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WO2014046199A1 - Méthode permettant d'obtenir des informations sur une tumeur du cerveau, marqueur et kit associés - Google Patents

Méthode permettant d'obtenir des informations sur une tumeur du cerveau, marqueur et kit associés Download PDF

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Publication number
WO2014046199A1
WO2014046199A1 PCT/JP2013/075336 JP2013075336W WO2014046199A1 WO 2014046199 A1 WO2014046199 A1 WO 2014046199A1 JP 2013075336 W JP2013075336 W JP 2013075336W WO 2014046199 A1 WO2014046199 A1 WO 2014046199A1
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Prior art keywords
methylation
brain tumor
dna
seq
primer set
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English (en)
Japanese (ja)
Inventor
綾子 酒井
玄太 永江
昌裕 梶田
油谷　浩幸
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Sysmex Corp
University of Tokyo NUC
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Sysmex Corp
University of Tokyo NUC
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Priority to JP2014536913A priority Critical patent/JP6269493B2/ja
Publication of WO2014046199A1 publication Critical patent/WO2014046199A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention relates to a method for acquiring information related to a brain tumor of a subject.
  • the present invention also relates to markers and kits used in the method.
  • Brain tumor is a general term for tumors that develop in the cranium, and is classified into primary brain tumors that arise from the brain and its surrounding tissues, and metastatic brain tumors that have metastasized to cancer in another organ.
  • primary brain tumors including malignant tumors and benign tumors.
  • gliomas also called gliomas
  • gliomas have a high incidence of about 30% of all brain tumors. .
  • AFP ⁇ -fetoprotein
  • HCG human chorionic ggonadotropin
  • a method based on information on DNA methylation examples include a method based on information on DNA methylation.
  • a CpG site (5 ′-(CG) -3 ′) in the base sequence of a predetermined gene is used as a marker.
  • information such as the presence or absence of cancer cells is obtained, and cancer is diagnosed using this information as an index.
  • the information obtained from the analysis results includes not only information on brain tumors but also other types. Information about other tumors may also be included. For example, even if information that tumor cells are present in a sample is obtained based on the analysis results, it is difficult to determine whether the tumor cells are derived from brain tumors or other types of tumors. It is.
  • the specificity of the markers to brain tumors is a very important factor. Therefore, it is desired to develop a novel marker specific to a brain tumor that makes it possible to specifically detect tumor cells derived from the brain tumor.
  • an object of the present invention is to provide a method for acquiring information related to a brain tumor by identifying such a novel marker and analyzing the methylation state of the marker. Moreover, an object of this invention is to provide the kit used suitably for the method.
  • the present inventors have identified, as a novel marker, a gene region in which methylation is specifically observed in DNA obtained from a cancerous tissue of a brain tumor. Based on the results obtained by analyzing the methylation status of these markers, the present inventors differed from tumor cells derived from brain tumors and other cells (normal tissue cells and brain tumors). The present invention was completed by discovering that tumor cells derived from different types of tumors can be clearly distinguished.
  • a step of preparing a DNA sample from a biological sample collected from a subject At least one gene selected from CREM (cAMP responsive element modulator), VRK2 (Vaccinia related kinase 2) and MYL12A (Myosin, light chain 12A, regulatory, non-sarcomeric) contained in the DNA sample obtained in the preparation process Analyzing the methylation status of the CpG site present in the promoter region;
  • a method for acquiring information related to a brain tumor is provided, including the step of acquiring information related to the brain tumor of the subject based on the analysis result obtained in the analysis step.
  • a marker for obtaining information on brain tumors by methylation analysis which is at least one selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A.
  • information on a brain tumor including a primer set for analyzing the state of at least one methylation selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A is obtained. Kits are provided for doing so.
  • the present invention can provide a novel marker that makes it possible to obtain information on brain tumors. Moreover, this invention can provide the method and kit for acquiring the information regarding a test subject's brain tumor by analyzing the methylation state about such a marker.
  • a DNA sample is prepared from a biological sample collected from a subject.
  • the brain tumor targeted by the present invention is not particularly limited as long as it is a primary brain tumor, and examples thereof include glioma, meningioma, pituitary adenoma, and schwannoma. Among them, glioma is particularly suitable as the object of the method of the present invention. Glioma is classified into glioblastoma, astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, etc., but is not particularly limited in the present invention.
  • the biological sample is not particularly limited as long as it is a biological sample containing DNA of a subject, but is preferably a sample containing genomic DNA, for example, a clinical specimen.
  • clinical specimens include body fluids, urine, tissues collected by surgery or biopsy, and the like.
  • body fluid include blood, serum, plasma, lymph, ascites, cerebrospinal fluid, bone marrow fluid, and nipple secretion.
