WO2019208739A1 - Method for assessing membership in good prognosis group after cancer treatment, and method for assessing membership in group at risk for developing cancer at young age - Google Patents

Method for assessing membership in good prognosis group after cancer treatment, and method for assessing membership in group at risk for developing cancer at young age Download PDF

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WO2019208739A1
WO2019208739A1 PCT/JP2019/017807 JP2019017807W WO2019208739A1 WO 2019208739 A1 WO2019208739 A1 WO 2019208739A1 JP 2019017807 W JP2019017807 W JP 2019017807W WO 2019208739 A1 WO2019208739 A1 WO 2019208739A1
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cancer
group
subject
polymorphism
gene
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PCT/JP2019/017807
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French (fr)
Japanese (ja)
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哲治 岡本
亮治 谷
浩一郎 徳丸
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国立大学法人広島大学
日本ケフィア株式会社
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Priority to JP2020515588A priority Critical patent/JP7399399B2/en
Publication of WO2019208739A1 publication Critical patent/WO2019208739A1/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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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

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  • the present invention relates to a method for determining whether a cancer belongs to a good prognosis group after cancer treatment and a method for determining whether a subject belongs to a juvenile cancer risk group.
  • the frequency of testing should be planned more effectively. It is desirable from the viewpoint of QOL and medical resource allocation.
  • the MICA gene (MHC class I chain-related gene A) product is a ligand for activated NK cell receptor NKG2D such as NK cells and ⁇ T cells, and activation signals mediated by the receptor are infection immunity and autoimmunity against viruses. It is said that it contributes to various immune responses such as diseases. For this reason, various therapeutic agents have been developed for the MICA gene or gene product thereof as a target for the action of the therapeutic agent (for example, Patent Document 1).
  • Non-patent Document 1 The presence of genetic polymorphism is known for the MICA gene, and it has been reported that there are five types of genetic polymorphism in exon5 encoding a transmembrane region (Non-patent Document 1).
  • an object of the present invention is to provide a novel method for determining whether or not a subject has a good prognosis after cancer treatment, and to provide a novel method for determining whether or not a subject belongs to a juvenile cancer risk group. is there.
  • the present inventor has been engaged in the clinical treatment of cancer and has been diligently researching the treatment results for many years. As a result, a human having A5.1 type as the MICA gene polymorphism has a young age of onset of oral cancer. In addition, the present inventors have found that the prognosis of treatment of oral cancer is good and reached the present invention.
  • the present invention includes the following (1) and below.
  • (1) A method of determining whether a subject belongs to a good prognosis group after cancer treatment by identifying a genetic polymorphism in exon 5 of the subject's MICA gene.
  • (2) The determination method according to (1), wherein the gene polymorphism in exon 5 of the MICA gene of the test subject is determined to belong to a good prognosis group when having A5.1 type as a homozygote.
  • the determination method according to any one of (1) to (2), wherein the good prognosis group is a group having a survival rate of 95% or more after 5 years.
  • (4) The determination method according to any one of (1) to (3), wherein the subject is a cancer patient or a healthy person.
  • the determination method according to any one of (1) to (4), wherein the cancer is human oral cancer.
  • (6) A method for determining whether a subject belongs to a juvenile cancer risk group by identifying a genetic polymorphism in exon 5 of the subject's MICA gene.
  • the subject has a homozygote of A5.1 type or a heterozygote of A5.1 type and A5 type as a genetic polymorphism in exon 5 of the MICA gene of the subject, the risk of developing juvenile cancer (6) The determination method according to (6).
  • 10 The determination method according to any one of (6) to (9), wherein the cancer is human oral cancer.
  • a prediction method, an estimation method, an evaluation method, a detection method, a determination method, an analysis method, an inspection method, a diagnosis method, a classification method, a classification method, and a selection method including the above-described determination method.
  • a prediction method, an estimation method, an evaluation method, a detection method, a determination method, an analysis method, an inspection method, a diagnosis method, a classification method, a classification method, and a selection method including the above-described determination method.
  • the step of identifying a genetic polymorphism in exon 5 of the subject's MICA gene can be performed using a sample collected from the subject, and the sample containing the gene from the subject prior to the step Can be performed.
  • the step of identifying a gene polymorphism in exon 5 of the subject's MICA gene can be performed by a known polymorphism identifying means, for example, by a polymorphism identifying means using a PCR method.
  • a step of performing PCR amplification of a gene in exon 5 of a subject's MICA gene using a primer at an appropriate position containing exon 5 and then identifying a polymorphism by using a PCR product based on a difference in the number of bases can be implemented.
  • the present invention it can be determined whether the cancer belongs to a good prognosis group after cancer treatment. Moreover, according to this invention, it can be determined whether a test subject belongs to the juvenile cancer onset risk group. As a result, it is possible to plan more effectively how often examinations should be performed, etc., which brings great benefits to individuals and society from the viewpoint of QOL and medical resource allocation.
  • FIG. 1 is an explanatory diagram of the MICA gene product.
  • FIG. 2 is an explanatory diagram showing five types of gene polymorphisms present in exon5 encoding the transmembrane region of the MICA gene.
  • FIG. 3 is an explanatory diagram of a polymorphism analysis chromatogram obtained by separating each PCR product with a capillary column based on the difference in the number of bases.
  • FIG. 4 shows the survival curve of oral cancer patients by Kaplan-Meier method with A5.1 / 5.1 homozygote and all other Genotype polymorphisms (non-A5.1 / 5.1). It is the graph shown by contrast.
  • Method to determine if cancer belongs to good prognosis after cancer treatment it is possible to determine whether a subject belongs to a good prognosis group after cancer treatment by identifying a genetic polymorphism in exon 5 of the subject's MICA gene.
  • the gene polymorphism in exon 5 of the subject's MICA gene belongs to a good prognosis group when it has A5.1 type homozygote.
  • the cancer is not particularly limited, but preferably can be suitably determined for human oral cancer.
  • human oral cancer include tongue cancer, gingival cancer, oral cavity cancer, buccal mucosa cancer, maxillary sinus cancer, oral pharyngeal cancer, and salivary gland cancer.
  • cancers of organs adjacent to human oral cancer include nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, and esophageal cancer.
  • the good prognosis group refers to a group having a good prognosis using the survival rate as an index, and can be expressed by, for example, a 5-year survival rate, a 10-year survival rate, or a 20-year survival rate.
  • a group in which the 5-year survival rate is 90% or more, 95% or more, or 99% or more can be mentioned, for example, the 10-year survival rate is 90% or more, 95% or more, 99%.
  • a group having a 20-year survival rate of 90% or more, 95% or more, or 99% or more can be given. As shown in the examples of the present invention, in a particularly preferred case, the 20-year survival rate was 100%.
  • the subject who determines whether or not it belongs to the good prognosis group after cancer treatment may be a cancer patient or a healthy person. If it is a cancer patient, the prognosis after cancer treatment can be determined, and if it is a healthy person, the prognosis after cancer treatment when cancer develops can be determined.
  • Method to determine if it belongs to juvenile cancer risk group According to the present invention, by identifying a genetic polymorphism in exon 5 of a subject's MICA gene, it can be determined whether the subject belongs to a juvenile cancer risk group.
  • the subject when the subject has the A5.1 type as a genetic polymorphism in exon 5 of the MICA gene, it can be determined that it belongs to the juvenile cancer risk group.
  • the polymorphism in exon 5 of the subject's MICA gene has a homozygote of A5.1 type or a heterozygote of A5.1 type and A5 type. It can be determined that it belongs to the juvenile cancer onset risk group.
  • the cancer is not particularly limited, but preferably can be suitably determined for human oral cancer.
  • human oral cancer include tongue cancer, gingival cancer, oral cavity cancer, buccal mucosa cancer, maxillary sinus cancer, oral pharyngeal cancer, and salivary gland cancer.
