WO2021192397A1 - Cancer examination method - Google Patents

Cancer examination method Download PDF

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WO2021192397A1
WO2021192397A1 PCT/JP2020/041986 JP2020041986W WO2021192397A1 WO 2021192397 A1 WO2021192397 A1 WO 2021192397A1 JP 2020041986 W JP2020041986 W JP 2020041986W WO 2021192397 A1 WO2021192397 A1 WO 2021192397A1
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cancer
dna
subject
methylation
pattern
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PCT/JP2020/041986
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French (fr)
Japanese (ja)
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雄喜 井上
舞子 脇田
奈央子 山口
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富士フイルム株式会社
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Publication of WO2021192397A1 publication Critical patent/WO2021192397A1/en

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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q1/6869—Methods for sequencing
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • This disclosure relates to cancer screening methods.
  • cancer-derived cells or nucleic acids are present in body fluids such as blood and saliva.
  • body fluids such as blood and saliva.
  • analyzing cells or nucleic acids contained in blood or saliva it is expected to know the presence or absence of cancer, the type of cancer, the therapeutic effect, and the like.
  • ctDNA circulating tumor DNA
  • ctDNA has a base sequence or methylation pattern peculiar to tumor cells, which is different from normal DNA, and this difference makes it possible to detect ctDNA.
  • the methylation pattern of ctDNA differs depending on the cancer type, and it is expected that the cancer type is estimated based on the difference in the methylation pattern.
  • a genetic test that analyzes the methylation pattern of ctDNA is less invasive as a cancer test, and may be able to detect cancer even in the early stages of onset. Genetic tests performed using plasma as a sample have already been published (for example, JP-A-2019-504642, JP-A-2018-508228, JP-A-2014-204728, and JP-A-2013-150611). ).
  • Cancer screening may be performed on patients who are subject to health examinations that they voluntarily perform, or who are suspected of developing cancer at a medical institution.
  • a general cancer test is a test targeting a specific cancer, such as an X-ray test or an endoscopy that limits the target site, or a blood test that detects a specific cancer marker.
  • PET-CT Pulsitron Emission Tomography-Computed Tomography
  • An object of the present disclosure is to provide a minimally invasive cancer screening method in which a plurality of types of cancers are analyzed at the same time and the primary tumor is estimated.
  • Step A of extracting the cell-free DNA contained in the blood sample of the subject step B of analyzing the methylation of the cell-free DNA to obtain the DNA methylation pattern of the subject, and step B of the subject.
  • a cancer testing method comprising step C of estimating the presence or absence of cancer and the primary lesion from a DNA methylation pattern.
  • step D for obtaining an answer to the questionnaire from the subject and step E for determining the cancer type to be focused on based on the answer are further included, and step B is focused on.
  • the cancer screening method according to ⁇ 1> which is performed by adjusting the analysis conditions according to the species.
  • the questionnaire contains one or more items selected from the group consisting of gender, age, physical characteristics, medical history, family history, lifestyle, past medical examination results, and personal genomic information.
  • Step C includes collating the DNA methylation pattern of the subject with a reference pattern in which a DNA methylation pattern characteristic of each cancer type is set, according to ⁇ 1> to ⁇ 3>.
  • the reference pattern is a set of DNA methylation patterns that are characteristic of each cancer type obtained by comparing the methylation between the DNA of cancer patients and the DNA of healthy subjects for each cancer type.
  • the cancer test method according to ⁇ 4> which is information.
  • step B cell-free DNA is bisulfite-treated, multiplex PCR is performed using a primer set for analyzing DNA methylation, which is a component of the reference pattern, and a sequencer is used.
  • step B is performed with appropriate reaction conditions for multiplex PCR.
  • a minimally invasive cancer screening method in which a plurality of types of cancers are analyzed at the same time and the primary tumor is estimated.
  • the numerical range indicated by using "-" in the present disclosure indicates a range including the numerical values before and after "-" as the minimum value and the maximum value, respectively.
  • the upper limit value or the lower limit value described in one numerical range may be replaced with the upper limit value or the lower limit value of another numerical range described stepwise. .. Further, in the numerical range described in the present disclosure, the upper limit value or the lower limit value of the numerical range may be replaced with the value shown in the examples.
  • process is included in this term as long as the intended purpose of the process is achieved even if it cannot be clearly distinguished from other processes as well as an independent process.
  • the cancer testing method of the present disclosure includes a step A of extracting cell-free DNA contained in a blood sample of a subject and a step B of analyzing methylation of the cell-free DNA to obtain a DNA methylation pattern of the subject. And step C of estimating the presence or absence of cancer and the primary lesion from the DNA methylation pattern of the subject.
  • the cancer screening method of the present disclosure can provide a minimally invasive cancer test by using a blood sample of a subject as a sample.
  • the cancer screening method of the present disclosure targets cell-free DNA contained in a blood sample, it is possible to analyze a plurality of types of cancer at the same time. Then, the presence or absence of cancer and the primary lesion can be estimated from the DNA methylation pattern of the subject.
  • the DNA methylation pattern of the subject was set as a set of DNA methylation patterns characteristic of each cancer type. It is realized by collating with a reference pattern (hereinafter referred to as "reference pattern").
  • the DNA methylation pattern is a pattern consisting of the methylated states of a plurality of methylation sites.
  • the DNA methylation pattern is, for example, a pattern in which the methylated site in the methylated state is shown in the first color, the methylated site in the non-methylated state is shown in the second color, and the pattern is color-coded in two dimensions; the methylated state.
  • the pattern is preferably a computer-processable pattern.
  • DNA methylation occurs in bases such as cytosine and adenine, and cytosine methylation is common.
  • the cancers and primary lesions to be tested by the cancer screening method disclosed in this disclosure are not particularly limited.
  • examples include cancer, cervical cancer, skin cancer, leukemia, myeloma, lymphoma and the like.
  • the cancer screening methods disclosed in the present disclosure include information that assists a doctor's diagnosis, grounds for a doctor or a subject to determine the necessity of a detailed examination (for example, imaging test), grounds for a doctor to select a treatment method or a therapeutic drug, and the like. It is useful as.
  • step D in which the subject answers the questionnaire and the cancer type to be focused on are determined based on the answers to the questionnaire obtained from the subject. It is preferable to carry out the step B by further including the step E and adjusting the analysis conditions according to the cancer type of interest.
  • FIG. 1 is a flowchart showing an example of an embodiment of the cancer screening method of the present disclosure.
  • steps D and E are performed in parallel with step A.
  • the cancer inspection method of the present disclosure is not limited to the form shown in FIG. 1, and steps D and E may be performed between step A and step B, and steps D and E may be performed before step A. It may be carried out, or the step D may be carried out before the step A, and the step E may be carried out after the step A.
  • the reference pattern is information that sets a characteristic DNA methylation pattern for each cancer type.
  • Examples of the morphology of the reference pattern include a reference pattern that covers cancer types in which abnormal DNA methylation is presumed to be related to the pathological condition, a reference pattern that sets cancer types that are susceptible to males, and a reference pattern that is susceptible to females.
  • a standard pattern that sets cancer types a standard pattern that sets cancer types that are easily affected by age group, a standard pattern that sets gastrointestinal cancer, a standard pattern that sets blood cancer, and a combination of these A reference pattern and the like can be mentioned.
  • the reference pattern is information that sets the characteristic DNA methylation patterns for each cancer type obtained by comparing the methylation between the DNA of a cancer patient and the DNA of a healthy person for each cancer type. Is preferable.
  • the reference pattern may be constructed based on the information of the existing cancer gene database (for example, The Cancer Genome Atlas, TCGA) or based on the result of the original measurement. The following is an example of how to construct a reference pattern.
  • cancers in which abnormal DNA methylation is presumed to be related to the pathological condition are extracted.
  • cancer A, cancer B, cancer C, and cancer D are cancers in which abnormal methylation is associated with the pathological condition.
  • Genome methylation is analyzed by the methylation array method (for example, Infinium MethylationEPIC BeadChip (Illumina)) to obtain methylation pattern A1 characteristic of cancer A.
  • the methylation array method for example, Infinium MethylationEPIC BeadChip (Illumina)
  • the methylation pattern A1 characteristic of cancer A there are more than 850,000 methylation sites in the entire human genome, and it is possible to comprehensively analyze the methylation sites in the entire human genome to obtain the methylation pattern A1 characteristic of cancer A. preferable. More than 100 cancer patients and 100 healthy individuals will be analyzed to eliminate individual-specific methylation. Furthermore, the information A2 of methylation detected in the cell-free DNA of the blood is obtained by using the blood of 100 or more cancer patients who became the test subjects as a sample. The methylation pattern A1 characteristic of cancer A is corrected by the information A2 to obtain a methylation pattern A characteristic of cancer A and detectable using blood as a sample.
  • methylation pattern B characteristic of cancer B and detectable using blood as a sample methylation pattern C characteristic of cancer C and detectable using blood as a sample
  • cancer D A characteristic and detectable methylation pattern D is obtained using blood as a sample. Then, the methylation pattern A of cancer A, the methylation pattern B of cancer B, the methylation pattern C of cancer C, and the methylation pattern D of cancer D are set as a set, and a reference pattern for examining gastrointestinal cancer. And.
  • Step A is a step of extracting the cell-free DNA contained in the blood sample of the subject.
  • the subject is a human being.
  • the subject is, for example, a person who has undergone a self-intentional health examination or a person who is suspected of having cancer at a medical institution.
  • Blood samples include blood itself and blood diluted with physiological saline; preserved blood obtained by adding additives such as glucose and anticoagulant to blood; these fractions (for example, plasma, serum); etc. include.
  • Extraction of cell-free DNA from blood samples may be performed according to a conventional method. Examples of embodiments are given below. For example, 5 mL to 20 mL of blood is collected from a subject in a blood collection tube containing an anticoagulant. The blood is centrifuged to separate the blood cell component and plasma, and the plasma is collected. Cell-free DNA in plasma is extracted using a commercially available cell-free DNA extraction kit (for example, QIAamp Circulating Nucleic Acid Kit (Qiagen)).
  • a commercially available cell-free DNA extraction kit for example, QIAamp Circulating Nucleic Acid Kit (Qiagen)
  • Step B is a step of analyzing the methylation of cell-free DNA to obtain a DNA methylation pattern of a subject.
  • cell-free DNA In addition to ctDNA (circulating tumor DNA), cell-free DNA also contains genomic DNA derived from normal cells, and in particular, a large amount of genomic DNA derived from blood cells.
