WO2015129916A1 - 腎細胞癌の予後判定方法 - Google Patents
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- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
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Definitions
- the present invention relates to a method for determining the prognosis of renal cell carcinoma using detection of methylated DNA.
- a CpG island is a region in which a two-base sequence of cytosine (C) -guanine (G) through a phosphodiester bond (p) appears frequently, and is often present in a promoter region upstream of a gene.
- C cytosine
- G guanine
- p phosphodiester bond
- Abnormal DNA methylation of CpG islands is involved in carcinogenesis through inactivation of tumor suppressor genes.
- Increased DNA methylation of CpG islands correlated with clinicopathological factors has been reported in colorectal cancer, gastric cancer, and the like (Non-Patent Documents 1 to 4). It is called CIMP) positive cancer.
- a method for analyzing methylated DNA there is a method using a bisulfite (bisulfite, bisulfite, bisulfite) reaction.
- This method is the most widely used method for analyzing methylated DNA.
- cytosine When single-stranded DNA is treated with bisulfite, cytosine is converted to uracil through sulfonation, hydrodeamination, and desulfonation.
- methylated cytosine has a very slow reaction rate of the first sulfonation, and thus remains methylated cytosine in the reaction time of the actual bisulfite treatment.
- PCR Polymerase Chain Reaction
- methylated cytosine remains cytosine, but unmethylated cytosine is amplified by replacing uracil with thymine.
- the methylation state is analyzed using the difference between the bases cytosine and thymine generated in the sequence of the PCR amplification product.
- MSP Methylation-Specific PCR
- COBRA Combined Bisulfite Restriction Analysis
- the MSP method performs PCR amplification using a methylated sequence-specific primer and an unmethylated sequence-specific primer after DNA bisulfite treatment, and agarose gel electrophoresis in order.
- This is a method for determining the DNA methylation state of a region.
- the COBRA method uses PCR amplification using a common primer for methylated and unmethylated DNA after treatment with bisulfite, and a restriction enzyme that recognizes different sequences between methylated and unmethylated DNA.
- the agarose gel electrophoresis are sequentially performed, and the DNA methylation state of the target region is determined based on the presence or absence of the restriction enzyme-treated fragment.
- Both methods are methylated DNA analysis methods that are widely used today because they allow quantitative analysis of methylated DNA without a special device. However, both methods are laborious in that they use electrophoresis for analysis. There was a problem of taking time.
- ion exchange chromatography is widely used as a simple and accurate method for separating and analyzing biopolymers such as nucleic acids, proteins, and polysaccharides in fields such as biochemistry and medicine.
- anion exchange chromatography that separates using the negative charge of phosphate contained in the nucleic acid molecule is used.
- Anion exchange chromatography columns packed with a column filler having a cationic functional group as an ion exchange group are already commercially available and used in various research fields.
- Renal cell carcinoma often occurs in the middle years of the working population, and there are many cases that can be cured by nephrectomy, but there are also cases of rapid metastasis. There is a big difference in the clinical course of both. Furthermore, there are known cases in which immunotherapy and molecular targeted therapeutic drugs are successful even after metastasis. Patients with a high probability of recurrence may closely follow up to diagnose recurrence early and add post-treatment to improve prognosis. However, there are cases of rapid metastasis while belonging to clear cell RCC, which is the most common histopathologically low histological type, and the prognosis prediction by existing clinicopathological factors is Have difficulty.
- Patent Document 4 the above-mentioned 17 is performed by a bead array method, mass spectrometry (MassARRAY method), pyrosequencing, methylation sensitive high resolution melting curve analysis, quantitative PCR, direct sequencing of a bisulfite-treated product, COBRA method and the like.
- a method for detecting the risk of poor prognosis of RCC by detecting the methylation level at the CpG site of a gene has been proposed.
- the value of the DNA methylation rate obtained by a conventionally used method such as MassARRAY method or pyrosequencing provides a DNA methylation rate as an average value of the entire sample subjected to the measurement. Therefore, when there are many cells with a low DNA methylation rate in the sample, even if cells having highly methylated DNA are mixed, the value of the obtained DNA methylation rate is low. As a result, there is a problem that the presence or absence of cells having highly methylated DNA cannot be determined, and further, there is an inevitable risk of estimating the DNA methylation rate low.
- cancer metastasis / recurrence can be predicted and detected early, immunotherapy and molecular targeted therapeutic agents can be expected to be effective against them, so a method capable of more accurate cancer prognosis is required. Further, there is a need for a quick and simple method for accurate cancer prognosis.
- the present inventors have found that methylated DNA can be detected quickly and easily by separating a PCR amplification product obtained by amplifying DNA treated with bisulfite by PCR using ion exchange chromatography. Furthermore, the present inventors analyze that the pattern of signals obtained by ion exchange chromatography differs between DNA obtained from CIMP positive cancer and DNA obtained from CIMP negative cancer, and the difference in the pattern. The inventors have found that a cancer with a poor prognosis can be determined, and have completed the present invention.
- a method for determining a tissue containing renal cell carcinoma comprising: (1) treating genomic DNA prepared from kidney tissue of a subject with bisulfite; (2) a step of amplifying the DNA treated with the bisulfite by PCR; (3) A step of subjecting the obtained PCR amplification product to ion exchange chromatography; (4) A step of obtaining a retention time of the detection signal obtained by the chromatography; (5) A step of determining that the tissue is a tissue containing renal cell carcinoma obtained from a renal cell carcinoma patient with a poor prognosis when the result of step (4) is earlier than a reference retention time; Including methods.
- a method for obtaining data for determining a tissue containing renal cell carcinoma (1) treating genomic DNA prepared from kidney tissue of a subject with bisulfite; (2) a step of amplifying the DNA treated with the bisulfite by PCR; (3) A step of subjecting the obtained PCR amplification product to ion exchange chromatography; (4) A step of obtaining a retention time of the detection signal obtained by the chromatography; (5) Whether the tissue is a tissue containing renal cell carcinoma obtained from a renal cell carcinoma patient with a poor prognosis is determined based on whether or not the result of step (4) is earlier than the reference retention time. Process to obtain as data for Including methods.
- a method for determining the prognosis of a renal cell carcinoma patient (1) treating genomic DNA prepared from kidney tissue of a subject with bisulfite; (2) a step of amplifying the DNA treated with the bisulfite by PCR; (3) A step of subjecting the obtained PCR amplification product to ion exchange chromatography; (4) A step of obtaining a retention time of the detection signal obtained by the chromatography; (5) a step of determining that the subject has a poor prognosis if the result of step (4) is earlier than a reference retention time; Including methods.
- DNA amplified in the step (2) is FAM150A, GRM6, ZNF540, ZFP42, ZNF154, RIMS4, PCDHAC1, KHDRBS2, ASCL2, KCNQ1, PRAC, WNT3A, TRH, FAM78A, ZNF671, N
- the present invention provides a rapid, simple and highly accurate cancer prognosis determination method. According to the present invention, the risk of recurrence of cancer patients can be determined more quickly, simply, and with high accuracy, so that early treatment resumption for cancer patients that require treatment becomes possible. Therefore, this invention contributes to the improvement of the survival rate of a cancer patient.
- renal cell carcinoma is a cancer of renal tubular epithelial cells, and, from its pathological characteristics, clear cell type, granule cell type, chromophore type, spindle type Cancers classified into cyst-associated type, cyst-derived type, cystic type, and papillary type.
- examples of the “subject” include a patient suffering from or suspected of having renal cell carcinoma, and a patient who has been treated for renal cell carcinoma by surgery or the like.
- “poor prognosis” of cancer includes, for example, a low prognosis (post-treatment) survival rate of a subject, and more specifically, a recurrence-free survival rate after 500 days after surgery.
- (Cancer-free survival rate) may be 50% or less, or the overall survival rate (overall survival rate) after 1500 days after surgery may be 70% or less.
- DNA methylation means a state in which the carbon at the 5-position of cytosine is methylated in DNA.
- detecting methylation of DNA means measuring the presence / absence or abundance of methylated DNA in the DNA, a ratio of the abundance, or a methylation rate of the DNA.
- the “DNA methylation rate” means the rate at which cytosine of CpG islands is methylated in the specific DNA to be detected.
- the CpG of the specific DNA to be detected It can be represented by the ratio of the number of methylated cytosines to the total number of cytosines (methylated cytosine and unmethylated cytosine) in the island.
- CpG site means a site where cytosine (C) and guanine (G) have a phosphodiester bond (p) in DNA.