  • cancers of organs adjacent to human oral cancer include nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, and esophageal cancer.
  • the juvenile cancer risk group refers to a group that has a high risk of developing cancer at a young age, and specifically refers to a group that has a high risk of cancer development at the age of 20 to 39 years.
  • the 20-39-year-old almost overlaps with the AYA (Adolescents and Young Adult) (adolescent and young adult) generation (15 years old and under 40 years old) advocated by the Ministry of Health, Labor and Welfare. Patients who have developed cancer in this age are particularly expected to return to society after cancer treatment, so predicting the onset of cancer in this age and starting appropriate treatment early is important. Especially significant.
  • the risk of developing cancer at 20-39 years is higher than the proportion of cancer onset at 20-39 years compared to other age groups such as 40-59 years, 60-79 years, 80-100 years And say significantly more.
  • MICA gene polymorphism As described in the examples described later, it has been reported that the MICA gene has five types of gene polymorphisms in exon5 encoding a transmembrane region. As described above, among these polymorphisms, in the present invention, the determination is made by identifying the A5.1 type.
  • a known technique can be used, for example, the means disclosed in the Examples can be used.
  • genomic DNA obtained from a cell is PCR amplified using a primer at an appropriate position including exon 5, and the obtained PCR product is applied to a capillary column with CEQ8000, and the difference in the number of bases is determined. Polymorph identification can be performed to identify the subject's Genotype.
  • MICA gene polymorphism MHC class I chain-related gene A (MICA) is a ligand of activated NK cell receptor NKG2D such as NK cells and ⁇ T cells, and activation signals mediated by the receptor are infectious immunity against viruses, tumor immunity, self It is said that it contributes to various immune responses such as immune diseases.
  • An explanatory diagram of this MICA gene product is shown in FIG.
  • the MICA gene has been reported to have 5 types of gene polymorphisms in exon5 encoding a transmembrane region. An explanatory diagram of these five gene polymorphisms is shown in FIG. As shown in FIG.
  • the base sequence in the polymorphisms A4, A5, A6, and A9, is [GCT] [GCT] in exon 5, followed by this last T (base 951 of exon 5). Furthermore, the base sequence continues with [GCT] [GCT].
  • the base sequence in the A5.1 polymorphism, is [GCT] [GCT] in exon 5, followed by this last T (base 951 of exon 5), and then G is inserted. The base sequence continues with [GCT] [GCT].
  • the codon frame is shifted, and the encoded amino acid sequence is different from other polymorphisms.
  • Genomic DNA extraction method using QIA amp (registered trademark) DNA Blood midi kit (QIAGEN) was performed according to the attached protocol.
  • the extracted DNA was quantified using Nano Drop (Nano Drop Technologies, Inc., USA).
  • genomic DNA was extracted from the OSCC tissue piece using QIA amp (registered trademark) DNA mini kit (QIAGEN) according to the attached protocol.
  • QIA amp registered trademark DNA mini kit
  • the extracted DNA was quantified using Nano Drop (Nano Drop Technologies, Inc., USA).
  • PCR Polymerase Chain Reaction
  • FIG. 3 shows, as an explanatory diagram, a polymorphism analysis chromatogram obtained by separating each PCR product with a capillary column using CEQ8000 based on the difference in the number of bases.
  • the obtained PCR product was used to purify the PCR product using a QlAquick (registered trademark) PCR Purification Kit (Qiagen).
  • QlAquick registered trademark
  • Qiagen PCR Purification Kit
  • Table 1-1 summarizes the results of MICA polymorphism comparison (by Phenotype) between healthy individuals and oral cancer patients by age group.
  • Table 1-2 shows the results of chi-square test (P value) corresponding to Table 1-1.
  • P value chi-square test
  • Table 1-1 summarizes the results of MICA polymorphism comparison (by Phenotype) between healthy individuals and oral cancer patients by age group.
  • Table 1-2 shows the results of chi-square test (P value) corresponding to Table 1-1.
  • P value chi-square test
  • Table 2-1 summarizes the results of MICA polymorphism comparison (by allele) between healthy individuals and oral cancer patients by age group.
  • Table 2-2 shows the results of chi-square test (P value) corresponding to Table 2-1.
  • P value chi-square test
  • Table 3 summarizes the results of MICA polymorphism comparison (by Genotype) between healthy individuals and oral cancer patients by age group. Table 3 further shows the results of chi-square test (P value).
  • * (asterisk) was attached
  • In comparison between healthy individuals and oral cancer patients there was a significant difference especially in the A5.1 / 5.1 Genotype group of 20-39 years old.
  • In comparison between healthy individuals and oral cancer patients there was a significant difference especially in the group of A5.1 / 5 Genotype 20-39 years old. That is, it was found that both Genotypes A5.1 / 5.1 and A5.1 / 5 are in the juvenile cancer risk group.
  • Table 4 summarizes the results of MICA polymorphism comparison (by Genotype) of oral cancer patients by age group. Table 4 further shows the results of chi-square test (P value). In Table 4, those having a significant difference are marked with * (asterisk). There was a significant difference between the A5.1 / 5.1 Genotype group and the A5.1 / 5 Genotype group in comparisons between 20-39 years old and other age-specific oral cancer patients. That is, it was found that Genotype of A5.1 / 5.1 in the 20-39 years of age has a higher risk of onset compared to other age groups of cancer patients.
  • Table 6 summarizes the results of treatment details for the A5.1 / 5.1 homozygote and all other Genotype polymorphisms (non-A5.1 / 5.1).
  • the abbreviations indicate CRT: chemotherapy + radiotherapy, C: chemotherapy, RT: radiation therapy, respectively.
  • C chemotherapy + radiotherapy
  • C chemotherapy
  • RT radiation therapy
  • the present invention provides a method for determining whether or not a subject has a good prognosis after cancer treatment and a method for determining whether or not a subject belongs to a juvenile cancer risk group.
  • the present invention is an industrially useful invention.

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Abstract

Provided are: a method for determining whether a subject is a member of a good prognosis group after cancer treatment by identifying the MICA-gene exon 5 genetic-polymorphism of the subject; and a method for assessing whether a subject is in a group at risk for developing cancer at a young age by identifying the MICA-gene exon 5 genetic-polymorphism of the subject.

Description

癌治療後に予後良好群に属するかを判定する方法、及び若年性癌発症リスク群に属するかを判定する方法Method for determining whether or not belonging to a good prognosis group after cancer treatment, and method for determining whether or not belonging to a juvenile cancer risk group
　本発明は、癌治療後に予後良好群に属するかを判定する方法、及び被験者が若年性癌発症リスク群に属するかを判定する方法に関する。 The present invention relates to a method for determining whether a cancer belongs to a good prognosis group after cancer treatment and a method for determining whether a subject belongs to a juvenile cancer risk group.
　癌治療の予後が良好か否かについて予測することができれば、癌治療後にどの程度の頻度で検査等を行うべきか等をより効果的に計画することができ、ＱＯＬの観点や医療資源の配分の観点から望ましい。 If we can predict whether the prognosis for cancer treatment is good or not, we can plan more effectively how often we should conduct tests after cancer treatment. From the viewpoint of.
　また、癌発症のリスクがどの程度あるか、あるいはそれが生涯のどの時期になるかについて、予測することができれば、どの程度の頻度で検査等を行うべきか等をより効果的に計画することができ、ＱＯＬの観点や医療資源の配分の観点から望ましい。 In addition, if the risk of cancer development or how long it will be in life can be predicted, the frequency of testing should be planned more effectively. It is desirable from the viewpoint of QOL and medical resource allocation.