  • the ctDNA varies depending on the type and degree of progression of the cancer, but in the case of early stage cancer, it is said to be about 1% to 5% of the cell-free DNA.
  • An example of an embodiment of the analysis of methylation of cell-free DNA is the bisulfite sequencing method.
  • Examples of embodiments of the bisulfite sequencer method include bisulfite treatment of cell-free DNA, multiplex PCR using a primer set for analyzing DNA methylation, which is a component of the reference pattern, and Includes sequence analysis of amplification products using a sequencer.
  • An embodiment of the bisulfite sequencing method will be described below.
  • DNA bisulfite treatment may be carried out according to a conventional method.
  • a commercially available bisulfite treatment kit for example, EZDNA Methylation Kit (Zymo Research), EZ DNA Methylation-Gold Kit (Zymo Research), EpiSight Bisulfite Conversion Kit Ver.2 (Fuji Film Wako Pure Chemical Industries, Ltd.)
  • Cell-free DNA may be processed.
  • multiplex PCR is performed using a primer set for analyzing DNA methylation, which is a component of the reference pattern.
  • the primer set is designed so that DNA methylation, which is a component of the reference pattern, can be comprehensively analyzed.
  • Multiplex PCR PCR reagents include Multiplex PCR Kit (Takara Bio Inc.), Multiplex PCR Kit ver2 (Takara Bio Inc.), KAPA library amplification Kit (KAPA Biosystems Inc.), Platinum Multiplex PCR master mix Kit (Thermo Fisher), Examples include KOD-Multi & Epi- (Toyo Spinning Co., Ltd.).
  • Multiplex PCR may be performed according to a conventional method. Repeat the PCR cycle until the amount of amplification product is as desired.
  • the primer set used in multiplex PCR contains many types of primer pairs, but the temperature at which the template DNA can be annealed differs for each primer. It is preferable to optimize the reaction conditions of multiplex PCR (ie, the temperature and time of each of denaturation, annealing and elongation) according to the primer pair for detecting the cancer type of interest. As a result, the accuracy of analysis of the cancer type of interest can be improved. If there are multiple cancer types of interest, multiplex PCR may be performed multiple times, each of which may be adapted to another cancer type.
  • Sequencer is a term that includes the first generation sequencer (capillary sequencer), the second generation sequencer (next generation sequencer), the third generation sequencer, the fourth generation sequencer, and the sequencer to be developed in the future.
  • the sequencer may be a capillary sequencer, a next-generation sequencer, or another sequencer.
  • the next-generation sequencer is preferable from the viewpoint of the speed of analysis, the large number of samples that can be processed at one time, and the like.
  • the next-generation sequencer refers to a sequencer that is classified in comparison with a capillary sequencer (called a first-generation sequencer) that uses the Sanger method.
  • the next-generation sequencer that is most widely used at present is a sequencer whose principle is to determine the base sequence by capturing fluorescence or luminescence linked to complementary strand synthesis by DNA polymerase or complementary strand binding by DNA ligase.
  • Specific examples thereof include MiSeq (Illumina), HiSeq2000 (Illumina and HiSeq are registered trademarks), Roche454 (Roche) and the like.
  • each read is arranged for each cancer type, the methylation state of each cytosine in DNA is determined, and the DNA methylation pattern of the subject is acquired. ..
  • Step C is a step of estimating the presence or absence of cancer and the primary lesion from the DNA methylation pattern of the subject.
  • An example of an embodiment of step C is to collate the DNA methylation pattern of the subject with a reference pattern to estimate the presence or absence of cancer and the primary lesion.
  • the measured quantitative value of methylation is expressed as a percentage as the degree of methylation for each gene, and the numerical values are compared.
  • the positive criteria are, for example, if there is a coincidence or similarity between the DNA methylation pattern of the subject and the reference pattern, the cancer type and primary lesion corresponding to the coincidence or similarity are positive.
  • the positive criterion is Species and primary lesions are positive.
  • Step D is a step of obtaining an answer to the questionnaire from the subject.
  • Step D is a step performed for the purpose of obtaining physical information and clinical information of the subject.
  • the questionnaire is a medium in which the question items are described and the answers of the subjects to the question items are entered.
  • the questionnaire may be paper or electronic media.
  • Question items are included in the questionnaire for the purpose of obtaining physical information and clinical information of the subject.
  • questions refer to the findings about cancer (for example, academic papers, patient surveys, cancer statistics, oncogene databases), and information or rationale for the causal relationship or correlation between the attributes of cancer patients and genetic abnormalities. It is preferable to obtain and prepare.
  • the question item may be a multiple-choice question or a descriptive question.
  • Question items include, for example, gender, age, physical characteristics (eg, height, weight, abdominal circumference, chest circumference), medical history, family history (eg, medical history of relatives within the third degree of kinship, such as parents, brothers, and sisters. , Grandparents, parents'brothers, and parents' sisters), lifestyle (eg smoking habits, drinking habits, eating habits, exercise habits), results of past health examinations (eg blood test values urine Test values of tests, findings of bacterial or viral infections, findings of imaging tests, findings of genetic tests), personal genome information can be mentioned.
  • physical characteristics eg, height, weight, abdominal circumference, chest circumference
  • medical history eg, medical history of relatives within the third degree of kinship, such as parents, brothers, and sisters. , Grandparents, parents'brothers, and parents' sisters
  • lifestyle eg smoking habits, drinking habits, eating habits, exercise habits
  • results of past health examinations eg blood test values urine Test values of tests, findings of bacterial or viral infections, findings of imaging tests, findings of genetic
  • the subject may read the questionnaire and fill in the answer by himself, or the questioner or a third party that the subject answered the questioner's question verbally. May be filled in.
  • Questioners are, for example, doctors, nurses, laboratory technicians, nurses, caregivers, and parents. Answers about past health test results (eg, blood test values, urinalysis test values, bacterial or viral infection findings, imaging test findings, genetic test findings) or personal genomic information are the test values.
  • a table, image, or electronic data may be attached to the questionnaire.
  • Step E is a step of determining the cancer type of interest based on the answers to the questionnaire obtained from the subject for the purpose of performing step B by optimizing the analysis conditions according to the cancer type of interest.
  • the decision criteria are preferably prepared with reference to statistical data prepared or edited by public research institutes or academic societies.
  • the determinant is preferably a form that covers the determinants of cancer and is a program executed by a computer.
  • the criteria for determination refer to, for example, statistical data prepared or edited by the National Cancer Center, literature such as "New Clinical Oncology” edited by the Japanese Society of Medical Oncology, or academic papers.
  • To create For example, if a hereditary cancer described in "New Clinical Oncology” is affected by a relative within the third degree of kinship, pay attention to the hereditary cancer regardless of the other attributes of the subject. Create the criteria for deciding to do.
  • step E will be described with reference to the flowchart shown in FIG. FIG. 2 is an example for explaining the concept of step E, and does not limit the embodiment.
  • FIG. 2 is a flowchart illustrating the flow using hepatocellular carcinoma as an example. This flow is based on the age of the subject, drinking habits, and the presence or absence of persistent hepatitis virus infection, which is the information obtained from the answers to the questionnaire as to whether or not hepatocellular carcinoma should be the focus of attention. decide.
  • the process proceeds to the stage shown in S514, and hepatocellular carcinoma is determined as a target of interest.
  • the process proceeds to the stage shown in S514, and hepatocellular carcinoma is determined as a target of interest.
  • step shown in S514 or the step shown in S515 After the step shown in S514 or the step shown in S515 is completed, the process returns to step E in FIG.
  • the computer comprehensively processes the flow as shown in FIG. 2 according to the program covering the cancer types.
  • M represents the molar concentration
  • 1M 1 mol / L.
  • % is based on mass unless otherwise specified.
  • Example 1 [Selection of characteristic methylation sites for each cancer type and construction of reference patterns] Information registered in the public database of genome, epigenome, and transcriptome about normal tissue and cancer tissue (about 40 samples of normal tissue, about 400 samples of cancer tissue) is compared and analyzed, and it is characteristic for each cancer type. The methylation site was extracted. Then, for pancreatic cancer, colon cancer, and hepatocellular carcinoma, which are highly significant for early detection, methylation patterns were created for each, and these were set as a reference pattern.
  • Each primer pair for 88 methylation sites constituting the above reference pattern was designed, and these were combined to form a primer set.
  • the 88 selected primer pairs were synthesized by Fasmac. Equal amounts of each primer (100 ⁇ M) were mixed in a single tube, and the mixture was diluted to 1 ⁇ M to prepare a primer set.
  • cell-free DNA was extracted from plasma derived from a subject and plasma derived from a healthy subject according to the protocol of the kit. 200 ⁇ L of QIAGEN Proteinase K was added to a 50 mL centrifuge tube. Then 2 mL of plasma was added to the tube. 1.6 mL of Buffer ACL (containing 1.0 ⁇ g of carrier RNA) was added, the lid was closed and mixed with pulse vortex for 30 seconds. After incubation at 60 ° C.
  • Buffer ACL containing 1.0 ⁇ g of carrier RNA
  • M-Wash buffer washing solution
  • M-Elution buffer 10 ⁇ L eluate
  • PCR was performed for 40 cycles of 3 steps of 94 ° C./2 minutes for 1 cycle, 98 ° C./10 seconds, 58 ° C./30 seconds, and 68 ° C./15 seconds.
  • the amplification reaction solution was purified by AM Pure XP Kit (Beckman Coulter, Inc.) and recovered in a 40 ⁇ L TE buffer. Index-added PCR for use in NGS was reacted using the Multiplex PCR Assay kit (Takara Bio Inc.). 1 ⁇ L of each of 1.25 ⁇ M PCR primers, 0.125 ⁇ L of Multiplex PCR Mix 1 and 12.5 ⁇ L of Multiplex PCR Mix 2 were added to the 8-unit PCR tube, and the mixture was adjusted to 25 ⁇ L with water.
  • PCR was denatured at 94 ° C./3 minutes, followed by 5 cycles of 3 steps of 94 ° C./45 seconds, 50 ° C./60 seconds, and 72 ° C./30 seconds, and 94 ° C./45 seconds, 55 ° C./60 seconds. Three steps of 72 ° C./30 seconds were performed for 11 cycles.
  • the Miseq Reagent Kit v2 300 Cycle was used at both ends of the DNA fragment obtained by multiplex PCR.
  • Two types of adapters were added, including a flow cell binding sequence (P5 or P7 sequence), a sample identification index sequence, and a sequence primer binding sequence.
