JP7481804B2 - 高度膵異形成の検出 - Google Patents
高度膵異形成の検出 Download PDFInfo
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- JP7481804B2 JP7481804B2 JP2018554339A JP2018554339A JP7481804B2 JP 7481804 B2 JP7481804 B2 JP 7481804B2 JP 2018554339 A JP2018554339 A JP 2018554339A JP 2018554339 A JP2018554339 A JP 2018554339A JP 7481804 B2 JP7481804 B2 JP 7481804B2
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Description
配列番号45および46からなる、BMP3に対するプライマーの組、
配列番号47および48からなる、NDRG4に対するプライマーの組、
配列番号1および2からなる、ABCB1に対するプライマーの組、
配列番号3および4からなる、AK055957に対するプライマーの組、
配列番号7および8からなる、C13ORF18に対するプライマーの組、
配列番号43および44からなる、CD1Dに対するプライマーの組、
配列番号9および10からなる、CLEC11Aに対するプライマーの組、
配列番号11および12からなる、DLX4に対するプライマーの組、
配列番号13および14からなる、ELMO1に対するプライマーの組、
配列番号15および16からなる、EMX1に対するプライマーの組、
配列番号17および18からなる、FER1L4に対するプライマーの組、
配列番号19および20からなる、FRMD4Aに対するプライマーの組、
配列番号21および22からなる、GRIN2Dに対するプライマーの組、
配列番号23および24からなる、HOXA1に対するプライマーの組、
配列番号25および26からなる、LRRC4に対するプライマーの組、
配列番号29および30からなる、PRKCBに対するプライマーの組、
配列番号31および32からなる、SP9に対するプライマーの組、
配列番号33および34からなる、ST6GAL2に対するプライマーの組、
配列番号35および36からなる、ST8SIA1に対するプライマーの組、
配列番号37および38からなる、TBX15に対するプライマーの組、
配列番号39および40からなる、VWC2に対するプライマーの組、および
配列番号41および42からなる、ZNF781に対するプライマーの組。
本願技術の理解を促進するために、多数の用語およびフレーズを、以下に定義する。追加の定義は、詳細な説明中に記載する。
膵臓がんは、本来的に成長の速い悪性腫瘍であり、必然的に高い死亡率を伴うという従来の見方とは対照的に、分子的な開始から、前がんへの発達、がんへの発展、および転移へと進行するのに、平均で20年かかる可能性があり(Yachida, S., et al., Nature, 2010. 467(7319): p. 1114-1117参照)、結腸直腸新生物の成長率および自然歴とよく似ている(Stryker, S.J., et al., Gastroenterology, 1987. 93(5): p. 1009-13;Bozic, I., et al., Proceedings of the National Academy of Sciences of the United States of America, 2010. 107(43): p. 18545-50参照)ということが、最近の計算モデルによって示唆されており。前がんステージおよびがんの初期ステージT1における症候前の長い滞留時間によって、実際には、スクリーン検出が可能な限られた機会を比較的長くできる可能性がある。
いくつかの実施形態において、上記技術は、表2および表6に示す二つ以上のDMR(例えば、DMR番号1~96の内の二つ以上のDMR)を含むマーカーの組み合わせのメチル化状態(state)を評価することに関する。いくつかの実施形態において、二つ以上のマーカーのメチル化状態(state)を評価することで、被検体における高度膵異形成(IPMN-HGD、PanIN-3、またはPDAC)の存在を同定するためのスクリーニングまたは診断の特異性および/または感度が増大する。