  • a culture obtained by culturing cells or tissues collected from a subject can also be used as a biological sample.
  • Preparation of a DNA sample can be performed by extracting DNA from a biological sample.
  • Methods for extracting DNA from biological samples are known in the art.
  • a biological sample and a treatment solution containing a surfactant eg, sodium cholate, sodium dodecyl sulfate, etc.
  • a surfactant eg, sodium cholate, sodium dodecyl sulfate, etc.
  • physical treatment eg.g, sodium cholate, sodium dodecyl sulfate, etc.
  • DNA can be extracted by performing ultrasonic crushing or the like to release DNA contained in the biological sample into the mixed solution.
  • the obtained supernatant may be purified by a method known in the art.
  • the extraction and purification of DNA from a biological sample can also be performed using a commercially available kit.
  • the preparation step preferably further includes a step of fragmenting the extracted DNA.
  • the methylated DNA immunoprecipitation (MeDIP) method and the unmethylated cytosine conversion treatment described later can be performed efficiently.
  • DNA fragmentation can be performed by ultrasonic treatment, alkali treatment, restriction enzyme treatment, or the like.
  • alkali treatment a DNA solution is fragmented by adding a sodium hydroxide solution to the DNA solution to a final concentration of 0.1 to 1.0 N and incubating at 10 to 40 ° C. for 5 to 15 minutes. It becomes.
  • restriction enzyme treatment the restriction enzyme to be used is appropriately selected based on the base sequence of the DNA, such as MseI or BamHI.
  • the methylation state of the CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A contained in the DNA obtained in the above preparation step is analyzed.
  • the “CpG site” means a site of a sequence in which cytosine (C) and guanine (G) in the base sequence are adjacent in this order in the 5 ′ to 3 ′ direction.
  • the letter “p” in CpG represents a phosphodiester bond between cytosine and guanine.
  • analyzing the methylation state means analyzing the presence or absence of methylation of a CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A, or It means analyzing the frequency of methylation of the promoter region.
  • the base sequences of promoter regions of CREM, VRK2 and MYL12A genes are known. These nucleotide sequences are publicly known such as a database (http://www.ncbi.nlm.nih.gov/) provided by the National Center for Biological Information (NCBI) of the US National Library of Medicine. You can know from the database. Various ID numbers of the above genes are shown in Table 1. In addition, the base sequence of the promoter region of each gene is shown as the sequence of SEQ ID NO: 1, 2 and 3, respectively (note that the base sequence of SEQ ID NO: 1 is a minus-strand sequence, and the base sequences of SEQ ID NO: 2 and 3 are Plus strand sequence).
  • the analyzing step is a step of analyzing the presence or absence of methylation of at least one CpG site among CpG sites existing in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A.
  • Presence / absence of methylation means whether cytosine at the CpG site present in the promoter region is methylated.
  • the number of CpG sites to be analyzed may be one, but it is preferable to analyze the presence or absence of methylation of a plurality of CpG sites.
  • the plurality of CpG sites may be selected from a promoter region of one gene, or may be selected from each of the promoter regions of a plurality of genes.
  • the analyzing step may be a step of analyzing the frequency of methylation in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A.
  • the “frequency of methylation” means the ratio of the number of methylated CpG sites to the number of CpG sites present in the promoter region.
  • the analysis target may be the entire promoter region described above or a part including at least one CpG site. Note that the analysis target may include only one CpG site, but preferably includes a plurality of CpG sites.
  • the analysis target may be determined from any one promoter region of the above genes, or may be determined from promoter regions of a plurality of genes.
  • the number of methylated CpG sites in the promoter region itself is It can be used as the frequency of methylation.
  • the above methylation frequency may be a “methylation score” obtained by analyzing the methylation state of a CpG site in DNA by mass spectrometry such as MassARRAY (registered trademark) described later.
  • MassARRAY registered trademark
  • a DNA fragment is measured, and a methylation score is calculated from an area ratio between a peak derived from a methylated DNA fragment and a peak derived from an unmethylated DNA fragment.
  • CpG site or a predetermined range including the CpG site in the promoter region of each gene of CREM, VRK2 and MYL12A is to be analyzed.
  • a method for analyzing the methylation state various methods are known in the art.
  • which analysis method is used is not particularly limited, but preferably, a step of distinguishing methylated DNA from unmethylated DNA, a step of amplifying DNA, methylated DNA and / or Detecting unmethylated DNA.
  • Examples of the step of distinguishing methylated DNA from unmethylated DNA include a step of performing methylation sensitive restriction enzyme treatment, MeDIP method, unmethylated cytosine conversion treatment, and the like.