  • the obtained PCR product was purified using AM Pure XP Kit (Beckman Coulter), and the concentration was measured using BioAnalyzer (Agilent Technology Co., Ltd.). For more accurate quantification of amplification products, quantification was performed using KAPA Library Quantification Kits (KAPA Biosystems), and a sequence library was obtained. The sequence library was sequenced using a next-generation sequencer (MiSeq (Illumina)). The obtained FastQ file was mapped to the human genome sequence (hg19) by BWA, the presence or absence of methylation was determined, and the DNA methylation pattern of the subject and the DNA methylation pattern of the healthy subject were obtained, respectively.

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Abstract

Provided is a cancer examination method including: step A for extracting cell-free DNA contained in a blood sample of a subject; step B for analyzing methylation of the cell-free DNA to obtain a DNA methylation pattern of the subject; and step C for estimating the presence or absence of cancer and the primary focus on the basis of the DNA methylation pattern of the subject.

Description

がん検査方法Cancer screening method
　本開示は、がん検査方法に関する。 This disclosure relates to cancer screening methods.
　血液及び唾液などの体液中にがんに由来する細胞又は核酸が存在することが知られている。血液又は唾液に含まれる細胞又は核酸を解析することによって、がんの存否、がんの種類、治療効果などを知ることが期待されている。 It is known that cancer-derived cells or nucleic acids are present in body fluids such as blood and saliva. By analyzing cells or nucleic acids contained in blood or saliva, it is expected to know the presence or absence of cancer, the type of cancer, the therapeutic effect, and the like.
　血漿中には細胞から放出されたセルフリーＤＮＡが存在しており、腫瘍細胞から血液中に放出されたＤＮＡをｃｔＤＮＡ（circulating tumor DNA）といい、有望ながんマーカーである。ｃｔＤＮＡは正常ＤＮＡとは異なる腫瘍細胞特有の塩基配列又はメチル化パターンを有しており、この違いによってｃｔＤＮＡの検出が可能である。また、ｃｔＤＮＡのメチル化パターンは、がん種毎に異なることが知られており、メチル化パターンの相違によってがん種を推定することも期待されている。ｃｔＤＮＡのメチル化パターンを解析する遺伝子検査は、がん検査としては侵襲性が低く、発症の初期段階でもがんを検出できる可能性がある。既に、血漿を試料にして行う遺伝子検査が公開されている（例えば、特表２０１９－５０４６４２号公報、特表２０１８－５０８２２８号公報、特開２０１４－２０４７２８号公報、及び特開２０１３－１５０６１１号公報）。 Cell-free DNA released from cells exists in plasma, and the DNA released from tumor cells into blood is called ctDNA (circulating tumor DNA), which is a promising cancer marker. ctDNA has a base sequence or methylation pattern peculiar to tumor cells, which is different from normal DNA, and this difference makes it possible to detect ctDNA. Further, it is known that the methylation pattern of ctDNA differs depending on the cancer type, and it is expected that the cancer type is estimated based on the difference in the methylation pattern. A genetic test that analyzes the methylation pattern of ctDNA is less invasive as a cancer test, and may be able to detect cancer even in the early stages of onset. Genetic tests performed using plasma as a sample have already been published (for example, JP-A-2019-504642, JP-A-2018-508228, JP-A-2014-204728, and JP-A-2013-150611). ).
　自らの意思で行う健康診断の被検者、又は、医療機関においてがん発症を疑われた者に対し、がん検査が行われることがある。一般的ながん検査は、対象部位を限定したＸ線検査又は内視鏡検査、特定のがんマーカーを検出する血液検査など、特定のがんを標的とした検査である。被検者の家族歴、年齢、症状などに基づき検査するがんの種類及び部位を特定するところ、検査すべき項目が除外されて初期のがんを見逃す懸念がある。一方で、がん細胞がブドウ糖を活発に取り込むことを利用して全身のがんを検査するＰＥＴ－ＣＴ（Positron Emission Tomography-Computed Tomography）があるが、ＰＥＴ－ＣＴは高額な検査であり、常に行われる検査ではない。簡便に且つ低額で行うことができる全身的ながん検査の実用化が望まれている。 Cancer screening may be performed on patients who are subject to health examinations that they voluntarily perform, or who are suspected of developing cancer at a medical institution. A general cancer test is a test targeting a specific cancer, such as an X-ray test or an endoscopy that limits the target site, or a blood test that detects a specific cancer marker. When identifying the type and site of cancer to be tested based on the subject's family history, age, symptoms, etc., there is a concern that the items to be tested will be excluded and the initial cancer will be overlooked. On the other hand, there is PET-CT (Positron Emission Tomography-Computed Tomography), which tests cancer throughout the body by utilizing the active uptake of glucose by cancer cells, but PET-CT is an expensive test and is always It is not a test performed. It is desired to put into practical use a systemic cancer test that can be performed easily and at a low cost.
　本開示の実施形態は、上記状況のもとになされた。
　本開示は、複数種のがんを同時に解析対象とし、原発巣の推定までを行う、低侵襲ながん検査方法を提供することを課題とする。 The embodiments of the present disclosure have been made under the above circumstances.
An object of the present disclosure is to provide a minimally invasive cancer screening method in which a plurality of types of cancers are analyzed at the same time and the primary tumor is estimated.
　課題を解決するための具体的手段には、下記の態様が含まれる。
＜１＞　被検者の血液試料に含まれるセルフリーＤＮＡを抽出する工程Ａと、セルフリーＤＮＡのメチル化を解析し、被検者のＤＮＡメチル化パターンを得る工程Ｂと、被検者のＤＮＡメチル化パターンからがんの存否及び原発巣を推定する工程Ｃと、を含むがん検査方法。
＜２＞　工程Ｂの前に、被検者から質問票への回答を得る工程Ｄと、回答に基づき注目するがん種を決定する工程Ｅと、をさらに含み、工程Ｂを、注目するがん種に応じて解析条件を適切化して行う、＜１＞に記載のがん検査方法。
＜３＞　質問票は、性別、年齢、身体的特徴、既往歴、家族歴、生活習慣、過去の健康診断の結果、及び個人ゲノム情報からなる群から選ばれる１つ以上の項目を含む、＜２＞に記載のがん検査方法。
＜４＞　工程Ｃが、被検者のＤＮＡメチル化パターンを、がん種毎に特徴的なＤＮＡメチル化パターンをセットにした基準パターンに照合することを含む、＜１＞～＜３＞のいずれか１項に記載のがん検査方法。
＜５＞　基準パターンが、がん種毎にがん患者のＤＮＡと健常者のＤＮＡとの間のメチル化を比較して得たがん種毎に特徴的なＤＮＡメチル化パターンをセットにした情報である、＜４＞に記載のがん検査方法。
＜６＞　工程Ｂが、セルフリーＤＮＡをバイサルファイト処理することと、基準パターンの構成要素であるＤＮＡメチル化を解析するためのプライマーセットを用いてマルチプレックスＰＣＲを行うことと、シーケンサーを用いて配列解析することと、を含む、＜４＞又は＜５＞に記載のがん検査方法。
＜７＞　工程Ｂを、マルチプレックスＰＣＲの反応条件を適切化して行う、＜６＞に記載のがん検査方法。 Specific means for solving the problem include the following aspects.
<1> Step A of extracting the cell-free DNA contained in the blood sample of the subject, step B of analyzing the methylation of the cell-free DNA to obtain the DNA methylation pattern of the subject, and step B of the subject. A cancer testing method comprising step C of estimating the presence or absence of cancer and the primary lesion from a DNA methylation pattern.
<2> Prior to step B, step D for obtaining an answer to the questionnaire from the subject and step E for determining the cancer type to be focused on based on the answer are further included, and step B is focused on. The cancer screening method according to <1>, which is performed by adjusting the analysis conditions according to the species.
<3> The questionnaire contains one or more items selected from the group consisting of gender, age, physical characteristics, medical history, family history, lifestyle, past medical examination results, and personal genomic information. The cancer screening method described in 2>.
<4> Step C includes collating the DNA methylation pattern of the subject with a reference pattern in which a DNA methylation pattern characteristic of each cancer type is set, according to <1> to <3>. The cancer screening method according to any one item.
<5> The reference pattern is a set of DNA methylation patterns that are characteristic of each cancer type obtained by comparing the methylation between the DNA of cancer patients and the DNA of healthy subjects for each cancer type. The cancer test method according to <4>, which is information.
<6> In step B, cell-free DNA is bisulfite-treated, multiplex PCR is performed using a primer set for analyzing DNA methylation, which is a component of the reference pattern, and a sequencer is used. The cancer testing method according to <4> or <5>, which comprises sequence analysis.
<7> The cancer screening method according to <6>, wherein step B is performed with appropriate reaction conditions for multiplex PCR.
　本開示によれば、複数種のがんを同時に解析対象とし、原発巣の推定までを行う、低侵襲ながん検査方法が提供される。 According to the present disclosure, a minimally invasive cancer screening method is provided in which a plurality of types of cancers are analyzed at the same time and the primary tumor is estimated.
本開示のがん検査方法の実施形態例を示すフローチャートである。It is a flowchart which shows the embodiment example of the cancer screening method of this disclosure. 本開示のがん検査方法において、被検者から得た質問票への回答に基づき注目するがんを決定する流れを説明するフローチャートである。It is a flowchart explaining the flow of determining the cancer to be focused on based on the answer to the questionnaire obtained from the subject in the cancer screening method of this disclosure.
　以下に、本開示の実施形態について説明する。これらの説明及び実施例は実施形態を例示するものであり、実施形態の範囲を制限するものではない。 Hereinafter, embodiments of the present disclosure will be described. These explanations and examples illustrate the embodiments and do not limit the scope of the embodiments.
　本開示において「～」を用いて示された数値範囲は、「～」の前後に記載される数値をそれぞれ最小値及び最大値として含む範囲を示す。
　本開示中に段階的に記載されている数値範囲において、一つの数値範囲で記載された上限値又は下限値は、他の段階的な記載の数値範囲の上限値又は下限値に置き換えてもよい。また、本開示中に記載されている数値範囲において、その数値範囲の上限値又は下限値は、実施例に示されている値に置き換えてもよい。 The numerical range indicated by using "-" in the present disclosure indicates a range including the numerical values before and after "-" as the minimum value and the maximum value, respectively.
In the numerical range described stepwise in the present disclosure, the upper limit value or the lower limit value described in one numerical range may be replaced with the upper limit value or the lower limit value of another numerical range described stepwise. .. Further, in the numerical range described in the present disclosure, the upper limit value or the lower limit value of the numerical range may be replaced with the value shown in the examples.
