JP5697448B2 - 膀胱癌の検出のための新規なマーカー - Google Patents
膀胱癌の検出のための新規なマーカー Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- A—HUMAN NECESSITIES
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- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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- C12Q2600/00—Oligonucleotides characterized by their use
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Description
本発明は、遺伝子のパネルのメチル化状態若しくは発現レベル又はその組合せを決定することを含む、癌細胞の存在、又は癌細胞由来のゲノムDNAの存在を検出するための方法及びキットに関する。特に、本発明は、膀胱癌の検出に関する。本発明はまた、癌に適した治療レジメンを決定するための薬理遺伝学的方法、及び癌患者を治療するための方法に関する。
分子的証明により、癌が、異常な遺伝子サイレンシング又は遺伝子活性化及び細胞機能障害をもたらし得る遺伝的及びエピジェネティックな異常の蓄積の段階的なプロセスであるという概念が支持されている。遺伝的プロセスとエピジェネティックなプロセスとの相乗効果は、腫瘍の進行及び悪性腫瘍を促進する。
特許請求の範囲に記載される本発明は、そのメチル化状態が膀胱癌に対する素因又は膀胱癌の発生に関連する、特異的な遺伝子及び遺伝子パネルの発見に基づくものである。膀胱癌を検出するためのこれらの遺伝子の使用は、特に適切な組織又は尿サンプルとの関連で、感度及び特異性が高い結果をもたらすことが示されている。
(a)NID2、TJP2、TWIST1、TNFRSF25、BMP7、RUNX3、CCNA1、APC、LOXL1、TUBB4、NTRK2、ARFGAP3、PDLIM4、RASSF1A、及びOSMRから選択される少なくとも1つの遺伝子におけるエピジェネティックな変化を検出するための手段と、
(b)尿サンプルを処理するための手段と
を含むキットに関する。
本発明は、NID2、TJP2、TWIST1、TNFRSF25、BMP7、RUNX3、CCNA1、APC、LOXL1、TUBB4、NTRK2、ARFGAP3、PDLIM4、RASSF1A、及びOSMRから選択される少なくとも1つの遺伝子におけるCpGジヌクレオチド内のシトシンが、ヒト組織癌/尿サンプルでは差次的にメチル化されており、正常なヒト組織/尿サンプルではメチル化されておらず、特に膀胱組織及び/又は尿サンプルにおいてはそれが顕著であるという発見に基づくものである。
S=センスプライマー(5’−3’)
AS=アンチセンスプライマー(5’−3’)
MB=モレキュラービーコン(修飾ビーコン:5’FAM、3’DABCYL)
という配列を含み得るか、それから本質的になり得るか、又はそれからなり得る。
RASSF1A_AS(配列番号2): CCCGTACTTCGCTAACTTTAAACG
RASSF1A_MB(配列番号3): CGTCTGCGTGGTTTCGTTCGGTTCGCGTTTGTTAGGCAGACG
APC(2)_AS(配列番号5): TCG ACG AAC TCC CGA CGA
APC(2)_MB(配列番号6): CGACATGCGTTGTGTAATTCGTTGGATGCGGATTAGGGCGGCATGTCG
CCNA1_gron_AS(配列番号8): CCAACCTAAAAAACGACCGA
CCNA1_gron_MB(配列番号9): CGACATGCACGACGCCCCCGAACCTAACGCATGTCG
TNFRSF25_1_AS(配列番号11): GCGTATTCTACTTAACCTATCCGC
TNFRSF25_1_MB(配列番号12): CGACATGCACGACCCCGCCTCCCCCCGCCGCATGTCG
TUBB4_2_AS(配列番号14): TACCTCAATTTCTCGATCCGC
TUBB4_2_MB(配列番号15): CGACATGCTGGGAGGGTTCGCGGTTATTGTAAGGAGCATGTCG
