JP6472075B2 - Diagnosis of prognosis or resistance to treatment of GIST - Google Patents
Diagnosis of prognosis or resistance to treatment of GIST Download PDFInfo
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Description
本発明は、消化管間質腫瘍（Ｇａｓｔｒｏｉｎｔｅｓｔｉｎａｌ ｓｔｒｏｍａｌ ｔｕｍｏｒ：ＧＩＳＴ）の予後又は化学療法抵抗性の診断方法に関する。 The present invention relates to a method for diagnosing the prognosis or chemoresistance of a gastrointestinal stromal tumor (GIST).
消化管に発生する間葉系腫瘍の中で最も頻度の高い腫瘍であるＧＩＳＴは、その約８５％にｃ−ｋｉｔ又はＰＤＧＦＲα遺伝子変異を有し、その変異によりｏｎｃｏｐｒｏｔｅｉｎであるＴｙｒｏｓｉｎｅ ｋｉｎａｓｅ ｒｅｃｅｐｔｏｒ（ＴＫＲ）がリガンド非依存性に恒常的に自己リン酸化され、腫瘍化することが分かっている（非特許文献１）。また、ＴＫ ｉｎｈｉｂｉｔｏｒであるイマニチブの投与に伴う耐性機構に、ｃ−ｋｉｔやＰＤＧＦＲαの二次性の遺伝子変異が関わっていることが示されている（非特許文献２）。ｃ−ｋｉｔやＰＤＧＦＲα遺伝子変異はＧＩＳＴ腫瘍発生には関与しているものの、進展や転移には直接の関与が明らかではないとされているが、ＴＫｉｎｈｉｂｉｔｏｒの投与による二次性の遺伝子変化が治療抵抗性に関与する（非特許文献１，２）など、その治療抵抗性への関与は明らかであり、その克服も急務となっている。 GIST, which is the most common tumor among the mesenchymal tumors occurring in the gastrointestinal tract, has a c-kit or PDGFRα gene mutation in about 85%, and the mutation causes an oncoprotein Tyrosine kinase receptor (TKR). Is constitutively autophosphorylated in a ligand-independent manner, and is known to become tumorous (Non-patent Document 1). In addition, it has been shown that secondary gene mutations of c-kit and PDGFRα are involved in the resistance mechanism associated with administration of imatinib, which is a TK inhibitor (Non-patent Document 2). Although c-kit and PDGFRα gene mutations are involved in GIST tumor development, direct involvement in development and metastasis is not clear, but secondary gene changes due to administration of TKinhibitor are resistant to treatment. Their involvement in treatment resistance, such as those related to sex (Non-Patent Documents 1 and 2), are clear, and there is an urgent need to overcome them.
前記のように、ＧＩＳＴの初期診断方法はある程度確立しているものの、その予後（進展や転移）及び治療抵抗性については何ら診断材料が示されていない。
従って、本発明の課題は、ＧＩＳＴの予後及び治療抵抗性を評価できる新たな手法を提供することにある。
As described above, although an initial diagnosis method for GIST has been established to some extent, no diagnostic material has been shown for its prognosis (progress or metastasis) and treatment resistance.
Accordingly, an object of the present invention is to provide a new technique capable of evaluating the prognosis and treatment resistance of GIST.
そこで、本発明者は、子宮体癌や卵巣癌などの他癌種において報告されているプロテインホスファターゼ２レギュレイトリーサブユニットＡα（Ｐｒｏｔｅｉｎ ｐｈｏｓｐｈａｔａｓｅ ２，ｒｅｇｕｌａｔｏｒｙ ｓｕｂｕｎｉｔ Ａ，ａｌｐｈａ：ＰＰＰ２Ｒ１Ａ）遺伝子の変異に着目して、ＧＩＳＴにおけるＰＰＰ２Ｒ１Ａ変異の有無と予後、治療抵抗性を検討した。その結果、ＰＰＰ２Ｒ１Ａのエクリン５及びエクソン６中の変異の存在が、ＧＩＳＴの予後、治療抵抗性と相関することを見出し、本発明を完成した。 Therefore, the present inventor has paid attention to the mutation of protein phosphatase 2 regulatory subunit Aα (Protein phosphatase 2, regulatory subunit, alpha: PPP2R1A) gene reported in other cancer types such as endometrial cancer and ovarian cancer. Then, the presence or absence of the PPP2R1A mutation in GIST, prognosis, and treatment resistance were examined. As a result, the present inventors have found that the presence of mutations in eccrine 5 and exon 6 of PPP2R1A correlates with the prognosis and treatment resistance of GIST, thereby completing the present invention.
すなわち、本発明は、次の〔１〕〜〔６〕を提供するものである。 That is, the present invention provides the following [1] to [6].
〔１〕被験者におけるＧＩＳＴの予後又は化学療法抵抗性を診断する目的で、被験者から得た試料中の、ＰＰＰ２Ｒ１Ａ遺伝子のエクソン５及び／又はエクソン６中の１又は２以上の変異の存在を検出する方法。
〔２〕前記変異が、次の（１）〜（２６）の変異から選ばれる１又は２以上である〔１〕記載の方法。
（１）エクソン５の１７３番目のＬｅｕがＰｒｏへの変異
（２）エクソン５の１８４番目のＡｌａがＡｓｐへの変異
（３）エクソン５の１９３番目のＡｌａがＴｈｒへの変異
（４）エクソン５の１９４番目のＬｙｓがＡｒｇへの変異
（５）エクソン５の１９５番目のＶａｌがＭｅｔへの変異
（６）エクソン５の１９７番目のＧｌｕがＧｌｙへの変異
（７）エクソン５の２０１番目のＶａｌがＡｌａへの変異
（８）エクソン５の２０１番目のＶａｌがＩｌｅへの変異
（９）エクソン５の２０４番目のＧｌｕがＶａｌへの変異
（10）エクソン５の２０４番目のＧｌｕがＧｌｙへの変異
（11）エクソン５の２０６番目のＩｌｅがＴｈｒへの変異
（12）エクソン５の２０８番目のＭｅｔがＴｈｒへの変異
（13）エクソン５の２０９番目のＰｈｅがＬｅｕへの変異
（14）エクソン５の２１１番目のＡｓｎがＡｓｐへの変異
（15）エクソン５の２１４番目のＳｅｒがＰｒｏへの変異
（16）エクソン６の２２５番目のＶａｌがＭｅｔへの変異
（17）エクソン６の２３２番目のＡｌａがＴｈｒへの変異
（18）エクソン６の２３３番目のＧｌｎがＨｉｓへの変異
（19）エクソン６の２３４番目のＬｅｕがＰｒｏへの変異
（20）エクソン６の２３８番目のＧｌｕがＬｙｓへの変異
（21）エクソン６の２４４番目のＶａｌがＭｅｔへの変異
（22）エクソン６の２４８番目のＬｅｕがＧｌｎへの変異
（23）エクソン６の２５２番目のＡｌａがＴｈｒへの変異
（24）エクソン６の２５７番目のＴｒｐが終止コドンへの変異
（25）エクソン６の２６１番目のＴｙｒがＨｉｓへの変異
（26）エクソン６の２６６番目のＬｙｓがＡｒｇへの変異
〔３〕ＰＰＰ２Ｒ１Ａ遺伝子のエクソン５及び／又はエクソン６を増幅できるように設計したプライマーセットを用いて、前記被験者から得た試料中の核酸を増幅させる工程を含む〔１〕記載の方法。
〔４〕次の（１）〜（２６）から選ばれる１又は２以上の変異を含む領域を増幅できるように設計したプライマーセットを用いる〔３〕記載の方法。