- CpG island refers to a region in which a two-base sequence of cytosine (C) -guanine (G) through a phosphodiester bond (p) appears with high frequency. CpG islands are often present in promoter regions upstream of genes.
- the CpG site or CpG island of a gene refers to a CpG island present at a position close to the coding region of the gene, or a CpG site contained in the CpG island, preferably It means a CpG site or CpG island present in the promoter region of a gene.
- a CpG site or CpG island of a specific gene can be identified based on a method such as MassARRAY method or pyrosequencing.
- the “standard retention time” (hereinafter also referred to as “standard retention time”) is an HPLC retention time that can separate a CIMP positive group and a CIMP negative group, or is prepared from renal cell carcinoma.
- the retention time of HPLC capable of separating a signal group derived from highly methylated DNA and a signal group derived from DNA having a low methylation degree is shown. That is, since a signal derived from highly methylated DNA is detected at a retention time earlier than the reference retention time (reference retention time), a sample having a detection signal at a retention time earlier than the reference retention time is It can be determined that the prognosis is poor.
- the above-described reference retention time may be set appropriately according to those conditions. In addition, it is preferable to set the reference retention time in consideration of the required clinical sensitivity.
- the present invention provides a method for determining a tissue containing renal cell carcinoma, comprising the following steps. (1) treating genomic DNA prepared from kidney tissue of a subject with bisulfite; (2) a step of amplifying the DNA treated with the bisulfite by PCR; (3) A step of subjecting the obtained PCR amplification product to ion exchange chromatography; (4) A step of obtaining a retention time of the detection signal obtained by the chromatography; (5) A step of determining that the tissue is a tissue containing renal cell carcinoma obtained from a renal cell carcinoma patient with a poor prognosis when the result of the step (4) is earlier than a reference retention time.
- the present invention provides a method for obtaining data for determining tissue containing renal cell carcinoma, comprising the following steps. (1) treating genomic DNA prepared from kidney tissue of a subject with bisulfite; (2) a step of amplifying the DNA treated with the bisulfite by PCR; (3) A step of subjecting the obtained PCR amplification product to ion exchange chromatography; (4) A step of obtaining a retention time of the detection signal obtained by the chromatography; (5) Determine whether the result of step (4) is earlier than the reference retention time, and whether the tissue is a tissue containing renal cell carcinoma obtained from a renal cell carcinoma patient with a poor prognosis The process of acquiring as data for doing.
- the present invention provides a prognosis determination method for a renal cell carcinoma patient, comprising the following steps. (1) treating genomic DNA prepared from kidney tissue of a subject with bisulfite; (2) a step of amplifying the DNA treated with the bisulfite by PCR; (3) A step of subjecting the obtained PCR amplification product to ion exchange chromatography; (4) A step of obtaining a retention time of the detection signal obtained by the chromatography; (5) A step of determining that the subject's renal cell carcinoma has a poor prognosis when the result of step (4) is earlier than the reference retention time.
- the present invention provides a method for determining a tissue containing renal cell carcinoma or a renal cell carcinoma patient, wherein the following steps are further performed before step (4).
- (1 ′) a step of treating unmethylated DNA corresponding to a PCR amplification region of genomic DNA prepared from kidney tissue of the subject with bisulfite;
- (2 ′) a step of amplifying the DNA treated with the bisulfite obtained in step (1 ′) by PCR;
- (3a) A step of obtaining difference data by subtracting the detection signal obtained by the chromatography of the step (3 ′) from the detection signal obtained by the chromatography of the step (3).
- the subject's kidney tissue may be a kidney tissue containing DNA or cells thereof, and examples thereof include tissues collected by biopsy, surgery, etc., and frozen or immobilized samples thereof. From the viewpoint of suppressing the degradation of genomic DNA and the like and more efficiently detecting DNA methylation, it is desirable to use frozen kidney tissue.
- the method for preparing sample DNA from the kidney tissue or cells is not particularly limited, and a known method can be appropriately selected and used.
- a known method for preparing DNA a phenol chloroform method or a commercially available DNA extraction kit such as QIAamp DNA Mini kit (manufactured by Qiagen), Clean Columns (manufactured by NexTec), AquaPure (manufactured by Bio-Rad), which will be described later.
- DNA extraction method using ZR Plant / Seed DNA Kit manufactured by Zymo Research
- prepGEM manufactured by ZyGEM
- BuccalQuick manufactured by TrimGen
- the extracted sample DNA is treated with bisulfite.
- a well-known method can be selected suitably and can be used.
- Known methods for the treatment of bisulfite include, for example, EpiTect Bisulfite Kit (48) (manufactured by Qiagen), Methyl Easy (manufactured by Human Genetics Pty), and Cells-to-CpG BisulfiteCv And a commercially available kit such as CpGenome Turbo Bisulfite Modification Kit (MERCK MILIPORE).
- the sample DNA treated with bisulfite is amplified by PCR.
- the PCR amplification method is not particularly limited, and a known method can be appropriately selected and used according to the sequence, length, amount, etc. of the DNA to be amplified.
- the target DNA to be PCR-amplified in the method of the present invention is preferably selected so that DNA methylation of CpG island in at least one gene selected from the group consisting of the 17 genes can be detected, more preferably , Selected so that methylation of the CpG site of the 17 genes can be detected.
- the target DNA is DNA encoding part or all of the coding region and / or promoter region of any of the 17 genes.
- it is DNA encoding part or all of the promoter region of any of the 17 genes. More preferably, it is a DNA encoding part or all of the CpG island of any one of the 17 genes.
- FAM150A is a gene encoding a protein specified by RefSeq ID: NP_997296
- GRM6 is a gene encoding a protein specified by RefSeq ID: NP_000834
- ZNF540 encodes a protein specified by RefSeq ID: NP_689819
- ZFP42 is a gene encoding a protein specified by RefSeq ID: NP_777560
- ZNF154 is a gene encoding a protein specified by RefSeq ID: NP_001078853
- RIMS4 is specified by RefSeq ID: NP_892015
- PCDHAC1 is specified by RefSeq ID: NP_061721
- KHDRBS2 is a gene encoding a protein specified by RefSeq ID: NP_689901
- ASCL2 is a gene encoding a protein specified by RefSeq ID: NP_005161
- FAM78A is RefSeq ID: NP_2 ZNF671 is a gene encoding a protein specified by RefSeq ID: NP — 079109
- SLC13A5 is a gene encoding a protein specified by RefSeq ID: NP — 808218
- NKX6 -2 is a gene encoding a protein specified by RefSeq ID: NP_769374.
- the CpG site of the 17 genes is present at the position on the chromosome described in Tables 1 to 4 with reference to the position on the NCBI database Genome Build 37, which is the reference human genome sequence.
- positions on the NCBI database Genome Build 37 which is a reference human genome sequence, are located at chromosomes 53, 478, 454, and 5th.
- the chain length of the PCR amplification product can be appropriately selected in consideration of factors such as shortening the PCR amplification time, shortening the analysis time in ion exchange chromatography, and maintaining separation performance.
- the PCR amplification product has a chain length of preferably 1000 bp or less, more preferably 700 bp or less, and even more preferably 500 bp or less.
- the chain length of the PCR amplification product when using sample DNA with few CpG islands is 30 to 40 bp which is the chain length of the PCR amplification product when using a primer near 15 mer that can avoid non-specific hybridization in PCR. Is the lower limit.
- cytosine at the CpG site is preferably contained at 2% or more, more preferably 5% or more, with respect to the chain length of the PCR amplification product.
- the ion exchange chromatography performed in the present invention is preferably anion exchange chromatography.
- the column packing used in the ion exchange chromatography performed in the present invention is not particularly limited as long as it is a base particle having a strong cationic group on the surface, but strong packing on the surface of the packing shown in Patent Document 2. Base particles having both groups and weak cationic groups are preferred.
- the strong cationic group means a cationic group that dissociates in a wide range of pH 1 to 14. That is, the strong cationic group can be kept dissociated (cationized) without being affected by the pH of the aqueous solution.
- the quaternary ammonium group is an example of the strong cationic group.
- Specific examples include trialkylammonium groups such as a trimethylammonium group, a triethylammonium group, and a dimethylethylammonium group.
- Examples of the counter ion of the strong cationic group include halide ions such as chloride ions, bromide ions, and iodide ions.