NTRK2_1_M_S(配列番号16): GTTAGAGCGCGTTTTTAGCGT
NTRK2_1_M_AS(配列番号17): CCGCAATACCTAACACTTCCG
NTRK2_1_MB(配列番号18): CGACATGCCCGACACGCTCCGAAACACCAGCATGTCG
OSMR_1_AS(配列番号20): GAAACGAACGTACAAAAACGA
OSMR_1_MB(配列番号21): CGACATGCCGAAACTATAAATCAACTACGAAACAAACGCGCATGTCG
TWIST1_3_AS(配列番号23): CCGTCGCCTTCCTCCGACGAA
TWIST1_3_MB(配列番号24): CGACATGCCGGCGGGGAAGGAAATCGTTTCGCATGTCG
LOXL1_29309_AS(配列番号26): ACAAAAACAAAAACGACGCCT
MB_LOXL1_29309b(配列番号27): CGACATGCCGGGTGTTGTTGGTCGGCGCGCATGTCG
TJP2_25301_AS(配列番号29): CCAACTTCCTACGACGCAT
TJP2_25301_MB(配列番号30): CGACATGCCTCCCAACCGCGCGACACAAGCATGTCG
Runx3_3_M_AS(配列番号32): TAACTTTTAACGAAATTACCCCG
RUNX3_3_MB2(配列番号33): CGACATGCCGGGTTAGGGGGGCGTAAAATTTTATTCGTTGCATGTCG
PDLIM4_4_M_AS(配列番号35): CGATCCCATATCTAAAACCGA
PDLIM4_4_MB(配列番号36): CGACATGCCTCGCGATCCGCCCGAAACGCATGTCG
BMP7_17911_AS(配列番号38): AAAACGATAACCCTTAAACCGA
MB_BMP7_17911(配列番号39): CGACATGCGCGGAGGGGTTAGCGTGGTTGCATGTCG
NID2_9091_AS(配列番号41): CTACGAAATTCCCTTTACGCT
MB_NID2_9091(配列番号42): CGACATGGGTTCGTAAGGTTTGGGGTAGCGGCCATGTCG
ARFGAP3_25342_A(配列番号44): GCCATTTCGCCTAACGAAC
ARFGAP3_25342_MB(配列番号45): CGACATGCACGCGCCCTCCTTCGACACGCATGTCG
・尿検査
・尿細胞診(癌性細胞を探すための尿の顕微鏡検査)
・膀胱鏡検査(膀胱の内部を見るための照明付き機器の使用。膀胱癌の診断及び病期診断は膀胱鏡検査で開始される)
・膀胱の生検(通常は膀胱鏡検査の際に行われる)
・静脈内腎盂像−IVP(通常のX線を用いて膀胱内のあらゆる腫瘍又は異常の良好な視覚化を可能にする色素を血流内に注射する。)
・画像化技術:上部尿路(尿管及び腎臓を含む)のX線画像化を行って、これらの構造のあらゆる関与を除外し得る。超音波を用いて腎臓を研究することができ、CTスキャンは、尿路の全長を研究する上で非常に好ましいことが多い。
・BTAアッセイ(Polymedco、前身はBard Dagnostics、USA)は、膀胱癌を有する患者の尿に存在するhCFHrp、すなわちヒト補因子H関連タンパク質を検出する。定量的及び定性的なBTA法の両方が利用可能である。
・NMP22試験キット(Matritech Inc.、Newton、MA)は、核マトリクスに豊富である核有糸分裂装置(NMA)タンパク質を検出する。膀胱腫瘍細胞において、NMAは増加し、検出可能なレベルで放出される。定量的及び定性的なNMP22法の両方が存在する。
・Vysis UroVysionアッセイ(Abbott Molecular Diagnostics)は、尿細胞診と分子(DNAに基づいた)技術とを組み合わせて、癌の再発を検出する。これは、蛍光in situハイブリダイゼーション(FISH)技術を用い、この技術は、DNAの特異的な領域を顕微鏡で同定するために、小さな蛍光標識したDNAプローブを用いる。
・ImmunoCyt(DiagnoCure)は、それまでに膀胱癌であると診断された患者の尿において、腫瘍細胞により発現されるムチン及びCEA抗原を検出するための、免疫細胞化学アッセイである。この免疫蛍光法は、膀胱癌の再発を初期に検出するために、尿細胞診と組み合わされる。ImmunoCytは定性的なアッセイである。