（１）エクソン５の１７３番目のＬｅｕがＰｒｏへの変異
（２）エクソン５の１８４番目のＡｌａがＡｓｐへの変異
（３）エクソン５の１９３番目のＡｌａがＴｈｒへの変異
（４）エクソン５の１９４番目のＬｙｓがＡｒｇへの変異
（５）エクソン５の１９５番目のＶａｌがＭｅｔへの変異
（６）エクソン５の１９７番目のＧｌｕがＧｌｙへの変異
（７）エクソン５の２０１番目のＶａｌがＡｌａへの変異
（８）エクソン５の２０１番目のＶａｌがＩｌｅへの変異
（９）エクソン５の２０４番目のＧｌｕがＶａｌへの変異
（10）エクソン５の２０４番目のＧｌｕがＧｌｙへの変異
（11）エクソン５の２０６番目のＩｌｅがＴｈｒへの変異
（12）エクソン５の２０８番目のＭｅｔがＴｈｒへの変異
（13）エクソン５の２０９番目のＰｈｅがＬｅｕへの変異
（14）エクソン５の２１１番目のＡｓｎがＡｓｐへの変異
（15）エクソン５の２１４番目のＳｅｒがＰｒｏへの変異
（16）エクソン６の２２５番目のＶａｌがＭｅｔへの変異
（17）エクソン６の２３２番目のＡｌａがＴｈｒへの変異
（18）エクソン６の２３３番目のＧｌｎがＨｉｓへの変異
（19）エクソン６の２３４番目のＬｅｕがＰｒｏへの変異
（20）エクソン６の２３８番目のＧｌｕがＬｙｓへの変異
（21）エクソン６の２４４番目のＶａｌがＭｅｔへの変異
（22）エクソン６の２４８番目のＬｅｕがＧｌｎへの変異
（23）エクソン６の２５２番目のＡｌａがＴｈｒへの変異
（24）エクソン６の２５７番目のＴｒｐが終止コドンへの変異
（25）エクソン６の２６１番目のＴｙｒがＨｉｓへの変異
（26）エクソン６の２６６番目のＬｙｓがＡｒｇへの変異
〔５〕ＰＰＰ２Ｒ１Ａ遺伝子のエクソン５及び／又はエクソン６中の次の（１）〜（２６）から選ばれる１又は２以上の変異を含む領域にストリンジェント条件でハイブリダイズすることができるように設計したプローブを、前記被験者から得た試料中の核酸にハイブリダイズさせる工程を含む、〔１〕記載の方法。
（１）エクソン５の１７３番目のＬｅｕがＰｒｏへの変異
（２）エクソン５の１８４番目のＡｌａがＡｓｐへの変異
（３）エクソン５の１９３番目のＡｌａがＴｈｒへの変異
（４）エクソン５の１９４番目のＬｙｓがＡｒｇへの変異
（５）エクソン５の１９５番目のＶａｌがＭｅｔへの変異
（６）エクソン５の１９７番目のＧｌｕがＧｌｙへの変異
（７）エクソン５の２０１番目のＶａｌがＡｌａへの変異
（８）エクソン５の２０１番目のＶａｌがＩｌｅへの変異
（９）エクソン５の２０４番目のＧｌｕがＶａｌへの変異
（10）エクソン５の２０４番目のＧｌｕがＧｌｙへの変異
（11）エクソン５の２０６番目のＩｌｅがＴｈｒへの変異
（12）エクソン５の２０８番目のＭｅｔがＴｈｒへの変異
（13）エクソン５の２０９番目のＰｈｅがＬｅｕへの変異
（14）エクソン５の２１１番目のＡｓｎがＡｓｐへの変異
（15）エクソン５の２１４番目のＳｅｒがＰｒｏへの変異
（16）エクソン６の２２５番目のＶａｌがＭｅｔへの変異
（17）エクソン６の２３２番目のＡｌａがＴｈｒへの変異
（18）エクソン６の２３３番目のＧｌｎがＨｉｓへの変異
（19）エクソン６の２３４番目のＬｅｕがＰｒｏへの変異
（20）エクソン６の２３８番目のＧｌｕがＬｙｓへの変異
（21）エクソン６の２４４番目のＶａｌがＭｅｔへの変異
（22）エクソン６の２４８番目のＬｅｕがＧｌｎへの変異
（23）エクソン６の２５２番目のＡｌａがＴｈｒへの変異
（24）エクソン６の２５７番目のＴｒｐが終止コドンへの変異
（25）エクソン６の２６１番目のＴｙｒがＨｉｓへの変異
（26）エクソン６の２６６番目のＬｙｓがＡｒｇへの変異
〔６〕ＰＰＰ２Ｒ１Ａ遺伝子のエクソン５及び／又はエクソン６中の次の（１）〜（２６）から選ばれる１又は２以上の変異を含む領域を増幅できるように設計したプライマーセット又は当該領域にストリンジェント条件でハイブリダイズすることができるように設計したプローブを含有するＧＩＳＴの予後又は化学療法抵抗性の診断薬。
（１）エクソン５の１７３番目のＬｅｕがＰｒｏへの変異
（２）エクソン５の１８４番目のＡｌａがＡｓｐへの変異
（３）エクソン５の１９３番目のＡｌａがＴｈｒへの変異
（４）エクソン５の１９４番目のＬｙｓがＡｒｇへの変異
（５）エクソン５の１９５番目のＶａｌがＭｅｔへの変異
（６）エクソン５の１９７番目のＧｌｕがＧｌｙへの変異
（７）エクソン５の２０１番目のＶａｌがＡｌａへの変異
（８）エクソン５の２０１番目のＶａｌがＩｌｅへの変異
（９）エクソン５の２０４番目のＧｌｕがＶａｌへの変異
（10）エクソン５の２０４番目のＧｌｕがＧｌｙへの変異
（11）エクソン５の２０６番目のＩｌｅがＴｈｒへの変異
（12）エクソン５の２０８番目のＭｅｔがＴｈｒへの変異
（13）エクソン５の２０９番目のＰｈｅがＬｅｕへの変異
（14）エクソン５の２１１番目のＡｓｎがＡｓｐへの変異
（15）エクソン５の２１４番目のＳｅｒがＰｒｏへの変異
（16）エクソン６の２２５番目のＶａｌがＭｅｔへの変異
（17）エクソン６の２３２番目のＡｌａがＴｈｒへの変異
（18）エクソン６の２３３番目のＧｌｎがＨｉｓへの変異
（19）エクソン６の２３４番目のＬｅｕがＰｒｏへの変異
（20）エクソン６の２３８番目のＧｌｕがＬｙｓへの変異
（21）エクソン６の２４４番目のＶａｌがＭｅｔへの変異
（22）エクソン６の２４８番目のＬｅｕがＧｌｎへの変異
（23）エクソン６の２５２番目のＡｌａがＴｈｒへの変異
（24）エクソン６の２５７番目のＴｒｐが終止コドンへの変異
（25）エクソン６の２６１番目のＴｙｒがＨｉｓへの変異
（26）エクソン６の２６６番目のＬｙｓがＡｒｇへの変異
[1] Detect the presence of one or more mutations in exon 5 and / or exon 6 of the PPP2R1A gene in a sample obtained from a subject for the purpose of diagnosing the prognosis of GIST or resistance to chemotherapy in the subject Method.
[2] The method according to [1], wherein the mutation is one or more selected from the following mutations (1) to (26).
(1) Mutation of 173rd Leu of exon 5 to Pro (2) Mutation of 184th Ala of exon 5 to Asp (3) Mutation of 193rd Ala of exon 5 to Thr (4) Exon 5 194 Lys mutation to Arg (5) Exon 5 195th Val mutation to Met (6) Exon 5 197th Glu mutation to Gly (7) Exon 5 201st Val Mutation to Ala (8) Mutation of 201st Val of Exon 5 to Ile (9) Mutation of 204th Glu of Exon 5 to Val (10) Mutation of 204th Glu of Exon 5 to Gly (11) Mutation of 206th Ile of exon 5 to Thr (12) Mutation of 208th Met of exon 5 to Thr (13) 209th Phe of exon 5 is Leu (14) Mutation of 211 of Asn in Exon 5 to Asp (15) Mutation of 214th Ser of Exon 5 to Pro (16) Mutation of Val 225 of Exon 6 to Met (17) Exon 6 232th Ala mutation to Thr (18) Exon 6 233rd Gln mutation to His (19) Exon 6 234th Leu mutation to Pro (20) Exon 6 238th mutation Mutation of Glu to Lys (21) Mutation of 244th Val of exon 6 to Met (22) Mutation of 248th Leu of exon 6 to Gln (23) Mutation of 252nd Ala of exon 6 to Thr (24) Mutation of 257th Trp of exon 6 to stop codon (25) Mutation of 261st Tyr of exon 6 to His (26) 266th Lys of exon 6 is A Mutation to g [3] Amplifying nucleic acid in a sample obtained from the subject using a primer set designed to amplify exon 5 and / or exon 6 of the PPP2R1A gene [1] Method.
[4] The method according to [3], wherein a primer set designed to amplify a region containing one or more mutations selected from the following (1) to (26) is used.