  • Examples of the step of amplifying DNA include a step of performing PCR method, quantitative PCR method, IVT (in vitro transcription) amplification method, SPIA (trademark) amplification method and the like.
  • Examples of the process for detecting methylated DNA and / or unmethylated DNA include a process for performing electrophoresis, sequence analysis, microarray analysis, mass spectrometry, Southern hybridization, and the like.
  • the MeDIP method is a method of concentrating methylated DNA contained in a biological sample by immunoprecipitation using an anti-methylated cytosine antibody, an anti-methylated cytidine antibody, or an antibody that specifically recognizes a methylated DNA-binding protein.
  • the analysis step may be a step of concentrating methylated DNA contained in the DNA obtained in the extraction step by the MeDIP method and analyzing the methylation state of the obtained methylated DNA.
  • methylated DNA concentrated by the MeDIP method can be amplified by the IVT amplification method or the like, and the methylated state of the obtained amplification product can be analyzed using a microarray. Such an analysis method is called the MeDIP on chip method.
  • Unmethylated cytosine conversion treatment is a reaction between a DNA extracted from a biological sample and an unmethylated cytosine converting agent to convert the unmethylated cytosine in the DNA to another base (uracil, thymine, adenine or guanine). It is processing to convert to.
  • the unmethylated cytosine converting agent is a substance that can react with DNA to convert unmethylated cytosine in the DNA into another base (uracil, thymine, adenine or guanine).
  • an unmethylated cytosine converting agent for example, bisulfite such as sodium sulfite, potassium salt, calcium salt, magnesium salt of bisulfite is preferably used.
  • the addition amount (concentration) of bisulfite is not particularly limited as long as it can sufficiently convert unmethylated cytosine in DNA.
  • the final concentration in a solution containing DNA is 1M.
  • the above is preferably 1 to 15 ⁇ M, more preferably 3 to 10 ⁇ M.
  • the incubation conditions (temperature and time) after the addition of bisulfite can be appropriately set according to the amount of bisulfite added. For example, when bisulfite is added at a final concentration of 6M, the incubation conditions are 50 to 80 ° C. Incubate for 10-90 minutes.
  • the methylation state of the CpG site contained in DNA can be analyzed by sequence analysis of bisulfite-treated DNA and detecting differences from the original base sequence. This method is called the bisulfite sequence method.
  • the methylation state of the CpG site can be analyzed by mass spectrometry. Specifically, using a bisulfite-treated DNA as a template, PCR amplification was performed using a primer set specific for the base sequence to be analyzed, and then the resulting PCR product was further IVT amplified to obtain methylated cytosine. And uracil become guanine (G) and adenine (A), respectively.
  • the obtained IVT amplification product is cleaved with RNase A, and the mass difference between G and A (16Da) between the obtained nucleic acid fragments is calculated using a MALDI-TOF (Matrix Assisted Laser Desorption / Ionization-Time of Flight) mass spectrometer.
  • MALDI-TOF Mass Assisted Laser Desorption / Ionization-Time of Flight
  • the site cleaved by RNase A in the IVT product is between any base and uracil (U) or thymine (T) adjacent to the base. Therefore, the base sequence and mass of the IVT product cleaved by RNase A can be predicted from the base sequence of the DNA used as a template. Therefore, for each peak obtained by MassARRAY (registered trademark), it can be identified from which part of the base sequence of DNA used as a template. For example, when one CpG site in the DNA fragment is methylated, the peak obtained with MassARRAY (registered trademark) is shifted 16 ⁇ Da toward the high mass side.
  • MassARRAY registered trademark
  • the methylation state of the CpG site can be analyzed by a methylation specific PCR (MSP) method.
  • MSP methylation specific PCR
  • the MSP method is a method of analyzing the methylation status (presence / absence of methylation) of the CpG site by performing PCR amplification on the bisulfite-treated DNA using the primer set described below and confirming the presence or absence of the PCR product. It is.
  • a base sequence in which the CpG site to be analyzed is methylated (ie, cytosine is not converted to uracil) can be amplified, but the CpG site is not methylated (ie, the cytosine is converted to uracil).
  • the CpG site to be analyzed is methylated when a PCR product is present.
  • the MSP method cannot be used to amplify a base sequence in which cytosine at the CpG site to be analyzed is not converted to uracil, but a primer set that can amplify a base sequence in which cytosine at the CpG site is converted to uracil should be used You can also. In this case, it can be seen that the CpG site to be analyzed is methylated when no PCR product is present.
  • Each primer included in the primer set used in the MSP method can be appropriately designed by those skilled in the art according to the base sequence including the CpG site to be analyzed, but the CpG site to be analyzed is located at or near the 3 ′ end of the primer. It is preferably designed to include cytosine.