配列番号49 − 5’−T46CGTCATCTGCCCCCACAGAG−3’
配列番号50 − 5’−T36TCTGCCCCCACAGAGCGCT−3’
配列番号51 − 5’−T28TCTGCCCCCACAGAGCGCT−3’
配列番号52 − 5’−T29GGTGGAGGCTGACGAGGCG−3’
配列番号53 − 5’−T43ACGAGGCGGGCAGTGTGT−3’
配列番号54 − 5’−T34CCTGTTCATCCTGGTGGTGG−3’
配列番号55 − 5’−T50GCACAACCTCGACTACTACAAG−3’
配列番号56 − 5’−T20CACAACCTCGACTACTACAAGA−3’
(a)NID2、TJP2、TWIST1、TNFRSF25、BMP7、RUNX3、CCNA1、APC、LOXL1、TUBB4、NTRK2、ARFGAP3、PDLIM4、RASSF1A、及びOSMRから選択される少なくとも1つの遺伝子、特にTWIST1における、エピジェネティックな変化を検出するための手段と、
(b)サンプルを処理するための手段と
を含むキットもまた提供される。
S=センスプライマー
AS=アンチセンスプライマー
MB=モレキュラービーコン
というヌクレオチド配列を含むか、それから本質的になるか、若しくはそれからなるプライマー及び/若しくはプローブを含むか、それから本質的になるか、又はそれからなるものから選択される。
RASSF1A_AS(配列番号2): CCCGTACTTCGCTAACTTTAAACG
RASSF1A_MB(配列番号3): 5’−FAM−CGTCTGCGTGGTTTCGTTCGGTTCGCGTTTGTTAGGCAGACG−3’−DABCYL
APC(2)_AS(配列番号5): TCG ACG AAC TCC CGA CGA
APC(2)_MB(配列番号6): 5’−FAM−CGACATGCGTTGTGTAATTCGTTGGATGCGGATTAGGGCGGCATGTCG−3’−DABCYL
CCNA1_gron_AS(配列番号8): CCAACCTAAAAAACGACCGA
CCNA1_gron_MB(配列番号9): 5’−FAM−CGACATGCACGACGCCCCCGAACCTAACGCATGTCG−3’−DABCYL
TNFRSF25_1_AS(配列番号11): GCGTATTCTACTTAACCTATCCGC
TNFRSF25_1_MB(配列番号12): 5’−FAM−CGACATGCACGACCCCGCCTCCCCCCGCCGCATGTCG−3’−DABCYL
TUBB4_2_AS(配列番号14): TACCTCAATTTCTCGATCCGC
TUBB4_2_MB(配列番号15): 5’−FAM−CGACATGCTGGGAGGGTTCGCGGTTATTGTAAGGAGCATGTCG−3’−DABCYL
NTRK2_1_M_S(配列番号16): GTTAGAGCGCGTTTTTAGCGT
NTRK2_1_M_AS(配列番号17): CCGCAATACCTAACACTTCCG
NTRK2_1_MB(配列番号18): 5’−FAM−CGACATGCCCGACACGCTCCGAAACACCAGCATGTCG−3’−DABCYL
OSMR_1_AS(配列番号20): GAAACGAACGTACAAAAACGA
OSMR_1_MB(配列番号21): 5’−FAM−CGACATGCCGAAACTATAAATCAACTACGAAACAAACGCGCATGTCG−3’−DABCYL
TWIST1_3_AS(配列番号23): CCGTCGCCTTCCTCCGACGAA
TWIST1_3_MB(配列番号24): 5’−FAM−CGACATGCCGGCGGGGAAGGAAATCGTTTCGCATGTCG−3’−DABCYL
LOXL1_29309_AS(配列番号26): ACAAAAACAAAAACGACGCCT
MB_LOXL1_29309b(配列番号27): 5’−FAM−CGACATGCCGGGTGTTGTTGGTCGGCGCGCATGTCG−3’−DABCYL
TJP2_25301_AS(配列番号29): CCAACTTCCTACGACGCAT
