WO2019156054A1 - Marqueur pour la détermination de groupe de méthylation mlh1 et procédé de détermination - Google Patents

Marqueur pour la détermination de groupe de méthylation mlh1 et procédé de détermination Download PDF

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WO2019156054A1
WO2019156054A1 PCT/JP2019/003992 JP2019003992W WO2019156054A1 WO 2019156054 A1 WO2019156054 A1 WO 2019156054A1 JP 2019003992 W JP2019003992 W JP 2019003992W WO 2019156054 A1 WO2019156054 A1 WO 2019156054A1
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methylation
cancer
mlh1
group
marker
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正人 河津
山本　陽子
博行 間野
佐藤　一仁
敏秀 上野
久恵 飯沼
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国立大学法人東京大学
学校法人帝京大学
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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
    • 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
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    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase

Definitions

  • the present invention relates to an MLH1 methylation group determination marker, a method for determining whether a subject belongs to an MLH1 methylation group, an MLH1 methylation group determination kit, and the like.
  • the large intestine is a gastrointestinal tract having a length of about 1.6 m in humans, and is roughly divided into three parts: the cecum, rectum, and colon.
  • the large intestine is an important organ responsible for functions such as fermentation of dietary fiber by bacteria and absorption of moisture.
  • Japan the number of patients with colorectal cancer has been increasing in recent years. As of 2014, it is estimated that the number of patients with colorectal cancer is approximately 250,000 or more, and the number of deaths is large. Detection methods and effective treatment methods are eagerly desired.
  • MSI-H colorectal cancers that have mutations in repetitive microsatellite sequences.
  • MSI-H colorectal cancer is further divided into the following three subtypes: (1) MLH1 (mutL homolog 1) gene expression is suppressed epigenetically (MLH1 methylation group), (2) heritability LS (Lynch syndrome: Lynch syndrome) that has a loss-of-function mutation in the germline of the MMR (mismatch repair) gene, and (3) in both (1) and (2) above It can be classified into LL (Lynch-like) which does not belong.
  • the cause and treatment target of the above three subtypes are different, if a determination method can be found, it can be useful for diagnosis and treatment of colorectal cancer.
  • the MLH1 methylation group can be found in cancers other than colorectal cancer (for example, Non-patent document 1 for gastric cancer and Non-patent documents 2 to 4 for endometrial cancer), the determination method thereof can be found. It can also help in the diagnosis and treatment of these cancers.
  • Non-Patent Document 5 describes the region of S: 38chr3: 36,992,827-36,992,855 and AS: chr3: 36,992,927-36,992,950 (hereinafter referred to as “Herman region”) on the EPM2AIP1 gene existing upstream of the MLH1 gene. ) Methylation is described as being effective in identifying the MLH1 methylation group.
  • 19 of 21 microsatellite stability (MSS) tumors examined in the Herman region were determined to be unmethylated groups, but 2 were classified as MLH1 methylated groups as false positives. There was a problem in terms of determination accuracy.
  • MSS microsatellite stability
  • Non-Patent Document 5 is problematic in terms of accuracy in determining the MLH1 methylation group, and it is effective to provide a more accurate marker for cancer diagnosis and treatment. It is.
  • An object of the present invention is to provide a new marker, method, kit, and the like that can determine the MLH1 methylation group with high accuracy.
  • hr38 chr3 36,993,202-36,993,865 (nucleic acid molecule consisting of the base sequence of SEQ ID NO: 1) as a marker that can accurately determine the MLH1 methylation group
  • hr38 chr3 36,993,202-36,993,865
  • the present invention includes the following aspects.
  • (1) MutL homolog 1 (MLH1) methylation group determination marker comprising the following (i) to (iii): (I) a nucleic acid molecule comprising the base sequence represented by SEQ ID NO: 1, (Ii) a nucleic acid molecule comprising a base sequence in which one or more bases have been added, deleted and / or substituted in SEQ ID NO: 1, and (iii) comprising one or more CpG sequences, (i) or (ii).
  • the marker comprising any of the fragments of the nucleic acid molecule of (2)
  • a method for determining whether a subject belongs to the MLH1 methylation group A detection step for detecting methylation of one or more cytosines in the marker described in (1) in genomic DNA or free DNA obtained from the subject, and if methylation is detected in the detection step, the subject The method comprising the step of determining that is belonging to the MLH1 methylation group.
  • the subject is colon cancer, small intestine cancer, endometrial cancer, ovarian cancer, gastric cancer, renal pelvis / ureter cancer, pancreatic cancer, biliary tract cancer, brain tumor, sebaceous adenoma and keratophyte cell tumor in Muir-Torre syndrome.
  • a cancer selected from the group consisting of malignant melanoma, non-small cell lung cancer, prostate cancer, and thyroid cancer .
  • the colon cancer is high-frequency microsatellite unstable colon cancer.