- the amount of the strong cationic group introduced onto the surface of the substrate particles is not particularly limited, but a preferable lower limit per dry weight of the filler is 1 ⁇ eq / g, and a preferable upper limit is 500 ⁇ eq / g.
- a preferable lower limit per dry weight of the filler is 1 ⁇ eq / g
- a preferable upper limit is 500 ⁇ eq / g.
- the amount of the strong cationic group is less than 1 ⁇ eq / g, the holding power is weak and the separation performance may be deteriorated.
- the amount of the strong cationic group exceeds 500 ⁇ eq / g, the holding power becomes too strong and the PCR amplification product cannot be easily eluted, and problems such as an excessive analysis time may occur.
- the weak cationic group means a cationic group having a pka of 8 or more. That is, the weak cationic group is affected by the pH of the aqueous solution, and the dissociation state changes. That is, when the pH is higher than 8, the protons of the weak cationic group are dissociated, and the proportion not having a positive charge increases. On the other hand, when the pH is lower than 8, the weak cationic group becomes protonated and the proportion of positive charges increases.
- Examples of the weak cationic group include a tertiary amino group, a secondary amino group, and a primary amino group. Of these, a tertiary amino group is desirable.
- the amount of the weak cationic group introduced onto the surface of the base particle is not particularly limited, but a preferable lower limit per dry weight of the filler is 0.5 ⁇ eq / g, and a preferable upper limit is 500 ⁇ eq / g.
- a preferable lower limit per dry weight of the filler is 0.5 ⁇ eq / g
- a preferable upper limit is 500 ⁇ eq / g.
- the amount of the weak cationic group is less than 0.5 ⁇ eq / g, the separation performance may not be improved because the amount is too small. If the amount of the weak cationic group exceeds 500 ⁇ eq / g, the holding power becomes too strong as in the case of the strong cationic group, so that the PCR amplification product cannot be easily eluted and the analysis time becomes too long. May occur.
- the amount of the strong cationic group or the weak cationic group on the surface of the substrate particle can be measured by quantifying the nitrogen atom contained in the amino group.
- An example of a method for quantifying nitrogen is the Kjeldahl method.
- nitrogen contained in the strong cationic group is quantified after polymerization, and then the strong cationic group and the weak cationic group after the introduction of the weak cationic group.
- the amount of the weak cationic group introduced later can be calculated. By quantifying in this manner, the amount of strong cationic group and the amount of weak cationic group can be adjusted within the above range when preparing the filler.
- the base particle for example, synthetic polymer fine particles obtained using a polymerizable monomer, inorganic fine particles such as silica, etc. can be used.
- the particles are desirable.
- the hydrophobic crosslinked polymer is a hydrophobic crosslinked polymer obtained by copolymerizing at least one hydrophobic crosslinking monomer and a monomer having at least one reactive functional group, and at least one hydrophobic crosslinked polymer. Any of the hydrophobic cross-linked polymers obtained by copolymerizing the polymerizable cross-linking monomer, the monomer having at least one reactive functional group and at least one hydrophobic cross-linking monomer may be used. .
- the hydrophobic crosslinkable monomer is not particularly limited as long as it has two or more vinyl groups in one monomer molecule.
- ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) Di (meth) acrylates such as acrylate, propylene glycol di (meth) acrylate, polypropylene glycol di (meth) acrylate, tri (meth) such as trimethylol methane tri (meth) acrylate, tetramethylol methane tri (meth) acrylate
- acrylic esters, tetra (meth) acrylic esters, and aromatic compounds such as divinylbenzene, divinyltoluene, divinylxylene, and divinylnaphthalene.
- the above (meth) acrylate means acrylate or methacrylate
- (meth) acryl means acryl or methacryl.
- Examples of the monomer having a reactive functional group include glycidyl (meth) acrylate and isocyanate ethyl (meth) acrylate.
- the hydrophobic non-crosslinkable monomer is not particularly limited as long as it is a non-crosslinkable polymerizable organic monomer having hydrophobic properties.
- methyl (meth) acrylate, ethyl (meth) acrylate examples thereof include (meth) acrylic acid esters such as butyl (meth) acrylate and t-butyl (meth) acrylate, and styrene monomers such as styrene and methylstyrene.
- the hydrophobic cross-linked polymer is obtained by copolymerizing the hydrophobic cross-linkable monomer and the monomer having a reactive functional group, the hydrophobic property in the hydrophobic cross-linked polymer
- the preferable lower limit of the content ratio of the segment derived from the crosslinkable monomer is 10% by weight, and the more preferable lower limit is 20% by weight.
- the filler for ion exchange chromatography of the present invention preferably has a polymer layer having the strong cationic group and the weak cationic group on the surface of the base particle.
- the strong cationic group and the weak cationic group are preferably derived from independent monomers.
- the filler for ion exchange chromatography of the present invention is a hydrophilic polymer having the above-mentioned hydrophobic crosslinked polymer particles and a strong cationic group copolymerized on the surface of the hydrophobic crosslinked polymer particles. It is preferable that a weak cationic group is introduced on the surface of the coated polymer particle comprising the above layer.
- the hydrophilic polymer having a strong cationic group is composed of a hydrophilic monomer having a strong cationic group, and is derived from a hydrophilic monomer having one or more strong cationic groups. What is necessary is just to contain a segment. That is, as a method for producing the hydrophilic polymer having a strong cationic group, a method of polymerizing a hydrophilic monomer having a strong cationic group alone, a hydrophilic property having two or more strong cationic groups. Examples thereof include a method of copolymerizing monomers, a method of copolymerizing a hydrophilic monomer having a strong cationic group and a hydrophilic monomer having no strong cationic group.
- the hydrophilic monomer having a strong cationic group is preferably one having a quaternary ammonium group.
- ethyl triethylammonium chloride ethyl dimethylethylammonium methacrylate, ethyl dimethylbenzylammonium methacrylate, ethyl dimethylbenzylammonium acrylate, ethyl triethylammonium acrylate, ethyl dimethylethylammonium acrylate
- Examples include chloride, acrylamidoethyltrimethylammonium chloride, acrylamidoethyltriethylammonium chloride, acrylamidoethyldimethylethylammonium chloride, and the like.
- a method for introducing the weak cationic group into the surface of the coated polymer particle a known method can be used. Specifically, for example, as a method of introducing a tertiary amino group as the weak cationic group, a hydrophobic crosslinked polymer particle comprising a hydrophobic crosslinked polymer having a segment derived from a monomer having a glycidyl group is used.
- the carboxy group produced by hydrolysis and the reagent having a tertiary amino group are then combined with the reagent. And a method such as condensation with Bojiimido.
- the hydrophilic monomer having a strong cationic group is copolymerized on the surface of a hydrophobic crosslinked polymer particle composed of a hydrophobic crosslinked polymer having a segment derived from a monomer having a glycidyl group, and then A method of reacting a reagent having a tertiary amino group with a glycidyl group, or the above strong cationic property on the surface of a hydrophobic crosslinked polymer particle comprising a hydrophobic crosslinked polymer having a segment derived from a monomer having an isocyanate group A method of copolymerizing a hydrophilic monomer having a group and then reacting a reagent having a tertiary amino group with an isocyanate group is preferred.
- the reagent having a tertiary amino group to be reacted with a reactive functional group is not particularly limited as long as the reagent has a functional group capable of reacting with the tertiary amino group and the reactive functional group.
- a functional group capable of reacting with the tertiary amino group and the reactive functional group include a primary amino group and a hydroxyl group. Of these, a group having a primary amino group at the terminal is preferable.
- Specific reagents having such functional groups include N, N-dimethylaminomethylamine, N, N-dimethylaminoethylamine, N, N-dimethylaminopropylamine, N, N-dimethylaminobutylamine, N, N- Diethylaminoethylamine, N, N-diethylaminopropylethylamine, N, N-diethylaminobutylamine, N, N-diethylaminopentylamine, N, N-diethylaminohexylamine, N, N-dipropylaminobutylamine, N, N-dibutylaminopropyl An amine etc. are mentioned.
- the relative position relationship between the strong cationic group, preferably a quaternary ammonium salt, and the weak cationic group, preferably a tertiary amino group is such that the strong cationic group is a substrate rather than the weak cationic group. It is preferable that it is located far from the surface of the particle, that is, outside. For example, it is preferable that the weak cationic group is within 30 mm from the surface of the base particle, and the strong cationic group is within 300 mm from the base particle surface, and is outside the weak cationic group.