TJP2_25301_MB(配列番号30): 5’−FAM−CGACATGCCTCCCAACCGCGCGACACAAGCATGTCG−3’−DABCYL
Runx3_3_M_AS(配列番号32): TAACTTTTAACGAAATTACCCCG
RUNX3_3_MB2(配列番号33): 5’−FAM−CGACATGCCGGGTTAGGGGGGCGTAAAATTTTATTCGTTGCATGTCG−3’−DABCYL
PDLIM4_4_M_AS(配列番号35): CGATCCCATATCTAAAACCGA
PDLIM4_4_MB(配列番号36): 5’−FAM−CGACATGCCTCGCGATCCGCCCGAAACGCATGTCG−3’−DABCYL
BMP7_17911_AS(配列番号38): AAAACGATAACCCTTAAACCGA
MB_BMP7_17911(配列番号39): 5’−FAM−CGACATGCGCGGAGGGGTTAGCGTGGTTGCATGTCG−3’−DABCYL
NID2_9091_AS(配列番号41): CTACGAAATTCCCTTTACGCT
MB_NID2_9091(配列番号42): 5’−FAM−CGACATGGGTTCGTAAGGTTTGGGGTAGCGGCCATGTCG−3’−DABCYL
ARFGAP3_25342_A(配列番号44): GCCATTTCGCCTAACGAAC
ARFGAP3_25342_MB(配列番号45): 5’−FAM−CGACATGCACGCGCCCTCCTTCGACACGCATGTCG−3’−DABCYL
β−アクチン_A(配列番号47): AACACACAATAACAAACACAAATTCAC
β−アクチン_MB(配列番号48): 5’−FAM−CGACTGCGTGTGGGGTGGTGATGGAGGAGGTTTAGGCAGTCG−3’−DABCYL
材料及び方法
マーカーの同定:膀胱癌細胞株の再発現プロフィールを用いて、候補遺伝子を同定した。プロモーター配列を遺伝子発現と関連させて、有効な、エピジェネティックにサイレンシングされた遺伝子を同定した。エピジェネティックに標的化された遺伝子の再発現分析のための、確立された、薬理学的なマスキングされていない戦略(5−アザ−2’−デオキシシチジン[DAC]及びトリコスタチンA[TSA])を、専売的(proprietary)な、高度なバイオインフォマティクスツールと組み合わせて、プロモーターをメチル化する傾向にある遺伝子を同定した。
β−アクチンのフォワードプライマー 5’−TAGGGAGTATATAGGTTGGGGAAGTT−3’
β−アクチンのリバースプライマー 5’−AACACACAATAACAAACACAAATTCAC−3’
ビーコン 5’−FAM−CGACTGCGTGTGGGGTGGTGATGGAGGAGGTTTAGGCAGTCG−3’−DABCYL
組織及び尿におけるアッセイの有効性の比率:130個のFFPE及び218個の尿サンプルを、リアルタイムMSPを用いて処理した(表2)。有効性の比率は、図1に示す基準に基づいたものである。リアルタイムMSPアッセイでは、96%のFFPEサンプル及び94%の尿サンプルにおいて有効な結果が得られた。
実施例1の5マーカーパネルを、「尿トレーニングセット2」と呼ばれる独立したサンプルセットを用いてさらに研究した。試験した遺伝子の数が減ったため(11から5)、サンプルのより大きなアリコートが、試験したそれぞれのアッセイに利用可能であり、その結果、適切なBT溶出容量及び結果を分析するための異なる決定木(比率よりもコピーに基づいたもの)が得られた(図4)。
マーカーの同定:実施例1において論じられるように、膀胱癌細胞株の再発現プロフィールを用いて、候補遺伝子を同定した。
TWIST1:77bp
RUNX3:127bp
NID2:99bp
ACTB:103bp
フォワードプライマー TWIST1: 5’− GTTAGGGTTCGGGGGCGTTGTT − 3’(配列番号22)
リバースプライマー TWIST1: 5’− CCGTCGCCTTCCTCCGACGAA − 3’(配列番号23)
フォワードプライマー RUNX3: 5’− CGTAGGGTTGTATTTGAGCGA − 3’(配列番号31)
リバースプライマー RUNX3: 5’− TAACTTTTAACGAAATTACCCCG − 3’(配列番号32)