5-メチルシトシンの存在のために核酸を分析する最も頻繁に使用されている方法は、Frommer et al.に記載のDNAにおける5-メチルシトシンを検出するための重亜硫酸塩方法(Frommer et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1827-31)またはその変形に基づく。5-メチルシトシンをマップする重亜硫酸塩方法は、5-メチルシトシンではなくシトシンと亜硫酸水素塩イオン(重亜硫酸塩としても知られる)との反応を観察することに基づく。当該反応は、通常、下記のステップにしたがって実施される:まず、シトシンと亜硫酸水素塩とを反応させてスルホン化シトシンを形成する。次に、スルホン化反応中間物の自発的脱アミノ反応によって、スルホン化ウラシルを得る。最後に、スルホン化ウラシル(uricil)をアルカリ状態下で脱スルホン化することによって、ウラシルを形成する。ウラシルはアデニンと共に塩基対を形成する(これにより、チミンのように作用する)一方、5-メチルシトシンはグアニンと塩基対を形成する(これにより、シトシンのように作用する)ので、検出が可能となる。これにより、例えば、重亜硫酸塩ゲノムシークエンシング(Grigg G, & Clark S, Bioessays (1994) 16: 431-36;Grigg G, DNA Seq. (1996) 6: 189-98)または、例えば、米国特許第5,786,146号に開示されるようなメチル化特異的PCR(MSP)によって、メチル化されたシトシンとメチル化されていないシトシンとの区別が可能になる。
上記技術のいくつかの実施形態において、以下のステップを含む方法を提供する。
2)高度膵異形成(IPMN-HGD、PanIN-3、またはPDAC)(例えば、80%以上の感度および80%以上の特異性を有する高度膵異形成)の欠如を検出すること。
2)高度膵異形成(IPMN-HGD、PanIN-3、またはPDAC)(例えば、80%以上の感度および80%以上の特異性を有する高度膵異形成)を分類すること。
膵管内乳頭粘液性腫瘍(IPMN)および膵上皮内腫瘍性病変(PanIN)は、膵管腺がん(PDAC)の鍵となる管状前駆体である。高度異形成(HGD)を有する前駆体の病変および初期PDACは外科的に治療するべきという共通認識がある一方、現在の撮像ツールおよびリスク予測モデルは不完全であり、そのような病変の検出に頻繁に失敗する。膵液、膵臓嚢胞液、または便等の媒体に適用される判別式分子マーカーが、高リスク病変のより正確な検出のために必要である。
異常膵臓組織像の程度が異なる8人の患者から採取したブラインド化嚢胞液試料に対してマーカーをテストした。本願のマーカーの内の7個を用いて、重亜硫酸塩変換された嚢胞液DNA上にMSPを行った。コピーを、LRRC4を分母としたメチル化パーセンテージに正規化した。4個のマーカーから得られた結果を表4に示す。
本実施例は、実施例1および2の材料、方法、および結果を記載する。
RRBSは、プロモーターおよびCpGアイランド(がん細胞において高メチル化され、正常な細胞において低メチル化されると知られている領域である)におけるエピジェネティックサインまたはメチル化サインの偏りのない観察を可能にする手法である。我々が行ってきた多数の研究は、同種の内で(がんの種類およびフィルタリング方法の両方において)初めてのものである。マーカー領域を同定およびランク付けするために我々が使用する基準は、臨床メチル化特異的増幅アッセイをデザインすることに最終目標を合わせており、そのことによって処理が独特なものとなっている。4つの主なフィルターは、1)定義されたCpG密集度基準およびベータ回帰モデリングの使用を要するDMR、2)ROC曲線分析、3)(正常な組織または正常な白血球のいずれかに対する)倍率変化、および4)(患者における)領域に対して実証されたCpG共メチル化および(コントロールにおける)共メチル化の欠如、である。
CpG含有モチーフを認識するメチル化特異的制限酵素であるMspIを10ユニット用いた消化によってゲノムDNA(300ng)を断片化し、それにより、試料のCpG濃度を増加させ、ゲノムの余剰領域を除去した。消化された断片の末端を修復し、5ユニットのクレノウ断片(3’-5’エキソ)によって末端にA鎖を付加し、(自身の試料IDをそれぞれの断片にリンクさせるための)バーコード配列を含むメチル化されたTruSeqアダプター(Illumina、カリフォルニア州サンディエゴ)と一晩かけて連結させた。Agencourt AMPure XP SPRIビーズ/緩衝液(Beckman Coulter、カリフォルニア州ブレーア)を用いて、160~340bp断片(40~220bpインサート)のサイズ選定を実施した。緩衝液のカットオフは、ビーズ/緩衝液の試料体積の0.7~1.1倍であった。最終溶出体積は22μL(EB緩衝液;Qiagen、メリーランド州ジャーマンタウン)であり、小さい試料アリコートにおけるライゲーション効率および断片品質の測定にはqPCRを用いた。改変Epi Tectプロトコル(Qiagen)を用いて、試料に重亜硫酸塩変換を施した(二回)。変換した試料アリコートを、qPCRおよび従来のPCR(PfuTurbo Cx hotstart、Agilent、カリフォルニア州サンタ・クララ)、続けてBioanalyzer 2100(Agilent)評価に供することによって、最終のライブラリー増幅の前に、最適なPCRサイクル数を決定した。最終PCRには、以下の条件を使用した:1)50μLの反応のそれぞれは、5μLの10倍緩衝液、1.25μLの10mM各デオキシリボヌクレオチド三リン酸(dNTP)、5μLのプライマーカクテル(~5μM)、15μLの鋳型(試料)、1μLのPfuTurbo Cx hotstart、および22.75の水、を含み;温度および時間は95℃で5分、98℃で30秒、12~16サイクルのそれぞれでは98℃で10秒、65℃で30秒、72℃で30秒、72℃で5分、および4℃で維持した。試料を、無作為化スキームに基づいて4-plexライブラリーへと(等モル)混合し、生体分析器を用いてテストすることにより最終サイズを確認し、ファイX基準およびアダプター特異的プライマーを用いたqPCRによってテストした。