　本開示において「工程」との語は、独立した工程だけでなく、他の工程と明確に区別できない場合であってもその工程の所期の目的が達成されれば、本用語に含まれる。 In the present disclosure, the term "process" is included in this term as long as the intended purpose of the process is achieved even if it cannot be clearly distinguished from other processes as well as an independent process.
　本開示において実施形態を、図面を参照して説明する場合、当該実施形態の構成は図面に示された構成に限定されない。 When the embodiment is described in the present disclosure with reference to the drawings, the configuration of the embodiment is not limited to the configuration shown in the drawings.
＜がん検査方法＞
　本開示のがん検査方法は、被検者の血液試料に含まれるセルフリーＤＮＡを抽出する工程Ａと、セルフリーＤＮＡのメチル化を解析し、被検者のＤＮＡメチル化パターンを得る工程Ｂと、被検者のＤＮＡメチル化パターンからがんの存否及び原発巣を推定する工程Ｃと、を含む。 <Cancer screening method>
The cancer testing method of the present disclosure includes a step A of extracting cell-free DNA contained in a blood sample of a subject and a step B of analyzing methylation of the cell-free DNA to obtain a DNA methylation pattern of the subject. And step C of estimating the presence or absence of cancer and the primary lesion from the DNA methylation pattern of the subject.
　本開示のがん検査方法は、被検者の血液試料を検体とすることによって、低侵襲ながん検査を提供することができる。 The cancer screening method of the present disclosure can provide a minimally invasive cancer test by using a blood sample of a subject as a sample.
　本開示のがん検査方法は、血液試料に含まれるセルフリーＤＮＡを解析対象とするので、複数種のがんを同時に解析対象とすることができる。そして、被検者のＤＮＡメチル化パターンから、がんの存否及び原発巣を推定することができる。 Since the cancer screening method of the present disclosure targets cell-free DNA contained in a blood sample, it is possible to analyze a plurality of types of cancer at the same time. Then, the presence or absence of cancer and the primary lesion can be estimated from the DNA methylation pattern of the subject.
　被検者のＤＮＡメチル化パターンからがんの存否及び原発巣を推定することは、例えば、被検者のＤＮＡメチル化パターンを、がん種毎に特徴的なＤＮＡメチル化パターンをセットにした基準パターン（以下「基準パターン」という。）に照合することによって実現される。 To estimate the presence or absence of cancer and the primary lesion from the DNA methylation pattern of the subject, for example, the DNA methylation pattern of the subject was set as a set of DNA methylation patterns characteristic of each cancer type. It is realized by collating with a reference pattern (hereinafter referred to as "reference pattern").
　ＤＮＡメチル化パターンとは、複数個のメチル化部位のメチル化状態からなるパターンである。ＤＮＡメチル化パターンは、例えば、メチル化状態にあるメチル化部位を第一の色で示し、メチル化状態にないメチル化部位を第二の色で示し、二次元に色分けしたパターン；メチル化状態にあるメチル化部位を第一の記号又は文字で示し、メチル化状態にないメチル化部位を第二の記号又は文字で示し、テキストとして示したパターン；などが挙げられる。パターンは、コンピュータが処理可能なパターンであることが好ましい。 The DNA methylation pattern is a pattern consisting of the methylated states of a plurality of methylation sites. The DNA methylation pattern is, for example, a pattern in which the methylated site in the methylated state is shown in the first color, the methylated site in the non-methylated state is shown in the second color, and the pattern is color-coded in two dimensions; the methylated state. A pattern in which the methylation site in the above is indicated by the first symbol or letter, the methylation site that is not in the methylated state is indicated by the second symbol or letter, and is shown as text; and the like. The pattern is preferably a computer-processable pattern.
　ＤＮＡのメチル化は、シトシン、アデニン等の塩基に起こり、シトシンのメチル化が一般的である。 DNA methylation occurs in bases such as cytosine and adenine, and cytosine methylation is common.
　本開示のがん検査方法の検査対象となるがん及び原発巣は、特に制限されない。具体的には、肺がん、食道がん、胃がん、大腸がん、膵臓がん、肝細胞がん、胆のうがん、胆管がん、膀胱がん、尿路がん、前立腺がん、乳がん、卵巣がん、子宮頸がん、皮膚がん、白血病、骨髄腫、リンパ腫などが挙げられる。 The cancers and primary lesions to be tested by the cancer screening method disclosed in this disclosure are not particularly limited. Specifically, lung cancer, esophageal cancer, gastric cancer, colon cancer, pancreatic cancer, hepatocellular carcinoma, cholecystic cancer, bile duct cancer, bladder cancer, urinary tract cancer, prostate cancer, breast cancer, ovary Examples include cancer, cervical cancer, skin cancer, leukemia, myeloma, lymphoma and the like.
　本開示のがん検査方法は、医師の診断を補助する情報、医師又は被検者が精密検査（例えば画像検査）の要否を判断する根拠、医師が治療方法又は治療薬を選択する根拠などとして有用である。 The cancer screening methods disclosed in the present disclosure include information that assists a doctor's diagnosis, grounds for a doctor or a subject to determine the necessity of a detailed examination (for example, imaging test), grounds for a doctor to select a treatment method or a therapeutic drug, and the like. It is useful as.
　本開示のがん検査方法は、工程Ｂの前に、被検者から質問票への回答を得る工程Ｄと、被検者から得た質問票への回答に基づき注目するがん種を決定する工程Ｅと、をさらに含み、注目するがん種に応じて解析条件を適切化して工程Ｂを行うことが好ましい。 In the cancer screening method of the present disclosure, prior to step B, step D in which the subject answers the questionnaire and the cancer type to be focused on are determined based on the answers to the questionnaire obtained from the subject. It is preferable to carry out the step B by further including the step E and adjusting the analysis conditions according to the cancer type of interest.
　図１は、本開示のがん検査方法の実施形態例を示すフローチャートである。図１に示す実施形態例においては、工程Ａと並行して工程Ｄ及び工程Ｅを行う。
　本開示のがん検査方法は、図１に示す形態に限定されず、工程Ａと工程Ｂとの間に工程Ｄ及び工程Ｅを行ってもよく、工程Ａの前に工程Ｄ及び工程Ｅを行ってもよく、工程Ａの前に工程Ｄを行い且つ工程Ａの後に工程Ｅを行ってもよい。 FIG. 1 is a flowchart showing an example of an embodiment of the cancer screening method of the present disclosure. In the embodiment shown in FIG. 1, steps D and E are performed in parallel with step A.
The cancer inspection method of the present disclosure is not limited to the form shown in FIG. 1, and steps D and E may be performed between step A and step B, and steps D and E may be performed before step A. It may be carried out, or the step D may be carried out before the step A, and the step E may be carried out after the step A.
　以下、基準パターン及び各工程を詳細に説明する。 The reference pattern and each process will be described in detail below.
［基準パターン］
　基準パターンは、がん種毎に特徴的なＤＮＡメチル化パターンをセットにした情報である。基準パターンの形態例としては、ＤＮＡのメチル化異常が病態に関連すると推定されるがん種を網羅した基準パターン、男性が罹患しやすいがん種をセットにした基準パターン、女性が罹患しやすいがん種をセットにした基準パターン、年代別に罹患しやすいがん種をセットにした基準パターン、消化器がんをセットにした基準パターン、血液がんをセットにした基準パターン、これらを組み合わせた基準パターンなどが挙げられる。 [Reference pattern]
The reference pattern is information that sets a characteristic DNA methylation pattern for each cancer type. Examples of the morphology of the reference pattern include a reference pattern that covers cancer types in which abnormal DNA methylation is presumed to be related to the pathological condition, a reference pattern that sets cancer types that are susceptible to males, and a reference pattern that is susceptible to females. A standard pattern that sets cancer types, a standard pattern that sets cancer types that are easily affected by age group, a standard pattern that sets gastrointestinal cancer, a standard pattern that sets blood cancer, and a combination of these A reference pattern and the like can be mentioned.
　基準パターンは、がん種毎にがん患者のＤＮＡと健常者のＤＮＡとの間のメチル化を比較して得たがん種毎に特徴的なＤＮＡメチル化パターンをセットにした情報であることが好ましい。基準パターンの構築は、既存のがん遺伝子データベース（例えば、The Cancer Genome Atlas，ＴＣＧＡ）の情報をもとに行ってもよく、独自の計測の結果に基づき行ってもよい。以下に、基準パターンの構築の仕方を例示する。 The reference pattern is information that sets the characteristic DNA methylation patterns for each cancer type obtained by comparing the methylation between the DNA of a cancer patient and the DNA of a healthy person for each cancer type. Is preferable. The reference pattern may be constructed based on the information of the existing cancer gene database (for example, The Cancer Genome Atlas, TCGA) or based on the result of the original measurement. The following is an example of how to construct a reference pattern.
　がん遺伝子データベース（例えばＴＣＧＡ）から、ＤＮＡのメチル化異常が病態に関連すると推定されるがんを抽出する。例えば、消化器がんのうち、がんＡ、がんＢ、がんＣ及びがんＤが、病態にメチル化異常が関連するがんであるとする。
　がんＡを発症しているがん患者１００人以上のがん化している組織Ａのゲノムメチル化と、がんＡを発症していない健常者１００人以上のがん化していない組織Ａのゲノムメチル化とを、メチル化アレイ法（例えば、Infinium MethylationEPIC BeadChip（イルミナ社））でそれぞれ解析し、がんＡに特徴的なメチル化パターンＡ１を得る。ここで、ヒトゲノム全体にはメチル化部位が８５万以上あると言われており、ヒトゲノム全体のメチル化部位を網羅的に解析して、がんＡに特徴的なメチル化パターンＡ１を得ることが好ましい。個人に特異的なメチル化を排除するために、がん患者及び健常者それぞれ１００人以上を解析する。
　さらに、上記の被検者となったがん患者１００人以上の血液を検体にして、血液のセルフリーＤＮＡにおいて検出されるメチル化の情報Ａ２を得る。
　がんＡに特徴的なメチル化パターンＡ１を情報Ａ２で補正し、がんＡに特徴的で且つ血液を検体にして検出可能なメチル化パターンＡを得る。
　同様にして、がんＢに特徴的で且つ血液を検体にして検出可能なメチル化パターンＢ、がんＣに特徴的で且つ血液を検体にして検出可能なメチル化パターンＣ、がんＤに特徴的で且つ血液を検体にして検出可能なメチル化パターンＤを得る。
　そして、がんＡのメチル化パターンＡ、がんＢのメチル化パターンＢ、がんＣのメチル化パターンＣ、がんＤのメチル化パターンＤをセットにし、消化器がんを検査する基準パターンとする。 From an oncogene database (eg, TCGA), cancers in which abnormal DNA methylation is presumed to be related to the pathological condition are extracted. For example, among gastrointestinal cancers, cancer A, cancer B, cancer C, and cancer D are cancers in which abnormal methylation is associated with the pathological condition.