フォワードプライマー NID2: 5’− GCGGTTTTTAAGGAGTTTTATTTTC − 3’(配列番号40)
リバースプライマー NID2: 5’− CTACGAAATTCCCTTTACGCT − 3’(配列番号41)
フォワードプライマー ACTB: 5’− TAGGGAGTATATAGGTTGGGGAAGTT −3’(配列番号46)
リバースプライマー ACTB: 5’ − AACACACAATAACAAACACAAATTCAC − 3’(配列番号47)
モレキュラービーコン TWIST1:
5’ − Fam−CGACATGCCGGCGGGGAAGGAAATCGTTTCGCATGTCG−Dabcyl − 3’(配列番号24)
モレキュラービーコン RUNX3:
5’ − Fam−CGACATGCCGGGTTAGGGGGGCGTAAAATTTTATTCGTTGCATGTCG−Dabcyl − 3’(配列番号33)
モレキュラービーコン NID2:
5’− Fam−CGACATGGGTTCGTAAGGTTTGGGGTAGCGGCCATGTCG−Dabcyl − 3’(配列番号42)
モレキュラービーコン ACTB:
5’ − Fam−CGACTGCGTGTGGGGTGGTGATGGAGGAGGTTTAGGCAGTCG−Dabcyl−3’(配列番号48)
TWIST1:7番染色体、19124120位と19124043位との間(RefSeq:NM_000474)
RUNX3:1番染色体、25128341位と25128468位との間(RefSeq:NM_004350)
NID2:14番染色体、51605816位と51605915位との間(RefSeq:NM_007361)
ACTB:7番染色体、5538428位と5538326位との間(RefSeq:NM_001101)
組織及び尿におけるアッセイの有効性の比率:130個のホルマリン固定したパラフィン包埋した(FFPE)組織サンプル及び415個の尿サンプルを、リアルタイムMSPを用いて処理した(表7)。リアルタイムMSPアッセイでは、96%のFFPEサンプル及び93%の尿サンプルにおいて有効な結果が得られた。
新たな尿ベースのDNAメチル化アッセイの感度をさらに増強させるために、本発明者らは、それらのメチル化試験を、尿サンプルにおける早期の膀胱癌を検出するための確立された方法と組み合わせることが実現可能であるかを調査した。従来の尿細胞診(現在の標準的治療手順)及びFGFR3突然変異分析の結果(再発の検出に特に有用である)を用いて、本発明の方法を補った。
サンプルの収集:実施例1及び2において記載される尿トレーニングセット1及びトレーニングセット2から得られたサンプルを用いて、メチル化アッセイを細胞診及び/又はFGFR3突然変異の結果と組み合わせることの有用性を実証した。
OMS尿ベースのDNAメチル化アッセイ:分析物(TWIST、RUNX3、NID2、及びACTB)の定量を、上記に詳述されるように、リアルタイムMSPアッセイによって実施した。
メチル化及び細胞診の結果の組合せ:
メチル化及び細胞診の結果(両方の尿トレーニングセットから得られたもの)を、同一の尿サンプルについて比較した(表11を参照されたい)。
メチル化パネルの感度は、細胞診の感度よりも有意に高かった(85%対37%)が、特異性はむしろ同様であった(93%対97%)。
メチル化パネル(3つの好ましいマーカーのパネル)の感度は、細胞診の感度よりも有意に高かった(91%対43%)が、特異性は同様であった(93%対94%)。
メチル化、細胞診、及びFGFR3突然変異の結果を、同一の尿サンプルについて比較した(表12)。全体で、61個のサンプルを両方の尿トレーニングセットからランダムに選択し(41件の癌の症例及び20個の対照サンプル)、上記に示したプライマー及び反応条件を用いて、3つの個別の方法を介して処理した。
典型的には、尿サンプルを収集の4時間以内に遠心分離して、DNAの分解を避ける。本発明者らは、尿サンプルに安定化緩衝液を加えると、尿収集の直後に遠心分離を行う必要がなくなることを示している(国際出願PCT/GB2008/002093を参照されたい)。サンプルは、安定化緩衝液を加えた後に、遠心分離をする必要なく、一方でDNAの完全性を維持しながら、最大72時間にわたり室温で保存することができる。
