ ID NO: 5 and 6 or a combination of their complementary strands; a primer set consisting of a combination of polynucleotides of SEQ ID NO: 7 and 8 or a combination of their complementary strands; a primer set consisting of a combination of polynucleotides of SEQ ID NO: 9 and 10 or a combination of their complementary strands; a primer set consisting of a combination of polynucleotides of SEQ ID NO: 11 and 12 or a combination of their complementary strands; a primer set consisting of a combination of polynucleotides of SEQ ID NO: 13 and 14 or a combination of their complementary strands; a primer set consisting of a combination of polynucleotides of SEQ ID NO: 15 and 16 or a combination of their complementary strands; a primer set consisting of polynucleotides of SEQ ID NO: 17 and 18 or a combination of their complementary strands; a primer set consisting of a combination of polynucleotides of SEQ ID NO: 19 and 20 or a combination of their complementary strands; and a primer set consisting of a combination of primers having at least 90% sequence identity with each of the polynucleotides contained in these primer sets, and amplifying DNA containing any of the nucleotide sequences of SEQ ID NO: 31 to 40. Other more preferred examples include a probe consisting of a nucleotide sequence of any one of SEQ ID NOs: 21 to 30 or a complementary strand thereof; and a probe consisting of a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 21 to 30 or a complementary strand thereof and capable of detecting a CpG site contained in a nucleotide sequence of any one of SEQ ID NOs: 31 to 40.

[0077] Furthermore, the present invention provides a kit for determining UTUC, comprising the primer or probe of the present invention. Preferably, the kit of the present invention is used for determining the presence or absence of UTUC in upper urothelial cells or a tissue containing the same, or in a subject, by any of the first to eighth techniques.

[0078] The kit of the present invention may contain components other than the primer or probe of the present invention. Examples of such components include reagents necessary for bisulfite treatment (e.g., sodium bisulfite solution, etc.), reagents necessary for PCR reaction (e.g., deoxyribonucleotides and heat-resistant DNA polymerase, etc.), reagents necessary for Infinium assay (e.g., nucleotides labeled with fluorescent substances), reagents necessary for MassARRAY (registered trademark) (e.g., RNase for base-specific cleavage reaction), reagents necessary for pyrosequencing (e.g., ATP sulfurylase, adenosine-5'-phosphosulfate, luciferase, luciferin for detecting pyrophosphate, streptavidin for separating single-stranded DNA, etc.), reagents necessary for MS-HRM method (e.g., intercalators that emit fluorescence when inserted between DNA double strands, etc.), reagents necessary for detection of labels (e.g., substrates and enzymes, positive and negative control samples, buffers used for diluting and washing samples (e.g., genomic DNA derived from tissues, etc.), etc.). In addition, the kit may contain instructions for use. EXAMPLES

[0079] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0080] Patients and tissue samples As the initial cohort, non-cancerous upper tract urothelial tissues (N) (n=31) and UTUC tissues (UTUC) (n=31) obtained from UTUC cases were used as samples. As a control sample, normal upper tract urothelial tissues (C) (n=26) obtained from non-urothelial carcinoma cases were used. In addition, bladder cancer tissue (BUC) samples (n=14) were obtained from bladder cancer patients. As the validation cohort, 41 UTUC samples and N samples (82 samples in total) were prepared. Tissue samples were obtained from patients who underwent surgery at the National Cancer Center Hospital. Written informed consent was obtained from all patients. In addition, all studies in this example were approved by the ethics committees of the National Cancer Center and Keio University School of Medicine.

[0081] The collected tissues were frozen and stored. The obtained fresh frozen tissue samples were treated with phenol-chloroform and then dialyzed to extract genomic DNA. 500 ng of the extracted DNA was methylated with EZ DNA Methylation-Gold. TM For pyrosequencing, 400 ng of extracted DNA was treated with bisulfite using the EpiTect Bisulfite Kit (Qiagen).

[0082] Example 1: Determination of UTUC assessment markers (Infinium Assay) The DNA methylation status at 485,764 CpG sites was analyzed at a resolution of 1 CpG site using the Infinium (registered trademark) HumanMethylation450K BeadChip (Illumina, Inc.). The Infinium (registered trademark) HumanMethylation450K BeadChip is said to cover 99% of RefSeq genes and 96% of CpG islands. More specifically, for the purpose of comprehensively analyzing DNA methylation status, the analysis targets include promoter regions, 5' untranslated regions, first exons, intragenic regions, and 3' untranslated regions, and is said to cover 99% of reference sequence genes.