  • Any one of (2) to (9), wherein the genomic DNA or free DNA is obtained from a sample selected from the group consisting of a biopsy tissue, a FFPE (formalin fixed paraffin embedded) sample, blood, and body fluid The method described in 1.
  • An MLH1 methylation group determination kit capable of detecting methylation of one or more cytosines in the marker described in (1), wherein methylation-sensitive restriction capable of cleaving a sequence containing a CpG sequence
  • the kit comprising at least one of an enzyme, a primer set capable of amplifying all or part of the marker, and a probe or antibody capable of detecting all or part of the marker.
  • the methylation sensitive restriction enzyme is selected from the group consisting of Sma I, Ehe I, Hin 6I, Hpa II, Acc II, and Hae II, and methylation sensitive isoschizomers of these enzymes,
  • the present invention makes it possible to determine the MLH1 methylation group with high accuracy. Since the three subtypes of MLH1 methylation group, Lynch syndrome, and Lynch-like in colorectal cancer have different causes and therapeutic targets, the present invention can be useful for diagnosis and treatment of cancer. In addition, determination of being an MLH1-methylated group for cancers other than colorectal cancer can be useful for cancer diagnosis and treatment.
  • FIG. 1 shows the region (Herman region) for identifying the MLH1 methylation group described in Herman JG. Et al (supra) in chr3 (hg38) and the region identified in Example 1 of the present application. It is a schematic diagram. The EPM2AIP1 gene and MLH1 gene are indicated by black boxes. The chromosomal location of the EPM2AIP1 gene and MLH1 gene is based on the NCBI database.
  • FIG. 2 shows a comparison between the methylation array result (A) and the methylation determination result (B) using a methylation sensitive restriction enzyme.
  • A methylation array result
  • B methylation determination result
  • the vertical axis indicates that different samples (# 1 to # 13) were used, and the horizontal axis indicates the methylation site analyzed by the methylation array from the 5 ′ side toward the 3 ′ side.
  • the color of each panel indicates the degree of methylation, and the darker the color, the higher the ⁇ value (the degree of methylation). NA is shaded.
  • FIG. 2A the Herman region and the region specified in Example 1 are indicated by dotted lines.
  • FIG. 2B shows the result of analyzing the same sample as that of FIG. 2A so as to correspond to FIG. 2A. That is, the result of electrophoresis in FIG. 2B corresponds to samples # 1 to # 13 in order from the top.
  • the bottom row is water
  • the bottom row is the negative control DLD1 (MSH6 mutant colorectal cancer cell line belonging to the MLH1 unmethylated group)
  • the bottom row is the positive control RKO (MLH1 methyl).
  • the results of the (Herogenetic colorectal cancer cell line) are shown.
  • BRAF is a control, and when a methylated MLH1 band is present, it can be determined that it is an MLH1 methylated group.
  • the determination result when using a methylation-sensitive restriction enzyme is shown on the right side of FIG. 2B (+ indicates an MLH1 methylation group, ⁇ indicates an MLH1 unmethylation group).
  • FIG. 3 shows the results of methylation determination using a methylation-sensitive restriction enzyme when frozen tissue or FFPE is used. 1 to 5 indicate that similar samples were used for FFPE and frozen tissue. FIG. 3 shows that similar results are obtained with frozen tissue and FFPE.
  • the present invention relates to a marker for determining MutL homolog 1 (MLH1) methylation group.
  • MLH1 refers to MutL homolog 1 located on the third chromosome.
  • MLH1 is a type of mismatch repair protein and is known to be associated with Lynch syndrome.
  • the “MLH1 methylation group” refers to a group in which the expression of the MLH1 gene is epigenetically suppressed by the methylation of the MLH1 gene.
  • MLH1 methylation group includes cancers such as colon cancer (eg, high frequency microsatellite instability (MSI-H) colon cancer), small intestine cancer, endometrial cancer, ovarian cancer, stomach cancer, renal pelvis / ureter cancer, It can also be found in other cancers such as pancreatic cancer, biliary tract cancer, brain tumor, Muir-Torre syndrome, malignant melanoma, non-small cell lung cancer, prostate cancer, and thyroid cancer.
  • MSI-H colorectal cancer refers to colorectal cancer in which a repetitive sequence of microsatellite is mutated, and is defined as a state showing MSI in BAT25 or BAT26, preferably both BAT25 and BAT26.
  • the MSI-H colorectal cancer accounts for about 10% of colorectal cancer and can be further classified into three subtypes: MLH1 methylation group, Lynch syndrome, and Lynch-like.
  • “Lynch syndrome” refers to a group having a loss-of-function mutation in any one or more of the mismatch repair genes in the germline.
  • the mismatch repair gene refers to MLH1, MSH2 (MutS protein homolog 2), MSH6 (MutS protein homolog 6), and PMS2 (PMS1 homolog 2).
  • Linch-like refers to a group of MSI-H colorectal cancers in which MLH1 expression is not epigenetically suppressed and neither is Lynch syndrome.