材料及び方法は、尿サンプルの調製を除き、上記の実施例2で記載されるものと同一である。4時間以内に尿サンプルを3000gで10分間遠心分離する代わりに、収集の時点で安定化剤(スタビラー(登録商標)錠剤、Cargille Laboratories、#40050、50mlの尿当たり5個の錠剤)を尿に加えた。尿サンプルを次に、最大72時間室温に保持し、その後遠心分離した。尿の沈渣を最大6カ月、−20℃で保管し、実施例2において記載されるようにさらに処理した。
組織及び尿におけるアッセイの有効性の比率:130個のホルマリン固定し、パラフィン包埋した(FFPE)組織サンプル及び495個の尿サンプルを、リアルタイムMSPを用いて処理した(表14)。リアルタイムMSPアッセイでは、96%のFFPEサンプル及び94%の尿サンプルにおいて有効であるという結果となった。
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Claims (16)
- サンプルにおける膀胱癌に対する素因又は膀胱癌の発生を検出する方法であって、TWIST1及びTJP2から選択される少なくとも1つの遺伝子におけるエピジェネティックな変化を検出することを含み、エピジェネティックな変化の検出が膀胱癌に対する素因又は膀胱癌の発生の指標である方法。
- 前記遺伝子の少なくとも1個又は2個を含む遺伝子パネルにおけるエピジェネティックな変化を検出することを含み、パネルにおける遺伝子の少なくとも1つにおけるエピジェネティックな変化の検出が、膀胱癌に対する素因又は膀胱癌の発生の指標である、請求項1に記載の方法。
- 遺伝子パネルが、TWIST1及びNID2を含む、若しくは、NID2、TWIST1及びRUNX3を含むか、それから本質的になるか、又はそれからなる、請求項2に記載の方法。
- サンプルが膀胱組織サンプル又は尿サンプルを含む、請求項1〜3のいずれか一項に記載の方法。
- 収集後の尿サンプルに安定化緩衝液を添加し、それにより、サンプルの遠心分離を必要とせずに、サンプルを最大72時間にわたり室温で保管することを可能にする、請求項4に記載の方法。
- エピジェネティックな変化がメチル化である、請求項1〜5のいずれか一項に記載の方法。
- 尿細胞診及び/又は突然変異分析と組み合わせて用いられる、請求項1〜6のいずれか一項に記載の方法。
- サンプルにおける膀胱癌に対する素因又は膀胱癌の発生を検出するためのキットであって、NID2、TWIST1及びRUNX3、又はTWIST1及びNID2のそれぞれのメチル化状態を決定するための少なくとも1つのプライマー対を含むキット。
- サンプルにおける膀胱癌に対する素因又は膀胱癌の発生を検出するためのキットであって、
(a)TWIST1及びTJP2から選択される少なくとも1つの遺伝子におけるエピジェネティックな変化を検出するための手段と、
(b)尿サンプルの収集のための密閉可能な容器及び安定化緩衝液から選択される、尿サンプルを処理するための手段とを含むキット。 - 前記遺伝子の少なくとも1個又は2個を含む遺伝子パネルにおけるエピジェネティックな変化を検出するための手段を含み、パネルにおける遺伝子の少なくとも1つにおけるエピジェネティックな変化の検出が、膀胱癌に対する素因又は膀胱癌の発生の指標である、請求項9に記載のキット。
- 遺伝子パネルが、TWIST1及びNID2、又は、NID2、TWIST1及びRUNX3を含む、請求項10に記載のキット。
- メチル化を検出するための手段がメチル化特異的PCR/増幅プライマーを含む、請求項9〜11のいずれか一項に記載のキット。
- FGFR3遺伝子における突然変異を検出するための1つ又は複数のプライマーをさらに含む、請求項9〜12のいずれか一項に記載のキット。
- TJP2遺伝子のメチル化状態を決定するためのプライマー又はプライマー対であって、配列番号28及び/又は29で示される1つ又は複数のヌクレオチド配列を含むプライマー又はプライマー対。
- TWIST1及びTJP2から選択される少なくとも1つの遺伝子のメチル化状態を決定するためのプローブであって、TWIST1では配列番号24、TJP2では配列番号30で示されるヌクレオチド配列を含むプローブ。
- 膀胱癌の再発又は膀胱癌の再発に対する素因を検出することを含む、請求項1〜7のいずれか一項に記載の方法。
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