(1) Mutation of 173rd Leu of exon 5 to Pro (2) Mutation of 184th Ala of exon 5 to Asp (3) Mutation of 193rd Ala of exon 5 to Thr (4) Exon 5 194 Lys mutation to Arg (5) Exon 5 195th Val mutation to Met (6) Exon 5 197th Glu mutation to Gly (7) Exon 5 201st Val Mutation to Ala (8) Mutation of 201st Val of Exon 5 to Ile (9) Mutation of 204th Glu of Exon 5 to Val (10) Mutation of 204th Glu of Exon 5 to Gly (11) Mutation of 206th Ile of exon 5 to Thr (12) Mutation of 208th Met of exon 5 to Thr (13) 209th Phe of exon 5 is Leu (14) Mutation of 211 of Asn in Exon 5 to Asp (15) Mutation of 214th Ser of Exon 5 to Pro (16) Mutation of Val 225 of Exon 6 to Met (17) Exon 6 232th Ala mutation to Thr (18) Exon 6 233rd Gln mutation to His (19) Exon 6 234th Leu mutation to Pro (20) Exon 6 238th mutation Mutation of Glu to Lys (21) Mutation of 244th Val of exon 6 to Met (22) Mutation of 248th Leu of exon 6 to Gln (23) Variation of 252nd Ala of exon 6 to Thr (24) Mutation of 257th Trp of exon 6 to stop codon (25) Mutation of 261st Tyr of exon 6 to His (26) 266th Lys of exon 6 is A Mutation to g [5] Hybridization under stringent conditions to a region containing one or more mutations selected from the following (1) to (26) in exon 5 and / or exon 6 of the PPP2R1A gene The method according to [1], comprising a step of hybridizing a probe designed so as to be able to hybridize to a nucleic acid in a sample obtained from the subject.
(1) Mutation of 173rd Leu of exon 5 to Pro (2) Mutation of 184th Ala of exon 5 to Asp (3) Mutation of 193rd Ala of exon 5 to Thr (4) Exon 5 194 Lys mutation to Arg (5) Exon 5 195th Val mutation to Met (6) Exon 5 197th Glu mutation to Gly (7) Exon 5 201st Val Mutation to Ala (8) Mutation of 201st Val of Exon 5 to Ile (9) Mutation of 204th Glu of Exon 5 to Val (10) Mutation of 204th Glu of Exon 5 to Gly (11) Mutation of 206th Ile of exon 5 to Thr (12) Mutation of 208th Met of exon 5 to Thr (13) 209th Phe of exon 5 is Leu (14) Mutation of 211 of Asn in Exon 5 to Asp (15) Mutation of 214th Ser of Exon 5 to Pro (16) Mutation of Val 225 of Exon 6 to Met (17) Exon 6 232th Ala mutation to Thr (18) Exon 6 233rd Gln mutation to His (19) Exon 6 234th Leu mutation to Pro (20) Exon 6 238th mutation Mutation of Glu to Lys (21) Mutation of 244th Val of exon 6 to Met (22) Mutation of 248th Leu of exon 6 to Gln (23) Mutation of 252nd Ala of exon 6 to Thr (24) Mutation of 257th Trp of exon 6 to stop codon (25) Mutation of 261st Tyr of exon 6 to His (26) 266th Lys of exon 6 is A Mutation to g [6] Primer set designed to amplify a region containing one or more mutations selected from the following (1) to (26) in exon 5 and / or exon 6 of the PPP2R1A gene: A diagnostic agent for GIST prognosis or chemoresistance comprising a probe designed to be able to hybridize to the region under stringent conditions.
(1) Mutation of 173rd Leu of exon 5 to Pro (2) Mutation of 184th Ala of exon 5 to Asp (3) Mutation of 193rd Ala of exon 5 to Thr (4) Exon 5 194 Lys mutation to Arg (5) Exon 5 195th Val mutation to Met (6) Exon 5 197th Glu mutation to Gly (7) Exon 5 201st Val Mutation to Ala (8) Mutation of 201st Val of Exon 5 to Ile (9) Mutation of 204th Glu of Exon 5 to Val (10) Mutation of 204th Glu of Exon 5 to Gly (11) Mutation of 206th Ile of exon 5 to Thr (12) Mutation of 208th Met of exon 5 to Thr (13) 209th Phe of exon 5 is Leu (14) Mutation of 211 of Asn in Exon 5 to Asp (15) Mutation of 214th Ser of Exon 5 to Pro (16) Mutation of Val 225 of Exon 6 to Met (17) Exon 6 232th Ala mutation to Thr (18) Exon 6 233rd Gln mutation to His (19) Exon 6 234th Leu mutation to Pro (20) Exon 6 238th mutation Mutation of Glu to Lys (21) Mutation of 244th Val of exon 6 to Met (22) Mutation of 248th Leu of exon 6 to Gln (23) Mutation of 252nd Ala of exon 6 to Thr (24) Mutation of 257th Trp of exon 6 to stop codon (25) Mutation of 261st Tyr of exon 6 to His (26) 266th Lys of exon 6 is A Mutation to g
本発明によれば、ＧＩＳＴの予後（進展、転移等）及び化学療法抵抗性が診断できることから、新たな治療指針の策定に有用である。 According to the present invention, since prognosis (progress, metastasis, etc.) and chemotherapy resistance of GIST can be diagnosed, it is useful for formulating new treatment guidelines.
本発明の検出方法は、被験者におけるＧＩＳＴの予後又は化学療法抵抗性を診断する目的で、被験者から得た試料中のＰＰＰ２Ｒ１Ａ遺伝子のエクソン５及び／又はエクソン６中の１又は２以上の変異の有無を検出する。 The detection method of the present invention is the presence or absence of one or more mutations in exon 5 and / or exon 6 of the PPP2R1A gene in a sample obtained from a subject for the purpose of diagnosing the prognosis of GIST or resistance to chemotherapy in the subject. Is detected.
被験者は、ＧＩＳＴ患者である。ＧＩＳＴ患者のうち、外科的治療及び／又は化学療法を受けた又は受けている患者であるのが好ましいが、診断目的で採取される生検検体のみを有する患者に対しても有効である。 The subject is a GIST patient. Of the GIST patients, those who have or have undergone surgical treatment and / or chemotherapy are preferred, but are also effective for patients with only biopsy specimens taken for diagnostic purposes.
被験者から得た試料としては、被験者からの採取物（生体から分離した試料）、具体的には、採取された細胞組織、体液（血液、口腔粘液等）、生検された試料等を用いるが、好適には生検された試料を用いる。採取した試料からゲノムＤＮＡもしくはＲＮＡを抽出して用いることができ、またその転写産物（例えば、ｍＲＮＡ、ｃＤＮＡ、蛋白質）を用いることもできる。特に、ｍＲＮＡ又はｃＤＮＡを調製して用いることが好ましい。 As a sample obtained from the subject, a sample collected from the subject (sample separated from the living body), specifically, a collected cell tissue, body fluid (blood, oral mucus, etc.), a biopsy sample, etc. are used. Preferably, a biopsy sample is used. Genomic DNA or RNA can be extracted from the collected sample and used, and its transcription product (eg, mRNA, cDNA, protein) can also be used. In particular, it is preferable to prepare and use mRNA or cDNA.
検出対象となる変異は、ＰＰＰ２Ｒ１Ａ遺伝子のエクソン５及び／又はエクソン６中の１又は２以上の変異である。当該ＰＰＰ２Ｒ１Ａ遺伝子のエクソン５又はエクソン６中の変異と、ＧＩＳＴの予後や治療抵抗性との関係は、従来全く知られていない。 The mutation to be detected is one or more mutations in exon 5 and / or exon 6 of the PPP2R1A gene. The relationship between the mutation in exon 5 or exon 6 of the PPP2R1A gene and the prognosis or treatment resistance of GIST has never been known.
当該エクソン５及び／又はエクソン６中の変異としては、次の（１）〜（２６）から選ばれる１又は２以上が挙げられる。 Examples of the mutation in exon 5 and / or exon 6 include one or more selected from the following (1) to (26).