　ＭＩＣＡ遺伝子（ＭＨＣ　ｃｌａｓｓ　Ｉ　ｃｈａｉｎ－ｒｅｌａｔｅｄ　ｇｅｎｅ　Ａ）産物は、ＮＫ細胞やγδＴ細胞などの活性化ＮＫ細胞レセプターＮＫＧ２Ｄのリガンドであり、同受容体を介した活性化シグナルはウイルスに対する感染免疫や自己免疫疾患などの多彩な免疫応答に寄与しているとされる。このため、ＭＩＣＡ遺伝子あるいはその遺伝子産物は、治療薬の作用の対象として、種々の治療薬の開発がなされている（例えば、特許文献１）。 The MICA gene (MHC class I chain-related gene A) product is a ligand for activated NK cell receptor NKG2D such as NK cells and γδT cells, and activation signals mediated by the receptor are infection immunity and autoimmunity against viruses. It is said that it contributes to various immune responses such as diseases. For this reason, various therapeutic agents have been developed for the MICA gene or gene product thereof as a target for the action of the therapeutic agent (for example, Patent Document 1).
　ＭＩＣＡ遺伝子は、遺伝子多型の存在が知られており、膜貫通領域をコードするｅｘｏｎ５内に５種類の遺伝子多型が存在することが報告されている（非特許文献１）。 The presence of genetic polymorphism is known for the MICA gene, and it has been reported that there are five types of genetic polymorphism in exon5 encoding a transmembrane region (Non-patent Document 1).
特表２０１６－５１２２２３号公報Special table 2016-512223 gazette
　したがって、本発明の目的は、癌治療後に予後良好群に属するかを判定する新規な方法を提供すること、被験者が若年性癌発症リスク群に属するかを判定する新規な方法を提供することにある。 Accordingly, an object of the present invention is to provide a novel method for determining whether or not a subject has a good prognosis after cancer treatment, and to provide a novel method for determining whether or not a subject belongs to a juvenile cancer risk group. is there.
　本発明者は、癌治療の臨床に携わり、その治療成績について長年の間、鋭意研究してきたところ、ＭＩＣＡ遺伝子多型としてＡ５．１型を有するヒトが、口腔癌発症年齢が若年となること、及び口腔癌の治療の予後が良好であることを見いだして、本発明に到達した。 The present inventor has been engaged in the clinical treatment of cancer and has been diligently researching the treatment results for many years. As a result, a human having A5.1 type as the MICA gene polymorphism has a young age of onset of oral cancer. In addition, the present inventors have found that the prognosis of treatment of oral cancer is good and reached the present invention.
　したがって、本発明は次の（１）以下を含む。
（１）
　被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型を同定することによって、被験者が、癌治療後に予後良好群に属するかを判定する方法。
（２）
　被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型として、Ａ５．１型をホモ接合体で有する場合に、予後良好群に属すると判定する、（１）に記載の判定方法。
（３）
　予後良好群が、５年後生存率９５％以上の群である、（１）～（２）のいずれかに記載の判定方法。
（４）
　被験者が、癌患者、又は健常人である、（１）～（３）のいずれかに記載の判定方法。
（５）
　癌がヒト口腔癌である、（１）～（４）のいずれかに記載の判定方法。
（６）
　被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型を同定することによって、被験者が、若年性癌発症リスク群に属するかを判定する方法。
（７）
　被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型として、Ａ５．１型を有する場合に、若年性癌発症リスク群に属すると判定する、（６）に記載の判定方法。
（８）
　被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型として、Ａ５．１型のホモ接合体を有するか、又はＡ５．１型とＡ５型とのヘテロ接合体を有する場合に、若年性癌発症リスク群に属すると判定する、（６）に記載の判定方法。
（９）
　若年性癌発症リスク群が、２０～３９歳での癌発症リスク群である、（６）～（８）のいずれかに記載の判定方法。
（１０）
　癌がヒト口腔癌である、（６）～（９）のいずれかに記載の判定方法。 Accordingly, the present invention includes the following (1) and below.
(1)
A method of determining whether a subject belongs to a good prognosis group after cancer treatment by identifying a genetic polymorphism in exon 5 of the subject's MICA gene.
(2)
The determination method according to (1), wherein the gene polymorphism in exon 5 of the MICA gene of the test subject is determined to belong to a good prognosis group when having A5.1 type as a homozygote.
(3)
The determination method according to any one of (1) to (2), wherein the good prognosis group is a group having a survival rate of 95% or more after 5 years.
(4)
The determination method according to any one of (1) to (3), wherein the subject is a cancer patient or a healthy person.
(5)
The determination method according to any one of (1) to (4), wherein the cancer is human oral cancer.
(6)
A method for determining whether a subject belongs to a juvenile cancer risk group by identifying a genetic polymorphism in exon 5 of the subject's MICA gene.
(7)
The determination method according to (6), wherein when the subject has polymorphism in exon 5 of the MICA gene having A5.1 type, it is determined that the subject belongs to a juvenile cancer risk group.
(8)
When the subject has a homozygote of A5.1 type or a heterozygote of A5.1 type and A5 type as a genetic polymorphism in exon 5 of the MICA gene of the subject, the risk of developing juvenile cancer (6) The determination method according to (6).
(9)
The determination method according to any one of (6) to (8), wherein the juvenile cancer risk group is a cancer risk group at the age of 20 to 39 years.
(10)
The determination method according to any one of (6) to (9), wherein the cancer is human oral cancer.
　好適な実施の態様において、上記の判定方法を含み、この判定方法からなる予測方法、推定方法、評価方法、検出方法、決定方法、分析方法、検査方法、診断方法、分類方法、分別方法、選別方法、識別方法、及び判別方法を含む。 In a preferred embodiment, a prediction method, an estimation method, an evaluation method, a detection method, a determination method, an analysis method, an inspection method, a diagnosis method, a classification method, a classification method, and a selection method including the above-described determination method. Including a method, an identification method, and a discrimination method.
　好適な実施の態様において、被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型を同定する工程を、被験者から採取した試料を用いて行うことができ、該工程に先立って、被験者から遺伝子を含む試料を採取する工程を行うことができる。 In a preferred embodiment, the step of identifying a genetic polymorphism in exon 5 of the subject's MICA gene can be performed using a sample collected from the subject, and the sample containing the gene from the subject prior to the step Can be performed.
　好適な実施の態様において、被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型を同定する工程を、公知の多型同定手段によって行うことができ、例えば、ＰＣＲ法を使用した多型同定手段によって行うことができ、例えば、被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子をエキソン５を含む適切な位置のプライマーを用いてＰＣＲ増幅した後にＰＣＲ産物を用いて塩基数の相違によって多型の同定を行う工程によって、実施することができる。 In a preferred embodiment, the step of identifying a gene polymorphism in exon 5 of the subject's MICA gene can be performed by a known polymorphism identifying means, for example, by a polymorphism identifying means using a PCR method. For example, a step of performing PCR amplification of a gene in exon 5 of a subject's MICA gene using a primer at an appropriate position containing exon 5 and then identifying a polymorphism by using a PCR product based on a difference in the number of bases Can be implemented.
　本発明によれば、癌治療後に予後良好群に属するかを判定することができる。また、本発明によれば、被験者が若年性癌発症リスク群に属するかを判定することができる。これによって、どの程度の頻度で検査等を行うべきか等をより効果的に計画することができ、ＱＯＬの観点や医療資源の配分の観点から個人と社会に大きな利益をもたらす。 According to the present invention, it can be determined whether the cancer belongs to a good prognosis group after cancer treatment. Moreover, according to this invention, it can be determined whether a test subject belongs to the juvenile cancer onset risk group. As a result, it is possible to plan more effectively how often examinations should be performed, etc., which brings great benefits to individuals and society from the viewpoint of QOL and medical resource allocation.