[0083] Specifically, the bisulfite-treated DNA was subjected to whole genome amplification (see Bibikova, M. et al., Epigenomics, 2009, vol. 1, pp. 177-200). The amplified DNA fragments were hybridized with the probes on the chip, and then fluorescently labeled bases were incorporated into the hybridized DNA by a single-base extension reaction. As a result, the methylation detection probe hybridized with the DNA fragment containing methylated CpG, and the unmethylation detection probe hybridized with the DNA fragment containing unmethylated CpG were each fluorescently labeled. Then, the fluorescent signal was measured using an Illumina iScan system (Illumina, Inc.) according to the manufacturer's protocol. The obtained data was analyzed using GenomeStudio methylation software (Illumina, Inc.).

[0084] (Calculation of DNA methylation level) For each CpG site, the relative ratio of the signal from the methylation detection probe to the sum of the signals from the methylation detection probe and the unmethylation detection probe was calculated. That is, the methylation level from each CpG site was expressed as a so-called β value (range: 0.00 to 1.00).

[0085] (Determination of marker CpG) 893 probes with call rates (detection P value < 0.01) below 90% for all tissue samples (26 C samples, and 31 N samples and 31 UTUC samples from the initial cohort) were excluded from the analysis because they may have been designed on polymorphic sites. In addition, 11,648 probes designed on sex chromosomes and 127 probes with β value deletions of 10% or more were also excluded from the analysis. Finally, DNA methylation data of 473,228 CpG sites were subjected to analysis. All statistical analyses were performed using the statistical program RStudio (RStudio Inc, [www.rstudio.com]) and R software (R Foundation for Statistical Computing, [www.r-project.org]).

[0086] The Infinium assay results for the 473,228 CpG sites for the C samples (n=26), N samples (n=31), and UTUC samples (n=31) were analyzed by principal component analysis and unsupervised hierarchical clustering (Euclidean distance, Ward method). The results of principal component analysis showed that the DNA methylation profile of the N samples tended to differ from that of the C samples, and the UTUC samples showed a DNA methylation profile that was clearly different from that of the N and C samples (Figure 1). Furthermore, the hierarchical clustering showed that the UTUC samples showed a DNA methylation profile that was clearly different from that of the N and C samples (Figure 2). These results suggested that UTUC could be distinguished from the N and C samples based on DNA methylation.

[0087] We performed a Welch t-test with Bonferroni correction to detect CpG sites with significantly different methylation levels between UTUC and C samples. Receiver operating characteristic (ROC) curves based on the sensitivity and specificity of the methylation levels were created for the detected CpG sites, and CpG sites with an area under the curve (AUC) of 1.0 were identified. Similar analyses were performed between UTUC and N samples, and between C and N samples. As a result, (a) 7,777 CpG sites with significantly different methylation levels between UTUC and C samples (P<0.05 and Δβ[UTUC-C]≧0.2) and with an AUC of the ROC curve of 1.0, (b) 2,723 CpG sites with significantly different methylation levels between UTUC and N samples (P<0.05 and Δβ[UTUC-N]≧0.2) and with an AUC of the ROC curve of 1.0, and (c) 22 CpG sites with significantly different methylation levels between C and N samples (P<0.05 and Δβ[NC]≧0.2) were identified. From these, 2,448 CpG sites were selected that met (a) and (b) but did not meet (c), i.e., the methylation levels showed a statistically significant difference between UTUC and C and N, but not between C and N.

[0088] From the 2,448 CpG sites, the top 100 sites with the largest difference in β value between UTUC and N were selected, and from these, 52 CpG sites located within CpG islands, island shores (2000 bp regions adjacent to CpG islands), or island shelves (2000 bp regions adjacent to island shores) were selected using the UCSC Genome Browser ([genome.ucsc.edu / ], provided by the University of California, Santa Cruz (UCSC)). Furthermore, from the 52 sites, 21 CpG sites that showed relatively stable methylation levels together with the surrounding CpG sites (the interquartile range of methylation levels of all CpG sites located within at least a 1000 bp region including the target CpG site was less than 20%) were selected using the integrative DNA methylation database iMETHYL ([imethyl.iwate-megabank.org], provided by the Iwate Tohoku Medical Megabank Organization).