（１）エクソン５の１７３番目のＬｅｕがＰｒｏへの変異
（２）エクソン５の１８４番目のＡｌａがＡｓｐへの変異
（３）エクソン５の１９３番目のＡｌａがＴｈｒへの変異
（４）エクソン５の１９４番目のＬｙｓがＡｒｇへの変異
（５）エクソン５の１９５番目のＶａｌがＭｅｔへの変異
（６）エクソン５の１９７番目のＧｌｕがＧｌｙへの変異
（７）エクソン５の２０１番目のＶａｌがＡｌａへの変異
（８）エクソン５の２０１番目のＶａｌがＩｌｅへの変異
（９）エクソン５の２０４番目のＧｌｕがＶａｌへの変異
（10）エクソン５の２０４番目のＧｌｕがＧｌｙへの変異
（11）エクソン５の２０６番目のＩｌｅがＴｈｒへの変異
（12）エクソン５の２０８番目のＭｅｔがＴｈｒへの変異
（13）エクソン５の２０９番目のＰｈｅがＬｅｕへの変異
（14）エクソン５の２１１番目のＡｓｎがＡｓｐへの変異
（15）エクソン５の２１４番目のＳｅｒがＰｒｏへの変異
（16）エクソン６の２２５番目のＶａｌがＭｅｔへの変異
（17）エクソン６の２３２番目のＡｌａがＴｈｒへの変異
（18）エクソン６の２３３番目のＧｌｎがＨｉｓへの変異
（19）エクソン６の２３４番目のＬｅｕがＰｒｏへの変異
（20）エクソン６の２３８番目のＧｌｕがＬｙｓへの変異
（21）エクソン６の２４４番目のＶａｌがＭｅｔへの変異
（22）エクソン６の２４８番目のＬｅｕがＧｌｎへの変異
（23）エクソン６の２５２番目のＡｌａがＴｈｒへの変異
（24）エクソン６の２５７番目のＴｒｐが終止コドンへの変異
（25）エクソン６の２６１番目のＴｙｒがＨｉｓへの変異
（26）エクソン６の２６６番目のＬｙｓがＡｒｇへの変異
(1) Mutation of 173rd Leu of exon 5 to Pro (2) Mutation of 184th Ala of exon 5 to Asp (3) Mutation of 193rd Ala of exon 5 to Thr (4) Exon 5 194 Lys mutation to Arg (5) Exon 5 195th Val mutation to Met (6) Exon 5 197th Glu mutation to Gly (7) Exon 5 201st Val Mutation to Ala (8) Mutation of 201st Val of Exon 5 to Ile (9) Mutation of 204th Glu of Exon 5 to Val (10) Mutation of 204th Glu of Exon 5 to Gly (11) Mutation of 206th Ile of exon 5 to Thr (12) Mutation of 208th Met of exon 5 to Thr (13) 209th Phe of exon 5 is Leu (14) Mutation of 211 of Asn in Exon 5 to Asp (15) Mutation of 214th Ser of Exon 5 to Pro (16) Mutation of Val 225 of Exon 6 to Met (17) Exon 6 232th Ala mutation to Thr (18) Exon 6 233rd Gln mutation to His (19) Exon 6 234th Leu mutation to Pro (20) Exon 6 238th mutation Mutation of Glu to Lys (21) Mutation of 244th Val of exon 6 to Met (22) Mutation of 248th Leu of exon 6 to Gln (23) Mutation of 252nd Ala of exon 6 to Thr (24) Mutation of 257th Trp of exon 6 to stop codon (25) Mutation of 261st Tyr of exon 6 to His (26) 266th Lys of exon 6 is A Mutation to g
本発明においては、検出対象変異を有するタンパク質又は検出対象変異を有するタンパク質をコードするポリヌクレオチドのいずれを検出対象としてもよい。 In the present invention, either a protein having a detection target mutation or a polynucleotide encoding a protein having a detection target mutation may be the detection target.
本発明においてポリヌクレオチドの変異の存在を検出する場合、被験者から得た試料のゲノムＤＮＡ中の変異ポリヌクレオチドの存在を検出すること、あるいは、被験者から得た試料から抽出したゲノムＤＮＡの転写産物（例えば、ｍＲＮＡ又はｃＤＮＡ）を調製し、変異ポリヌクレオチドに対応するｍＲＮＡ又はｃＤＮＡの存在を検出することにより実施できる。ゲノムＤＮＡの抽出、ｍＲＮＡ及びｃＤＮＡの調製は当該分野で公知の方法で行うことができ、市販のキットを用いて簡便に行うことができる。 In the present invention, when the presence of a polynucleotide mutation is detected, the presence of the mutated polynucleotide in the genomic DNA of a sample obtained from the subject is detected, or the transcript of genomic DNA extracted from the sample obtained from the subject ( For example, it can be carried out by preparing mRNA or cDNA) and detecting the presence of mRNA or cDNA corresponding to the mutated polynucleotide. Extraction of genomic DNA and preparation of mRNA and cDNA can be performed by methods known in the art, and can be easily performed using a commercially available kit.
この検出工程は、当該分野で公知の遺伝子変異解析方法を使用することができる。例えば、被験者から得た試料由来の核酸（例えば、ｍＲＮＡやｃＤＮＡ等）に対して公知の核酸増幅方法（ＰＣＲ法、ＲＴ−ＰＣＲ法、ＬＣＲ法（Ｌｉｇａｓｅ ｃｈａｉｎ Ｒｅａｃｔｉｏｎ）、ＳＤＡ法（Ｓｔｒａｎｄ ｄｉｓｐｌａｃｅｍｅｎｔ ａｍｐｌｉｆｉｃａｔｉｏｎ）、ＮＡＳＢＡ法（Ｎｕｃｌｅｉｃ ａｃｉｄ ｓｅｑｕｅｎｃｅ−ｂａｓｅｄ ａｍｐｌｉｆｉｃａｔｉｏｎ）、ＩＣＡＮ法（Ｉｓｏｔｈｅｒｍａｌ ａｎｄ ｃｈｉｍｅｒｉｃ ｐｒｉｍｅｒ−ｉｎｉｔｉａｔｅｄ ａｍｐｌｉｆｉｃａｔｉｏｎ ｏｆ ｎｕｃｌｅｉｃ ａｃｉｄｓ）、ＬＡＭＰ法（Ｌｏｏｐ−ｍｅｄｉａｔｅｄ ｉｓｏｔｈｅｒｍａｌ ａｍｐｌｉｆｉｃａｔｉｏｎ）、ＴＭＡ法（Ｇｅｎ−Ｐｒｏｂｅ’ｓ ＴＭＡ ｓｙｓｔｅｍ）等）を用いて、変異ポリヌクレオチド中の検出対象変異に対応する部分を含む領域を増幅させる工程を含み、増幅後、この増幅産物について配列決定を行うことによって変異の有無を確認することができる。このような配列決定法としては、ダイレクトシーケンス法等の当該分野で公知の方法を使用することができる。 For this detection step, a gene mutation analysis method known in the art can be used. For example, a known nucleic acid amplification method (PCR method, RT-PCR method, LCR method (Ligase chain Reaction), SDA method (Strand displacement amplification) for nucleic acid derived from a sample obtained from a subject (eg, mRNA, cDNA, etc.) , NASBA method (Nucleic acid sequence-based amplification), ICAN method (Isomal and chimeric-primed-amplified TM method), LAMP method (Loop method) Using the mutant polynucleotide Comprising the step of amplifying a region including a portion corresponding to the detected mutation in fault, after amplification, it is possible to confirm the presence or absence of a mutation by performing sequenced amplified product. As such a sequencing method, a method known in the art such as a direct sequence method can be used.
核酸増幅方法に用いられるプライマーは、変異ポリヌクレオチド中の検出対象変異領域を増幅できるものであれば、特には限定されず、変異ポリヌクレオチドの塩基配列に基づいて設計する。プライマー設計は、プライマー設計ソフトウェア（例えば、Ｐｒｉｍｅｒ Ｅｘｐｒｅｓｓ；ＰＥ Ｂｉｏｓｙｓｔｅｍｓ）等を利用してできる。また、増幅産物のサイズが大きくなると増幅効率が悪くなるため、センスプライマーとアンチセンスプライマーは、ｍＲＮＡ又はｃＤＮＡを対象に増幅したときの増幅産物の大きさが１ｋｂ以下になるように設定するのが適切である。 The primer used in the nucleic acid amplification method is not particularly limited as long as it can amplify the mutation region to be detected in the mutant polynucleotide, and is designed based on the base sequence of the mutant polynucleotide. Primer design can be performed using primer design software (for example, Primer Express; PE Biosystems). In addition, since the amplification efficiency decreases as the size of the amplification product increases, the sense primer and the antisense primer should be set so that the size of the amplification product when mRNA or cDNA is amplified is 1 kb or less. Is appropriate.