図１は、ＭＩＣＡ遺伝子産物についての説明図である。FIG. 1 is an explanatory diagram of the MICA gene product. 図２は、ＭＩＣＡ遺伝子の膜貫通領域をコードするｅｘｏｎ５内に存在する５種類の遺伝子多型を示す説明図である。FIG. 2 is an explanatory diagram showing five types of gene polymorphisms present in exon5 encoding the transmembrane region of the MICA gene. 図３は、塩基数の違いに基づき、キャピラリーカラムで各ＰＣＲ産物を分離した多型解析のクロマトグラムの説明図である。FIG. 3 is an explanatory diagram of a polymorphism analysis chromatogram obtained by separating each PCR product with a capillary column based on the difference in the number of bases. 図４は、Ｋａｐｌａｎ－Ｍｅｉｅｒ法による口腔癌患者の生存曲線を、Ａ５．１／５．１のホモ接合体とそれ以外の全てのＧｅｎｏｔｙｐｅ多型（ｎｏｎ－Ａ５．１／５．１）とを対比して示したグラフである。FIG. 4 shows the survival curve of oral cancer patients by Kaplan-Meier method with A5.1 / 5.1 homozygote and all other Genotype polymorphisms (non-A5.1 / 5.1). It is the graph shown by contrast.
　具体的な実施の形態をあげて、以下に本発明を詳細に説明する。本発明は、以下にあげる具体的な実施の形態に限定されるものではない。 The present invention will be described in detail below with specific embodiments. The present invention is not limited to the following specific embodiments.
［癌治療後に予後良好群に属するかを判定する方法］
　本発明によれば、被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型を同定することによって、被験者が、癌治療後に予後良好群に属するかを判定することができる。 [Method to determine if cancer belongs to good prognosis after cancer treatment]
According to the present invention, it is possible to determine whether a subject belongs to a good prognosis group after cancer treatment by identifying a genetic polymorphism in exon 5 of the subject's MICA gene.
　好適な実施の態様において、被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型として、Ａ５．１型をホモ接合体で有する場合に、予後良好群に属すると判定することができる。 In a preferred embodiment, it can be determined that the gene polymorphism in exon 5 of the subject's MICA gene belongs to a good prognosis group when it has A5.1 type homozygote.
　好適な実施の態様において、癌としては、特に制約はないが、好ましくはヒト口腔癌について好適に判定することができる。ヒト口腔癌として、例えば、舌癌、歯肉癌、口底癌、頬粘膜癌、上顎洞癌、口峡咽頭癌、唾液腺癌をあげることができる。ヒト口腔癌に隣接する臓器の癌として、例えば、上咽頭癌、中咽頭癌、下咽頭癌、食道癌をあげることができる。 In a preferred embodiment, the cancer is not particularly limited, but preferably can be suitably determined for human oral cancer. Examples of human oral cancer include tongue cancer, gingival cancer, oral cavity cancer, buccal mucosa cancer, maxillary sinus cancer, oral pharyngeal cancer, and salivary gland cancer. Examples of cancers of organs adjacent to human oral cancer include nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, and esophageal cancer.
［予後良好群］
　好適な実施の態様において、予後良好群は、生存率を指標として予後が良好である群を言い、例えば５年生存率、１０年生存率、２０年生存率によって表現することができる。好適な実施の態様において、例えば５年生存率が、９０％以上、９５％以上、９９％以上である群をあげることができ、例えば１０年生存率が、９０％以上、９５％以上、９９％以上である群をあげることができ、例えば２０年生存率が、９０％以上、９５％以上、９９％以上である群をあげることができる。本発明の実施例で示すように、特に好適な場合に、２０年生存率が１００％であった。 [Good prognosis group]
In a preferred embodiment, the good prognosis group refers to a group having a good prognosis using the survival rate as an index, and can be expressed by, for example, a 5-year survival rate, a 10-year survival rate, or a 20-year survival rate. In a preferred embodiment, for example, a group in which the 5-year survival rate is 90% or more, 95% or more, or 99% or more can be mentioned, for example, the 10-year survival rate is 90% or more, 95% or more, 99%. For example, a group having a 20-year survival rate of 90% or more, 95% or more, or 99% or more can be given. As shown in the examples of the present invention, in a particularly preferred case, the 20-year survival rate was 100%.
　好適な実施の態様において、癌治療後に予後良好群に属するかを判定する被験者としては、癌患者であってもよく、健常人であってもよい。癌患者であれば、癌治療後の予後を判定することができ、健常人であれば、癌を発症した場合における癌治療後の予後を判定することができる。 In a preferred embodiment, the subject who determines whether or not it belongs to the good prognosis group after cancer treatment may be a cancer patient or a healthy person. If it is a cancer patient, the prognosis after cancer treatment can be determined, and if it is a healthy person, the prognosis after cancer treatment when cancer develops can be determined.
［若年性癌発症リスク群に属するかを判定する方法］
　本発明によれば、被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型を同定することによって、被験者が、若年性癌発症リスク群に属するかを判定することができる。 [Method to determine if it belongs to juvenile cancer risk group]
According to the present invention, by identifying a genetic polymorphism in exon 5 of a subject's MICA gene, it can be determined whether the subject belongs to a juvenile cancer risk group.
　好適な実施の態様において、被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型として、Ａ５．１型を有する場合に、若年性癌発症リスク群に属すると判定することができる。 In a preferred embodiment, when the subject has the A5.1 type as a genetic polymorphism in exon 5 of the MICA gene, it can be determined that it belongs to the juvenile cancer risk group.
　好適な実施の態様において、被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型として、Ａ５．１型のホモ接合体を有するか、又はＡ５．１型とＡ５型とのヘテロ接合体を有する場合に、若年性癌発症リスク群に属すると判定することができる。 In a preferred embodiment, the polymorphism in exon 5 of the subject's MICA gene has a homozygote of A5.1 type or a heterozygote of A5.1 type and A5 type. It can be determined that it belongs to the juvenile cancer onset risk group.
　好適な実施の態様において、癌としては、特に制約はないが、好ましくはヒト口腔癌について好適に判定することができる。ヒト口腔癌として、例えば、舌癌、歯肉癌、口底癌、頬粘膜癌、上顎洞癌、口峡咽頭癌、唾液腺癌をあげることができる。ヒト口腔癌に隣接する臓器の癌として、例えば、上咽頭癌、中咽頭癌、下咽頭癌、食道癌をあげることができる。 In a preferred embodiment, the cancer is not particularly limited, but preferably can be suitably determined for human oral cancer. Examples of human oral cancer include tongue cancer, gingival cancer, oral cavity cancer, buccal mucosa cancer, maxillary sinus cancer, oral pharyngeal cancer, and salivary gland cancer. Examples of cancers of organs adjacent to human oral cancer include nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, and esophageal cancer.
［若年性癌発症リスク群］
　好適な実施の態様において、若年性癌発症リスク群は、若年における癌発症のリスクが大きい群を言い、具体的には２０～３９歳での癌発症リスクが大きな群を言う。この２０～３９歳は、厚生労働省の提唱するＡＹＡ（Ａｄｏｌｅｓｃｅｎｔｓ　ａｎｄ　Ｙｏｕｎｇ　Ａｄｕｌｔ）（思春期・若年成人）世代（１５歳以上４０歳未満）とほぼ重なっている。この年代で癌を発症した患者は、癌治療の後に社会に復帰することが特に大きく期待されているから、この年代での癌発症を予測して、適切な治療を早期に始めることは、社会的な意義が特に大きい。２０～３９歳での癌発症リスクが大きいとは、２０～３９歳での癌発症の割合が、他の年代、例えば４０～５９歳、６０～７９歳、８０～１００歳の患者群と比較して有意に多いことを言う。 [Young cancer risk group]
In a preferred embodiment, the juvenile cancer risk group refers to a group that has a high risk of developing cancer at a young age, and specifically refers to a group that has a high risk of cancer development at the age of 20 to 39 years. The 20-39-year-old almost overlaps with the AYA (Adolescents and Young Adult) (adolescent and young adult) generation (15 years old and under 40 years old) advocated by the Ministry of Health, Labor and Welfare. Patients who have developed cancer in this age are particularly expected to return to society after cancer treatment, so predicting the onset of cancer in this age and starting appropriate treatment early is important. Especially significant. The risk of developing cancer at 20-39 years is higher than the proportion of cancer onset at 20-39 years compared to other age groups such as 40-59 years, 60-79 years, 80-100 years And say significantly more.