  • the marker for determining the MLH1 methylation group of the present invention includes (i) a nucleic acid molecule comprising the base sequence represented by SEQ ID NO: 1, (ii) one or more bases added, deleted, and / or SEQ ID NO: 1. It consists of either a nucleic acid molecule comprising a substituted base sequence and (iii) a fragment of the nucleic acid molecule of (i) or (ii) comprising one or more CpG sequences.
  • the nucleotide sequence represented by SEQ ID NO: 1 refers to the sequence of hg38 chr3: 36,993,202-36,993,865 (37,034,693-37,035,356 for hg19) of the human genome.
  • the nucleic acid molecule containing the base sequence can be effective for identifying the MLH1 methylation group.
  • the nucleic acid molecule (i) includes other sequences in addition to the base sequence represented by SEQ ID NO: 1, for example, a sequence of 1000 bases or less, 500 bases or less, 300 bases or less, or 100 bases or less derived from the human genome. Also good.
  • the nucleic acid molecule of (i) consists essentially of the base sequence shown in SEQ ID NO: 1 or consists of the base sequence shown in SEQ ID NO: 1.
  • the range of “one or more” in the nucleic acid molecule (ii) is, for example, 1 to 10, preferably 1 to 7, more preferably 1 to 5, particularly preferably 1 to 3, or 1 Or two. Accordingly, the nucleic acid molecule (ii) includes a mutant of the nucleic acid molecule (i), a single nucleotide polymorphism (SNP), and the like.
  • the length of the nucleic acid molecule fragment of (iii) above is not particularly limited as long as it can be used for MLH1 methylation group determination.
  • the length of the fragment of the nucleic acid molecule (iii) may be 10 bases or more, 15 bases or more, 20 bases or more, 30 bases or more, 40 bases or more, or 50 bases or more, and 600 bases or less, It may be 500 bases or less, 400 bases or less, 300 bases or less, 200 bases or less, or 100 bases or less.
  • the nucleic acid molecule fragment (iii) can be obtained, for example, by extracting genomic DNA or free DNA from a subject and digesting it with a restriction enzyme or nuclease.
  • the invention relates to a method for determining whether a subject belongs to the MLH1 methylation group.
  • This method includes a detection step and a determination step as essential steps.
  • the present method may include a genomic DNA or free DNA extraction step as an optional step before the detection step.
  • this method may include a gene mutation detection step as an optional step after the determination step.
  • genomic DNA or free DNA extraction process is a step of extracting genomic DNA or free DNA from a subject.
  • subject refers to a subject to which the method of the present invention is applied, and is in principle a human.
  • the subject is a cancer (eg, solid cancer), eg, colon cancer, small intestine cancer, endometrial cancer, ovarian cancer, gastric cancer, renal pelvis / ureteral cancer, pancreatic cancer, biliary tract cancer, brain tumor, Muir- Suffering from or at risk for a cancer selected from the group consisting of sebaceous adenoma and keratophyte tumor in Torre syndrome, malignant melanoma, non-small cell lung cancer, prostate cancer, and thyroid cancer.
  • the subject is suffering from or at risk of, for example, colon cancer, particularly high frequency microsatellite instability (MSI-H) colon cancer.
  • MSI-H microsatellite instability
  • the subject may or may not have been examined for microsatellite instability. Whether a subject suffers from or is at risk for these cancers is determined by conventional methods known to those skilled in the art, blood tests, diagnostic imaging, X-ray examinations, MRI, endoscopy, and pathological
  • free DNA refers to DNA that may be present in blood and / or body fluid, and examples thereof include cell-free DNA (cell free DNA, cfDNA) and circulating tumor DNA (circulating tumor DNA, ctDNA). Is mentioned.
  • blood includes whole blood, serum, and plasma.
  • body fluid include tissue fluid and lymph fluid.
  • the sample for extracting genomic DNA or free DNA is not limited.
  • the sample may be a biopsy tissue, a FFPE (formalin fixed paraffin embedded) sample, blood or body fluid, or a cell.
  • Genomic DNA is preferably derived from a biopsy tissue or FFPE sample
  • free DNA is preferably derived from a sample such as blood or body fluid of a cancer-bearing patient.
  • the sample is preferably a cancer (eg solid cancer), eg, colon cancer, small intestine cancer, endometrial cancer, ovarian cancer, gastric cancer, renal pelvis / ureter cancer, pancreatic cancer, biliary tract cancer, brain tumor, sebaceous gland in Muir-Torre syndrome It is derived from a cancer selected from the group consisting of tumor and keratinous pheochromocytoma, malignant melanoma, non-small cell lung cancer, prostate cancer, and thyroid cancer, more preferably from a colon cancer tissue or cell.