核酸増幅方法を用いる別の公知の遺伝子変異解析方法として、ＲＦＬＰ法（制限酵素断片長多型解析法）、ＴａｑＭａｎ法、アレル特異的プライマーＰＣＲ（ＡＳＰ−ＰＣＲ）法、ＳＳＣＰ法等のＰＣＲに基づく方法も使用可能である。ＲＦＬＰ法は、ＰＣＲにより検出対象変異領域を増幅した後、変異に基づき制限酵素認識部位に変化が生じる場合に、その制限酵素処理後の核酸断片の差異を電気泳動により解析する。ＴａｑＭａｎ法は、５’末端に蛍光物質（ＦＡＭ、ＶＩＣ等）、３’末端にクエンチャー（消光）物質で修飾したプローブ（ＴａｑＭａｎプローブ）をＰＣＲ反応系に添加する方法である。本方法では、検出対象変異領域に対してＴａｑＭａｎプローブをハイブリダイズさせ、プライマーからのＰＣＲ反応を行うと、伸長反応におけるＤＮＡポリメラーゼの５’→３’エキソヌクレアーゼ活性によりＴａｑＭａｎプローブが分解され、それにより蛍光物質がプローブより遊離し、クエンチャーによる抑制が解除されて蛍光物質から蛍光が発し、これにより目的の変異が検出される。ＡＳＰ−ＰＣＲ法は、３’末端に検出対象変異部位を有するようにプライマーを設計し、ＰＣＲによる核酸増幅の有無を検出する方法である。ＳＳＣＰ法では、検出対象変異領域をＰＣＲ増幅した後、一本鎖ＤＮＡに分離し、これを非変性ゲルでの電気泳動により分離する。変異によりＤＮＡの高次構造が変化し、これがゲル上の移動度の差異を反映する。 Other known gene mutation analysis methods using nucleic acid amplification methods are based on PCR such as RFLP method (restriction enzyme fragment length polymorphism analysis method), TaqMan method, allele-specific primer PCR (ASP-PCR) method, SSCP method, etc. A method can also be used. In the RFLP method, after a mutation region to be detected is amplified by PCR, when a change occurs in a restriction enzyme recognition site based on the mutation, a difference in nucleic acid fragments after the restriction enzyme treatment is analyzed by electrophoresis. The TaqMan method is a method of adding a probe (TaqMan probe) modified with a fluorescent substance (FAM, VIC, etc.) at the 5 ′ end and a quencher (quenching) substance at the 3 ′ end to the PCR reaction system. In this method, when a TaqMan probe is hybridized to the mutation region to be detected and a PCR reaction is performed from the primer, the TaqMan probe is degraded by the 5 ′ → 3 ′ exonuclease activity of the DNA polymerase in the extension reaction, thereby The fluorescent substance is released from the probe, the suppression by the quencher is released, and fluorescence is emitted from the fluorescent substance, whereby the target mutation is detected. The ASP-PCR method is a method of detecting the presence or absence of nucleic acid amplification by PCR by designing a primer so as to have a detection target mutation site at the 3 'end. In the SSCP method, a mutation region to be detected is amplified by PCR and then separated into single-stranded DNA, which is separated by electrophoresis on a non-denaturing gel. Mutation changes the higher order structure of the DNA, which reflects the difference in mobility on the gel.
また別の遺伝子変異解析方法として、プローブハイブリダイゼーションに基づく方法も使用可能であり、例えば、ＤＮＡチップ法、Ｉｎｖａｄｅｒ法、融解曲線解析法等が挙げられる。ＤＮＡチップ法では、検出対象変異領域を含むＤＮＡを基板上に配置し、被験者由来の核酸をＤＮＡチップにハイブリダイズさせ、ハイブリダイズの有無を確認する。Ｉｎｖａｄｅｒ法では、検出対象変異部位の前後にハイブリダイズする２種の核酸（Ｉｎｖａｄｅｒオリゴ及びシグナルプローブ）を被験者由来の核酸に反応させた後、形成されるＤＮＡの三重鎖構造を認識するＣｌｅｖａｓｅ酵素を作用させ、シグナルプローブ中のＦｌａｐを遊離させる。次いで、Ｆｌａｐが検出用ＦＲＥＴ Ｐｒｏｂｅとハイブリダイズし、これに同酵素が再度作用し、ＦＲＥＴ Ｐｒｏｂｅから遊離された蛍光物質の蛍光を検出する。融解曲線解析法は、検出対象変異領域に蛍光標識プローブをハイブリダイズさせた後、徐々に温度を上昇させ、温度上昇に伴う蛍光の変化を測定して融解曲線を作成する。これにより、変異の有無に伴う融解温度の変化を確認する。 As another gene mutation analysis method, a method based on probe hybridization can also be used, and examples thereof include a DNA chip method, an Invader method, and a melting curve analysis method. In the DNA chip method, DNA containing a mutation region to be detected is placed on a substrate, nucleic acid derived from a subject is hybridized to the DNA chip, and the presence or absence of hybridization is confirmed. In the Invader method, two types of nucleic acids (Invader oligo and signal probe) that hybridize before and after the mutation site to be detected are reacted with a nucleic acid derived from a subject, and then a Cleavease enzyme that recognizes the triplex structure of the DNA to be formed is used. Act to release the flap in the signal probe. Next, the flap hybridizes with the detection FRET probe, and the enzyme acts again on this to detect the fluorescence of the fluorescent substance released from the FRET probe. In the melting curve analysis method, after a fluorescently labeled probe is hybridized to the mutation region to be detected, the temperature is gradually raised, and a change in fluorescence accompanying the temperature rise is measured to create a melting curve. This confirms the change in melting temperature with or without mutation.
また別の遺伝子変異解析方法として、プライマーエクステンションに基づく方法も使用可能であり、例えば、ＳＮａＰｓｈｏｔ法、Ｐｙｒｏｓｅｑｕｅｎｃｉｎｇ法が挙げられる。ＳＮａＰｓｈｏｔ法では、検出対象変異部位に隣接するプライマーと蛍光標識したｄｄＮＴＰを用い、プライマーから一塩基のみを伸長させて取り込まれた塩基を検出する。Ｐｙｒｏｓｅｑｕｅｎｃｉｎｇ法では、プライマー伸長反応において、１種類ずつｄＮＴＰを添加し、ポリメラーゼの反応によりｄＮＴＰが取り込まれるとピロリン酸が生じる。生じたピロリン酸をルシフェラーゼによる蛍光反応で検出し、発光ピークパターンから塩基配列を決定する。 As another gene mutation analysis method, a method based on primer extension can be used, and examples thereof include SNaPshot method and Pyrosequencing method. In the SNaPshot method, a primer adjacent to the mutation site to be detected and a fluorescently labeled ddNTP are used, and the incorporated base is detected by extending only one base from the primer. In the pyrosequencing method, pyrophosphoric acid is generated when dNTPs are added one by one in the primer extension reaction and dNTPs are incorporated by the polymerase reaction. The resulting pyrophosphate is detected by a fluorescence reaction with luciferase, and the base sequence is determined from the emission peak pattern.
当業者であれば、使用する遺伝子変異解析方法に基づいて、使用されるプライマー及びプローブを適宜設計することができ、特に限定されることはないが、例えば、化学合成法によって製造することができる。 A person skilled in the art can appropriately design primers and probes to be used based on the gene mutation analysis method to be used, and is not particularly limited. For example, it can be produced by a chemical synthesis method. .
本発明により、前記の変異の１又は２以上が検出された場合、当該被験者の予後が悪い、及び／又は化学療法抵抗性があると診断できる。 According to the present invention, when one or more of the above mutations are detected, it can be diagnosed that the subject has a poor prognosis and / or chemoresistance.
本発明に用いるプライマーセットは、前記エクソン５及び／又はエクソン６の変異領域を増幅できるように設計したセンスプライマー及びアンチセンスプライマーを含む。アンチセンスプライマーは、変異ポリヌクレオチドにストリンジェントな条件下（好ましくは、よりストリンジェントな条件下）でハイブリダイズする核酸分子（好ましくは、少なくとも１６塩基の核酸分子）からなり、センスプライマーは、変異ポリヌクレオチドの相補鎖にストリンジェントな条件（好ましくは、よりストリンジェントな条件下）でハイブリダイズする核酸分子（好ましくは、少なくとも１６塩基の核酸分子）からなる。 The primer set used in the present invention includes a sense primer and an antisense primer designed so that the mutated region of exon 5 and / or exon 6 can be amplified. The antisense primer is composed of a nucleic acid molecule (preferably a nucleic acid molecule of at least 16 bases) that hybridizes with a mutant polynucleotide under stringent conditions (preferably under more stringent conditions). It consists of a nucleic acid molecule (preferably a nucleic acid molecule of at least 16 bases) that hybridizes with a complementary strand of a polynucleotide under stringent conditions (preferably under more stringent conditions).