［ＭＩＣＡ遺伝子多型］
　後述する実施例のなかでも説明しているように、ＭＩＣＡ遺伝子には、膜貫通領域をコードするｅｘｏｎ５内に５種類の遺伝子多型が存在することが報告されている。上述のように、これらの多型のなかで、本発明ではＡ５．１型を有することを同定することによって、判定を行っている。ＭＩＣＡ遺伝子の多型の同定には、公知の技術を使用することができ、例えば、実施例に開示した手段を使用することができる。典型的な手法として、例えば、細胞から得たゲノムＤＮＡを、エキソン５を含む適切な位置のプライマーを用いてＰＣＲ増幅して、得られたＰＣＲ産物をＣＥＱ８０００にてキャピラリーカラムにかけて、塩基数の違いによって多型の同定を行って、被験者のＧｅｎｏｔｙｐｅを特定することができる。 [MICA gene polymorphism]
As described in the examples described later, it has been reported that the MICA gene has five types of gene polymorphisms in exon5 encoding a transmembrane region. As described above, among these polymorphisms, in the present invention, the determination is made by identifying the A5.1 type. For identification of the polymorphism of the MICA gene, a known technique can be used, for example, the means disclosed in the Examples can be used. As a typical technique, for example, genomic DNA obtained from a cell is PCR amplified using a primer at an appropriate position including exon 5, and the obtained PCR product is applied to a capillary column with CEQ8000, and the difference in the number of bases is determined. Polymorph identification can be performed to identify the subject's Genotype.
　以下に実施例をあげて、本発明を詳細に説明する。本発明は、以下に例示する実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples. The present invention is not limited to the examples illustrated below.
［臨床研究］
　広島大学病院の顎・口腔外科を受診した口腔扁平上皮癌（ＯＳＣＣ）患者３８６名より、同意のもと、腫瘍組織または血液を採取した。対照として１０３名の健常人ボランティアの血液も同様に同意を得て採取した。本研究は広島大学ヒトゲノム・遺伝子解析研究倫理審査委員会の承認済み（許可番号：第ヒー５７号）である。 [Clinical research]
Tumor tissue or blood was collected with consent from 386 patients with oral squamous cell carcinoma (OSCC) who had undergone jaw and oral surgery at Hiroshima University Hospital. As a control, blood samples from 103 healthy volunteers were collected with similar consent. This study has been approved by Hiroshima University Human Genome / Gene Analysis Research Ethics Review Board (permission number: Hee 57).
［ＭＩＣＡ遺伝子多型］
　ＭＨＣ　ｃｌａｓｓ　Ｉ　ｃｈａｉｎ－ｒｅｌａｔｅｄ　ｇｅｎｅ　Ａ（ＭＩＣＡ）は、ＮＫ細胞やγδＴ細胞などの活性化ＮＫ細胞レセプターＮＫＧ２Ｄのリガンドであり、同受容体を介した活性化シグナルはウイルスに対する感染免疫や腫瘍免疫、自己免疫疾患などの多彩な免疫応答に寄与しているとされる。このＭＩＣＡ遺伝子産物についての説明図を図１に示す。
　ＭＩＣＡ遺伝子には、膜貫通領域をコードするｅｘｏｎ５内に５種類の遺伝子多型が存在することが報告されている。この５種類の遺伝子多型の説明図を図２に示す。図２に示されるように、Ａ４、Ａ５、Ａ６、Ａ９の多型では、エキソン５中において塩基配列が［ＧＣＴ］［ＧＣＴ］と続いてこの最後のＴ（エキソン５の９５１番塩基）の次に、さらに塩基配列が［ＧＣＴ］［ＧＣＴ］と続いている。一方、Ａ５．１の多型では、エキソン５中において塩基配列が［ＧＣＴ］［ＧＣＴ］と続いてこの最後のＴ（エキソン５の９５１番塩基）の次に、Ｇが挿入されて、その後に塩基配列が［ＧＣＴ］［ＧＣＴ］と続いている。この結果、Ａ５．１多型では、コドンのフレームがシフトして、コードされるアミノ酸配列が他の多型とは異なったものとなっている。 [MICA gene polymorphism]
MHC class I chain-related gene A (MICA) is a ligand of activated NK cell receptor NKG2D such as NK cells and γδT cells, and activation signals mediated by the receptor are infectious immunity against viruses, tumor immunity, self It is said that it contributes to various immune responses such as immune diseases. An explanatory diagram of this MICA gene product is shown in FIG.
The MICA gene has been reported to have 5 types of gene polymorphisms in exon5 encoding a transmembrane region. An explanatory diagram of these five gene polymorphisms is shown in FIG. As shown in FIG. 2, in the polymorphisms A4, A5, A6, and A9, the base sequence is [GCT] [GCT] in exon 5, followed by this last T (base 951 of exon 5). Furthermore, the base sequence continues with [GCT] [GCT]. On the other hand, in the A5.1 polymorphism, the base sequence is [GCT] [GCT] in exon 5, followed by this last T (base 951 of exon 5), and then G is inserted. The base sequence continues with [GCT] [GCT]. As a result, in the A5.1 polymorphism, the codon frame is shifted, and the encoded amino acid sequence is different from other polymorphisms.
［ゲノムＤＮＡの抽出］
　血液からのゲノムＤＮＡの抽出は、ＱＩＡ　ａｍｐ（登録商標）　ＤＮＡ　Ｂｌｏｏｄ　ｍｉｄｉ　ｋｉｔ（ＱＩＡＧＥＮ）を用いて行った。また、ＯＳＣＣ組織片からのゲノムＤＮＡの抽出は、ＱＩＡ　ａｍｐ（登録商標）　ＤＮＡ　ｍｉｎｉ　ｋｉｔ（ＱＩＡＧＥＮ）を用いて行った。 [Extraction of genomic DNA]
Extraction of genomic DNA from blood was performed using QIA amp (registered trademark) DNA Blood midi kit (QIAGEN). Extraction of genomic DNA from the OSCC tissue piece was performed using QIA amp (registered trademark) DNA mini kit (QIAGEN).
　ＱＩＡ　ａｍｐ（登録商標）　ＤＮＡ　Ｂｌｏｏｄ　ｍｉｄｉ　ｋｉｔ（ＱＩＡＧＥＮ）を用いたゲノムＤＮＡ抽出法は添付プロトコールに準じて行った。抽出したＤＮＡはＮａｎｏ　Ｄｒｏｐ（Ｎａｎｏ　Ｄｒｏｐ　Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｉｎｃ．，ＵＳＡ）を用いて定量した。 Genomic DNA extraction method using QIA amp (registered trademark) DNA Blood midi kit (QIAGEN) was performed according to the attached protocol. The extracted DNA was quantified using Nano Drop (Nano Drop Technologies, Inc., USA).
　また、ＯＳＣＣ組織片からのゲノムＤＮＡの抽出は、ＱＩＡ　ａｍｐ（登録商標）　ＤＮＡ　ｍｉｎｉ　ｋｉｔ（ＱＩＡＧＥＮ）を用い添付プロトコールに準じて行った。抽出したＤＮＡはＮａｎｏ　Ｄｒｏｐ（Ｎａｎｏ　Ｄｒｏｐ　Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｉｎｃ．，ＵＳＡ）を用いて定量した。 In addition, the genomic DNA was extracted from the OSCC tissue piece using QIA amp (registered trademark) DNA mini kit (QIAGEN) according to the attached protocol. The extracted DNA was quantified using Nano Drop (Nano Drop Technologies, Inc., USA).