[0089] We performed a Welch t-test on the 21 CpG sites and detected 18 CpG sites with significantly different methylation levels between the 31 UTUC samples and the 14 BUC samples. These 18 CpG sites were considered to be candidate markers for the determination of UTUC, which can distinguish UTUC from not only C and N but also BUC.

[0090] The DNA methylation levels of the selected candidate markers were confirmed by pyrosequencing. For the 10 CpG sites for which the pyrosequencing conditions were successfully optimized, the methylation levels were quantified in 26 C samples, as well as 31 N samples and 30 UTUC samples from the initial cohort (one UTUC sample was excluded due to insufficient DNA). Table 2 shows the target CpG sites and conditions for pyrosequencing.

[0091] [Table 2]

[0092] The quantitative results of methylation levels by pyrosequencing for 10 CpG sites are shown in Figure 3. There was no difference in the methylation levels of any CpG site between C and N, and UTUC was statistically significantly different from C and N. A statistically significant correlation was observed between the DNA methylation levels by the Infinium assay and those by pyrosequencing for all 10 CpG sites (Pearson's product moment correlation coefficient r>0.913, P<7.02×10 -35 ). Therefore, it was confirmed that these 10 CpG sites can be used as markers for the diagnosis of UTUC.

[0093] Furthermore, as shown in Table 2, the pyrosequencing target sequence contained CpG sites other than the 10 CpG sites detected by the Infinium assay. As predicted based on the iMETHYL database, these adjacent CpG sites were predicted to be usable as UTUC determination markers, similar to the above 10 CpG sites. In fact, the 10 CpG sites detected by the Infinium assay also contained adjacent CpG sites (probe IDs: cg10874111 and cg14302471). The chromosomal locations of the above 10 CpG sites and their adjacent CpG sites contained in the pyrosequencing target sequence, as well as the associated gene names, are shown in Table 3.

[0094] [Table 3]

[0095] Example 2: Evaluation of UTUC assessment markers The usefulness of the above 10 CpG sites and their adjacent CpG sites as UTUC diagnosis markers was evaluated. For the methylation levels of each of the 10 CpG sites and the adjacent CpG sites, ROC curves of sensitivity and specificity for distinguishing the UTUC samples (n=30) from the initial cohort and the N samples (n=31) were created. In addition, ROC curves were similarly created for the average methylation levels of each of the 10 CpG sites and their adjacent CpG sites. The AUC of the obtained ROC curve was calculated, and the diagnostic threshold (cutoff value) was determined by the top left method.

[0096] The ROC curves for each CpG site are shown in Figures 4-5. The ROC curves for the 10 CpG sites detected by the Infinium assay all showed excellent AUC values ​​(0.988-1.000). In addition, the AUC values ​​for adjacent CpG sites were 0.959-1.000, and the AUC values ​​for each of the 10 CpG sites and the average value of the adjacent CpG sites were 0.986-1.000, both of which were high values.

[0097] We set up 37 markers (see Table 4) including the methylation levels of CpG sites detected by the Infinium assay, the methylation levels of adjacent CpG sites, and their average values, and calculated the sensitivity and specificity of UTUC diagnosis. The sensitivity of UTUC diagnosis was 86.6%-100%, and the specificity was 93.5%-100%.

[0098] To verify the reliability of the 37 markers, the methylation levels of the UTUC samples and N samples (n=41 each) in the validation cohort were examined by pyrosequencing, and the sensitivity and specificity of UTUC diagnosis were calculated based on the cutoff values ​​determined above. The sensitivity of the validation cohort was 73.1%-97.5%, and the specificity was 87.8%-100%. The reliability of the 37 markers was confirmed.

[0099] For the 37 markers, the methylation status in UTUC, AUC, cutoff value, and sensitivity and specificity for UTUC determination in the initial cohort and validation cohort are shown in Table 4. In addition, the clinicopathological parameters (gender, age, malignancy, stage, and lymph node metastasis) of the initial cohort and validation cohort are shown in Table 5. In both the initial cohort and validation cohort, whether or not the 37 markers determined UTUC positive showed no correlation with the clinicopathological parameters. It was shown that the 37 markers can diagnose UTUC regardless of clinicopathological characteristics.