- the average particle diameter of the base particles used in the filler for ion exchange chromatography of the present invention is not particularly limited, but a preferred lower limit is 0.1 ⁇ m and a preferred upper limit is 20 ⁇ m. If the average particle size is less than 0.1 ⁇ m, the inside of the column may become too high, resulting in poor separation. When the average particle diameter exceeds 20 ⁇ m, the dead volume in the column becomes too large, which may cause poor separation.
- the average particle diameter indicates a volume average particle diameter, and can be measured using a particle size distribution measuring apparatus (such as AccuSize 780 / Particle Sizing Systems).
- composition of the eluent used in the ion exchange chromatography performed in the present invention known conditions can be used.
- buffers or organic solvents containing known salt compounds it is preferable to use buffers or organic solvents containing known salt compounds. Specifically, for example, Tris-HCl buffer, TE buffer consisting of Tris and EDTA, Tris And a TBA buffer solution composed of boric acid and EDTA.
- the pH of the eluent is not particularly limited, but the preferred lower limit is 5 and the preferred upper limit is 10. By setting in this range, it is considered that the weak cationic group also effectively acts as an ion exchange group (anion exchange group).
- the more preferable lower limit of the pH of the eluent is 6, and the more preferable upper limit is 9.
- Examples of the salt contained in the eluent include salts consisting of halides such as sodium chloride, potassium chloride, sodium bromide, potassium bromide and alkali metals; calcium chloride, calcium bromide, magnesium chloride, magnesium bromide. Salts composed of halides such as alkaline earth metals and the like; inorganic acid salts such as sodium perchlorate, potassium perchlorate, sodium sulfate, potassium sulfate, ammonium sulfate, sodium nitrate, and potassium nitrate can be used. Moreover, organic acid salts, such as sodium acetate, potassium acetate, sodium succinate, potassium succinate, can also be used. Any of the above salts may be used alone or in combination.
- the salt concentration of the eluent may be appropriately adjusted according to the analysis conditions, but the preferred lower limit is 10 mmol / L, the preferred upper limit is 2000 mmol / L, the more preferred lower limit is 100 mmol / L, and the more preferred upper limit is 1500 mmol / L. L.
- the eluent used in the ion exchange chromatography of the present invention contains anti-chaotropic ions to further improve the separation performance.
- Anti-chaotropic ions have a property opposite to that of kaorotopic ions and have a function of stabilizing the hydration structure. Therefore, there is an effect of strengthening the hydrophobic interaction between the filler and the nucleic acid molecule.
- the main interaction of the ion exchange chromatography of the present invention is an electrostatic interaction, but in addition, the separation performance is enhanced by utilizing the action of hydrophobic interaction.
- Anti-chaotropic ions contained in the eluent include phosphate ions (PO 4 3 ⁇ ), sulfate ions (SO 4 2 ⁇ ), ammonium ions (NH 4 + ), potassium ions (K + ), sodium ions (Na + )). Among these ion combinations, sulfate ions and ammonium ions are preferably used.
- the anti-chaotropic ions can be used either alone or in combination.
- a part of the above-mentioned antichaotropic ion includes a salt or a buffer component contained in the eluent. When such a component is used, it has both a property as a salt or a buffer capacity contained in the eluent and a property as an anti-chaotropic ion, which is preferable for the present invention.
- the concentration of anti-chaotropic ions in the eluent for ion-exchange chromatography according to the present invention may be appropriately adjusted according to the analysis target, but is preferably 2000 mmol / L or less as the anti-chaotropic salt.
- a method of performing gradient elution with the concentration of the antichaotropic salt in the range of 0 to 2000 mmol / L Therefore, the concentration of the antichaotropic salt at the start of the analysis need not be 0 mmol / L, and the concentration of the antichaotropic salt at the end of the analysis need not be 2000 mmol / L.
- the gradient elution method may be a low pressure gradient method or a high pressure gradient method, but a method of eluting while performing precise concentration adjustment by the high pressure gradient method is preferred.
- the anti-chaotropic ion may be added to only one type of eluent used for elution, or may be added to a plurality of types of eluent.
- the anti-chaotropic ion may have both the role of enhancing the hydrophobic interaction between the packing material and the PCR amplification product or the buffering capacity, and the effect of eluting the PCR amplification product from the column.
- the column temperature when analyzing PCR amplification products by ion exchange chromatography performed in the present invention is preferably 30 ° C. or higher, more preferably 40 ° C. or higher, and further preferably 45 ° C. or higher.
- the column temperature of the ion exchange chromatography is less than 30 ° C., the hydrophobic interaction between the packing material and the PCR amplification product becomes weak, and it becomes difficult to obtain a desired separation effect.
- the column temperature of ion exchange chromatography is less than 45 ° C.
- the PCR amplification product (methylated DNA sample) of methylated DNA treated with bisulfite and the PCR amplified product of unmethylated DNA treated with bisulfite The difference in retention time from the unmethylated DNA sample is small.
- the column temperature is 60 ° C. or higher, the difference in retention time between the methylated DNA sample and the non-methylated DNA sample is further widened, and each peak becomes clearer. Can be detected.
- both of the methylated DNA sample and the unmethylated DNA sample are clearly separated when the column temperature of the ion exchange chromatography is increased, both of the methylated DNA sample and the unmethylated DNA sample are present according to the abundance ratio of the methylated DNA and the unmethylated DNA in the sample DNA. A difference tends to occur in the peak area or peak height of the holding time. Therefore, the higher the column temperature, the presence of each of methylated and unmethylated DNA in the sample DNA based on the area or height of the retention time peak between the methylated and unmethylated DNA samples It becomes easier to measure the quantity and the abundance ratio.
- the column temperature of ion exchange chromatography is 90 ° C. or higher, the double strands of the nucleic acid molecules in the PCR amplification product are dissociated, which is not preferable for analysis. Furthermore, if the column temperature is 100 ° C. or higher, the eluent may be boiled, which is not preferable for analysis. Therefore, the column temperature when analyzing the PCR amplification product by ion exchange chromatography performed in the present invention may be 30 ° C. or higher and lower than 90 ° C., preferably 40 ° C. or higher and lower than 90 ° C., more preferably 45 ° C. It is more than 55 degreeC and less than 90 degreeC, More preferably, it is 55 degreeC or more and 85 degrees C or less, More preferably, it is 60 degreeC or more and 85 degrees C or less.
- the amount of sample injected into the ion exchange chromatography column is not particularly limited, and may be appropriately adjusted according to the ion exchange capacity and sample concentration of the column.
- the flow rate is preferably from 0.1 mL / min to 3.0 mL / min, more preferably from 0.5 mL / min to 1.5 mL / min. If the flow rate is slow, improvement of the separation can be expected. However, if the flow rate is too slow, it may take a long time for the analysis, or the separation performance may be lowered due to broad peaks. Conversely, an increase in the flow rate has an advantage in terms of shortening the analysis time, but the peak is compressed, leading to a decrease in separation performance.
- the holding time of each sample can be determined in advance by conducting a preliminary experiment on each sample.
- a liquid feeding method a known liquid feeding method such as a linear gradient elution method or a stepwise elution method can be used, but a linear gradient elution method is preferred as the liquid feeding method in the present invention.
- the size of the gradient may be appropriately adjusted in accordance with the separation performance of the column and the characteristics of the analyte (here, PCR amplification product) in the range of 0 to 100% of the eluent used for elution.
- DNA methylation in sample DNA is detected by subjecting a PCR amplification product of DNA treated with bisulfite in the above-described procedure to ion exchange chromatography.
- the detection signal from the PCR amplification product of the sample DNA treated with bisulfite is the same as that of the sample DNA but the DNA amplification product of the bisulfite treated product of DNA that is not methylated (hereinafter, negative control).
- negative control the DNA amplification product of the bisulfite treated product of DNA that is not methylated
- positive control a PCR amplification product of a bisulfite treatment product of DNA having the same base sequence as the sample DNA and a known methylation rate (eg, 100%).
- the abundance of methylated DNA in the sample DNA and unmethylated DNA by comparing the detection signal from the PCR amplification product of the bisulfite treatment of the sample DNA with the detection signal from the negative and positive controls
- the abundance ratio can be measured.
- detection signals from a plurality of PCR amplification products hereinafter referred to as standards
- a bisulfite-treated product of a plurality of DNAs having the same base sequence as the sample DNA and a known methylation rate Measure the methylation rate, abundance, and ratio of abundance with unmethylated DNA in the sample DNA by comparing the detection signal from the PCR amplification product of the bisulfite-treated product of Can do.