好ましくは、本発明のプライマーセットは、前記の（１）〜（２６）から選ばれる１又は２以上の変異を含む領域を増幅できるように設計したセンスプライマー及びアンチセンスプライマーを含む。 Preferably, the primer set of the present invention includes a sense primer and an antisense primer designed to amplify a region containing one or more mutations selected from the above (1) to (26).
本発明に用いるプローブは、前記のエクソン５及び／又はエクソン６の変異領域にハイブリダイズすることができるように設計したプローブである。本発明のプローブは、変異ポリヌクレオチド又はその相補鎖中の検出対象変異領域にストリンジェントな条件下（好ましくは、よりストリンジェントな条件下）でハイブリダイズする核酸分子（好ましくは、少なくとも１６塩基の核酸分子）からなる。 The probe used in the present invention is a probe designed so that it can hybridize to the mutation region of exon 5 and / or exon 6 described above. The probe of the present invention comprises a nucleic acid molecule (preferably having at least 16 bases) that hybridizes under stringent conditions (preferably under more stringent conditions) to a mutant polynucleotide or a mutant region to be detected in its complementary strand. Nucleic acid molecule).
好ましくは、本発明のプローブは、前記（１）〜（２６）から選ばれる１又は２以上を含む領域にハイブリダイズするように設計される。 Preferably, the probe of the present invention is designed to hybridize to a region containing one or more selected from the above (1) to (26).
前記「ストリンジェントな条件」とは、ハイブリダイゼーションのための条件として、「５×ＳＳＰＥ、５×Ｄｅｎｈａｒｄｔ’ｓ液、０．５％ＳＤＳ、５０％ホルムアミド、２００μｇ／ｍＬ鮭精子ＤＮＡ、４２℃オーバーナイト」、洗浄のための条件として、「０．５×ＳＳＣ、０．１％ＳＤＳ、４２℃」の条件である。「よりストリンジェントな条件」とは、ハイブリダイゼーションのための条件として、「５×ＳＳＰＥ、５×Ｄｅｎｈａｒｄｔ’ｓ液、０．５％ＳＤＳ、５０％ホルムアミド、２００μｇ／ｍＬ鮭精子ＤＮＡ、４２℃オーバーナイト」、洗浄のための条件として、「０．２×ＳＳＣ、０．１％ＳＤＳ、６５℃」の条件である。 The “stringent conditions” are as follows: “5 × SSPE, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 200 μg / mL sperm DNA, over 42 ° C. “Night” is a condition for cleaning, “0.5 × SSC, 0.1% SDS, 42 ° C.”. “More stringent conditions” means “5 × SSPE, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 200 μg / mL sperm DNA, over 42 ° C.” “Night”, the conditions for cleaning are “0.2 × SSC, 0.1% SDS, 65 ° C.”.
本発明のプライマー及びプローブは、通常、１５〜４０塩基、好ましくは１６〜２４塩基、更に好ましくは１８〜２４塩基、特に好ましくは２０〜２４塩基の鎖長を有する。 The primer and probe of the present invention usually have a chain length of 15 to 40 bases, preferably 16 to 24 bases, more preferably 18 to 24 bases, and particularly preferably 20 to 24 bases.
本発明のプライマーセット及びプローブは、例えば、化学合成法によって当業者に容易に製造することができ、また、検出方法に応じて、蛍光標識等で標識化してもよい。 The primer set and probe of the present invention can be easily produced by those skilled in the art, for example, by chemical synthesis, and may be labeled with a fluorescent label or the like depending on the detection method.
本発明の診断薬は、ＧＩＳＴの予後又は化学療法抵抗性の診断薬であり、前記のプライマーセット又はプローブの少なくとも１つを含む。 The diagnostic agent of the present invention is a diagnostic agent having a prognosis of GIST or resistance to chemotherapy, and includes at least one of the aforementioned primer set or probe.
次に実施例を挙げて本発明を更に詳細に説明する。 EXAMPLES Next, an Example is given and this invention is demonstrated still in detail.
実施例１
（１）ＧＩＳＴ患者由来のホルマリン固定パラフィン包埋検体より腫瘍部のゲノムＤＮＡを抽出し、ＰＰＰ２Ｒ１Ａ遺伝子のエクソン５、エクソン６について、エクソン５は２種類、エクソン６は１種類のプライマーを用いてＰＣＲを施行し、両方向性のダイレクトシークエンス法で配列を確定し解析した。ＰＣＲは、９４℃２分後、９４℃３０秒、５５℃３０秒、７２℃３０秒を４０サイクル、その後７２℃２分で行った。
Example 1
(1) Genomic DNA in a tumor site is extracted from a formalin-fixed paraffin-embedded specimen derived from a GIST patient, and the PCR2 exon 5 and exon 6 of the PPP2R1A gene are subjected to PCR using two types of exons 5 and one type of exons 6 The sequence was determined and analyzed by the bidirectional direct sequencing method. PCR was performed at 94 ° C. for 2 minutes, followed by 40 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 30 seconds, and then 72 ° C. for 2 minutes.
用いたプライマーセットを以下に示す。
（１）PPP2R1A exon5 F1/R1（Product size: 219bp）
F:5’-AAAACCTGGACCCACACAAC-3’（配列番号１）
R:5’-TTGGAGAACATGGGGATGAT-3’（配列番号２）
（２）PPP2R1A exon5 F1/R1（Product size: 165bp）
F: 5’-AAAACCTGGACCCACACAAC-3’（配列番号３）
R: 5’-TTGGAGAACATGGGGATGAT-3’（配列番号４）
（３）PPP2R1A exon6 F1/R1（Product size: 219bp）
F: 5’-CTCTCCTCTCCCTAGGACTCG-3’（配列番号５）
R: 5’-GTGTCAGTGTCCCCACCAGT-3’ （配列番号６）
The primer sets used are shown below.
(1) PPP2R1A exon5 F1 / R1 (Product size: 219bp)
F: 5'-AAAACCTGGACCCACACAAC-3 '(SEQ ID NO: 1)
R: 5'-TTGGAGAACATGGGGATGAT-3 '(SEQ ID NO: 2)
(2) PPP2R1A exon5 F1 / R1 (Product size: 165bp)
F: 5'-AAAACCTGGACCCACACAAC-3 '(SEQ ID NO: 3)
R: 5'-TTGGAGAACATGGGGATGAT-3 '(SEQ ID NO: 4)
(3) PPP2R1A exon6 F1 / R1 (Product size: 219bp)
F: 5'-CTCTCCTCTCCCTAGGACTCG-3 '(SEQ ID NO: 5)
R: 5'-GTGTCAGTGTCCCCACCAGT-3 '(SEQ ID NO: 6)
（１）のプライマーセットのＰＣＲ産物を配列番号７に、（２）のプライマーセットのＰＣＲ産物を配列番号８に、（３）のプライマーセットのＰＣＲ産物を配列番号９にそれぞれ示す。 The PCR product of the primer set of (1) is shown in SEQ ID NO: 7, the PCR product of the primer set of (2) is shown in SEQ ID NO: 8, and the PCR product of the primer set of (3) is shown in SEQ ID NO: 9.
（２）その結果、ＧＩＳＴ患者９６症例のうち、１７症例に表１に記載の変異が検出された。 (2) As a result, mutations described in Table 1 were detected in 17 cases among 96 cases of GIST patients.
遺伝子変異のあった１７症例のうち、１個の変異を有する症例は１０例、変異を複数有する症例が７例あり、そのうち３個変異を有する症例が３症例、２個の症例が４例であった。 Of the 17 cases with gene mutations, there are 10 cases with 1 mutation, 7 cases with multiple mutations, 3 cases with 3 mutations and 4 cases with 2 cases there were.
９６症例の臨床経過より無病生存期間、生存期間との相関を統計解析したところ、変異陽性症例は、１７症例（１７．７％）であり、無病生存期間はｐ＝０．０２１＜０．０５、生存期間はｐ＝０．００２＜０．０１であった（図１、図２）。
この結果より、ＧＩＳＴ患者におけるＰＰＰ２Ｒ１Ａ遺伝子中のエクソン５及びエクソン６中のいずれか１個以上の変異がある症例は、予後が悪いことが明らかとなり、当該変異の検出はＧＩＳＴの予後の診断に有用であることが判明した。
From the clinical course of 96 cases, the disease-free survival time and the correlation with the survival time were statistically analyzed. As a result, 17 mutation-positive cases (17.7%) were found, and the disease-free survival time was p = 0.021 <0.05. The survival time was p = 0.002 <0.01 (FIGS. 1 and 2).