  • a cancer eg solid cancer
  • colon cancer eg, small intestine cancer, endometrial cancer, ovarian cancer, gastric cancer, renal pelvis / ureter cancer, pancreatic cancer, biliary tract cancer, brain tumor, sebaceous gland in Muir-Torre syndrome
  • a cancer selected from the group consisting of tumor and keratinous pheochromocytoma, malignant
  • genomic DNA and free DNA are known to those skilled in the art. For example, methods described in Green and Sambrook, Molecular Cloning, 4th Ed (2012), Cold Spring Harbor Laboratory Press may be referred to. Specifically, a general phenol chloroform extraction method, a commercially available kit capable of extracting genomic DNA or free DNA, such as PureLink (registered trademark) Genomic DNA (Thermo Fisher Scientific), and Mag Extractor TM ⁇ For Genome- (TOYOBO) and free DNA, DNA can be extracted using a commercially available kit such as NucleoSnap (registered trademark) DNA Plasma. The extracted DNA can be used in the following detection step as it is or after purification.
  • Detection process in the detection step, in genomic DNA or free DNA obtained from a subject, ⁇ 1. Methylation of one or more cytosines in the MLH1 methylation group determination marker described in MLH1 methylation group determination marker> is detected.
  • the position of cytosine for detecting methylation in the detection step is not particularly limited as long as it is a cytosine in the CpG sequence of the base sequence shown in SEQ ID NO: 1.
  • Examples of cytosine for detecting methylation in the detection step include any CpG in the base sequence of SEQ ID NO: 1, and as the position of cytosine for detecting methylation, for example, positions 1 and 7 of the base sequence of SEQ ID NO: 1, 38, 47, 61, 78, 85, 95, 97, 103, 122, 133, 148, 217, 240, 264, 272, 305, 305, 320 , 340, 351, 355, 371, 374, 382, 398, 403, 405, 408, 425, 466, 476, 481, 500, 508, 513 515, 528, 530, 536, 548, 567, 580, 583, 590, 593, 605, 631, 638, 653, and 663 Can be mentioned
  • the position of cytosine for detecting methylation is preferably 1, position 38, position 47, position 95, position 122, position 133, position 148, position 217, position 305, position 340, position 371, position 398 of SEQ ID NO: 1.
  • Methylated cytosine may be detected at one of these positions, or methylated cytosine may be detected at one or more, for example, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, or all.
  • methylation-sensitive restriction enzyme means a restriction enzyme whose digestion is inhibited when the CpG sequence is methylated.
  • the CpG sequence contained in the base sequence represented by SEQ ID NO: 1 Examples of methylation sensitive restriction enzymes that can be recognized include Sma I, Ehe I, and Hin 6I, Hpa II, Acc II, and Hae II, and methylation sensitive isoschizomers of these enzymes.
  • methylated cytosine that can be detected by the methylation-sensitive restriction enzyme are as follows: SEQ ID NO: 1, 7, 95, 97, 103, 340, 403, 405, 481, 481, 513, 515 , 528 position, 530 position, 548 position, 567 position, and 593 position. Methylated cytosine may be detected at one of these positions, or methylated cytosine may be detected at one or more, eg, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, or all.
  • the genomic DNA or free DNA to be tested in the detection step may be obtained from biopsy tissue, FFPE (formalin fixed paraffin embedded) sample, blood, body fluid or cells.
  • Genomic DNA is preferably in cancer (eg solid cancer), for example, colon cancer, small intestine cancer, endometrial cancer, ovarian cancer, gastric cancer, renal pelvis / ureteral cancer, pancreatic cancer, biliary tract cancer, brain tumor, Muir-Torre syndrome It is derived from a cancer selected from the group consisting of sebaceous adenoma and keratophyte cell carcinoma, malignant melanoma, non-small cell lung cancer, prostate cancer, and thyroid cancer, and more preferably from a colon cancer tissue or cell.
  • Genomic DNA is preferably derived from a biopsy tissue or FFPE sample, and free DNA is preferably derived from a sample such as blood or body fluid of a cancer-bearing patient.
  • the detection method in the detection step is not limited, but can be performed by, for example, a methylation sensitive restriction enzyme cleavage method, a methylation array, a bisulfite method, or a method using a methylated DNA-specific binding protein. Each method will be described in detail below.
  • the methylation-sensitive restriction enzyme cleavage method is particularly preferred because it can be detected easily and in a short time and is inexpensive.
  • the “methylation sensitive restriction enzyme cleavage method” is a method for detecting the presence or absence of methylation using a methylation sensitive restriction enzyme that inhibits digestion when the CpG sequence is methylated.
  • detection can be performed using PCR or a probe after cleaving with a methylation-sensitive restriction enzyme.
  • a methylation-sensitive restriction enzyme is allowed to act on genomic DNA obtained from a subject, followed by PCR using a primer that can amplify a nucleic acid molecule containing a position for detecting methylation. I do.
  • the CpG sequence is not digested with a methylation sensitive restriction enzyme, so a PCR amplification product is obtained, and when not methylated, the CpG sequence is digested with a methylation sensitive restriction enzyme, PCR amplification product cannot be obtained.