［ポリメラーゼ連鎖反応（Ｐｏｌｙｍｅｒａｓｅ　Ｃｈａｉｎ　Ｒｅａｃｔｉｏｎ；　ＰＣＲ）］
　ＭＩＣＡ遺伝子の膜貫通領域（エキソン５）を含む染色体ＤＮＡ断片を増幅するプライマーを、以下のように設計した。
プライマー：
ＭＩＣＡ５Ｆ：５’－ＣＣＴＴＴＴＴＴＴＣＡＧＧＧＡＡＡＧＴＧＣ－３’
ＭＩＣＡ５Ｒ：５’－ＣＣＴＴＡＣＣＡＴＣＴＣＣＡＧＡＡＡＣＴＧＣ－３’ [Polymerase Chain Reaction (PCR)]
A primer for amplifying a chromosomal DNA fragment containing the transmembrane region (exon 5) of the MICA gene was designed as follows.
Primer:
MICA5F: 5'-CCTTTTTTCAGGGAAAGTGC-3 '
MICA5R: 5'-CCTTACCATCTCCAGAAACTGC-3 '
　抽出したゲノムＤＮＡに、上記のプライマー対と、ＫＯＤ　ＦＸ　Ｎｅｏ（Ｔｏｙｏｂｏ，Ｏｓａｋａ，Ｊａｐａｎ）を用いて、サーマルサイクラー（ＰＴＣ－０２２０　ＤＮＡ　Ｅｎｇｉｎｅ　Ｄｙａｄ（登録商標）：　ＭＪ　Ｊａｐａｎ，Ｔｏｋｙｏ）を使用し、変性反応は９８℃；１０秒間、アニーリングは５８℃；３０秒間、伸長反応は６８℃；３０秒間の条件で行い、これを１サイクルとして３５サイクル行い、ＰＣＲ産物を得た。このＰＣＲ産物を１．５％アガロースゲルにて電気泳動後、ＳＹＢＲ　Ｓａｆｅ　ＤＮＡ　ｇｅｌ　ｓｔａｉｎ（Ｉｎｖｉｔｒｏｇｅｎ）にて可視化しＭＩＣＡ遺伝子エキソン５領域のＰＣＲ増幅物の有無を確認した。 Using the above primer pair and KOD FX Neo (Toyobo, Osaka, Japan) to the extracted genomic DNA, using a thermal cycler (PTC-0220 DNA Engineer Dyad (registered trademark): MJ Japan, Tokyo) The reaction was performed at 98 ° C. for 10 seconds, annealing at 58 ° C. for 30 seconds, and the extension reaction at 68 ° C. for 30 seconds. This was performed as 35 cycles for obtaining a PCR product. This PCR product was electrophoresed on a 1.5% agarose gel and then visualized with SYBR Safe DNA gel stain (Invitrogen) to confirm the presence or absence of a PCR amplification product of the MICA gene exon 5 region.
　ＭＩＣＡ遺伝子の各アレルによってＰＣＲ産物の大きさが異なるため、ＭＩＣＡ遺伝子多型の解析は、塩基数によっても判別が可能となる。図３に、塩基数の違いに基づき、ＣＥＱ８０００にてキャピラリーカラムで各ＰＣＲ産物を分離した多型解析のクロマトグラムを、説明図として示す。 Since the size of the PCR product differs depending on the allele of the MICA gene, the analysis of the MICA gene polymorphism can be discriminated also by the number of bases. FIG. 3 shows, as an explanatory diagram, a polymorphism analysis chromatogram obtained by separating each PCR product with a capillary column using CEQ8000 based on the difference in the number of bases.
［塩基配列の決定(ダイレクトシークエンス)］
　得られたＰＣＲ産物を用い、ＱｌＡｑｕｉｃｋ（登録商標）　ＰＣＲ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（Ｑｉａｇｅｎ）を使用して、ＰＣＲ産物の精製を行った。得られた産物を、サンガーシーケンス法にてＭＩＣＡ遺伝子のエキソン５領域の塩基配列を決定した。詳細は下記のとおりである。 [Determination of nucleotide sequence (direct sequence)]
The obtained PCR product was used to purify the PCR product using a QlAquick (registered trademark) PCR Purification Kit (Qiagen). The nucleotide sequence of the exon 5 region of the MICA gene was determined for the obtained product by the Sanger sequencing method. Details are as follows.
　Ｔｈｅｒｍａｌ　Ｃｙｃｌｅｒ（ＰＴＣ－０２２０　ＤＮＡ　Ｅｎｇｉｎｅ　Ｄｙａｄ：ＭＪ　Ｊａｐａｎ）を使用し、ＤＴＣＳ　Ｑｕｉｃｋ　Ｓｔａｒｔ　Ｍａｓｔｅｒ　Ｍｉｘ　Ｋｉｔ（Ｂｅｃｋｍａｎ　Ｃｏｕｌｔｅｒ）を用いて、変性反応は９６℃；２０秒間、アニーリングは５０℃；２０秒間、伸長反応は６０℃；２分間の条件でシークエンス反応を行い、これを１サイクルとして４０サイクル行い、ＰＣＲ産物を得た。ＰＣＲ反応終了後は、エタノール沈殿を行った後、ＳＬＳ容液（Ｓａｍｐｌｅ　Ｌｏａｄｉｎｇ　Ｓｏｌｕｔｉｏｎ）にて溶解し、遺伝子解析システムＣＥＱ８０００　Ｇｅｎｅｔｉｃ　Ａｎａｌｙｚｅｒ　（Ｂｅｃｋｍａｎ　Ｃｏｕｌｔｅｒ，ＵＳＡ）にて解析を行った。 Using Thermal Cycler (PTC-0220 DNA Engine Dyad: MJ Japan), DTCS Quick Start Master Mix Kit (Beckman Coulter), denaturation reaction at 96 ° C; 20 seconds, annealing at 50 ° C; The sequence reaction was carried out at 60 ° C. for 2 minutes, and this was regarded as one cycle for 40 cycles to obtain a PCR product. After completion of the PCR reaction, ethanol precipitation was performed, followed by dissolution with an SLS solution (Sample Loading Solution), and analysis was performed with a gene analysis system CEQ8000 Genetic Analyzer (Beckman Coulter, USA).
［ＭＩＣＡ遺伝子アレルの解析法（フラグメント解析）］
　フラグメント解析は　ＣＥＱ８０００　Ｇｅｎｅｔｉｃ　Ａｎａｌｙｚｅｒ（Ｂｅｃｋｍａｎ　Ｃｏｕｌｔｅｒ，　ＵＳＡ）を用いて行った。上記と同じ配列のＭＩＣＡ５Ｒプライマーに　ＣＥＱ８０００専用のＢｅｃｋｍａｎ　Ｄｙｅ４（登録商標）で標識した蛍光　ＥｃｏＲＩ－セレクティブプライマーを作製し、上述した方法に準じてＰＣＲ反応を行った。Ｄｙｅ４で標識したプライマーは蛍光強度が強いため、ＰＣＲ産物をＳＬＳで１０倍希釈し、希釈ＰＣＲ産物２μｌと、ＳＬＳおよび分子量マーカーとなるｓｔａｎｄａｒｄ４００を混合したベース液３０μｌを、サンプルプレートの各ウェルに加え、ＣＥＱ８０００にてキャピラリーカラムで各ＰＣＲ産物を塩基数の差を利用して分離することで解析し、付属のフラグメント解析ソフトを用いて、ＭＩＣＡ遺伝子多型を決定した。 [MICA gene allele analysis method (fragment analysis)]
Fragment analysis was performed using a CEQ8000 Genetic Analyzer (Beckman Coulter, USA). A fluorescent EcoRI-selective primer labeled with Beckman Dye4 (registered trademark) exclusively for CEQ8000 was prepared on the MICA5R primer having the same sequence as described above, and a PCR reaction was performed according to the method described above. Since the primer labeled with Dye4 has strong fluorescence intensity, the PCR product is diluted 10-fold with SLS, and 2 μl of the diluted PCR product and 30 μl of the base solution mixed with standard 400 serving as a molecular weight marker are added to each well of the sample plate. Then, each PCR product was analyzed using CEQ8000 with a capillary column using the difference in the number of bases, and MICA gene polymorphism was determined using the attached fragment analysis software.