From this result, it is clear that cases with any one or more mutations in exon 5 and exon 6 in the PPP2R1A gene in GIST patients have a poor prognosis, and the detection of such mutations is useful for the diagnosis of the prognosis of GIST. It turned out to be.
Claims (4)
前記変異が、次の（１）〜（２６）の変異から選ばれる１又は２以上である遺伝子検出方法。
（１）エクソン５の１７３番目のＬｅｕがＰｒｏへの変異
（２）エクソン５の１８４番目のＡｌａがＡｓｐへの変異
（３）エクソン５の１９３番目のＡｌａがＴｈｒへの変異
（４）エクソン５の１９４番目のＬｙｓがＡｒｇへの変異
（５）エクソン５の１９５番目のＶａｌがＭｅｔへの変異
（６）エクソン５の１９７番目のＧｌｕがＧｌｙへの変異
（７）エクソン５の２０１番目のＶａｌがＡｌａへの変異
（８）エクソン５の２０１番目のＶａｌがＩｌｅへの変異
（９）エクソン５の２０４番目のＧｌｕがＶａｌへの変異
（10）エクソン５の２０４番目のＧｌｕがＧｌｙへの変異
（11）エクソン５の２０６番目のＩｌｅがＴｈｒへの変異
（12）エクソン５の２０８番目のＭｅｔがＴｈｒへの変異
（13）エクソン５の２０９番目のＰｈｅがＬｅｕへの変異
（14）エクソン５の２１１番目のＡｓｎがＡｓｐへの変異
（15）エクソン５の２１４番目のＳｅｒがＰｒｏへの変異
（16）エクソン６の２２５番目のＶａｌがＭｅｔへの変異
（17）エクソン６の２３２番目のＡｌａがＴｈｒへの変異
（18）エクソン６の２３３番目のＧｌｎがＨｉｓへの変異
（19）エクソン６の２３４番目のＬｅｕがＰｒｏへの変異
（20）エクソン６の２３８番目のＧｌｕがＬｙｓへの変異
（21）エクソン６の２４４番目のＶａｌがＭｅｔへの変異
（22）エクソン６の２４８番目のＬｅｕがＧｌｎへの変異
（23）エクソン６の２５２番目のＡｌａがＴｈｒへの変異
（24）エクソン６の２５７番目のＴｒｐが終止コドンへの変異
（25）エクソン６の２６１番目のＴｙｒがＨｉｓへの変異
（26）エクソン６の２６６番目のＬｙｓがＡｒｇへの変異 And detecting the presence of one or more mutations of the examiner from the sample obtained, protein phosphatase 2 regulated late Lee subunit Aα exon 5 and / or exon 6 in the gene, between the gastrointestinal tract in a subject A genetic mutation detection method for prognosis or resistance to chemotherapy of a mass tumor,
The method for detecting a gene, wherein the mutation is one or more selected from the following mutations (1) to (26).
(1) Mutation of 173rd Leu of Exon 5 to Pro
(2) Mutation of 184th Ala of Exon 5 to Asp
(3) Mutation of 193rd Ala of exon 5 to Thr
(4) Mutation of 194th Lys of exon 5 to Arg
(5) Mutation of 195th Val of exon 5 to Met
(6) Mutation of 197th Glu of exon 5 to Gly
(7) Mutation of Exon 5 at 201st Val to Ala
(8) Mutation of exon 5 at 201st Val to Ile
(9) Mutation of exon 5 at 204th Glu to Val
(10) Mutation of 204th Glu of exon 5 to Gly
(11) Mutation of 206th Ile of Exon 5 to Thr
(12) Mutation of exon 5 at 208th Met to Thr
(13) Mutation of 209th Phe of exon 5 to Leu
(14) Mutation of Asn at 211st Asn in Exon 5
(15) Mutation of the 214th Ser of exon 5 to Pro
(16) Mutation of Exon 6 at 225th Val to Met
(17) Mutation of 232nd Ala of exon 6 to Thr
(18) Mutation of 233rd Gln of exon 6 to His
(19) Mutation of Exon 6 Leu at position 234 to Pro
(20) Mutation of 238th Glu of exon 6 to Lys
(21) Mutation of exon 6 at 244th Val to Met
(22) Mutation of 248th Leu of exon 6 to Gln
(23) Mutation of 252nd Ala of exon 6 to Thr
(24) Mutation of 257th Trp of exon 6 to stop codon
(25) Mutation of 261st Tyr of Exon 6 to His
(26) Mutation of Exon 6 at 266th Lys to Arg
プロテインホスファターゼ２レギュレイトリーサブユニットＡα遺伝子のエクソン５及び／又はエクソン６中の次の（１）〜（２６）から選ばれる１又は２以上の変異を含む領域を増幅できるように設計したプライマーセットを用いる方法。
（１）エクソン５の１７３番目のＬｅｕがＰｒｏへの変異
（２）エクソン５の１８４番目のＡｌａがＡｓｐへの変異
（３）エクソン５の１９３番目のＡｌａがＴｈｒへの変異
（４）エクソン５の１９４番目のＬｙｓがＡｒｇへの変異
（５）エクソン５の１９５番目のＶａｌがＭｅｔへの変異
（６）エクソン５の１９７番目のＧｌｕがＧｌｙへの変異
（７）エクソン５の２０１番目のＶａｌがＡｌａへの変異
（８）エクソン５の２０１番目のＶａｌがＩｌｅへの変異
（９）エクソン５の２０４番目のＧｌｕがＶａｌへの変異
（10）エクソン５の２０４番目のＧｌｕがＧｌｙへの変異
（11）エクソン５の２０６番目のＩｌｅがＴｈｒへの変異
（12）エクソン５の２０８番目のＭｅｔがＴｈｒへの変異
（13）エクソン５の２０９番目のＰｈｅがＬｅｕへの変異
（14）エクソン５の２１１番目のＡｓｎがＡｓｐへの変異
（15）エクソン５の２１４番目のＳｅｒがＰｒｏへの変異
（16）エクソン６の２２５番目のＶａｌがＭｅｔへの変異
（17）エクソン６の２３２番目のＡｌａがＴｈｒへの変異
（18）エクソン６の２３３番目のＧｌｎがＨｉｓへの変異
（19）エクソン６の２３４番目のＬｅｕがＰｒｏへの変異
（20）エクソン６の２３８番目のＧｌｕがＬｙｓへの変異
（21）エクソン６の２４４番目のＶａｌがＭｅｔへの変異
（22）エクソン６の２４８番目のＬｅｕがＧｌｎへの変異
（23）エクソン６の２５２番目のＡｌａがＴｈｒへの変異
（24）エクソン６の２５７番目のＴｒｐが終止コドンへの変異
（25）エクソン６の２６１番目のＴｙｒがＨｉｓへの変異
（26）エクソン６の２６６番目のＬｙｓがＡｒｇへの変異 The method according to claim 1, further comprising the step of amplifying the nucleic acid in the sample obtained from the subject using a primer set designed to amplify exon 5 and / or exon 6 of the protein phosphatase 2 regulatory subunit Aα gene. A method ,
A primer set designed to amplify a region containing one or more mutations selected from the following (1) to (26) in exon 5 and / or exon 6 of protein phosphatase 2 regulatory subunit Aα gene The method to use.