  • the methylation state of the CpG site can also be analyzed using a microarray.
  • the analysis microarray can be prepared by immobilizing on the substrate a nucleic acid probe complementary to the nucleotide sequence of the promoter region of each gene of CREM, VRK2 and MYL12A.
  • Such a microarray can be produced by a method known in the art.
  • DNA extracted from a biological sample is preferably labeled with a labeling substance known in the art. Therefore, it is preferable that the determination method of the present invention further includes a step of labeling the extracted DNA. Since this labeling step can label all DNA in the biological sample, it is advantageous to be performed after the DNA amplification step.
  • labeling substances include fluorescent substances, haptens such as biotin, radioactive substances, and the like.
  • the fluorescent substance include Cy3, Cy5, FITC, Alexa Fluor (trademark) and the like.
  • the above signal may be an appropriate signal depending on the type of microarray.
  • the signal may be an electrical signal generated when a DNA fragment hybridized with each probe of the microarray is present, or when the DNA to be analyzed is labeled as described above, a labeling substance Signals such as fluorescence and luminescence generated from The signal can be detected by a scanner provided in a normal microarray measurement apparatus. Examples of the scanner include GeneChip (registered trademark) Scanner 3000-7G (Affymetrix), Illumina (registered trademark) BeadArray Reader (Illumina).
  • information on the brain tumor of the subject is acquired based on the analysis result obtained in the above analysis step.
  • the information regarding the brain tumor is not particularly limited as long as it is information that can serve as an index for diagnosis of the brain tumor, but is preferably information indicating the occurrence and / or state of the brain tumor in the subject.
  • Such information includes, for example, the presence or absence of brain tumor-derived tumor cells in a biological sample collected from a subject, the possibility that a brain tumor has occurred in the subject, and the risk that a brain tumor will occur in the subject.
  • information on the brain tumor includes the prognosis of the subject, the degree of progression of the tumor (stage), and the like.
  • the information regarding the brain tumor acquired based on the analysis result obtained by said analysis process does not contain the information regarding the kind of tumor different from a brain tumor substantially. This is because the promoter region of the above gene used as a marker in the method of the present invention is specifically methylated in tumor cells derived from a brain tumor.
  • the analysis step when an analysis result indicating that there is a methylated CpG site is obtained, information indicating that a brain tumor has occurred, or the state of the brain tumor is poor (or , Bad) information can be acquired.
  • the methylation frequency obtained in the analysis step is higher than a predetermined threshold or the same result as the threshold is obtained, such information may be acquired. it can. More specifically, information that tumor cells derived from brain tumors are present in the biological sample can be acquired. Alternatively, information that the subject has a high risk of developing a brain tumor or information that a brain tumor has already occurred can be acquired. Further, if a brain tumor has occurred in the subject, information that the prognosis of the subject is poor (or bad), or information that the tumor state is a more advanced stage can be acquired.
  • the analysis process when the result that there is no methylated CpG site is obtained, information indicating that no brain tumor has occurred or information indicating that the brain tumor is in good condition can be obtained. .
  • information can be acquired when a result that the frequency of methylation obtained in the analysis step is lower than a predetermined threshold is obtained. More specifically, information that tumor cells derived from brain tumors are not present in the biological sample can be acquired. Alternatively, information that the subject has a low risk of developing a brain tumor or information that a brain tumor has not yet occurred can be acquired. Further, if a brain tumor has occurred in the subject, information that the prognosis of the subject is good or information that the tumor state is not so advanced can be acquired.
  • the predetermined threshold is not particularly limited, and can be set empirically by accumulating data on various biological samples.
  • a predetermined threshold value may be set as follows. First, for DNA extracted from each of biological samples (normal brain tissue or normal brain cells) that have been confirmed in advance not to contain tumor cells derived from brain tumors, and biological samples containing tumor cells derived from brain tumors, Analyze methylation frequency. Next, based on the obtained analysis result, a threshold value is set from a range that is higher than the methylation frequency of the biological sample that does not include tumor cells and lower than the methylation frequency of the biological sample that includes tumor cells. Preferably, a threshold value is set as a value capable of accurately distinguishing between a biological sample not containing tumor cells and a biological sample containing tumor cells.
  • the scope of the present invention includes a marker (hereinafter, also simply referred to as “marker”) for acquiring information related to a brain tumor by methylation analysis.
  • the marker of the present invention is at least one selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A.
  • the methylation state of a marker in a DNA sample prepared from a biological sample collected from a subject can be analyzed, and information related to the brain tumor of the subject can be acquired based on the obtained analysis result.
  • the analysis of methylation status and the acquisition of brain tumor information are the same as described above.