Genome methylation of cancerous tissue A in 100 or more cancer patients who have developed cancer A, and non-cancerous tissue A in 100 or more healthy individuals who have not developed cancer A. Genome methylation is analyzed by the methylation array method (for example, Infinium MethylationEPIC BeadChip (Illumina)) to obtain methylation pattern A1 characteristic of cancer A. Here, it is said that there are more than 850,000 methylation sites in the entire human genome, and it is possible to comprehensively analyze the methylation sites in the entire human genome to obtain the methylation pattern A1 characteristic of cancer A. preferable. More than 100 cancer patients and 100 healthy individuals will be analyzed to eliminate individual-specific methylation.
Furthermore, the information A2 of methylation detected in the cell-free DNA of the blood is obtained by using the blood of 100 or more cancer patients who became the test subjects as a sample.
The methylation pattern A1 characteristic of cancer A is corrected by the information A2 to obtain a methylation pattern A characteristic of cancer A and detectable using blood as a sample.
Similarly, methylation pattern B characteristic of cancer B and detectable using blood as a sample, methylation pattern C characteristic of cancer C and detectable using blood as a sample, and cancer D A characteristic and detectable methylation pattern D is obtained using blood as a sample.
Then, the methylation pattern A of cancer A, the methylation pattern B of cancer B, the methylation pattern C of cancer C, and the methylation pattern D of cancer D are set as a set, and a reference pattern for examining gastrointestinal cancer. And.
［工程Ａ］
　工程Ａは、被検者の血液試料に含まれるセルフリーＤＮＡを抽出する工程である。 [Step A]
Step A is a step of extracting the cell-free DNA contained in the blood sample of the subject.
　被検者とは、ヒトである。被検者は、例えば、自らの意思で行う健康診断の受診者、医療機関においてがんを疑われた者である。 The subject is a human being. The subject is, for example, a person who has undergone a self-intentional health examination or a person who is suspected of having cancer at a medical institution.
　血液試料は、血液そのもの、及び、生理食塩水で希釈した血液；血液にグルコース、抗血液凝固剤等の添加剤を加えた保存血液；これらの分画物（例えば、血漿、血清）；などを含む。 Blood samples include blood itself and blood diluted with physiological saline; preserved blood obtained by adding additives such as glucose and anticoagulant to blood; these fractions (for example, plasma, serum); etc. include.
　血液試料からのセルフリーＤＮＡの抽出は、常法に従って実施してよい。以下に実施形態例を挙げる。
　被検者から例えば５ｍＬ～２０ｍＬの血液を、抗凝固剤入りの採血管に採取する。血液に遠心分離を施し、血球成分と血漿とに分離し、血漿を回収する。市販のセルフリーＤＮＡ抽出キット（例えば、QIAamp Circulating Nucleic Acid Kit（キアゲン社））を用いて血漿中のセルフリーＤＮＡを抽出する。 Extraction of cell-free DNA from blood samples may be performed according to a conventional method. Examples of embodiments are given below.
For example, 5 mL to 20 mL of blood is collected from a subject in a blood collection tube containing an anticoagulant. The blood is centrifuged to separate the blood cell component and plasma, and the plasma is collected. Cell-free DNA in plasma is extracted using a commercially available cell-free DNA extraction kit (for example, QIAamp Circulating Nucleic Acid Kit (Qiagen)).
［工程Ｂ］
　工程Ｂは、セルフリーＤＮＡのメチル化を解析し、被検者のＤＮＡメチル化パターンを得る工程である。 [Step B]
Step B is a step of analyzing the methylation of cell-free DNA to obtain a DNA methylation pattern of a subject.
　セルフリーＤＮＡには、ｃｔＤＮＡ（circulating tumor DNA）のほかに、正常細胞由来のゲノムＤＮＡも含まれており、特に血液細胞由来のゲノムＤＮＡが多く含まれている。ｃｔＤＮＡは、がんの種類や進行度により異なるが、初期のがんの場合は、セルフリーＤＮＡの１％～５％程度といわれている。ｃｔＤＮＡのメチル化情報を得るために、ＰＣＲ（polymerase chain reaction）によってセルフリーＤＮＡを増幅し、増幅産物の配列解析を行うことが好ましい。 In addition to ctDNA (circulating tumor DNA), cell-free DNA also contains genomic DNA derived from normal cells, and in particular, a large amount of genomic DNA derived from blood cells. The ctDNA varies depending on the type and degree of progression of the cancer, but in the case of early stage cancer, it is said to be about 1% to 5% of the cell-free DNA. In order to obtain methylation information of ctDNA, it is preferable to amplify the cell-free DNA by PCR (polymerase chain reaction) and perform sequence analysis of the amplification product.
　セルフリーＤＮＡのメチル化の解析の実施形態例として、バイサルファイトシーケンス法が挙げられる。バイサルファイトシーケンス法のある実施形態例は、セルフリーＤＮＡをバイサルファイト処理することと、基準パターンの構成要素であるＤＮＡメチル化を解析するためのプライマーセットを用いてマルチプレックスＰＣＲを行うことと、シーケンサーを用いて増幅産物の配列解析することと、を含む。以下に、バイサルファイトシーケンス法の実施形態例を説明する。 An example of an embodiment of the analysis of methylation of cell-free DNA is the bisulfite sequencing method. Examples of embodiments of the bisulfite sequencer method include bisulfite treatment of cell-free DNA, multiplex PCR using a primer set for analyzing DNA methylation, which is a component of the reference pattern, and Includes sequence analysis of amplification products using a sequencer. An embodiment of the bisulfite sequencing method will be described below.
－セルフリーＤＮＡをバイサルファイト処理すること－
　ＤＮＡ中の非メチル化シトシンはバイサルファイト処理によってウラシルに変換されるが、ＤＮＡ中のメチル化シトシンはバイサルファイト処理の影響を受けない。したがって、バイサルファイト処理によってメチル化の有無が配列の相違に変換され、配列解析によってメチル化の有無が検出可能となる。 -Treatment of cell-free DNA with bisulfite-
Unmethylated cytosine in DNA is converted to uracil by bisulfite treatment, whereas methylated cytosine in DNA is unaffected by bisulfite treatment. Therefore, the presence or absence of methylation is converted into a sequence difference by bisulfite treatment, and the presence or absence of methylation can be detected by sequence analysis.
　ＤＮＡのバイサルファイト処理は、常法に従って実施してよい。市販のバイサルファイト処理キット（例えば、EZ DNA Methylation Kit（ザイモリサーチ社）、EZ DNA Methylation-Gold Kit（ザイモリサーチ社）、EpiSight Bisulfite Conversion Kit Ver.2（富士フイルム和光純薬株式会社））を用いてセルフリーＤＮＡを処理してよい。 DNA bisulfite treatment may be carried out according to a conventional method. Using a commercially available bisulfite treatment kit (for example, EZDNA Methylation Kit (Zymo Research), EZ DNA Methylation-Gold Kit (Zymo Research), EpiSight Bisulfite Conversion Kit Ver.2 (Fuji Film Wako Pure Chemical Industries, Ltd.)) Cell-free DNA may be processed.
－基準パターンの構成要素であるＤＮＡメチル化を解析するためのプライマーセットを用いてマルチプレックスＰＣＲを行うこと－
　複数種のがんを同時に解析対象とするために、基準パターンの構成要素であるＤＮＡメチル化を解析するためのプライマーセットを用いて、マルチプレックスＰＣＲを行う。プライマーセットは、基準パターンの構成要素であるＤＮＡメチル化を網羅的に解析可能に設計されている。 -Perform multiplex PCR using a primer set to analyze DNA methylation, which is a component of the reference pattern-
In order to analyze multiple types of cancer at the same time, multiplex PCR is performed using a primer set for analyzing DNA methylation, which is a component of the reference pattern. The primer set is designed so that DNA methylation, which is a component of the reference pattern, can be comprehensively analyzed.
　マルチプレックスＰＣＲのＰＣＲ試薬としては、Multiplex PCR Kit（タカラバイオ社）、Multiplex PCR Kit ver2（タカラバイオ社）、KAPA library amplification Kit（KAPA Biosystems社）、Platinum Multiplex PCR master mix Kit（サーモフィッシャー社）、KOD-Multi & Epi-（東洋紡社）等が挙げられる。 Multiplex PCR PCR reagents include Multiplex PCR Kit (Takara Bio Inc.), Multiplex PCR Kit ver2 (Takara Bio Inc.), KAPA library amplification Kit (KAPA Biosystems Inc.), Platinum Multiplex PCR master mix Kit (Thermo Fisher), Examples include KOD-Multi & Epi- (Toyo Spinning Co., Ltd.).
　マルチプレックスＰＣＲは、常法に従って実施してよい。増幅産物が所望の量になるまでＰＣＲサイクルを繰り返す。 Multiplex PCR may be performed according to a conventional method. Repeat the PCR cycle until the amount of amplification product is as desired.
　マルチプレックスＰＣＲにおいて使用するプライマーセットは、多種類のプライマー対を含むところ、プライマーごとにテンプレートＤＮＡにアニーリング可能な温度が異なる。マルチプレックスＰＣＲの反応条件（つまり、変性、アニーリング及び伸長それぞれの温度及び時間）を、注目するがん種を検出するためのプライマー対に合わせて適切化することが好ましい。これによって、注目するがん種の解析精度を上げることができる。注目するがん種が複数ある場合は、マルチプレックスＰＣＲを複数回行い、各回をそれぞれ別の一つのがん種に合わせて適切化してもよい。 The primer set used in multiplex PCR contains many types of primer pairs, but the temperature at which the template DNA can be annealed differs for each primer. It is preferable to optimize the reaction conditions of multiplex PCR (ie, the temperature and time of each of denaturation, annealing and elongation) according to the primer pair for detecting the cancer type of interest. As a result, the accuracy of analysis of the cancer type of interest can be improved. If there are multiple cancer types of interest, multiplex PCR may be performed multiple times, each of which may be adapted to another cancer type.
－シーケンサーを用いて配列解析すること－
　マルチプレックスＰＣＲの増幅産物について、シーケンサーを用いて配列解析をする。 -Sequence analysis using a sequencer-
Sequence analysis is performed on the amplification product of multiplex PCR using a sequencer.