  • electrophoresis or the like the presence or absence of methylation can be detected.
  • methylation-sensitive restriction enzymes capable of recognizing the CpG sequence contained in the base sequence represented by SEQ ID NO: 1 include Sma I, Ehe I, and Hin 6I, Hpa II, Acc II, and Hae II, and these Examples include the methylation sensitive isoschizomer of the enzyme.
  • the methylation sensitive restriction enzyme is Sma I or Hin 6I.
  • Methods of methylation array is a technique for examining the degree of methylation of DNA using microarray analysis.
  • the methylated array include MIAMI method using a methylation sensitive restriction enzyme, MeDIP method using an anti-methylated cytosine antibody, and a methylated array using bisulfite.
  • methylated cytosine is detected using bisulfite.
  • Bisulfite treatment converts unmethylated cytosine to uracil, whereas methylated cytosine is not converted to uracil, which can distinguish between methylated and unmethylated cytosine.
  • loss of DNA in the bisulfite treatment process is often a problem.
  • Examples of the bisulfite method include bisulfite sequencing, pyrosequencing, methylation specific PCR, and methylation specific real-time PCR.
  • a bisulfite treatment is performed on a sample, and then a sequence reaction is performed. Unmethylated cytosine converted to uracil is indicated as thymine. Based on this, the presence or absence of methylation is detected by comparing the sequence data of genomic DNA before and after bisulfite treatment.
  • the Pyrosequencing method is a method that converts the bisulfite sequencing method into a quantitative real-time sequence.
  • genomic DNA is treated with bisulfite, followed by polymerase base extension to decode the base sequence. Since pyrophosphoric acid generated during the extension reaction and the luminescence intensity are in a proportional relationship, highly accurate quantitative analysis can be performed.
  • a primer for detecting methylated DNA and a primer for detecting unmethylated DNA are designed at the base site where unmethylated cytosine can be converted to uracil by bisulfite treatment.
  • PCR or real-time PCR eg, using SYBR Green
  • a TaqMan TM probe for detecting methylated DNA and a TaqMan TM probe for detecting unmethylated DNA can be designed at the base site where unmethylated cytosine can be converted to uracil by bisulfite treatment.
  • a primer common to DNA may be designed at a position sandwiching this base site, and real-time PCR may be performed to identify methylation or unmethylation depending on the presence or absence or degree of amplification.
  • a methylated DNA-specific binding protein In the “method using a methylated DNA-specific binding protein”, only DNA that has been methylated with a methylated DNA-specific binding protein such as an anti-methylated cytosine antibody or MBD (Methyl-CpG-binding domain) is selected. Separate. Next, the isolated DNA is subjected to PCR, real-time PCR, hybridization using a probe, or the like to detect methylated cytosine.
  • a methylated DNA-specific binding protein such as an anti-methylated cytosine antibody or MBD (Methyl-CpG-binding domain) is selected. Separate. Next, the isolated DNA is subjected to PCR, real-time PCR, hybridization using a probe, or the like to detect methylated cytosine.
  • the determination step it is determined that the subject belongs to the MLH1 methylation group when methylation is detected in the detection step.
  • MSI-H colorectal cancer if it belongs to the MLH1 methylation group, it becomes clear that it is not Lynch syndrome or Lynch-like, so it is necessary to perform further diagnosis to determine whether it is Lynch syndrome or Lynch-like Disappear.
  • the “MLH1 unmethylated group” refers to a group in which the expression of the MLH1 gene is not suppressed epigenetically.
  • This step is an optional step for further classifying the subject after the determination step.
  • the MLH1 methylation group can be further classified into, for example, BRAF (V600E), KRAS mutant, fusion kinase, and four other subtypes in MSI-H colorectal cancer. Therefore, the subject can be further classified by detecting the mutation of these genes for the subject determined as the MLH1 methylation group in the determination step.
  • Each of these subtypes can have a different therapeutic agent (for example, if it is a fusion kinase, an inhibitor of the kinase can be a therapeutic agent), and by making such a determination, it can be useful for treatment of a disease.
  • MLH1 unmethylated group is further classified into Lynch syndrome and Lynch-like. Therefore, a genetic mutation detection step may be performed on a subject determined to be an MLH1 unmethylated group in the determination step in order to determine whether the subject belongs to Lynch syndrome or Lynch-like. For example, in any one or more of mismatch repair genes MLH1, MSH2, MSH6, and PMS2, it is examined whether there is a loss-of-function mutation in the germline, and if there is a mutation, in Lynch syndrome, If there is no mutation, it can be classified as Lynch-like.
  • the gene mutation detection step is performed by a known gene analysis method (for example, a PCR method, RT-PCR method, Real-time PCR method, LCR (Ligase chain reaction), LAMP (Loop-mediated isothermal amplification) commonly used as a gene detection method) , Microarray method, Northern hybridization method, dot blot method, fluorescent in situ hybridization (FISH) method, analysis by next-generation sequencer, etc.).