- a sample containing the PCR amplification product of the bisulfite-treated product of the sample DNA, the negative control or the positive control, or the standard sample are individually separated.
- the sample adsorbed on the column is eluted with a gradient using a plurality of eluents, so that the PCR amplification product of the bisulfite-treated product of the sample DNA and the negative control or positive control or standard are converted according to the DNA methylation rate. Elute with different retention times.
- the detection signal from the negative control was obtained by performing bisulfite treatment and PCR according to the procedure described above using DNA having the same base sequence as that of the sample DNA but not methylated in place of the sample DNA.
- the product can be obtained by ion exchange chromatography.
- the detection signal from the positive control was obtained by performing bisulfite treatment and PCR according to the procedure described above using DNA having the same base sequence as the sample DNA and a known methylation rate (for example, 100%) instead of the sample DNA.
- the obtained PCR amplification product can be obtained by subjecting it to ion exchange chromatography.
- a detection signal from the negative or positive control may be obtained by subjecting the above-described synthetic DNA or commercially available DNA to ion exchange chromatography as a negative or positive control.
- the detection signal from the standard is obtained by performing bisulfite treatment and PCR according to the procedure described above using a plurality of DNAs having the same base sequence as the sample DNA and a known methylation rate instead of the sample DNA.
- a plurality of PCR amplification products can be obtained by subjecting each product to ion exchange chromatography. Further, a calibration curve may be created from each obtained detection signal.
- the detection signal from the standard may be obtained by subjecting the above-described synthetic DNA or commercially available DNA to the ion exchange chromatography as a standard.
- the detection signal from the PCR amplification product of the bisulfite-treated sample DNA obtained by the above chromatography is compared with the detection signal from the negative or positive control or standard. Based on the difference between the two detection signals, methylation of the sample DNA can be detected.
- the retention time of the peak of the detection signal obtained from the PCR amplification product of the sample DNA treated with bisulfite is different from the retention time of the negative control peak, it can be determined that the sample DNA is methylated. . Furthermore, at this time, it can be estimated that the greater the shift in retention time, the greater the methylation rate. Conversely, as the retention time of the peak of the detection signal obtained from the PCR amplification product of the sample DNA treated with bisulfite is shifted from the retention time of the peak of the 100% methylation positive control, the methylation of the sample DNA is increased. The rate can be estimated to be smaller.
- a calibration curve can be created based on the retention times of a plurality of peaks obtained from a standard with a known methylation rate, and the methylation rate of the sample DNA can be determined based on this calibration curve (FIG. 1 and FIG. 1). 2).
- the above-mentioned calibration curve can correlate the DNA methylation rate and the retention time. Therefore, based on the calibration curve, a DNA methylation rate corresponding to the reference retention time (hereinafter also referred to as a reference DNA methylation rate) can be obtained. Once the reference DNA methylation rate is obtained, even if the HPLC equipment and analysis conditions are changed, the reference DNA methylation rate is added to the calibration curve newly created under the changed equipment and conditions. By applying, a new reference holding time can be easily calculated.
- the peak height or peak area of the detection signal obtained from the PCR amplification product of the sample DNA treated with bisulfite is treated with the bisulfite treatment of DNA with a known methylation rate and mixing ratio of the methylated DNA.
- the abundance ratio of methylated DNA in the sample DNA for example, the abundance ratio of unmethylated DNA or methyl at a specific ratio
- the abundance ratio of the converted DNA, etc. can be determined.
- LCsolution Shiadzu Corporation
- GRAMS / AI Thermo Fisher Scientific
- Igor Pro Peak detection using WaveMetrics
- the parameter “WIDTH” is set to be larger than the half-width of the unnecessary peak
- the parameter “SLOPE” is set to be larger than the rising slope of the unnecessary peak
- the setting of the parameter “DRIFT” is changed so that the low-separation peak is vertically For example, selecting whether to divide or to divide the baseline. Since different chromatograms can be obtained as parameter values depending on the analysis conditions, the type of gene marker selected, the amount of specimen, etc., appropriate values may be set according to the chromatogram.
- Retention time ie peak top time
- the chromatogram is first-order differentiated, and the time when the differential coefficient changes from positive to negative can be acquired as the peak top time.
- the retention time of the detection signal by chromatography is examined.
- the sample is determined to be a sample obtained from a renal cell carcinoma patient with a poor prognosis.
- FIGS. 3A and 3B the separation of the peak of the high methylation rate DNA and the peak of the low methylation rate DNA is good, and in the case of bimodalization, the effect of unmethylated DNA is removed by the separation of the peaks. It is possible to accurately determine that the sample is obtained from a renal cell carcinoma patient with a poor prognosis.
- difference data obtained by subtracting the detection signal obtained from the negative control from the detection signal obtained from the PCR amplification product of the bisulfite-treated sample DNA can be obtained.
- the difference data it is possible to remove the signal (noise) from the unmethylated DNA from the detection signal as the whole sample DNA and extract only the signal from the methylated DNA.
- the difference data corresponds to a detection signal by methylated DNA in the sample DNA.
- the difference data holding time is compared with the reference holding time. If the result is earlier than the reference retention time, the sample is determined to be a sample obtained from a renal cell carcinoma patient with a poor prognosis.
- sample DNA having a low abundance ratio of methylated DNA or sample DNA containing methylated DNA having a low methylation rate In addition, it becomes possible to detect and analyze methylated DNA.
- various patterns of chromatograms are obtained, such as a shouldered peak and a plurality of peaks overlapping. In such a case, since only the signal of the sample DNA having a high methylation rate can be extracted by obtaining the difference data, the cancer prognosis can be determined with high accuracy.
- the procedure of the cancer prognosis determination method of the present invention when using the difference data is basically the same as that of the data before the difference described above.
- the retention time of the detection signal by chromatography is examined, and when the detection signal is obtained at a retention time earlier than the reference retention time, the sample is obtained from a renal cell carcinoma patient with a poor prognosis. Judge that there is.
- the use of the difference data is very effective.
- Samples collected for clinical examination include normal cells such as non-cancer epithelial cells and stromal cells that have not undergone DNA methylation, or precancerous conditions in which DNA methylation has not progressed so much.
- cancer cells with various DNA methylation rates may exist in various ratios.
- methylated DNA and unmethylated DNA in a sample can be separated and detected, so that even if the sample contains many normal cells, the presence of methylated DNA and The methylation rate can be detected with high accuracy, and accurate prognosis can be determined.
- the method of the present invention it is possible to accurately determine a prognosis even for a subject who has not been determined to be CIMP positive despite a poor prognosis in a conventional test.
- 109 cancer tissue (T) samples and corresponding 107 non-cancerous renal cortical tissue (N) samples were surgically removed from 110 patients with primary clear cell renal cell carcinoma No significant histological change was observed in the N sample. These patients have not received preoperative treatment and have undergone nephrectomy at the National Cancer Center Hospital. It consists of 79 men and 31 women, with an average age of 62.8 ⁇ 10.3 years (mean ⁇ standard deviation, 36-85 years).
- HCC hepatocellular carcinoma
- Type 3 (multi-nodule type) HCC is less histologically differentiated than type 1 (single-nodule type) and type 2 (peri-nodule growth type) HCC, and the incidence of intrahepatic metastasis is High (see Kanai, T. et al., Cancer, 1987, 60, 810-819).
- the presence or absence of vascular invasion was examined by observing a slide stained with hematoxylin-eosin and elastica one-Geeson with a microscope.
- the presence of tumor thrombus in the main trunk of the renal vein was examined by visual observation.
- the MassARRAY method amplifies the DNA after bisulfite treatment, transcribes it into RNA, further cleaves with RNAase in a base-specific manner, and then uses a mass spectrometer to determine the molecular weight of methylated DNA fragments and unmethylated DNA fragments. This is a method for detecting a difference.
- MassARRAY primer design was performed on the CpG island including the CpG site using EpiDesigner (manufactured by SEQUENOM, PrimeARRAY primer design software).
- a fresh frozen tissue sample obtained from the patient was treated with phenol-chloroform and then subjected to dialysis to extract high molecular weight DNA (Sambrook, J. et al., Molecular Cloning: Experimental Manual 3rd Edition, Cold Spring Harbor Publishing, NY, see pages 6.14-6.15). Then, 500 ng of DNA was subjected to bisulfite treatment using an EZ DNA Methylation-Gold TM kit (manufactured by Zymo Research). Bisulfite-treated genomic DNA was amplified by PCR and subjected to in vitro transcription reaction.