　シーケンサーは、第一世代シーケンサー（キャピラリーシーケンサー）、第二世代シーケンサー（次世代シーケンサー）、第三世代シーケンサー、第四世代シーケンサー、及び今後開発されるシーケンサーを含む用語である。シーケンサーは、キャピラリーシーケンサーでもよく、次世代シーケンサーでもよく、その他のシーケンサーでもよい。シーケンサーとしては、解析の速さ、１度に処理可能な試料数の多さ等の観点から、次世代シーケンサーが好ましい。次世代シーケンサー（next generation sequencer，ＮＧＳ）とは、サンガー法を利用したキャピラリーシーケンサー（第一世代シーケンサーと呼ばれる。）に対比して分類されるシーケンサーを指す。現時点で最も普及している次世代シーケンサーは、ＤＮＡポリメラーゼによる相補鎖合成又はＤＮＡリガーゼによる相補鎖結合に連動した蛍光又は発光をとらえ塩基配列を決定する原理のシーケンサーである。具体的には、MiSeq（イルミナ社）、HiSeq2000（イルミナ社、HiSeqは登録商標）、Roche454（ロシュ社）等が挙げられる。 Sequencer is a term that includes the first generation sequencer (capillary sequencer), the second generation sequencer (next generation sequencer), the third generation sequencer, the fourth generation sequencer, and the sequencer to be developed in the future. The sequencer may be a capillary sequencer, a next-generation sequencer, or another sequencer. As the sequencer, the next-generation sequencer is preferable from the viewpoint of the speed of analysis, the large number of samples that can be processed at one time, and the like. The next-generation sequencer (NGS) refers to a sequencer that is classified in comparison with a capillary sequencer (called a first-generation sequencer) that uses the Sanger method. The next-generation sequencer that is most widely used at present is a sequencer whose principle is to determine the base sequence by capturing fluorescence or luminescence linked to complementary strand synthesis by DNA polymerase or complementary strand binding by DNA ligase. Specific examples thereof include MiSeq (Illumina), HiSeq2000 (Illumina and HiSeq are registered trademarks), Roche454 (Roche) and the like.
　シーケンサー（好ましくはＮＧＳ）で取得した塩基配列データに基づき、各リードをがん種毎に並べ、さらにＤＮＡ中の各シトシンのメチル化状態を判別し、被検者のＤＮＡメチル化パターンを取得する。 Based on the nucleotide sequence data acquired by a sequencer (preferably NGS), each read is arranged for each cancer type, the methylation state of each cytosine in DNA is determined, and the DNA methylation pattern of the subject is acquired. ..
［工程Ｃ］
　工程Ｃは、被検者のＤＮＡメチル化パターンからがんの存否及び原発巣を推定する工程である。工程Ｃの実施形態例としては、被検者のＤＮＡメチル化パターンを基準パターンに照合し、がんの存否及び原発巣を推定することが挙げられる。
　照合の方法は、例えば、計測したメチル化の定量値を、遺伝子ごとにメチル化度として百分率（パーセンテージ）で表し、数値を比較する。
　陽性の判断基準は、例えば、被検者のＤＮＡメチル化パターンと、基準パターンとの間に一致点又は類似点があれば、その一致点又は類似点に相当するがん種及び原発巣を陽性とする。陽性の判断基準は、より具体的には、例えば、被験者のＤＮＡメチル化パターンと、あるがん種に特徴的なＤＮＡメチル化パターンとが５０％以上の遺伝子で類似している場合、そのがん種及び原発巣を陽性とする。 [Step C]
Step C is a step of estimating the presence or absence of cancer and the primary lesion from the DNA methylation pattern of the subject. An example of an embodiment of step C is to collate the DNA methylation pattern of the subject with a reference pattern to estimate the presence or absence of cancer and the primary lesion.
In the collation method, for example, the measured quantitative value of methylation is expressed as a percentage as the degree of methylation for each gene, and the numerical values are compared.
The positive criteria are, for example, if there is a coincidence or similarity between the DNA methylation pattern of the subject and the reference pattern, the cancer type and primary lesion corresponding to the coincidence or similarity are positive. And. More specifically, for example, when the DNA methylation pattern of a subject and the DNA methylation pattern characteristic of a certain cancer type are similar in 50% or more of genes, the positive criterion is Species and primary lesions are positive.
　陽性の結果が出たがん種及び原発巣については、がん種及び原発巣に応じた適切な二次スクリーニング（例えば、画像検査、生体組織検査）を行うことが望ましい。 For cancer types and primary lesions that give a positive result, it is desirable to perform appropriate secondary screening (for example, imaging test, biopsy test) according to the cancer type and primary lesion.
［工程Ｄ］
　工程Ｄは、被検者から質問票への回答を得る工程である。工程Ｄは、被検者の身体情報及び臨床情報を得る目的で行う工程である。 [Step D]
Step D is a step of obtaining an answer to the questionnaire from the subject. Step D is a step performed for the purpose of obtaining physical information and clinical information of the subject.
　質問票とは、質問項目が記載されており、質問項目に対する被検者の回答が記入される媒体である。質問票は、紙でもよく、電子媒体でもよい。 The questionnaire is a medium in which the question items are described and the answers of the subjects to the question items are entered. The questionnaire may be paper or electronic media.
　質問項目は、被検者の身体情報及び臨床情報を得る目的で質問票に盛り込まれる。質問項目は、がんについての知見（例えば、学術論文、患者調査、がん統計、がん遺伝子データベース）を参照し、がん患者の属性と遺伝子異常との因果関係又は相関関係の情報又は根拠を得て作成することが好ましい。質問項目は、選択式質問でもよく、記述式質問でもよい。 Question items are included in the questionnaire for the purpose of obtaining physical information and clinical information of the subject. For questions, refer to the findings about cancer (for example, academic papers, patient surveys, cancer statistics, oncogene databases), and information or rationale for the causal relationship or correlation between the attributes of cancer patients and genetic abnormalities. It is preferable to obtain and prepare. The question item may be a multiple-choice question or a descriptive question.
　質問項目としては、例えば、性別、年齢、身体的特徴（例えば、身長、体重、腹囲、胸囲）、既往歴、家族歴（例えば、三親等内の血族の既往歴。例えば、父母、兄弟、姉妹、祖父母、父母の兄弟、及び父母の姉妹の各既往歴）、生活習慣（例えば、喫煙習慣、飲酒習慣、食習慣、運動習慣）、過去の健康診断の結果（例えば、血液検査の検査値　尿検査の検査値、細菌又はウイルスの感染の所見、画像検査の所見、遺伝子検査の所見）、個人ゲノム情報が挙げられる。 Question items include, for example, gender, age, physical characteristics (eg, height, weight, abdominal circumference, chest circumference), medical history, family history (eg, medical history of relatives within the third degree of kinship, such as parents, brothers, and sisters. , Grandparents, parents'brothers, and parents' sisters), lifestyle (eg smoking habits, drinking habits, eating habits, exercise habits), results of past health examinations (eg blood test values urine Test values of tests, findings of bacterial or viral infections, findings of imaging tests, findings of genetic tests), personal genome information can be mentioned.
　質問票への記入は、被検者が自分で質問票を読み自分で回答を記入してもよく、被検者が質問者の質問に対して口頭で回答したことを質問者又は第三者が記入してもよい。質問者は、例えば、医師、看護師、検査技師、看護者、介護者、親である。
　過去の健康診断の結果（例えば、血液検査の検査値　尿検査の検査値、細菌又はウイルスの感染の所見、画像検査の所見、遺伝子検査の所見）又は個人ゲノム情報についての回答は、検査値の表、画像、又は電子データを質問票に添付することでもよい。 To fill out the questionnaire, the subject may read the questionnaire and fill in the answer by himself, or the questioner or a third party that the subject answered the questioner's question verbally. May be filled in. Questioners are, for example, doctors, nurses, laboratory technicians, nurses, caregivers, and parents.
Answers about past health test results (eg, blood test values, urinalysis test values, bacterial or viral infection findings, imaging test findings, genetic test findings) or personal genomic information are the test values. A table, image, or electronic data may be attached to the questionnaire.
［工程Ｅ］
　工程Ｅは、注目するがん種に応じて解析条件を適切化して工程Ｂを行う目的で、被検者から得た質問票への回答に基づき注目するがん種を決定する工程である。 [Step E]
Step E is a step of determining the cancer type of interest based on the answers to the questionnaire obtained from the subject for the purpose of performing step B by optimizing the analysis conditions according to the cancer type of interest.
　被検者の回答に基づき注目するがん種を決定することは、予め用意した決定基準に従って行うことが好ましい。決定基準は、公的研究機関又は学術団体が作成又は編集した統計資料などを参照して作成することが好ましい。決定基準は、がんの決定基準を網羅した形態であって、コンピュータが実行するプログラムである形態が好ましい。 It is preferable to determine the type of cancer to be noted based on the answers of the subjects according to the determination criteria prepared in advance. The decision criteria are preferably prepared with reference to statistical data prepared or edited by public research institutes or academic societies. The determinant is preferably a form that covers the determinants of cancer and is a program executed by a computer.
　日本人を被検者とする場合、決定基準は、例えば、国立がん研究センターが作成又は編集した統計資料、日本臨床腫瘍学会編集「新臨床腫瘍学」等の文献、又は学術論文を参照して作成する。例えば、「新臨床腫瘍学」に記載されている遺伝性がんを三親等内の血族が罹患していた場合には、被検者のその他の属性にかかわらず、その遺伝性がんに注目する、との決定基準を作成する。 When Japanese subjects are selected, the criteria for determination refer to, for example, statistical data prepared or edited by the National Cancer Center, literature such as "New Clinical Oncology" edited by the Japanese Society of Medical Oncology, or academic papers. To create. For example, if a hereditary cancer described in "New Clinical Oncology" is affected by a relative within the third degree of kinship, pay attention to the hereditary cancer regardless of the other attributes of the subject. Create the criteria for deciding to do.
　以下、工程Ｅの流れについて、図２に示すフローチャートを用いて説明する。図２は、工程Ｅの概念を説明するための例示であり、実施形態を限定するものではない。 Hereinafter, the flow of step E will be described with reference to the flowchart shown in FIG. FIG. 2 is an example for explaining the concept of step E, and does not limit the embodiment.
　図２は、肝細胞がんを例にして流れを説明するフローチャートである。本フローは、肝細胞がんを注目対象とするか否かを、質問票への回答から得た情報であるところの、被検者の年齢、飲酒習慣及び肝炎ウイルスの持続感染の有無に基づき決定する。 FIG. 2 is a flowchart illustrating the flow using hepatocellular carcinoma as an example. This flow is based on the age of the subject, drinking habits, and the presence or absence of persistent hepatitis virus infection, which is the information obtained from the answers to the questionnaire as to whether or not hepatocellular carcinoma should be the focus of attention. decide.