  • a known gene analysis method for example, a PCR method, RT-PCR method, Real-time PCR method, LCR (Ligase chain reaction), LAMP (Loop-mediated isothermal amplification) commonly used as a gene detection method) , Microarray method, Northern hybridization method, dot blot method, fluorescent in situ hybridization (FISH) method, analysis by next-generation sequencer, etc.
  • a nucleic acid extracted from a sample derived from a subject for example, mRNA
  • a gene amplification technique using an appropriate primer
  • the present invention provides ⁇ 2.
  • a step of determining whether or not the subject belongs to the MLH1 methylation group by the MLH1 methylation group determination method described in the method for determining whether or not the subject belongs to the MLH1 methylation group, and an anticancer agent It relates to a method of treatment comprising the step of administering to a subject.
  • the type of the anticancer agent can be appropriately selected based on the results of the MLH1 methylation group determination method, and examples thereof include anti-EGFR antibodies, anti-VEGF antibodies, and kinase inhibitors such as tyrosine kinase inhibitors.
  • the present invention provides ⁇ 1.
  • the present invention relates to an MLH1 methylation group determination kit capable of detecting methylation of one or more cytosines in the MLH1 methylation group determination marker described in MLH1 methylation group determination marker>.
  • the kit of the present invention includes a methylation-sensitive restriction enzyme capable of cleaving a sequence containing one or more CpG sequences in the base sequence represented by SEQ ID NO: 1, a primer set capable of amplifying all or part of the marker, and It includes at least one of a probe or an antibody capable of detecting all or part of the marker.
  • methylation sensitive restriction enzymes examples include Sma I, Ehe I, Hin 6I, Hpa II, Acc II, and Hae II, and methylation sensitive isoschizomers of these enzymes. It is done.
  • the methylation sensitive restriction enzyme is Sma I or Hin 6I.
  • the primer set that can be included in the kit of the present invention is not particularly limited as long as it can specifically detect the MLH1 methylation group determination marker described in the present specification.
  • the forward primer and reverse primer in the primer set are regions of hg38 chr3: 36,993,202-36,993,865 (SEQ ID NO: 1) or its vicinity (eg, 500 bp or less, 300 bp or less, 100 bp or less, or 50 bp or less outside the region)
  • the forward primer is composed of nucleotides comprising 14 to 30 consecutive nucleotides of SEQ ID NO: 1, for example, 16 to 28 nucleotides, preferably 18 to 26 nucleotides
  • the reverse primer is a sequence complementary to the sequence of SEQ ID NO: 1.
  • the forward primer comprises a nucleotide comprising a sequence of 14 to 30 bases, such as 16 to 28 bases, preferably 18 to 26 bases, which hybridizes under stringent conditions to a nucleic acid comprising a complementary sequence of SEQ ID NO: 1.
  • the reverse primer may comprise a nucleotide comprising a sequence of 14 to 30 bases, such as 16 to 28 bases, preferably 18 to 26 bases, which hybridizes to the nucleic acid comprising the sequence of SEQ ID NO: 1 under stringent conditions.
  • the “stringent condition” means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed.
  • known hybridization conditions can be used. For example, it may be appropriately determined with reference to Green and Sambrook, Molecular Cloning, 4th Ed (2012), Cold Spring Harbor Laboratory Press.
  • stringent conditions may be set according to the hybridization method temperature and the salt concentration contained in the solution, and the temperature and the salt concentration contained in the solution in the washing step of the hybridization method. More detailed stringent conditions include, for example, a sodium concentration of 25 to 500 mM, preferably 25 to 300 mM, and a temperature of 42 to 68 ° C., preferably 42 to 65 ° C. More specifically, 5 ⁇ SSC (750 mM NaCl, 75 mM sodium citrate) is appropriately diluted at a temperature of 42 ° C.
  • the probe that can be included in the kit of the present invention is not particularly limited as long as it can detect the marker, and chr3: 36,993,202-36,993,865 (base sequence of SEQ ID NO: 1) of hg38 or the vicinity thereof (for example, outside the region) 500 bp or less, 300 bp or less, 100 bp or less, or 50 bp or less).
  • the probe may be a polynucleotide comprising 14 or more consecutive nucleotide sequences of SEQ ID NO: 1, for example, 20 or more, 30 or more, 50 or more, or 100 or more nucleotide sequences, or a complementary sequence thereof.
  • the probe is a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising 14 or more consecutive nucleotide sequences of SEQ ID NO: 1, for example, 20 or more, 30 or more, 50 or more, or 100 or more, or a complementary sequence thereof. It may be.
  • the kit of the present invention may contain at least one of, for example, a buffer, an enzyme, and an instruction manual in addition to the primer set, probe, or antibody.