- RNA was specifically cleaved at the uracil site by RNAase, and fragments having different lengths according to the presence or absence of methylation of the genomic DNA of each sample were generated.
- the obtained RNA fragment was subjected to MALDI-TOF MAS (manufactured by SEQUENOM, MassARRAY Analyzer 4) capable of detecting a difference in mass of a single base, and mass spectrometry was performed.
- MALDI-TOF MAS manufactured by SEQUENOM, MassARRAY Analyzer 4
- the obtained mass spectrometry result was aligned with the reference sequence, and the mass ratio of the RNA fragment derived from methylated DNA to the RNA fragment derived from unmethylated DNA From this, the methylation level was calculated.
- the sequences of the primers used in this analysis and the sequences of the PCR products amplified using the set of primers are shown in Tables 5 and 6 and the Sequence Listing.
- the mixture was heated with stirring and polymerized at 80 ° C. for 1 hour in a nitrogen atmosphere.
- 100 g of ethyl trimethyl ammonium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) as a hydrophilic monomer having a strong cationic group was dissolved in ion-exchanged water. This was added to the same reactor and polymerized in the same manner at 80 ° C. for 2 hours under stirring in a nitrogen atmosphere.
- 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 obtained coated polymer particles were measured using a particle size distribution analyzer (Accumizer 780 / Particle Sizing Systems), and the average particle size was 10 ⁇ m.
- the above-mentioned packing material for ion exchange chromatography was packed into a stainless steel column (column size: inner diameter 4.6 mm ⁇ length 20 mm) of a liquid chromatography system.
- PCR The bisulfite-treated genomic DNA obtained in (1) was PCR amplified.
- PCR is 10 ng of template DNA, GeneAmp 1 ⁇ PCR buffer (manufactured by Life Technologies), 200 ⁇ mol / L GeneAmp dNTP Mix (manufactured by Life Technologies), 0.75U AmpliTold Gold Polymol Polymol PolyGol DNATolgo Molmol
- the reaction was performed in 25 ⁇ L of a reaction solution containing L forward and reverse primers. In PCR, after initial heat denaturation at 95 ° C. for 5 minutes, 35 cycles of 94 ° C. 30 seconds ⁇ 59 ° C. (when using F3-R3 primer) 30 seconds ⁇ 72 ° C.
- Example 1 Variation in chromatographic retention time depending on the DNA methylation rate DNA in which all 39 CpG sites are methylated based on the DNA sequence of the 384 bp region having 39 CpG sites in the FAM150A gene promoter
- Eight DNAs with different methylation rates were synthesized from (100% methylated DNA) to DNA not methylated at all (0% methylated DNA).
- For 50% methylated DNA three patterns of DNA were synthesized with the methylation positions closer to the 5 'side, closer to the 3' side, and closer to the center.
- Table 8 shows the methylation rate of each synthetic DNA and the methylation number of CpG islands.
- the eight synthetic DNAs were subjected to bisulfite treatment, PCR, and HPLC according to the procedure of Reference Example 3.
- the HPLC chromatogram for 5 (75% methylation) is shown in FIG.
- the retention time was shortened according to the DNA methylation rate.
- the data which plotted the retention time of HPLC with respect to methylation rate about 8 DNA are shown in FIG.
- HPLC retention time showed a very high correlation with DNA methylation rate.
- 50% methylated DNA showed almost the same retention time regardless of the methylation position. From this, it was shown that the retention time was determined depending on the methylation rate, regardless of the methylation position in the DNA. Therefore, the methylation rate contained in the sample DNA can be measured by measuring the retention time of HPLC.
- Example 2 DNA methylation analysis and prognosis determination in renal cell carcinoma Among renal cell carcinomas determined in CIMP in Reference Example 1, CIMP positive renal cell carcinoma from 13 patients and CIMP negative kidney cells from 5 Genomic DNA was prepared from cancer. The DNA was subjected to bisulfite treatment, PCR, and HPLC according to the procedure of Reference Example 3 (1) to (3). In PCR, a 384 bp region in the FAM150A gene promoter was amplified. Furthermore, HPLC analysis was also performed on DNA having a methylation rate of 0% (negative control) and 100% (positive control) in the PCR amplification region by the same procedure.
- FIG. 3 shows HPLC chromatograms obtained from CIMP positive and CIMP negative samples. Also shown in FIG. 3 are chromatograms of unmethylated DNA (negative control) and 100% methylated DNA (positive control). 3A and 3B are chromatograms of a CIMP positive sample. The peak of unmethylated DNA (negative control) and the peak having different retention times clearly appear, indicating the presence of methylated DNA. FIG. 3C is a chromatogram of a CIMP negative sample, the peak of which is almost indistinguishable from the peak of unmethylated DNA (negative control), indicating that there is almost no methylated DNA. FIG.
- 3D is a chromatogram of a CIMP negative sample, where a peak of unmethylated DNA (negative control) and a peak having a different retention time clearly appear, indicating the presence of methylated DNA. That is, even if it is determined that the CIMP is negative, it may indicate a sample with a poor prognosis.
- FIG. 4 is a graph plotting HPLC chromatogram peak retention times for all 18 CIMP negative / positive samples examined in this example. Since the distribution of retention time is clearly different between the CIMP negative sample and the positive sample, it is clear that the CIMP of the cancer determined by the MassARRAY method can be accurately determined by the method of the present invention by the method of the present invention. It was made.
- the CIMP positive group and the CIMP negative group are separated around the retention time of 9.3 min.
- the above-mentioned reference retention time may be set to an appropriate reference retention time because the chromatogram changes depending on the HPLC analysis conditions, the genomic DNA region, or the type of gene marker. In addition, it is preferable to set the reference retention time in consideration of the required clinical sensitivity.
- the retention time of the positive control (DNA methylation rate 100%) was about 9.0 minutes and the retention time of the negative control (DNA methylation rate 0%) was about 9.35 minutes.
- the reference DNA methylation rate resulting in a reference retention time was calculated to be about 17%. Therefore, in the FAM150A gene promoter 384 bp region used in this example, a sample having a DNA methylation rate higher than the reference DNA methylation rate (about 17%) was shown to have a poor prognosis.
- the average value of the DNA methylation rate obtained by MassARRAY is compared with the DNA methylation rate obtained by the present invention.
- the detection signal obtained by the present invention is calculated from the retention time (the retention time of the earlier elution is a and the retention time of the later is b) for the bimodal peaks as shown in FIGS. 3A and 3B.
- FIG. 5A shows a plot of the DNA methylation rate and comparison with the DNA methylation rate obtained by MassARRAY. In FIG. 5A, the DNA methylation rates obtained by each method do not appear to be correlated.
- the DNA methylation rate obtained from the synthetic peak C (retention time c) calculated from the average of the retention time a and the retention time b is plotted, and similarly compared with the DNA methylation rate obtained by MassARRAY. Shown in FIG. 5B. As shown in FIG. 5B, the DNA methylation rate calculated from the synthetic peak C correlates well with the average value of the DNA methylation rate obtained by the MassARRAY method.
- a sample in which equal amounts of 30% methylated DNA and 70% methylated DNA are mixed with a sample that is all 50% methylated DNA can be clearly distinguished on the chromatogram.
- the MassARRAY method can only obtain information on 50% methylated DNA as an average value for any sample.
- signals of various DNA methylation rates contained in a sample can be separated and detected, so that an unaveraged DNA methylation rate can be obtained.
- the value of the DNA methylation rate obtained by the method such as MassARRAY method or pyrosequencing provides the DNA methylation rate as an average value of the whole sample subjected to the measurement, and as a result, highly methylated DNA.
- the present invention even if it is determined that the CIMP is negative, it is possible to remove the influence of non-methylated DNA and detect whether or not it contains hypermethylated DNA, thereby avoiding the risk of underestimating the DNA methylation rate. . Furthermore, a sample suspected of having a poor prognosis can be determined.
- CIMP determination by the method described in Patent Document 4 methylation of CpG sites was examined for CpG islands of a plurality of gene markers, and CIMP positive or CIMP negative was determined. According to the present invention, it was suggested that prognosis determination of renal cell carcinoma equivalent to CIMP determination can be easily performed by analyzing a detection signal obtained by HPLC even with a single gene marker. Furthermore, it was suggested that the prognosis determination could be improved by analyzing the detection signals by HPLC for a plurality of gene markers.