　まず、Ｓ５１１に示す段階において、被検者に肝炎ウイルスの持続感染があるか否かを確認する。被検者に肝炎ウイルスの持続感染がある場合は、Ｓ５１４に示す段階へ進み、肝細胞がんを注目対象に決定する。被検者に肝炎ウイルスの持続感染がない場合は、Ｓ５１２に示す段階へ進む。 First, at the stage shown in S511, it is confirmed whether or not the subject has persistent hepatitis virus infection. If the subject has persistent hepatitis virus infection, proceed to the stage shown in S514 and determine hepatocellular carcinoma as the target of attention. If the subject does not have persistent hepatitis virus infection, the process proceeds to the stage shown in S512.
　Ｓ５１２に示す段階において、被検者の年齢が５０歳以上であるか否かを確認する。被検者の年齢が５０歳以上である場合は、Ｓ５１４に示す段階へ進み、肝細胞がんを注目対象に決定する。 At the stage shown in S512, it is confirmed whether or not the subject's age is 50 years or older. When the subject's age is 50 years or older, the process proceeds to the stage shown in S514, and hepatocellular carcinoma is determined as a target of interest.
　Ｓ５１２に示す段階において被検者の年齢が５０歳未満である場合は、Ｓ５１３に示す段階へ進み、飲酒の頻度及び量を確認する。飲酒の頻度及び量が所定基準以上である場合は、Ｓ５１４に示す段階へ進み、肝細胞がんを注目対象に決定する。 If the subject's age is less than 50 years old at the stage shown in S512, proceed to the stage shown in S513 and check the frequency and amount of drinking. If the frequency and amount of alcohol drinking is equal to or higher than the predetermined standard, the process proceeds to the stage shown in S514, and hepatocellular carcinoma is determined as a target of interest.
　Ｓ５１３に示す段階において飲酒の頻度及び量が所定基準未満である場合は、Ｓ５１５に示す段階へ進み、肝細胞がんを注目対象にしないと決定する。 If the frequency and amount of alcohol consumption is less than the predetermined standard at the stage shown in S513, the process proceeds to the stage shown in S515, and it is decided not to focus on hepatocellular carcinoma.
　Ｓ５１４に示す段階又はＳ５１５に示す段階の完了後、図１における工程Ｅに戻る。 After the step shown in S514 or the step shown in S515 is completed, the process returns to step E in FIG.
　工程Ｅの実施形態例としては、がん種を網羅したプログラムに従って、図２に示すような流れをコンピュータが網羅的に処理する形態が好ましい。 As an example of the embodiment of the step E, it is preferable that the computer comprehensively processes the flow as shown in FIG. 2 according to the program covering the cancer types.
　以下、実施例により発明の実施形態をさらに説明するが、発明の実施形態は、これら実施例に何ら限定されるものではない。 Hereinafter, embodiments of the invention will be further described with reference to Examples, but the embodiments of the invention are not limited to these Examples.
　下記において、物質濃度に関し「Ｍ」はモル濃度を表し、１Ｍ＝１ｍｏｌ／Ｌである。物質濃度に関し、特に断りのない限り、「％」は質量基準である。 In the following, regarding the substance concentration, "M" represents the molar concentration, and 1M = 1 mol / L. Regarding the substance concentration, "%" is based on mass unless otherwise specified.
＜実施例１＞
［がん種毎に特徴的なメチル化部位の選定、及び基準パターンの構築］
　正常組織とがん組織とに関するゲノム・エピゲノム・トランスクリプトームの公共データベースに登録済みの情報（正常組織約４０検体、がん組織約４００検体）を比較解析し、がん種毎に特徴的なメチル化部位を抽出した。そして、早期発見の意義が高い膵臓がん、大腸がん及び肝細胞がんについて、それぞれのメチル化パターンを作成し、これらをセットにして基準パターンとした。 <Example 1>
[Selection of characteristic methylation sites for each cancer type and construction of reference patterns]
Information registered in the public database of genome, epigenome, and transcriptome about normal tissue and cancer tissue (about 40 samples of normal tissue, about 400 samples of cancer tissue) is compared and analyzed, and it is characteristic for each cancer type. The methylation site was extracted. Then, for pancreatic cancer, colon cancer, and hepatocellular carcinoma, which are highly significant for early detection, methylation patterns were created for each, and these were set as a reference pattern.
［プライマーの設計及び準備］
　上記の基準パターンを構成するメチル化部位８８ヵ所に対する各プライマー対を設計し、これらを合わせてプライマーセットとした。
　選定した８８ヵ所のプライマー対の合成はファスマック社にて行った。各プライマー（１００μＭ）を単一チューブに等量混合し、混合液を１μＭに希釈したものをプライマーセットとした。 [Primer design and preparation]
Each primer pair for 88 methylation sites constituting the above reference pattern was designed, and these were combined to form a primer set.
The 88 selected primer pairs were synthesized by Fasmac. Equal amounts of each primer (100 μM) were mixed in a single tube, and the mixture was diluted to 1 μM to prepare a primer set.
［被検者から質問票への回答を得る工程］
　以下の質問項目を記載した質問票（媒体は紙である。）を被検者に渡した。被検者が回答を記入した質問票を回収した。
１．性別、年齢
２．身長、体重、腹囲、胸囲
３．既往歴
４．家族歴（父母、兄弟、姉妹、祖父母、父母の兄弟、及び父母の姉妹の各既往歴）
５．生活習慣（喫煙の有無及び量、飲酒の頻度及び量、食習慣、運動習慣）
６．過去の健康診断の結果（血液検査、画像所見） [Process of obtaining answers to questionnaires from subjects]
A questionnaire containing the following question items (the medium is paper) was given to the subject. A questionnaire was collected in which the subject filled in the answers.
1. 1. Gender, age 2. Height, weight, abdominal circumference, chest circumference 3. Medical history 4. Family history (history of parents, siblings, sisters, grandparents, parents'brothers, and parents' sisters)
5. Lifestyle (presence / absence and amount of smoking, frequency and amount of drinking, eating habits, exercise habits)
6. Results of past health examinations (blood tests, imaging findings)
［被検者の回答に基づき注目するがん種を決定する工程］
　被検者の回答から、男性、年齢４０代、肥満という特徴が得られた。「科学的根拠に基づくがんリスク評価とがん予防ガイドライン提言に関する研究」（国立がん研究センター）を参照し、肥満との関連が示唆されている大腸がんを注目対象に決定した。 [Process of determining the type of cancer to be noted based on the answers of the subjects]
From the answers of the subjects, the characteristics of male, age 40s, and obesity were obtained. With reference to "Scientific evidence-based cancer risk assessment and research on cancer prevention guideline recommendations" (National Cancer Center), colorectal cancer, which has been suggested to be associated with obesity, was selected as the focus of attention.
［セルフリーＤＮＡの抽出］
　QIAamp Circulating Nucleic Acid Kit（キアゲン社）を用いて、当該キットのプロトコルに従って、被検者由来の血漿と健常者由来の血漿からそれぞれセルフリーＤＮＡの抽出を行った。
　５０ｍＬ遠心チューブに２００μＬのQIAGEN Proteinase Kを添加した。次いで、チューブに血漿を２ｍＬ添加した。１．６ｍＬのBuffer ACL（１．０μｇのキャリア　ＲＮＡを含む。）を添加し、蓋を閉めてパルスボルテックスで３０秒間混和した。６０℃で３０分間インキュベートの後、３．６ｍＬのBuffer ACBをチューブ中のライセートに添加し蓋を閉め、１５～３０秒間パルスボルテックスして完全に混和した。氷上で５分間インキュベート後に、QIAvac 24 PlusにVacConnectorとQIAamp Mini Columnを用いて抽出ＤＮＡの洗浄を行い、２０μＬの溶出液（Buffer AVE）によりセルフリーＤＮＡを回収した。 [Extraction of cell-free DNA]
Using the QIAamp Circulating Nucleic Acid Kit (Qiagen), cell-free DNA was extracted from plasma derived from a subject and plasma derived from a healthy subject according to the protocol of the kit.
200 μL of QIAGEN Proteinase K was added to a 50 mL centrifuge tube. Then 2 mL of plasma was added to the tube. 1.6 mL of Buffer ACL (containing 1.0 μg of carrier RNA) was added, the lid was closed and mixed with pulse vortex for 30 seconds. After incubation at 60 ° C. for 30 minutes, 3.6 mL of Buffer ACB was added to the lysate in the tube, the lid was closed, and pulse vortexed for 15-30 seconds for complete mixing. After incubation on ice for 5 minutes, the extracted DNA was washed with QIAvac 24 Plus using VacConnector and QIAamp Mini Column, and cell-free DNA was recovered with 20 μL of eluate (Buffer AVE).
［ＤＮＡのバイサルファイト処理］
　バイサルファイト処理は、EZ DNA Methylation-Gold Kit（ザイモリサーチ社）を用いて、当該キットのプロトコルに従って行った。
　８連ＰＣＲチューブに１０ｎｇのセルフリーＤＮＡを入れ、水で２０μＬにメスアップした。次に、１３０μＬのLightning Conversion Reagentを添加し混和した。そのうち７５μＬを新しいチューブに移し、９８℃／８分、次いで５４℃／６０分、インキュベートした。反応液を、キット付属のスピンカラムに添加し洗浄液（M-Wash buffer）で洗浄後、１０μＬの溶出液（M-Elution buffer）で回収した。 [DNA bisulfite treatment]
Bisulfite treatment was performed using the EZ DNA Methylation-Gold Kit (Zymo Research) according to the protocol of the kit.
10 ng of cell-free DNA was placed in an 8-unit PCR tube, and the volume was adjusted to 20 μL with water. Next, 130 μL of Lightning Conversion Reagent was added and mixed. 75 μL of this was transferred to a new tube and incubated for 98 ° C / 8 min and then 54 ° C / 60 min. The reaction solution was added to the spin column attached to the kit, washed with a washing solution (M-Wash buffer), and then recovered with a 10 μL eluate (M-Elution buffer).