  • Example 1 Identification of specific region showing MLH1 methylation> (Materials and methods)
  • tissue samples were collected from 93 Microsatellite instability-high (MSI-H, high frequency microsatellite instability) colorectal cancer patients exhibiting microsatellite instability in the BAT25 and BAT26 regions, and about 2 from frozen tissue.
  • MSI-H Microsatellite instability-high
  • QIAamp DNA Mini Kit Qiagen
  • Methylation array analysis was performed according to the method described in Pidsley® R. et al., “BMC Genomics,” 14: 293, 2013.
  • the method of methylation array is briefly as follows. First, 500 ng genomic DNA from each sample was treated with sodium bisulfite using the EZ96 DNA methylation kit (Zymo Research, CA, USA) according to the manufacturer's standard protocol. Subsequently, genomic-scale DNA methylation was assessed using an Illumina Inmanium Methylation450 BeadChip (Illumina Inc, CA, USA) according to the manufacturer's instructions. Using Illumina GenomeStudio software, raw data of the signal intensity of each probe was obtained. Computer and statistical analyzes were performed as described in Pidsley R. et al (supra).
  • Herman JG. Et al., Proc. Natl. Acad. Sci., 95, 6870-6875, 1998 include the S: chr3: 36,992,827-36,992,855 and AS: chr3: 36,992,927-36,992,950 regions on the EPM2AIP1 gene ( Hereinafter, it is described that methylation of the Herman region) is effective for specifying MLH1 methylation.
  • S: chr3: 36,992,827-36,992,855 and AS: chr3: 36,992,927-36,992,950 regions on the EPM2AIP1 gene Hereinafter, it is described that methylation of the Herman region
  • MLH1 methylation was determined using methylation in the Herman region as an index, 2 cases were considered to be false positives based on comparison with all exon analysis results.
  • False positive 2 Occupied site is rectal sigmoid, and even if only VarScan calls are included, there are not many mutations, including 1 base substitution (SNVs) 125 and insertion / deletion mutation (Indels) 9. Cases considered to be satellite stability (MSS) were classified into the MLH1 methylation group based on the Herman region (data not shown).
  • Example 2 Specific detection method of MLH1 methylation> (Materials and methods)
  • a methylation-sensitive restriction enzyme a restriction enzyme that inhibits digestion when the CpG sequence is methylated. It was investigated.
  • Cytosine and guanine contiguous sequence (CpG) in which methylation can occur is underlined, of which the site where methylation was detected in the methylation array in Example 2 is bold, as an example of a recognition sequence for a methylation sensitive restriction enzyme,
  • the Sma (I recognition sequence (CCCGGG) and the Ehe I recognition sequence (GGCGCC) are boxed, and the Hin6I recognition sequence (GCGC) is shown in italics.
  • the sites where methylation was detected with the methylation array in Example 2 were the 1st, 38th, 47th, 95th, 122th, 133th, 148th and 217th positions of the base sequence of SEQ ID NO: 1. 305, 371, 398, 425, 466, 476, 476, 513, 515, 528, 530, 536, 590, and 663.
  • each primer used is as follows: MLH1 Primer 1: 5′-ATCCTTCTAGGTAGCGGGCA-3 ′ (SEQ ID NO: 2) MLH1 Primer 2: 5′-CGGTCTGCGGAAAAGGAGAA-3 ′ (SEQ ID NO: 3) BRAF Primer 1: 5′-ATAGGTGATTTTGGTCTAGCTACA-3 ′ (SEQ ID NO: 4) BRAF Primer 2: 5′-AACTCAGCAGCATCTCAGGG-3 ′ (SEQ ID NO: 5)
  • the BRAF amplification product as a control is a nucleic acid molecule having a length of 147 bases that does not contain the Sma I recognition sequence. After heating at 94 ° C. for 1 minute, PCR was performed by performing 25 cycles of 98 ° C. for 10 seconds, 62 ° C. for 15 seconds, and 68 ° C. for 32 seconds.
  • the presence or absence of BRAF mutation was also determined according to the following procedure. First, the product obtained by the above PCR reaction was reacted with the following reaction solution at 37 ° C. for 4 minutes and cleaned up by heat denaturation at 80 ° C. for 1 minute.
  • ExoSAP-IT-treated sample was subjected to concentration measurement using Qubit® dsDNA® HS assay (Thermofisher® Scientific).
  • the DNA concentration varied from 0.806 ng / ⁇ l to 3.08 ng / ⁇ l depending on the presence or absence of the MLH1 band, but the BRAF PCR product had almost the same band density, so the lowest 0.806 ng / ⁇ l (almost BRAF (Same band only) was used as a template for the sequence reaction.
  • the sequence was performed with the following reaction solution, heated at 96 ° C for 1 minute, and then subjected to a cycle of 96 ° C for 10 seconds, 50 ° C for 10 seconds, and 60 ° C for 2 minutes 25 times.