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Abstract
Description
〔1〕腎細胞癌を含む組織の判定方法であって:
(1)被験体の腎臓組織から調製されたゲノムDNAを亜硫酸水素塩で処理する工程;
(2)該亜硫酸水素塩によって処理されたDNAをPCRによって増幅する工程;
(3)得られたPCR増幅産物をイオン交換クロマトグラフィーにかける工程;
(4)該クロマトグラフィーで得られた検出シグナルの保持時間を得る工程;
(5)工程(4)の結果が基準となる保持時間より早い場合に、該組織を、予後不良の腎細胞癌患者から得られた腎細胞癌を含む組織であると判定する工程、
を含む方法。
(1)被験体の腎臓組織から調製されたゲノムDNAを亜硫酸水素塩で処理する工程;
(2)該亜硫酸水素塩によって処理されたDNAをPCRによって増幅する工程;
(3)得られたPCR増幅産物をイオン交換クロマトグラフィーにかける工程;
(4)該クロマトグラフィーで得られた検出シグナルの保持時間を得る工程;
(5)工程(4)の結果が基準となる保持時間より早いか否かを、該組織が予後不良の腎細胞癌患者から得られた腎細胞癌を含む組織であるか否かを判定するためのデータとして取得する工程、
を含む方法。
(1)被験体の腎臓組織から調製されたゲノムDNAを亜硫酸水素塩で処理する工程;
(2)該亜硫酸水素塩によって処理されたDNAをPCRによって増幅する工程;
(3)得られたPCR増幅産物をイオン交換クロマトグラフィーにかける工程;
(4)該クロマトグラフィーで得られた検出シグナルの保持時間を得る工程;
(5)工程(4)の結果が基準となる保持時間より早い場合に、該被験体の腎細胞癌を予後不良と判定する工程、
を含む方法。
(1’)前記被験体の腎臓組織から調製されたゲノムDNAのPCR増幅領域に相当するメチル化していないDNAを亜硫酸水素塩で処理する工程;
(2’)工程(1’)で得られた亜硫酸水素塩によって処理されたDNAをPCRによって増幅する工程;
(3’)工程(2’)で得られたPCR増幅産物をイオン交換クロマトグラフィーにかける工程;
(3a)工程(3)のクロマトグラフィーで得られた検出シグナルから、工程(3’)のクロマトグラフィーで得られた検出シグナルを差し引いて差分データを得る工程、
を含む、〔1〕~〔6〕のいずれか1項記載の方法。
(1)被験体の腎臓組織から調製されたゲノムDNAを亜硫酸水素塩で処理する工程;
(2)該亜硫酸水素塩によって処理されたDNAをPCRによって増幅する工程;
(3)得られたPCR増幅産物をイオン交換クロマトグラフィーにかける工程;
(4)該クロマトグラフィーで得られた検出シグナルの保持時間を得る工程;
(5)工程(4)の結果が基準となる保持時間より早い場合に、該組織を、予後不良の腎細胞癌患者から得られた腎細胞癌を含む組織であると判定する工程。
(1)被験体の腎臓組織から調製されたゲノムDNAを亜硫酸水素塩で処理する工程;
(2)該亜硫酸水素塩によって処理されたDNAをPCRによって増幅する工程;
(3)得られたPCR増幅産物をイオン交換クロマトグラフィーにかける工程;
(4)該クロマトグラフィーで得られた検出シグナルの保持時間を得る工程;
(5)工程(4)の結果が基準となる保持時間より早い場合か否かを、該組織が予後不良の腎細胞癌患者から得られた腎細胞癌を含む組織であるか否かを判定するためのデータとして取得する工程。
(1)被験体の腎臓組織から調製されたゲノムDNAを亜硫酸水素塩で処理する工程;
(2)該亜硫酸水素塩によって処理されたDNAをPCRによって増幅する工程;
(3)得られたPCR増幅産物をイオン交換クロマトグラフィーにかける工程;
(4)該クロマトグラフィーで得られた検出シグナルの保持時間を得る工程;
(5)工程(4)の結果が基準となる保持時間より早い場合に、該被験体の腎細胞癌を予後不良と判定する工程。
(1’)上記被験体の腎臓組織から調製されたゲノムDNAのPCR増幅領域に相当するメチル化していないDNAを亜硫酸水素塩で処理する工程;
(2’)工程(1’)で得られた亜硫酸水素塩によって処理されたDNAをPCRによって増幅する工程;
(3’)工程(2’)で得られたPCR増幅産物をイオン交換クロマトグラフィーにかける工程;
(3a)工程(3)のクロマトグラフィーで得られた検出シグナルから、工程(3’)のクロマトグラフィーで得られた検出シグナルを差し引いて差分データを得る工程。
109の癌組織(T)サンプル及び対応する107の非癌腎皮質組織(N)サンプルは、原発性の淡明細胞型腎細胞癌を罹患している110人の患者から手術によって摘出された試料から得たものであり、Nサンプルには顕著な組織学的変化は認められていない。なお、これら患者は、術前の治療は受けておらず、国立がん研究センター病院にて腎摘出術を受けた患者である。79名の男性と31名の女性とからなり、平均年齢は62.8±10.3歳(平均±標準偏差、36~85歳)である。
従来法によるCIMP陰性/陽性判定は、特許文献4に記載のMassARRAY法(実施例5)に従って行った。メチル化DNA検出方法の1つであるMassARRAY法にて、17遺伝子(FAM150A、GRM6、ZNF540、ZFP42、ZNF154、RIMS4、PCDHAC1、KHDRBS2、ASCL2、KCNQ1、PRAC、WNT3A、TRH、FAM78A、ZNF671、SLC13A5及びNKX6-2)のCpGサイト(表1~4)についてのDNAメチル化レベルを検出した。
攪拌機付き反応器中の3重量%ポリビニルアルコール(日本合成化学社製)水溶液2000mLに、テトラエチレングリコールジメタアクリレート(新中村化学工業社製)200g、トリエチレングリコールジメタアクリレート(新中村化学工業社製)100g、グリシジルメタクリレート(和光純薬工業社製)100gおよび過酸化ベンゾイル(キシダ化学社製)1.0gの混合物を添加した。攪拌しながら加熱し、窒素雰囲気下にて80℃で1時間重合した。次に、強カチオン性基を有する親水性単量体として、メタクリル酸エチルトリメチルアンモニウムクロリド(和光純薬工業社製)100gをイオン交換水に溶解した。これを同じ反応器に添加して、同様にして、攪拌しながら窒素雰囲気下にて80℃で2時間重合した。得られた重合組成物を水およびアセトンで洗浄することにより、4級アンモニウム基を有する親水性重合体の層を表面に有する被覆重合体粒子を得た。得られた被覆重合体粒子について、粒度分布測定装置(AccuSizer780/Particle Sizing Systems社製)を用いて測定したところ、平均粒子径は10μmであった。
(1)ゲノムDNAの抽出と亜硫酸水素塩処理
患者から得た新鮮凍結組織サンプルを、フェノール-クロロホルムにて処理し、次いで透析を施すことによって、高分子量DNAを抽出した(Sambrook,J.ら、モレキュラークローニング:実験マニュアル 第3版、コールドスプリングハーバー出版、NY、6.14~6.15ページ 参照)。DNA500ngを、EZ DNA Methylation-GoldTMキット(Zymo Research社製)を用い、亜硫酸水素塩処理に供した。
(1)で得られた亜硫酸水素塩処理ゲノムDNAをPCR増幅した。PCRは、鋳型DNA 10ng、GeneAmp 1×PCR buffer(Life Technologies社製)、200μmol/L GeneAmp dNTP Mix(Life Technologies社製)、0.75U AmpliTaq Gold DNA Polymerase(Life Technologies社製)、0.25μmol/L forwardおよびreverseプライマーを含んだ25μLの反応液で行った。PCRでは、95℃5分間初期熱変性を行った後、94℃30秒→59℃(F3-R3プライマー使用時)30秒→72℃40秒を1サイクルとして35サイクル続け、さらに72℃10分の伸張反応を行った。PCR終了後、予めethidium bromideを添加した3%アガロースゲルに、反応液5μLにloading dye solution 1μLを混ぜた後アプライして電気泳動し、PCR増幅産物を観察して目的のPCR増幅産物が得られたことを確認した。各プライマーの配列は、表7に示した。
参考例2で準備したアニオン交換カラムを用いて、以下の条件でイオン交換クロマトグラフィーを行い、(2)で得られた各PCR増幅産物を分離検出した。
システム:LC-20Aシリーズ(島津製作所社製)