  • kits for acquiring information on brain tumors comprising a primer set for analyzing the state of at least one methylation selected from CpG sites present in the promoter region of each gene of CREM, VRK2 and MYL12A.
  • the primer set included in the kit is used to determine the methylation state of the CpG site by an analysis method involving PCR amplification such as mass spectrometry such as MassARRAY (registered trademark), MSP method, bisulfite sequencing method, etc.
  • mass spectrometry such as MassARRAY (registered trademark), MSP method, bisulfite sequencing method, etc.
  • a primer set for analysis may be used, but a primer set used in mass spectrometry such as MassARRAY (registered trademark) or MSP method is preferable.
  • the base sequence of each primer included in the primer set can be appropriately set by those skilled in the art according to the base sequence of the promoter region.
  • a primer set that is the base sequence of SEQ ID NO: 4 and SEQ ID NO: 5 a primer set that is the base sequence of SEQ ID NO: 6 and SEQ ID NO: 7, a primer set that is the base sequence of SEQ ID NO: 8 and SEQ ID NO: 9, and SEQ ID NO: 12
  • a primer set that is the base sequence of SEQ ID NO: 13 a primer set that is the base sequence of SEQ ID NO: 14 and SEQ ID NO: 15, and a primer set that is the base sequence of SEQ ID NO: 16 and SEQ ID NO: 17.
  • the present invention also includes a system suitable for providing information on brain tumors in a subject.
  • a system suitable for providing information on brain tumors in a subject is, for example, as follows.
  • a computer comprising a processor and memory under control of the processor;
  • the memory has the following steps: Obtaining an analysis result of the methylation state of the CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A contained in the subject-derived DNA sample; Based on the obtained analysis results, a computer program for causing the computer to execute the step of providing information on the brain tumor of the subject is recorded.
  • the present invention also includes a computer program product for causing a computer to provide information related to a brain tumor in a subject.
  • a computer program product for example, as follows.
  • a computer-readable medium includes the following steps: Obtaining an analysis result of the methylation state of the CpG site present in the promoter region of at least one gene selected from CREM, VRK2 and MYL12A contained in the subject-derived DNA sample; Based on the obtained analysis results, a computer program for causing the computer to execute the step of providing information on the brain tumor of the subject is recorded.
  • a computer program product that causes a computer to provide information about brain tumors in a subject.
  • FIG. 7 is a schematic diagram illustrating an example of a determination device for providing information related to a brain tumor of a subject.
  • the determination apparatus 1 shown in FIG. 7 includes a measurement apparatus 2 and a computer system 3 connected to the measurement apparatus 2.
  • the measuring device 2 is a MALDI-TOF type mass spectrometer. This measuring device 2 acquires mass spectrometry information such as the flight time and mass-to-charge ratio (m / z value) of the test substance.
  • mass spectrometry information such as the flight time and mass-to-charge ratio (m / z value) of the test substance.
  • the measurement device 2 acquires mass spectrometry information of the nucleic acid contained in the measurement sample, and the obtained mass spectrometry information is stored in the computer system 3. Send to.
  • the measuring device 2 may be a gel imaging device such as a fluorescence image scanner.
  • the measuring device 2 detects the amplification product. Then, the measuring device 2 acquires the band intensity data of the amplification product and transmits the obtained data to the computer system 3.
  • the computer system 3 includes a computer main body 3a, an input device 3b, and a display unit 3c that displays sample information, determination results, and the like.
  • the computer system 3 receives mass spectrometry information from the measuring device 2. Then, the processor of the computer system 3 executes a program that provides information related to the brain tumor in the subject based on the mass spectrometry information.
  • FIG. 8 is a block diagram showing a functional configuration of the determination apparatus shown in FIG.
  • the computer system 3 includes an acquisition unit 301, a storage unit 302, a calculation unit 303, a determination unit 304, and an output unit 305.
  • the acquisition unit 301 is connected to the measurement apparatus 2 via a network so that communication is possible.
  • the calculation unit 303 and the determination unit 304 constitute a control unit 306.
  • the acquisition unit 301 acquires information transmitted from the measurement device 2.
  • the storage unit 302 stores a threshold and a formula for calculating a methylation score necessary for determination.
  • the calculation unit 303 calculates the methylation score according to the formula stored in the storage unit 302 using the information acquired by the acquisition unit 301.
  • the determination unit 304 determines whether the methylation score calculated by the calculation unit 303 is lower than the threshold value stored in the storage unit 302.
  • the output unit 305 outputs the determination result by the determination unit 304 as information on the brain tumor of the subject (for example, the presence or absence of cancer cells derived from the brain tumor in the biological sample collected from the subject).
  • FIG. 9 is a block diagram showing a hardware configuration of the determination apparatus shown in FIG.