［統計学的解析］
　ＯＳＣＣ患者および対照とした健常人ボランティアにおける、ＭＩＣＡ遺伝子のエキソン５内の遺伝子多型について、表現形質（Ｐｈｅｎｏｔｙｐｅ）別、各アレル（Ａｌｌｅｌｅ）別、Ｇｅｎｏｔｙｐｅ別に集計し、統計学的解析（カイ２乗検定）を行った。さらに、口腔癌患者の初診時年齢を４群（２０～３９歳、４０～５９歳、６０～７９歳、８０～１００歳）に区分し、各年齢群における、表現形質（Ｐｈｅｎｏｔｙｐｅ）別、各アレル（Ａｌｌｅｌｅ）別、Ｇｅｎｏｔｙｐｅ別にＭＩＣＡ遺伝子多型を集計し、対照群と統計学的解析（カイ２乗検定）を行った。
　さらに、ＭＩＣＡ多型別の治療内容の内訳を検討するとともに、ＭＩＣＡ遺伝子多型別（５．１／５．１ホモ接合体とそれ以外の遺伝子多型群）の口腔癌患者の生存曲線の算出をＫａｐｌａｎ－Ｍｅｉｅｒ法で行い、生存率の有意差はｌｏｇ－ｒａｎｋ検定で行なった。 [Statistical analysis]
For polymorphisms in exon 5 of the MICA gene in OSCC patients and healthy volunteers as controls, statistical analysis (chi-square) was performed by counting by phenotype (Phenotype), by allele (Allele), and by Genotype. Test). Furthermore, the age at the first visit of oral cancer patients is divided into 4 groups (20-39 years old, 40-59 years old, 60-79 years old, 80-100 years old), and each age group is classified by phenotype (Phenotype). MICA gene polymorphisms were tabulated by Allele and Genotype, and statistical analysis (chi-square test) was performed with the control group.
In addition, we examined the breakdown of treatment by MICA polymorphism and calculated survival curves for oral cancer patients by MICA gene polymorphism (5.1 / 5.1 homozygote and other gene polymorphism groups). Was performed by the Kaplan-Meier method, and a significant difference in the survival rate was performed by the log-rank test.
　表１－１に健常人と各年代別口腔癌患者のＭＩＣＡ多型比較（Ｐｈｅｎｏｔｙｐｅ別）の結果をまとめて示す。表１－２に、表１－１に対応するカイ２乗検定（Ｐ値）の結果を示す。Ｐｈｅｎｏｔｙｐｅ別の比較においては、該当する多型を有している場合には、それぞれＡ４、Ａ５、Ａ５．１、Ａ６、Ａ９のいずれかに分類して計数した。ヘテロ接合体は１人が２回計数されている。ホモ接合体ではＰｈｅｎｏｔｙｐｅは１つであるとして１人が１回だけ計数されている。表１－２において有意差のあるものには＊（アスタリスク）を付した。健常人と口腔癌患者の比較において、特にＡ５．１型の２０～３９歳の群について有意差があった。 Table 1-1 summarizes the results of MICA polymorphism comparison (by Phenotype) between healthy individuals and oral cancer patients by age group. Table 1-2 shows the results of chi-square test (P value) corresponding to Table 1-1. In the comparison by Phenotype, when the corresponding polymorphism was present, it was classified into any of A4, A5, A5.1, A6, and A9 and counted. Heterozygotes are counted twice per person. In a homozygote, one person is counted only once, assuming that there is one Phenotype. Those with significant difference in Table 1-2 are marked with * (asterisk). In comparison between healthy individuals and oral cancer patients, there was a significant difference, especially in the A5.1 type 20-39 year old group.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表２－１に健常人と各年代別口腔癌患者のＭＩＣＡ多型比較（アレル別）の結果をまとめて示す。表２－２に、表２－１に対応するカイ２乗検定（Ｐ値）の結果を示す。アレル別の比較においては、Ｇｅｎｏｔｙｐｅのいずれかのアレルとして該当する多型を有している場合には、それぞれＡ４、Ａ５、Ａ５．１、Ａ６、Ａ９のいずれかに分類して計数した。すなわち、ホモ接合体とヘテロ接合体のいずれにおいても１人がアレルごとに２回計数されている。表２－２において有意差のあるものには＊（アスタリスク）を付した。 Table 2-1 summarizes the results of MICA polymorphism comparison (by allele) between healthy individuals and oral cancer patients by age group. Table 2-2 shows the results of chi-square test (P value) corresponding to Table 2-1. In the comparison by allele, when it had a polymorphism corresponding to any allele of Genotype, it was classified into A4, A5, A5.1, A6, A9 and counted. That is, one person is counted twice for each allele in both the homozygote and the heterozygote. Those with significant differences in Table 2-2 are marked with * (asterisk).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　表３に健常人と各年代別口腔癌患者のＭＩＣＡ多型比較（Ｇｅｎｏｔｙｐｅ別）の結果をまとめて示す。表３にさらにカイ２乗検定（Ｐ値）の結果を示す。表３において有意差のあるものには＊（アスタリスク）を付した。健常人と口腔癌患者の比較において、特にＡ５．１／５．１のＧｅｎｏｔｙｐｅの２０～３９歳の群について有意差があった。また、健常人と口腔癌患者の比較において、特にＡ５．１／５のＧｅｎｏｔｙｐｅの２０～３９歳の群について有意差があった。すなわち、Ａ５．１／５．１及びＡ５．１／５のいずれのＧｅｎｏｔｙｐｅも若年性癌発症リスク群であることがわかった。また、健常人と口腔癌患者の比較において、特にＡ４／６のＧｅｎｏｔｙｐｅの４０～５９歳の群について有意差があった。 Table 3 summarizes the results of MICA polymorphism comparison (by Genotype) between healthy individuals and oral cancer patients by age group. Table 3 further shows the results of chi-square test (P value). In Table 3, * (asterisk) was attached | subjected to what has a significant difference. In comparison between healthy individuals and oral cancer patients, there was a significant difference especially in the A5.1 / 5.1 Genotype group of 20-39 years old. In comparison between healthy individuals and oral cancer patients, there was a significant difference especially in the group of A5.1 / 5 Genotype 20-39 years old. That is, it was found that both Genotypes A5.1 / 5.1 and A5.1 / 5 are in the juvenile cancer risk group. In comparison between healthy individuals and oral cancer patients, there was a significant difference especially in the A4 / 6 Genotype group of 40 to 59 years old.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　表４に各年代別口腔癌患者のＭＩＣＡ多型比較（Ｇｅｎｏｔｙｐｅ別）の結果をまとめて示す。表４にさらにカイ２乗検定（Ｐ値）の結果を示す。表４において有意差のあるものには＊（アスタリスク）を付した。２０～３９歳と他の各年代別の口腔癌患者との比較において、Ａ５．１／５．１のＧｅｎｏｔｙｐｅの群とＡ５．１／５のＧｅｎｏｔｙｐｅの群について有意差があった。すなわち、２０－３９歳代のＡ５．１／５．１のＧｅｎｏｔｙｐｅは他の年代の癌患者群と比較して発症リスクが高いことがわかった。 Table 4 summarizes the results of MICA polymorphism comparison (by Genotype) of oral cancer patients by age group. Table 4 further shows the results of chi-square test (P value). In Table 4, those having a significant difference are marked with * (asterisk). There was a significant difference between the A5.1 / 5.1 Genotype group and the A5.1 / 5 Genotype group in comparisons between 20-39 years old and other age-specific oral cancer patients. That is, it was found that Genotype of A5.1 / 5.1 in the 20-39 years of age has a higher risk of onset compared to other age groups of cancer patients.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　表５に全てのＭＩＣＡ多型別の治療内容内訳の結果をまとめて示す。略号はそれぞれ、ＣＲＴ：化学療法＋放射線療法、Ｃ：化学療法、ＲＴ：放射線療法を示す。表５において、カイ２乗検定（Ｐｅａｒｓｏｎ）ｐ＝０．７５を行ったが、各ＭＩＣＡ多型別において実施された治療方法に有意差はなかった。 