  • the product of the above reaction was purified with Sam Solution 45 ⁇ l / well and X Terminator 10 ⁇ l / well in BigDye XTerminator TM Purification Kit (ThermoFisher Scientific, MA, USA), and capillary sequencer ABI 3130 genetic analyzer (Applied Biosystems, Japan) Sequence analysis was performed.
  • FIG. 2 shows a comparison between the methylation array results and the methylation determination results using the methylation-sensitive restriction enzyme of this example.
  • the sites where methylation arraying was performed were performed in order from the left side: 1st, 38th, 47th, 95th, 122th, 133th, 148th, 217 305, 371, 398, 425, 466, 476, 476, 513, 515, 528, 530, 536, 590, and 663, the sites where SmaI acts , Sequence containing cytosine at position 548, indicated by arrows.
  • the MLH1 methylation group determination result using restriction enzyme was defined as ( ⁇ ) when the band was almost invisible as a result of electrophoresis, and (+) when the band was visible.
  • the band in the case of (-) is clearly thinner than the BRAF band which is the internal control, so whether it is positive or negative by migrating with RKO which is positive control and DLD1 which is negative control Objective determination is possible.
  • the multiplex PCR product used for the determination of MLH1 methylation could be used without any problem as a sequence template for examining the presence or absence of BRAF (V600E) mutation (data not shown). Therefore, further examination of BRAF mutations showed that subjects belonging to the MLH1 methylation group could be further classified based on BRAF mutations.
  • Example 3 Specific detection method of MLH1 methylation of DNA extracted from paraffin-embedded (FFPE) section> (Materials and methods)
  • Example 2 shows a method using DNA extracted from cryopreserved tissue
  • FFPE paraffin-embedded
  • the base length is shorter and the yield is lower than DNA obtained from frozen tissue. Therefore, in this example, it was examined whether or not methylation of DNA extracted from paraffin-embedded (FFPE) sections could be detected using the methylation-sensitive restriction enzyme in the region specified in Example 1.
  • FFPE paraffin-embedded
  • GeneRead DNA FFPE Kit Qiagen
  • QIAamp DNA FFPE Tissue Kit Qiagen
  • genomic DNA was extracted and digested with a restriction enzyme at 37 ° C. for 120 minutes in a reaction solution having the following composition, followed by heat denaturation at 80 ° C. for 20 minutes.
  • frozen tissue-derived DNA extracted by the same method as in Example 1 was used as a comparative control.
  • each primer used is as follows: MLH1 Primer 3: 5'-AGCCGGGCTCACTTAAGGG-3 '(SEQ ID NO: 6) MLH1 Primer 4: 5′-CATGCGCTGTACATGCCTCT-3 ′ (SEQ ID NO: 7) BRAF Primer 1: 5′-ATAGGTGATTTTGGTCTAGCTACA-3 ′ (SEQ ID NO: 4) BRAF Primer 2: 5′-AACTCAGCAGCATCTCAGGG-3 ′ (SEQ ID NO: 5)
  • the BRAF amplification product as a control is a nucleic acid molecule having a length of 147 bases that does not contain the Hin6I recognition sequence.
  • PCR was performed at 94 ° C. for 1 minute, followed by 30 cycles of 98 ° C. for 10 seconds, 62 ° C. for 15 seconds, and 68 ° C. for 9 seconds.
  • 12.5 ⁇ l of the PCR product was mixed with 2.5 ⁇ l of 6 ⁇ Loading Buffer Orange G (Nippon Gene), electrophoresed on a 3% agarose / TBE gel, the photograph was inverted in black and white, and used for determination.
  • As the DNA size marker 2.5 ⁇ l of 50 bp ladder (Nippon Genetics, Japan) was used.

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Abstract

Selon un mode de réalisation, la présente invention concerne un marqueur, un procédé, un kit, etc., nouveaux, permettant de déterminer un groupe de méthylation MLH1 avec une précision élevée. Selon un mode de réalisation, un marqueur permettant de déterminer un groupe de méthylation de l'homologue 1 de mutL (MLH1) selon la présente invention comprend un élément quelconque parmi (i) à (iii) : (i) une molécule d'acide nucléique comprenant une séquence de bases représentée par SEQ ID No : 1, (ii) une molécule d'acide nucléique comprenant une séquence de bases dans laquelle une ou plusieurs bases ont été ajoutées, supprimées et/ou substituées dans SEQ ID No : 1, et (iii) un fragment d'une molécule d'acide nucléique de (i) ou (ii) comprenant une ou plusieurs séquences CpG.
PCT/JP2019/003992 2018-02-06 2019-02-05 Marqueur pour la détermination de groupe de méthylation mlh1 et procédé de détermination WO2019156054A1 (fr)

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WO2021192396A1 (fr) * 2020-03-25 2021-09-30 富士フイルム株式会社 Procédé de test génique
WO2024090265A1 (fr) * 2022-10-28 2024-05-02 株式会社biomy Dispositif de traitement d'informations, procédé de traitement d'informations et programme

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