溶離液:溶離液A 25mmol/Lトリス塩酸緩衝液(pH7.5)
溶離液B 25mmol/Lトリス塩酸緩衝液(pH7.5)+1mol/L硫酸アンモニウム
分析時間:分析時間は15分
溶出法:以下のグラジエント条件により溶離液Bの混合比率を直線的に増加させた。
0分(溶離液B40%)→10分(溶離液B100%)
検体:(2)で得られたPCR増幅産物
流速:1.0mL/min
検出波長:260nm
試料注入量:5μL
カラム温度:70℃
FAM150A遺伝子プロモーターにおける、39個のCpGサイトを有する384bp領域のDNA配列に基づいて、39個のCpGサイト全てがメチル化されているDNA(100%メチル化DNA)から全くメチル化されていないDNA(0%メチル化DNA)まで、メチル化率の異なる8つのDNAを合成した。なお50%メチル化DNAについては、メチル化位置を5'側寄り、3’側寄り、および中央寄りの3パターンのDNAを合成した。各合成DNAのメチル化率およびCpGアイランドのメチル化数を表8に示す。
参考例1でCIMP判定された腎細胞癌のうち、患者13人からのCIMP陽性腎細胞癌と、5人からのCIMP陰性腎細胞癌からゲノムDNAを調製した。参考例3(1)~(3)の手順に従って、該DNAを亜硫酸水素塩処理、PCR、およびHPLCにかけた。PCRでは、FAM150A遺伝子プロモーターにおける、384bp領域を増幅した。
さらに、上記PCR増幅領域におけるメチル化率が0%(陰性対照)および100%(陽性対照)のDNAについても、それぞれ同様の手順でHPLC分析した。
Claims (15)
- 腎細胞癌を含む組織の判定方法であって:
(1)被験体の腎臓組織から調製されたゲノムDNAを亜硫酸水素塩で処理する工程;
(2)工程(1)で得られた亜硫酸水素塩によって処理されたDNAをPCRによって増幅する工程;
(3)工程(2)で得られたPCR増幅産物をイオン交換クロマトグラフィーにかける工程;
(4)該クロマトグラフィーで得られた検出シグナルの保持時間を得る工程;
(5)工程(4)の結果が基準となる保持時間より早い場合に、該組織を、予後不良の腎細胞癌患者から得られた腎細胞癌を含む組織であると判定する工程、
を含む方法。 - 前記工程(2)において、PCR増幅されるDNAが、FAM150A、GRM6、ZNF540、ZFP42、ZNF154、RIMS4、PCDHAC1、KHDRBS2、ASCL2、KCNQ1、PRAC、WNT3A、TRH、FAM78A、ZNF671、SLC13A5及びNKX6-2からなる群より選択される少なくとも1つの遺伝子におけるCpGアイランドを含む、請求項1記載の方法。
- 前記工程(2)においてPCR増幅されるDNAが、FAM150A遺伝子プロモーター領域を含む、請求項1記載の方法。
- 前記工程(2)のPCRにおいて、配列番号51及び52で示されるPCRプライマーが使用される、請求項1記載の方法。
- 前記工程(4)の前に、さらに以下の工程:
(1’)前記被験体の腎臓組織から調製されたゲノムDNAのPCR増幅領域に相当するメチル化していないDNAを亜硫酸水素塩で処理する工程;
(2’)工程(1’)で得られた亜硫酸水素塩によって処理されたDNAをPCRによって増幅する工程;
(3’)工程(2’)で得られたPCR増幅産物をイオン交換クロマトグラフィーにかける工程;
(3a)工程(3)のクロマトグラフィーで得られた検出シグナルから、工程(3’)のクロマトグラフィーで得られた検出シグナルを差し引いて差分データを得る工程、
を含む、請求項1~4のいずれか1項記載の方法。 - 腎細胞癌患者の予後判定方法であって:
(1)被験体の腎臓組織から調製されたゲノムDNAを亜硫酸水素塩で処理する工程;
(2)該亜硫酸水素塩によって処理されたDNAをPCRによって増幅する工程;
(3)得られたPCR増幅産物をイオン交換クロマトグラフィーにかける工程;
(4)該クロマトグラフィーで得られた検出シグナルの保持時間を得る工程;
(5)工程(4)の結果が基準となる保持時間より早い場合に、該被験体の腎細胞癌を予後不良と判定する工程、
を含む方法。 - 前記工程(2)において、PCR増幅されるDNAが、FAM150A、GRM6、ZNF540、ZFP42、ZNF154、RIMS4、PCDHAC1、KHDRBS2、ASCL2、KCNQ1、PRAC、WNT3A、TRH、FAM78A、ZNF671、SLC13A5及びNKX6-2からなる群より選択される少なくとも1つの遺伝子におけるCpGアイランドを含む、請求項6記載の方法。
- 前記工程(2)においてPCR増幅されるDNAが、FAM150A遺伝子プロモーター領域を含む、請求項6記載の方法。
- 前記工程(2)のPCRにおいて、配列番号51及び52で示されるPCRプライマーが使用される、請求項6記載の方法。
- 前記工程(4)の前に、さらに以下の工程:
(1’)前記被験体の腎臓組織から調製されたゲノムDNAのPCR増幅領域に相当するメチル化していないDNAを亜硫酸水素塩で処理する工程;
(2’)工程(1’)で得られた亜硫酸水素塩によって処理されたDNAをPCRによって増幅する工程;
(3’)工程(2’)で得られたPCR増幅産物をイオン交換クロマトグラフィーにかける工程;
(3a)工程(3)のクロマトグラフィーで得られた検出シグナルから、工程(3’)のクロマトグラフィーで得られた検出シグナルを差し引いて差分データを得る工程、
を含む、請求項6~9のいずれか1項記載の方法。 - 腎細胞癌を含む組織を判定するためのデータを得る方法であって:
(1)被験体の腎臓組織から調製されたゲノムDNAを亜硫酸水素塩で処理する工程;
(2)該亜硫酸水素塩によって処理されたDNAをPCRによって増幅する工程;
(3)得られたPCR増幅産物をイオン交換クロマトグラフィーにかける工程;
(4)該クロマトグラフィーで得られた検出シグナルの保持時間を得る工程;
(5)工程(4)の結果が基準となる保持時間より早いか否かを、該組織が予後不良の腎細胞癌患者から得られた腎細胞癌を含む組織であるか否かを判定するためのデータとして取得する工程、
を含む方法。 - 前記工程(2)において、PCR増幅されるDNAが、FAM150A、GRM6、ZNF540、ZFP42、ZNF154、RIMS4、PCDHAC1、KHDRBS2、ASCL2、KCNQ1、PRAC、WNT3A、TRH、FAM78A、ZNF671、SLC13A5及びNKX6-2からなる群より選択される少なくとも1つの遺伝子におけるCpGアイランドを含む、請求項11記載の方法。
- 前記工程(2)においてPCR増幅されるDNAが、FAM150A遺伝子プロモーター領域を含む、請求項11記載の方法。
- 前記工程(2)のPCRにおいて、配列番号51及び52で示されるPCRプライマーが使用される、請求項11記載の方法。
- 前記工程(4)の前に、さらに以下の工程:
(1’)前記被験体の腎臓組織から調製されたゲノムDNAのPCR増幅領域に相当するメチル化していないDNAを亜硫酸水素塩で処理する工程;
(2’)工程(1’)で得られた亜硫酸水素塩によって処理されたDNAをPCRによって増幅する工程;
(3’)工程(2’)で得られたPCR増幅産物をイオン交換クロマトグラフィーにかける工程;
(3a)工程(3)のクロマトグラフィーで得られた検出シグナルから、工程(3’)のクロマトグラフィーで得られた検出シグナルを差し引いて差分データを得る工程、
を含む、請求項11~14のいずれか1項記載の方法。
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EP3112475A4 (en) | 2017-10-18 |
US20180340236A1 (en) | 2018-11-29 |
JPWO2015129916A1 (ja) | 2017-03-30 |
CN106062215A (zh) | 2016-10-26 |
EP3112475A1 (en) | 2017-01-04 |
EP3112475B1 (en) | 2018-08-15 |
JP5897228B2 (ja) | 2016-03-30 |
CN106062215B (zh) | 2020-09-22 |
CN111057752A (zh) | 2020-04-24 |
US20170058355A1 (en) | 2017-03-02 |
US10190172B2 (en) | 2019-01-29 |
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