  • the computer main body 3 a includes a CPU (Central Processing Unit) 30, a ROM (Read Only Memory) 121, a ROM 32, a hard disk 33, an input / output interface 34, a reading device 35, and a communication.
  • An interface 36 and an image output interface 37 are provided.
  • a CPU 30, a ROM 31, a RAM (Random Access Memory) 32, a hard disk 33, an input / output interface 34, a reading device 35, a communication interface 36 and an image output interface 37 are connected by a bus 38 so that data communication is possible.
  • the CPU 30 can execute a computer program stored in the ROM 31 and a computer program loaded in the ROM 32. Each functional block described above is realized by the CPU 30 executing the application program. Thereby, a computer system functions as a terminal as a determination apparatus for providing the information regarding a test subject's brain tumor.
  • the ROM 31 is configured by a mask ROM, PROM, EPROM, EEPROM, or the like.
  • the ROM 31 stores a computer program executed by the CPU 30 and data used therefor.
  • the ROM 32 is configured by SRAM, DRAM or the like.
  • the ROM 32 is used for reading computer programs recorded in the ROM 31 and the hard disk 33.
  • the ROM 32 is also used as a work area for the CPU 30 when executing these computer programs.
  • the hard disk 33 is installed with an operating system to be executed by the CPU 30, a computer program such as an application program (a computer program for providing information on a brain tumor of a subject), and data used for executing the computer program.
  • a computer program such as an application program (a computer program for providing information on a brain tumor of a subject), and data used for executing the computer program.
  • the reading device 35 includes a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, and the like.
  • the reading device 35 can read the computer program or data recorded on the portable recording medium 40.
  • the input / output interface 34 includes, for example, a serial interface such as USB, IEEE1394, RS-232C, a parallel interface such as SCSI, IDE, IEEE1284, and an analog interface including a D / A converter, an A / D converter, and the like. It is configured.
  • An input device 3 b such as a keyboard and a mouse is connected to the input / output interface 34. The operator can input data to the computer main body 3a by using the input device 3b.
  • the communication interface 36 is, for example, an Ethernet (registered trademark) interface.
  • the computer system 3 can transmit print data to the printer via the communication interface 36.
  • the image output interface 37 is connected to a display unit 3c composed of an LCD, a CRT, or the like. Thereby, the display part 3c can output the video signal according to the image data given from CPU30.
  • the display unit 3c displays an image (screen) according to the input video signal.
  • FIG. 10 is a flowchart for providing information related to a brain tumor using the determination apparatus shown in FIG.
  • the peak area is calculated from the mass spectrometry information of the nucleic acid contained in the measurement sample prepared from the subject-derived DNA sample
  • the methylation score is calculated from the obtained peak area
  • the obtained methylation score is the threshold value.
  • step S1-1 the acquisition unit 301 of the determination apparatus 1 acquires mass spectrometry information from the measurement apparatus 2.
  • step S ⁇ b> 1-2 the calculation unit 303 calculates a peak area from the mass spectrometry information acquired by the acquisition unit 301 and transmits it to the storage unit 302.
  • step S ⁇ b> 1-3 the calculation unit 303 calculates a methylation score according to the formula stored in the storage unit 302 based on the peak area stored in the storage unit 302.
  • step S1-4 the determination unit 304 determines whether or not the methylation score calculated by the calculation unit 303 is lower than the threshold value stored in the storage unit 302.
  • the process proceeds to step S1-5, and the determination unit 304 displays a determination result indicating that the biological sample collected from the subject has no brain tumor-derived cancer cells.
  • the data is transmitted to the output unit 305.
  • the output unit 305 outputs a determination result indicating that the biological sample collected from the subject has brain tumor-derived cancer cells. Send to.
  • step S1-7 the output unit 305 outputs the determination result as information on the brain tumor of the subject, and displays it on the display unit 3c or prints it on a printer. Thereby, it is possible to provide a doctor or the like with information that assists in determining whether the subject has a brain tumor or not.
  • Reference Example 1 Identification of a new marker using methylation data of cancerous tissue and normal brain tissue of brain tumor (1) Acquisition of methylation data
  • cancerous tissue (283 samples) of brain tumor and normal brain tissue (2 specimens)
  • Infinium HumanMethylation27 BeadChip (Illumina) methylation data published on TCGA (The Cancer Genome Atlas: http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp) I got it.
  • the gene methylation positive rate is calculated by “(number of methylation positive samples / total number of cancer tissue samples among cancer tissue samples) ⁇ 100”.
  • the gene was used as a marker for methylation observed in the cancer tissue.
  • the promoter region of each gene of CREM, VRK2 and MYL12A was identified as a marker with high methylation observed in the cancerous tissue of brain tumor (FIG. 1A- C).