Table 5 summarizes the results of treatment details by all MICA polymorphisms. The abbreviations indicate CRT: chemotherapy + radiotherapy, C: chemotherapy, RT: radiation therapy, respectively. In Table 5, chi-square test (Pearson) p = 0.75 was performed, but there was no significant difference in the treatment method performed for each MICA polymorphism.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　表６にＡ５．１／５．１のホモ接合体と、それ以外の全てのＧｅｎｏｔｙｐｅ多型（ｎｏｎ－Ａ５．１／５．１）についての治療内容内訳の結果をまとめて示す。略号はそれぞれ、ＣＲＴ：化学療法＋放射線療法、Ｃ：化学療法、ＲＴ：放射線療法を示す。表６において、カイ２乗検定（Ｐｅａｒｓｏｎ）ｐ＝０．９１を行ったが、Ａ５．１／５．１の患者群と非Ａ５．１／５．１の患者群において実施された治療方法に有意差はなかった。 Table 6 summarizes the results of treatment details for the A5.1 / 5.1 homozygote and all other Genotype polymorphisms (non-A5.1 / 5.1). The abbreviations indicate CRT: chemotherapy + radiotherapy, C: chemotherapy, RT: radiation therapy, respectively. In Table 6, chi-square test (Pearson) p = 0.91 was performed, but the treatment method implemented in the A5.1 / 5.1 patient group and the non-A5.1 / 5.1 patient group There was no significant difference.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　Ｋａｐｌａｎ－Ｍｅｉｅｒ法による口腔癌患者の生存曲線を、Ａ５．１／５．１のホモ接合体と、それ以外の全てのＧｅｎｏｔｙｐｅ多型（ｎｏｎ－Ａ５．１／５．１）とを対比して、図４に示す。Ａ５．１／５．１ホモ接合体個体群の予後は、それ以外の多型のＭＩＣＡ多型個体群より有意に良好であった（ｌｏｇ－ｒａｎｋ検定　ｐ＝０．０１８５）。 The survival curve of oral cancer patients by Kaplan-Meier method was compared with A5.1 / 5.1 homozygote and all other Genotype polymorphisms (non-A5.1 / 5.1). As shown in FIG. The prognosis of the A5.1 / 5.1 homozygous population was significantly better than the other polymorphic MICA polymorphic populations (log-rank test p = 0.0185).
　本発明は、癌治療後に予後良好群に属するかの判定方法、及び被験者が若年性癌発症リスク群に属するかの判定方法を提供する。本発明は、産業上有用な発明である。 The present invention provides a method for determining whether or not a subject has a good prognosis after cancer treatment and a method for determining whether or not a subject belongs to a juvenile cancer risk group. The present invention is an industrially useful invention.

Claims (10)

  1. 　被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型を同定することによって、被験者が、癌治療後に予後良好群に属するかを判定する方法。 A method of determining whether a subject belongs to a good prognosis group after cancer treatment by identifying a genetic polymorphism in exon 5 of the subject's MICA gene.
  2. 　被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型として、Ａ５．１型をホモ接合体で有する場合に、予後良好群に属すると判定する、請求項１に記載の判定方法。 The determination method according to claim 1, wherein when the subject has a A5.1 type homozygote as a genetic polymorphism in exon 5 of the MICA gene, it is determined that the subject belongs to a good prognosis group.
  3. 　予後良好群が、５年後生存率９５％以上の群である、請求項１～２のいずれかに記載の判定方法。 3. The determination method according to claim 1, wherein the good prognosis group is a group having a survival rate of 95% or more after 5 years.
  4. 　被験者が、癌患者、又は健常人である、請求項１～３のいずれかに記載の判定方法。 4. The determination method according to claim 1, wherein the subject is a cancer patient or a healthy person.
  5. 　癌がヒト口腔癌である、請求項１～４のいずれかに記載の判定方法。 The determination method according to any one of claims 1 to 4, wherein the cancer is human oral cancer.
  6. 　被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型を同定することによって、被験者が、若年性癌発症リスク群に属するかを判定する方法。 A method for determining whether a subject belongs to a juvenile cancer risk group by identifying a genetic polymorphism in exon 5 of the subject's MICA gene.
  7. 　被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型として、Ａ５．１型を有する場合に、若年性癌発症リスク群に属すると判定する、請求項６に記載の判定方法。 The determination method according to claim 6, wherein when the subject has polymorphism in exon 5 of the MICA gene having A5.1 type, it is determined that the subject belongs to the juvenile cancer risk group.
  8. 　被験者のＭＩＣＡ遺伝子のエキソン５内の遺伝子多型として、Ａ５．１型のホモ接合体を有するか、又はＡ５．１型とＡ５型とのヘテロ接合体を有する場合に、若年性癌発症リスク群に属すると判定する、請求項６に記載の判定方法。 When the subject has a homozygote of A5.1 type or a heterozygote of A5.1 type and A5 type as a genetic polymorphism in exon 5 of the MICA gene of the subject, the risk of developing juvenile cancer The determination method according to claim 6, wherein the determination method is determined to belong.
  9. 　若年性癌発症リスク群が、２０～３９歳での癌発症リスク群である、請求項６～８のいずれかに記載の判定方法。 The determination method according to any one of claims 6 to 8, wherein the juvenile cancer risk group is a cancer risk group at the age of 20 to 39 years.
  10. 　癌がヒト口腔癌である、請求項６～９のいずれかに記載の判定方法。 The determination method according to any one of claims 6 to 9, wherein the cancer is human oral cancer.
PCT/JP2019/017807 2018-04-25 2019-04-25 Method for assessing membership in good prognosis group after cancer treatment, and method for assessing membership in group at risk for developing cancer at young age WO2019208739A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002053018A2 (en) * 2001-01-08 2002-07-11 Genomics Collaborative, Inc. Method of detecting polymorphisms associated with breast carcinoma in mhc genes
US20150218635A1 (en) * 2011-12-14 2015-08-06 Fundación Pública Andaluza Progreso Y Salud Method for obtaining data that can be used for the diagnosis and prognosis of neurosensory hypoacusis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002053018A2 (en) * 2001-01-08 2002-07-11 Genomics Collaborative, Inc. Method of detecting polymorphisms associated with breast carcinoma in mhc genes
US20150218635A1 (en) * 2011-12-14 2015-08-06 Fundación Pública Andaluza Progreso Y Salud Method for obtaining data that can be used for the diagnosis and prognosis of neurosensory hypoacusis

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEN, DAN ET AL.: "A variant upstream of HLA-DRB1 and multiple variants in MICA influence susceptibility to cervical cancer in a Swedish population", CANCER MEDICINE, vol. 3, no. 1, 2014, pages 190 - 198 *

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