[0100] [Table 4]

[0101] [Table 5]

[0102] Example 3: Determination of UTUC using urine samples Urine is useful as a sample for determining UTUC because it can contain upper urothelial cells and can be collected non-invasively and easily. In this example, UTUC patients were detected based on the methylation level of a UTUC determination marker contained in a urine sample. The DNA methylation level was analyzed by high performance liquid chromatography (HPLC). The marker used for determining UTUC was a marker whose methylation level is increased in UTUC (marker No. 37 in Table 4).

[0103] (Patient and sample processing) Urine samples were collected from catheterized urine samples from 7 UTUC patients and 55 non-UTUC patients (bladder cancer cases and non-UTUC patients who visited a urology clinic). Tissue samples were prepared from UTUC cases (n=10) and non-UTUC tissue samples (n=20) consisting of bladder cancer tissue and non-cancerous tissue from UTUC or bladder cancer cases. Urine and tissue samples were frozen and stored until DNA extraction.

[0104] 4 mL to 55 mL of urine sample was returned to room temperature by natural thawing, and DNA was extracted using QIAamp DNA Micro Kit (Qiagen) according to the manufacturer's protocol. DNA extraction from tissue samples was performed in the same manner as in Example 1. The entire amount of extracted DNA was bisulfite treated in the same manner as in Example 1, and then PCR amplified using the primer set of SEQ ID NO: 19 and 20 under the same conditions as in Table 2. The obtained PCR product was purified and subjected to HPLC. The column used for HPLC and the HPLC conditions are described below.

[0105] (Preparation of anion exchange column) A mixture of 200g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), 100g of triethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), 100g of glycidyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) and 1.0g of benzoyl peroxide (manufactured by Kishida Chemical Co., Ltd.) was added to 2000mL of 3% by weight aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Co., Ltd.) in a reactor equipped with a stirrer. The mixture was heated with stirring and polymerized at 80°C for 1 hour under a nitrogen atmosphere. Next, 100g of ethyl methacrylate trimethylammonium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in ion-exchanged water as a hydrophilic monomer having a strong cationic group. This was added to the same reactor and polymerized at 80°C for 2 hours under a nitrogen atmosphere with stirring in the same manner. The obtained polymerization composition was washed with water and acetone to obtain coated polymer particles having a hydrophilic polymer layer having a quaternary ammonium group on the surface. The coated polymer particles obtained were measured using a particle size distribution analyzer (AccuSizer780 / Particle Sizing Systems), and the average particle diameter was 10 μm. 10 g of the coated polymer particles obtained was dispersed in 100 mL of ion-exchanged water to prepare a pre-reaction slurry. Next, while stirring the slurry, 10 mL of N,N-dimethylaminopropylamine (Wako Pure Chemical Industries, Ltd.), a reagent having a weak cationic group, was added and reacted at 70 ° C. for 4 hours. After the reaction was completed, the supernatant was removed using a centrifuge (Hitachi, Ltd., "Himac CR20G") and washed with ion-exchanged water. After washing, the supernatant was removed using a centrifuge. This washing with ion-exchanged water was repeated four more times to obtain a packing material for ion-exchange chromatography having a quaternary ammonium group and a tertiary amino group on the surface of the base particle. The obtained packing material for ion-exchange chromatography was packed into a stainless steel column (column size: inner diameter 4.6 mm × length 150 mm) of a liquid chromatography system.

[0106] (HPLC conditions) System: LC-20A series (Shimadzu Corporation) Column: Anion exchange column (prepared above) Eluent: Eluent A 25mM MES-NaOH (pH6.0) Eluent B 25mM MES-NaOH (pH6.0) +2M Guanidine sulfate Analysis time: 15 minutes Elution method: The mixing ratio of eluent B was increased linearly under the following gradient conditions. 0 minutes (eluent B 30%) → 10 minutes (eluent B 50%) Sample: PCR amplified product obtained in 3) Flow rate: 1.0mL / min Detection wavelength: 260nm Sample injection volume: 5 μL Column temperature: 70℃