  • these markers are also called markers of the present invention.
  • Reference Example 2 Comparison of methylation data in multiple types of cancer / tumor-derived cancer / tumor tissue samples, non-cancerous tissue samples and normal tissue samples (1) Acquisition of methylation data
  • reference example 1 In addition to the indicated cancerous tissue (283 samples) and normal brain tissue (2 samples) of the brain tumor, methylation of 9 types of cancer / tumor tissue samples, 7 types of non-cancerous tissue samples and 12 types of normal tissue samples The data were compared. The number of specimens in each tissue is shown in the following table.
  • cancer tissue specimens derived from gastric cancer cancer tissue specimens derived from hepatocellular carcinoma, non-cancerous tissue specimens derived from hepatocellular carcinoma, normal tissue specimens derived from normal liver, 11 types of normal tissue specimens.
  • methylation data measured by Infinium Methylation Assay using Infinium HumanMethylation27 BeadChip (Illumina) was obtained.
  • TCGA The Cancer Genome Atlas: http://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp
  • lung cancer prostate cancer
  • 93 healthy human peripheral blood specimens 93 healthy human peripheral blood specimens below.
  • the methylation data referred to here is the methylation rate (mCpG) of the CpG region of CREM, VRK2 and MYL12A obtained as described in (2) of Reference Example 1 above.
  • all the markers of the present invention are not highly methylated in other types of cancer, and normal human tissues and normal human blood (healthy human peripheral blood, Salhia B et al.). It was found to be specifically and highly methylated.
  • Comparative Example 1 Comparison of methylation positive rates in multiple types of cancer / tumor-derived cancer / tumor tissue samples, non-cancerous tissue samples, and normal tissue samples It is already known that the tumor cells derived from brain tumors are methylated For each of the MGMT and CDKN2A genes (hereinafter referred to as “known markers”), the methylation positive rate in each tissue was calculated in the same manner as in Reference Example 2. The results are shown in FIGS. 3A and B.
  • both MGMT and CDKN2A have a high positive rate of cases in brain tumors, but methylation is detected in other types of cancer and other non-cancerous tissues.
  • the known marker has high sensitivity as a brain tumor marker, but has low specificity. Therefore, known markers have many problems in performance as diagnostic markers for brain tumors. That is, it was shown that it is necessary to conduct a marker search in consideration of specificity in other types of cancer and other non-cancerous tissues.
  • Example 1 Comparison of methylation data (MassARRAY) in normal brain and brain tumor (1) Biological sample
  • normal brain tissue (1 sample) and cancerous tissue of brain tumor (8 samples) are used as the biological sample. It was.
  • Genomic DNA was extracted from each of the above tissues using the QIAamp DNA Mini Kit (QIAGEN). Genomic DNA contained in the obtained DNA solution was fragmented with Bioruptor (COSMO BIO). Further, in order to prepare a calibration curve in mass spectrometry, human peripheral blood lymphocyte genomic DNA was used as a control genomic DNA. The genomic DNA of this human peripheral blood lymphocyte was amplified using GenomiPhi v2DNA amplification kit (GE Healthcare Life Science). The obtained amplification product consists of unmethylated DNA.
  • the amplified product was fragmented by Bioruptor (manufactured by COSMO BIO) to obtain a solution of an unmethylated DNA fragment (0% methylated DNA).
  • a portion of the solution of this unmethylated DNA fragment is taken and reacted with SssI methylase (New England Biolab) to methylate all cytosines in the CG sequence to obtain a methylated DNA fragment (100% methyl). Solution).
  • SssI methylase New England Biolab
  • a 0% methylated DNA solution and a 100% methylated DNA solution were mixed at a predetermined ratio to obtain 25%, 50% and 75% methylated DNA solutions.
  • Table 5 shows the primer set sequences for the markers of the present invention. Further, regarding the promoter regions of the CREM, VRK2 and MYL12A genes, the nucleotide sequences of the regions analyzed with the primer sets shown in Table 5 are shown in SEQ ID NOs: 18 to 20, respectively (the nucleotide sequence of SEQ ID NO: 18 is a minus strand) And the base sequences of SEQ ID NOs: 19 and 20 are positive strand sequences).
  • Tag array AGGAAGAGAG (SEQ ID NO: 10) -T7 promoter sequence: CAGTAATACGACTCACTATAGGGAGAAGGCT (SEQ ID NO: 11)
  • a PCR reaction solution was prepared by mixing the following reagents. 10x Hot Star buffer (QIAGEN) 0.5 ⁇ L 25 mM dNTP mix 0.04 ⁇ L Hot Star Taq (5U / ⁇ l) (QIAGEN) 0.04 ⁇ L Primer mix 2.0 ⁇ L DNA solution 1.0 ⁇ L 1.42 ⁇ L water Total 5 ⁇ L
  • PCR reaction was performed on condition of the following. 1 cycle at 94 ° C for 15 minutes, 45 cycles of 94 ° C for 20 seconds, 52 ° C for 30 seconds and 72 ° C for 1 minute, 3 minutes at 72 ° C.