(1) Mutation of 173rd Leu of Exon 5 to Pro
(2) Mutation of 184th Ala of Exon 5 to Asp
(3) Mutation of 193rd Ala of exon 5 to Thr
(4) Mutation of 194th Lys of exon 5 to Arg
(5) Mutation of 195th Val of exon 5 to Met
(6) Mutation of 197th Glu of exon 5 to Gly
(7) Mutation of Exon 5 at 201st Val to Ala
(8) Mutation of exon 5 at 201st Val to Ile
(9) Mutation of exon 5 at 204th Glu to Val
(10) Mutation of 204th Glu of exon 5 to Gly
(11) Mutation of 206th Ile of Exon 5 to Thr
(12) Mutation of exon 5 at 208th Met to Thr
(13) Mutation of 209th Phe of exon 5 to Leu
(14) Mutation of Asn at 211st Asn in Exon 5
(15) Mutation of the 214th Ser of exon 5 to Pro
(16) Mutation of Exon 6 at 225th Val to Met
(17) Mutation of 232nd Ala of exon 6 to Thr
(18) Mutation of 233rd Gln of exon 6 to His
(19) Mutation of Exon 6 Leu at position 234 to Pro
(20) Mutation of 238th Glu of exon 6 to Lys
(21) Mutation of exon 6 at 244th Val to Met
(22) Mutation of 248th Leu of exon 6 to Gln
(23) Mutation of 252nd Ala of exon 6 to Thr
(24) Mutation of 257th Trp of exon 6 to stop codon
(25) Mutation of 261st Tyr of Exon 6 to His
(26) Mutation of Exon 6 at 266th Lys to Arg
（１）エクソン５の１７３番目のＬｅｕがＰｒｏへの変異
（２）エクソン５の１８４番目のＡｌａがＡｓｐへの変異
（３）エクソン５の１９３番目のＡｌａがＴｈｒへの変異
（４）エクソン５の１９４番目のＬｙｓがＡｒｇへの変異
（５）エクソン５の１９５番目のＶａｌがＭｅｔへの変異
（６）エクソン５の１９７番目のＧｌｕがＧｌｙへの変異
（７）エクソン５の２０１番目のＶａｌがＡｌａへの変異
（８）エクソン５の２０１番目のＶａｌがＩｌｅへの変異
（９）エクソン５の２０４番目のＧｌｕがＶａｌへの変異
（10）エクソン５の２０４番目のＧｌｕがＧｌｙへの変異
（11）エクソン５の２０６番目のＩｌｅがＴｈｒへの変異
（12）エクソン５の２０８番目のＭｅｔがＴｈｒへの変異
（13）エクソン５の２０９番目のＰｈｅがＬｅｕへの変異
（14）エクソン５の２１１番目のＡｓｎがＡｓｐへの変異
（15）エクソン５の２１４番目のＳｅｒがＰｒｏへの変異
（16）エクソン６の２２５番目のＶａｌがＭｅｔへの変異
（17）エクソン６の２３２番目のＡｌａがＴｈｒへの変異
（18）エクソン６の２３３番目のＧｌｎがＨｉｓへの変異
（19）エクソン６の２３４番目のＬｅｕがＰｒｏへの変異
（20）エクソン６の２３８番目のＧｌｕがＬｙｓへの変異
（21）エクソン６の２４４番目のＶａｌがＭｅｔへの変異
（22）エクソン６の２４８番目のＬｅｕがＧｌｎへの変異
（23）エクソン６の２５２番目のＡｌａがＴｈｒへの変異
（24）エクソン６の２５７番目のＴｒｐが終止コドンへの変異
（25）エクソン６の２６１番目のＴｙｒがＨｉｓへの変異
（26）エクソン６の２６６番目のＬｙｓがＡｒｇへの変異 Hybridization under stringent conditions to a region containing one or more mutations selected from the following (1) to (26) in exon 5 and / or exon 6 of protein phosphatase 2 regulatory subunit Aα gene The method according to claim 1, comprising hybridizing a probe designed to be able to nucleic acid in a sample obtained from the subject.
(1) Mutation of 173rd Leu of exon 5 to Pro (2) Mutation of 184th Ala of exon 5 to Asp (3) Mutation of 193rd Ala of exon 5 to Thr (4) Exon 5 194 Lys mutation to Arg (5) Exon 5 195th Val mutation to Met (6) Exon 5 197th Glu mutation to Gly (7) Exon 5 201st Val Mutation to Ala (8) Mutation of 201st Val of Exon 5 to Ile (9) Mutation of 204th Glu of Exon 5 to Val (10) Mutation of 204th Glu of Exon 5 to Gly (11) Mutation of 206th Ile of exon 5 to Thr (12) Mutation of 208th Met of exon 5 to Thr (13) 209th Phe of exon 5 is Leu (14) Mutation of 211 of Asn in Exon 5 to Asp (15) Mutation of 214th Ser of Exon 5 to Pro (16) Mutation of Val 225 of Exon 6 to Met (17) Exon 6 232th Ala mutation to Thr (18) Exon 6 233rd Gln mutation to His (19) Exon 6 234th Leu mutation to Pro (20) Exon 6 238th mutation Mutation of Glu to Lys (21) Mutation of 244th Val of exon 6 to Met (22) Mutation of 248th Leu of exon 6 to Gln (23) Mutation of 252nd Ala of exon 6 to Thr (24) Mutation of 257th Trp of exon 6 to stop codon (25) Mutation of 261st Tyr of exon 6 to His (26) 266th Lys of exon 6 is A Mutation to g
（１）エクソン５の１７３番目のＬｅｕがＰｒｏへの変異
（２）エクソン５の１８４番目のＡｌａがＡｓｐへの変異
（３）エクソン５の１９３番目のＡｌａがＴｈｒへの変異
（４）エクソン５の１９４番目のＬｙｓがＡｒｇへの変異
（５）エクソン５の１９５番目のＶａｌがＭｅｔへの変異
（６）エクソン５の１９７番目のＧｌｕがＧｌｙへの変異
（７）エクソン５の２０１番目のＶａｌがＡｌａへの変異
（８）エクソン５の２０１番目のＶａｌがＩｌｅへの変異
（９）エクソン５の２０４番目のＧｌｕがＶａｌへの変異
（10）エクソン５の２０４番目のＧｌｕがＧｌｙへの変異
（11）エクソン５の２０６番目のＩｌｅがＴｈｒへの変異
（12）エクソン５の２０８番目のＭｅｔがＴｈｒへの変異
（13）エクソン５の２０９番目のＰｈｅがＬｅｕへの変異
（14）エクソン５の２１１番目のＡｓｎがＡｓｐへの変異
（15）エクソン５の２１４番目のＳｅｒがＰｒｏへの変異
（16）エクソン６の２２５番目のＶａｌがＭｅｔへの変異
（17）エクソン６の２３２番目のＡｌａがＴｈｒへの変異
（18）エクソン６の２３３番目のＧｌｎがＨｉｓへの変異
（19）エクソン６の２３４番目のＬｅｕがＰｒｏへの変異
（20）エクソン６の２３８番目のＧｌｕがＬｙｓへの変異
（21）エクソン６の２４４番目のＶａｌがＭｅｔへの変異
（22）エクソン６の２４８番目のＬｅｕがＧｌｎへの変異
（23）エクソン６の２５２番目のＡｌａがＴｈｒへの変異
（24）エクソン６の２５７番目のＴｒｐが終止コドンへの変異
（25）エクソン６の２６１番目のＴｙｒがＨｉｓへの変異
（26）エクソン６の２６６番目のＬｙｓがＡｒｇへの変異 A primer set designed to amplify a region containing one or more mutations selected from the following (1) to (26) in exon 5 and / or exon 6 of protein phosphatase 2 regulatory subunit Aα gene: A diagnostic agent for prognosis or chemoresistance of a gastrointestinal stromal tumor containing a probe designed to be able to hybridize to the region under stringent conditions.
(1) Mutation of 173rd Leu of exon 5 to Pro (2) Mutation of 184th Ala of exon 5 to Asp (3) Mutation of 193rd Ala of exon 5 to Thr (4) Exon 5 194 Lys mutation to Arg (5) Exon 5 195th Val mutation to Met (6) Exon 5 197th Glu mutation to Gly (7) Exon 5 201st Val Mutation to Ala (8) Mutation of 201st Val of Exon 5 to Ile (9) Mutation of 204th Glu of Exon 5 to Val (10) Mutation of 204th Glu of Exon 5 to Gly (11) Mutation of 206th Ile of exon 5 to Thr (12) Mutation of 208th Met of exon 5 to Thr (13) 209th Phe of exon 5 is Leu (14) Mutation of 211 of Asn in Exon 5 to Asp (15) Mutation of 214th Ser of Exon 5 to Pro (16) Mutation of Val 225 of Exon 6 to Met (17) Exon 6 232th Ala mutation to Thr (18) Exon 6 233rd Gln mutation to His (19) Exon 6 234th Leu mutation to Pro (20) Exon 6 238th mutation Mutation of Glu to Lys (21) Mutation of 244th Val of exon 6 to Met (22) Mutation of 248th Leu of exon 6 to Gln (23) Mutation of 252nd Ala of exon 6 to Thr (24) Mutation of 257th Trp of exon 6 to stop codon (25) Mutation of 261st Tyr of exon 6 to His (26) 266th Lys of exon 6 is A Mutation to g
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