［マルチプレックスＰＣＲ］
　注目対象が大腸がんであることから、大腸がんのメチル化パターンに対する各プライマーが反応しやすいＰＣＲ条件を検討し、反応温度、反応時間、及びプライマー濃度を最適化した。
　マルチプレックスＰＣＲは、KOD-Multi & Epi-（東洋紡（株）社）を用いて、当該キットのプロトコルに従って行った。
　８連ＰＣＲチューブに、2x PCR Buffer for KOD-Multi & Epi-を２５μＬ、KOD-Multi & Epi-を１μＬ、１μＭプライマーミックスを１５μＬ、バイサルファイト済みＤＮＡを８．５μＬ、水を０．５μＬ添加した。ＰＣＲは、９４℃／２分を１サイクル、９８℃／１０秒、５８℃／３０秒、６８℃／１５秒の３ステップを４０サイクル行った。増幅反応液は、AMPure XP Kit（ベックマン・コールター社）により精製し、４０μＬのTE bufferに回収した。
　ＮＧＳに供するためのＩｎｄｅｘ付加ＰＣＲは、Multiplex PCR Assay kit（タカラバイオ社）を用いて反応を行った。８連ＰＣＲチューブに、１．２５μＭ　ＰＣＲプライマーを各１μＬ、Multiplex PCR Mix1を０．１２５μＬ、Multiplex PCR Mix2を１２．５μＬ添加し、水で２５μＬにメスアップした。ＰＣＲは、９４℃／３分で変性した後、９４℃／４５秒、５０℃／６０秒、７２℃／３０秒の３ステップを５サイクル行い、９４℃／４５秒、５５℃／６０秒、７２℃／３０秒の３ステップを１１サイクル行った。
　次世代シーケンサー（MiSeq（イルミナ社））を用いてデュアルインデックス・シーケンシングを行うため、Miseq Reagent Kit v2 300 Cycle（イルミナ社）を用いて、マルチプレックスＰＣＲによって得られたＤＮＡ断片の両端にそれぞれ、フローセル結合用配列（Ｐ５配列又はＰ７配列）、サンプル識別用のインデックス配列、及びシークエンスプライマー結合用配列を含む２種類のアダプターを付加した。 [Multiplex PCR]
Since the subject of interest is colorectal cancer, the PCR conditions under which each primer easily reacts with the methylation pattern of colorectal cancer were examined, and the reaction temperature, reaction time, and primer concentration were optimized.
Multiplex PCR was performed using KOD-Multi & Epi- (Toyobo Co., Ltd.) according to the protocol of the kit.
To 8 consecutive PCR tubes, 25 μL of 2x PCR Buffer for KOD-Multi & Epi-, 1 μL of KOD-Multi & Epi-, 15 μL of 1 μM primer mix, 8.5 μL of bisulfite DNA, and 0.5 μL of water were added. .. PCR was performed for 40 cycles of 3 steps of 94 ° C./2 minutes for 1 cycle, 98 ° C./10 seconds, 58 ° C./30 seconds, and 68 ° C./15 seconds. The amplification reaction solution was purified by AM Pure XP Kit (Beckman Coulter, Inc.) and recovered in a 40 μL TE buffer.
Index-added PCR for use in NGS was reacted using the Multiplex PCR Assay kit (Takara Bio Inc.). 1 μL of each of 1.25 μM PCR primers, 0.125 μL of Multiplex PCR Mix 1 and 12.5 μL of Multiplex PCR Mix 2 were added to the 8-unit PCR tube, and the mixture was adjusted to 25 μL with water. PCR was denatured at 94 ° C./3 minutes, followed by 5 cycles of 3 steps of 94 ° C./45 seconds, 50 ° C./60 seconds, and 72 ° C./30 seconds, and 94 ° C./45 seconds, 55 ° C./60 seconds. Three steps of 72 ° C./30 seconds were performed for 11 cycles.
In order to perform dual index sequencing using the next-generation sequencer (MiSeq (Illumina)), the Miseq Reagent Kit v2 300 Cycle (Illumina) was used at both ends of the DNA fragment obtained by multiplex PCR. Two types of adapters were added, including a flow cell binding sequence (P5 or P7 sequence), a sample identification index sequence, and a sequence primer binding sequence.
［ＮＧＳを用いたシーケンス］
　得られたＰＣＲ産物は、AMPure XP Kit（ベックマン・コールター社）を用いて精製し、BioAnalyzer（アジレント・テクノロジー株式会社）を用いて濃度を測定した。より正確な増幅産物の定量として、KAPA Library Quantification Kits（KAPA Biosystems社）を用いて定量を行い、シーケンスライブラリを取得した。次世代シーケンサー（MiSeq（イルミナ社））を用いてシーケンスライブラリのシーケンスを行った。得られたＦａｓｔＱファイルをＢＷＡにてヒトゲノム配列（ｈｇ１９）へマッピングを行い、メチル化の有無を判定し、被検者のＤＮＡメチル化パターン、健常者のＤＮＡメチル化パターンをそれぞれ取得した。 [Sequence using NGS]
The obtained PCR product was purified using AM Pure XP Kit (Beckman Coulter), and the concentration was measured using BioAnalyzer (Agilent Technology Co., Ltd.). For more accurate quantification of amplification products, quantification was performed using KAPA Library Quantification Kits (KAPA Biosystems), and a sequence library was obtained. The sequence library was sequenced using a next-generation sequencer (MiSeq (Illumina)). The obtained FastQ file was mapped to the human genome sequence (hg19) by BWA, the presence or absence of methylation was determined, and the DNA methylation pattern of the subject and the DNA methylation pattern of the healthy subject were obtained, respectively.
［がんの存否と原発巣の推定］
　被検者のＤＮＡメチル化パターン及び健常者のＤＮＡメチル化パターンを、先述の基準パターンと照合した。その結果、被検者が大腸がんであると推定された。 [Presence or absence of cancer and estimation of primary lesion]
The DNA methylation pattern of the subject and the DNA methylation pattern of the healthy subject were collated with the above-mentioned reference pattern. As a result, it was estimated that the subject had colorectal cancer.
　２０２０年３月２５日に出願された日本国出願番号第２０２０－０５５１１８号の開示は、その全体が参照により本明細書に取り込まれる。
　本明細書に記載された全ての文献、特許出願、及び技術規格は、個々の文献、特許出願、及び技術規格が参照により取り込まれることが具体的かつ個々に記された場合と同程度に、本明細書中に参照により取り込まれる。 The disclosure of Japanese Application No. 2020-055118, filed March 25, 2020, is incorporated herein by reference in its entirety.
All documents, patent applications, and technical standards described herein are to the same extent as if the individual documents, patent applications, and technical standards were specifically and individually stated to be incorporated by reference. Incorporated herein by reference.

Claims (7)

  1. 　被検者の血液試料に含まれるセルフリーＤＮＡを抽出する工程Ａと、
    　前記セルフリーＤＮＡのメチル化を解析し、前記被検者のＤＮＡメチル化パターンを得る工程Ｂと、
    　前記被検者のＤＮＡメチル化パターンからがんの存否及び原発巣を推定する工程Ｃと、
    　を含むがん検査方法。     Step A to extract cell-free DNA contained in the blood sample of the subject, and
    Step B, in which the methylation of the cell-free DNA is analyzed to obtain the DNA methylation pattern of the subject,
    Step C of estimating the presence or absence of cancer and the primary lesion from the DNA methylation pattern of the subject, and
    Cancer screening methods including.
  2. 　前記工程Ｂの前に、前記被検者から質問票への回答を得る工程Ｄと、前記回答に基づき注目するがん種を決定する工程Ｅと、をさらに含み、
    　前記工程Ｂを、前記注目するがん種に応じて解析条件を適切化して行う、請求項１に記載のがん検査方法。     Prior to the step B, a step D of obtaining an answer to the questionnaire from the subject and a step E of determining the cancer type of interest based on the answer are further included.
    The cancer screening method according to claim 1, wherein the step B is performed with appropriate analysis conditions according to the cancer type of interest.
  3. 　前記質問票は、性別、年齢、身体的特徴、既往歴、家族歴、生活習慣、過去の健康診断の結果、及び個人ゲノム情報からなる群から選ばれる１つ以上の項目を含む、請求項２に記載のがん検査方法。 The questionnaire includes one or more items selected from the group consisting of gender, age, physical characteristics, medical history, family history, lifestyle, past medical examination results, and personal genomic information. Cancer screening method described in.
  4. 　前記工程Ｃが、前記被検者のＤＮＡメチル化パターンを、がん種毎に特徴的なＤＮＡメチル化パターンをセットにした基準パターンに照合することを含む、請求項１～請求項３のいずれか１項に記載のがん検査方法。 Any of claims 1 to 3, wherein step C collates the DNA methylation pattern of the subject with a reference pattern in which a DNA methylation pattern characteristic of each cancer type is set. The cancer testing method described in item 1.
  5. 　前記基準パターンが、がん種毎にがん患者のＤＮＡと健常者のＤＮＡとの間のメチル化を比較して得たがん種毎に特徴的なＤＮＡメチル化パターンをセットにした情報である、請求項４に記載のがん検査方法。 The reference pattern is information that sets the characteristic DNA methylation patterns for each cancer type obtained by comparing the methylation between the DNA of a cancer patient and the DNA of a healthy person for each cancer type. The cancer testing method according to claim 4.
  6. 　前記工程Ｂが、
    　前記セルフリーＤＮＡをバイサルファイト処理することと、
    　前記基準パターンの構成要素であるＤＮＡメチル化を解析するためのプライマーセットを用いてマルチプレックスＰＣＲを行うことと、
    　シーケンサーを用いて配列解析することと、
    　を含む、請求項４又は請求項５に記載のがん検査方法。     The step B
    By treating the cell-free DNA with bisulfite,
    Performing multiplex PCR using a primer set for analyzing DNA methylation, which is a component of the reference pattern,
    Sequence analysis using a sequencer and
    4. The cancer screening method according to claim 4 or 5.
  7. 　前記工程Ｂを、前記マルチプレックスＰＣＲの反応条件を適切化して行う、請求項６に記載のがん検査方法。 The cancer screening method according to claim 6, wherein the step B is performed by optimizing the reaction conditions of the multiplex PCR.
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Citations (2)

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WO2019028556A1 (en) * 2017-08-09 2019-02-14 Enrich Bioscience Inc. Method and system for analysis of dna methylation and use of same to detect cancer
JP2019504642A (en) * 2016-01-29 2019-02-21 エピゲノミクス・アクチェンゲゼルシャフトＥｐｉｇｅｎｏｍｉｃｓ ＡＧ Method for detecting CpG methylation of tumor-derived DNA in a blood sample

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JP2019504642A (en) * 2016-01-29 2019-02-21 エピゲノミクス・アクチェンゲゼルシャフトＥｐｉｇｅｎｏｍｉｃｓ ＡＧ Method for detecting CpG methylation of tumor-derived DNA in a blood sample
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