  • each reaction product obtained was purified by Clean Resin (SEQUENOM) to obtain a sample for mass spectrometry.
  • SEQUENOM Clean Resin
  • the methylation score was calculated from the area ratio of the peak of the fragment containing the methylated CpG site and the peak of the fragment not containing the methylated CpG site. The results are shown in FIGS.
  • FIG. 4 shows that in the analysis of the methylation frequency using the marker of the present invention, it is possible to set a threshold value capable of distinguishing between a brain tumor specimen and a normal brain tissue specimen.
  • Example 2 Comparison of methylation data (MSP) in normal brain and brain tumor (1) Biological sample
  • MSP methylation data
  • Biological sample as a biological sample, the same normal brain tissue (one specimen) and brain tumor cancer as in Example 1 above. Tissue (8 specimens) was used.
  • Genomic DNA was extracted from each of the above tissues using the QIAamp DNA Mini Kit (QIAGEN). Genomic DNA contained in the obtained DNA solution was fragmented with Bioruptor (COSMO BIO). Further, human peripheral blood lymphocyte genomic DNA was used as a control genomic DNA. The genomic DNA of this human peripheral blood lymphocyte was amplified using GenomiPhi v2DNA amplification kit (GE Healthcare Life Science). The obtained amplification product consists of unmethylated DNA. Subsequently, the amplified product was fragmented by Bioruptor (manufactured by COSMO BIO) to obtain a solution of an unmethylated DNA fragment (0% methylated DNA).
  • Bioruptor manufactured by COSMO BIO
  • MSP Methylation specific PCR
  • Table 6 shows primer sets used in the above MSP. These are primer sets that can obtain an amplification product when the DNA of the region to be amplified is methylated.
  • a primer set for determining whether or not the bisulfite treatment was appropriately performed was also used as a quality control primer set (see Table 7).
  • the base sequences of the regions analyzed with the primer sets shown in Table 6 are shown in SEQ ID NOs: 23 to 25, respectively (the base sequence of SEQ ID NO: 23 is a minus strand) And the base sequences of SEQ ID NOs: 24 and 25 are positive strand sequences).
  • ⁇ PCR reaction conditions > 6 minutes at 95 ° C Y cycle for 30 seconds at 95 °C, 30 seconds at X °C, 30 seconds at 72 °C, 7 minutes at 72 ° C Leave at 16 ° C.
  • “X” and “Y” represent the annealing temperature and the number of cycles shown in Tables 6 and 7, respectively.
  • MSP methylation specific PCR
  • Example 3 Methylation Analysis by MSP Method Using Blood Sample methylation analysis was performed on CpG sites in CREM and VRK2 promoter regions by MSP using peripheral blood as a biological sample.
  • peripheral blood 8 samples collected from a brain tumor patient was used as the biological sample.
  • healthy human peripheral blood (3 samples) was used as a control sample.
  • MSP Qualitative MSP was performed using the measurement sample obtained in (2) above.
  • the composition of PCR reagents used and the PCR conditions are shown below.
  • the base sequences of the primer sets (CREM, VRK2, and accuracy control) used in this example are shown in Table 6 and Table 7.
  • ⁇ PCR reaction conditions > 6 minutes at 95 ° C Y cycle for 30 seconds at 95 °C, 30 seconds at X °C, 30 seconds at 72 °C, 7 minutes at 72 ° C Leave at 16 ° C.
  • “X” and “Y” represent the annealing temperature and the number of cycles shown in Table 8, respectively.
  • FIG. 6C shows that bands were detected in all samples by PCR using the primer set for quality control. This indicates that the bisulfite treatment of the sample was properly performed.
  • the marker of the present invention showed a high methylation tendency in brain tumor patients even when peripheral blood was used as a biological sample, and methylation was not detected in healthy individuals. Therefore, it was suggested that the marker of the present invention is a marker highly specific for brain tumors, and is useful for determining the presence or absence of cancer cells derived from brain tumors in blood samples.

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JP2022078120A (ja) * 2015-01-18 2022-05-24 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 癌の状態を判定する方法とシステム
CN112626184A (zh) * 2020-12-31 2021-04-09 深圳市第二人民医院(深圳市转化医学研究院) 一种检测mgmt基因启动子甲基化状态的方法

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