[0107] (Calculation of methylation levels of UTUC markers) FIG. 6 shows an example of a chromatogram of DNA derived from a urine sample of a UTUC patient. In addition to UTUC cells, urine samples from UTUC patients may contain non-cancerous epithelial cells, blood cells, and the like. When DNA methylation levels are analyzed by HPLC using a sample containing a mixture of cells of different lineages, the chromatogram often shows bimodal peaks, shoulder peaks as shown in FIG. 6, or broad peaks, and it is estimated that peaks derived from cells of different lineages are mixed in the chromatogram. Therefore, in this embodiment, the original peaks of the chromatogram obtained by HPLC for the urine sample were separated into peaks derived from cells of different lineages, i.e., peaks with different DNA methylation levels, and the methylation levels of the UTUC determination markers were calculated. That is, fitting was performed on the assumption that multiple normal distribution peaks exist for the original peaks derived from the urine sample, and the peaks included were estimated. The retention time of each estimated peak was detected, and the methylation level of the estimated peak was calculated from a calibration curve created from the retention time of the unmethylated peak (methylation rate 0%) and the retention time of the methylated peak (methylation rate 100%) measured at the same time. If the methylation level of the estimated peak was higher than the cutoff value of the UTUC determination marker (cutoff value of 35.17% for marker No. 37 in Table 4), the estimated peak was estimated to be a peak of DNA derived from UTUC cells. Urine samples in which a peak of DNA derived from UTUC cells was detected were determined to be urine samples from UTUC patients.

[0108] (UTUC judgment) Figure 7 shows the original peaks of the chromatogram obtained from the urine sample of the UTUC patient shown in Figure 6 and the peaks estimated as its components. A peak derived from hypermethylated DNA with a methylation level of 72% (estimated peak 1) was detected and was estimated to be a peak of DNA derived from UTUC cells. The presence of this hypermethylated DNA peak confirmed that the patient was UTUC positive.

[0109] In the same manner, for all urine samples, putative peaks were detected from the original peaks of the chromatogram, and the methylation levels of the putative peaks were calculated to determine whether the urine samples were from UTUC patients. For tissue samples, the DNA methylation levels were calculated based on the original peaks. From the results, the sensitivity and specificity of UTUC determination were calculated for each of the urine samples and tissue samples, as well as for all samples including the urine samples and tissue samples.

[0110] The results are shown in Table 6. For urine samples, the sensitivity for detecting samples from UTUC patients was approximately 83%, and the specificity was approximately 91% (Table 6(a)). On the other hand, for tissue samples, the sensitivity and specificity were both 100% (Table 6(b)), and for all samples, the sensitivity and specificity were both approximately 94% (Table 6(c)). These results demonstrate that it is possible to detect UTUC determination markers and perform UTUC determination with high accuracy based on their methylation levels not only in tissue samples but also in urine samples.

[0111] [Table 6]

Claims

1. A method for determining whether a cell or tissue has upper tract urothelial carcinoma, comprising: In genomic DNA derived from upper urinary tract epithelial cells or tissues containing same, positions 158,799,748, 158,799,765, 158,799,775, 158,799,777, and 158,799,789 on chromosome 7; positions 111,813,685, 111,813,690, and 111,813,698 on chromosome 1; position 240,375,464 on chromosome 1; positions 28,956,374, 28,956,381, 28,956,384, and 28,956,387 on chromosome 6; positions 91,182,528 and 91,182,528 on chromosome 1 detecting the DNA methylation level of at least one CpG site selected from the group consisting of positions 2,534 on chromosome 1; positions 119,535,921, 119,535,925, 119,535,928, and 119,535,937 on chromosome 1; positions 4,850,072, 4,850,075, and 4,850,090 on chromosome 7; positions 31,846,849, 31,846,844, 31,846,839, and 31,846,833 on chromosome 11; and positions 106,187,192 and 106,187,210 on chromosome 14; determining whether the cell or tissue has upper tract urothelial carcinoma based on the detected DNA methylation level; The method includes:

2. The method according to claim 1 , wherein the detection of the DNA methylation level comprises detecting the DNA methylation level of the at least one CpG site using the genomic DNA that has been bisulfite-treated.

3. The method according to claim 2, wherein the detection of the DNA methylation level is carried out using pyrosequencing, mass spectrometry, bead array or ion exchange chromatography.

4. The method according to any one of claims 1 to 3, wherein the genomic DNA is DNA derived from upper urothelial cells contained in urine.

5. A probe for determining upper tract urothelial carcinoma, which is a probe consisting of a polynucleotide consisting of any of the nucleotide sequences of SEQ ID NOs: 21 to 30 or a complementary strand thereof.

Images