内部アッセイコントロール用のさらなるレーンを用意するため無作為レーン割当て法にしたがって試料をフローセルに充填した。Mayo Clinic Medical Genome Facilityの次世代シークエンシングコア(Next Generation Sequencing Core)によって、Illumina HiSeq 2000を用いて配列決定を行った。101回のサイクルにおいて、読み取りは一方向で行った。各フローセルレーンでは、整列された配列用の30~50倍のシークエンシング深度(CpG毎のリード数)のカバレッジの中央値に十分である、100,000,000~120,000,000のリードを生成した。標準Illuminaパイプラインソフトウェアでは、fastqフォーマットにおいて、塩基を呼び出し、リード生成をシークエンシングした。配列アラインメントおよびメチル化抽出には低発現量重亜硫酸塩シークエンシング用流線分析およびアノテーションパイプライン(SAAP-RRBS)を用いた。RRBS発見段階では、各マップ化CpGにおける、膵臓新生物患者と、膵臓コントロール/白血球コントロール/(ある用途では)結腸コントロールとの間のメチル化の差異が主な比較対象であった。CpGアイランドは、期待値に対する観察値CpG比>0.6によって生物化学的に定義される。しかしながら、このモデルでは、CpG分析のタイルユニット「可変メチル化領域(DMR)」が、染色体それぞれにおけるCpG部位の位置間の距離に基づいて生成された。単一のCpGのみを有するアイランドは除外した。疾患グループ毎のカバレッジの全体深度が200リード以上(平均が一被検体につき10リード)であって、メチル化パーセンテージの分散が0よりも大きい(非有益なCpGは除外した)場合にのみ、個々のCpG部位が層別解析の対象となった。リード‐深度基準は、それぞれのグループにおける試料サイズが18個体である二つのグループ間のメチル化パーセンテージにおける10%の差異を検出するための望ましい統計的検出力に基づいた。統計的有意性は、リード数に基づいて、DMR毎のメチル化割合のロジスティック回帰によって決定した。個々の被検体にわたって異なるリード深度を考慮するために、過度に分散したロジスティック回帰モデルを使用し、当該モデルにおいて、分散パラメータは適合モデルからの残余のピアソン・カイ二乗統計値を用いて推定した。その有意レベルに応じてランク付けされたDMRは、さらに、組織および白血球コントロールの組み合わせにおけるメチル化パーセンテージが患者において1%以下10%以上である場合に考慮の対象とした。この結果、94個のマーカーが得られた(表2)。全てのマーカーはAUCが0.85よりも高く、患者対正常な膵臓組織におけるシグナル倍率変化が25よりも大きく、患者対コントロール(低度および中度異形成試料を含む)におけるシグナル倍率変化が5よりも大きく、p値が0.001未満であった。
組織における候補マーカー(通常、一部位につき上位20~30個のRRBS DMR)をメチル化特異的PCRによって確認する。最初は発見試料に対して行い、次により大きな個別試料集合に対して行う。
28個の上位のマーカーに特異的なプライマーを設計および発注した(IDT、アイオワ州コーラルビル)。設計は、Methprimerソフトウェア(University of California、カリフォルニア州サンフランシスコ)またはMSPプライマー(Johns Hopkins University、メリーランド州ボルチモア)のいずれかを用いて行った。アッセイは、普遍的にメチル化されたゲノムDNAおよびメチル化されていないゲノムDNAコントロールの希釈液上で、SYBRグリーンを用いたqPCRによってテストおよび最適化した。
発見試料に対して定量的MSP反応を行い、パフォーマンスを確認した。ゲノムワイド次世代(genome wide next gen)シークエンシング研究は、高い発見失敗率を被る場合があるため、高い分析的感度および特異性を有するプラットフォームにおいてDMR候補をテストすることは重要である。確認は、患者グループおよびコントロールグループから得られた別々のマイクロ切開組織およびメチル化特異的PCRを用いて選択されたマーカーのブラインド化アッセイに基づいて行い、マーカーレベルは、膵臓上皮組織用マーカーであるメチル化されたLRRC4について標準化した。患者グループ(n=53)はIPMN-HGD(19)、PanIN-3(4)、およびPDAC(30)を含んだ。コントロールグループ(n=111)は組織学的に正常な膵臓組織(31)、IPMN-LGD(36)、PanIN-1(34)、およびPanIN-2(10)を含んだ。AUCはロジスティック回帰によってそれぞれのマーカーについて算出し、マーカーの組み合わせによる区別を探った。
本実施例では、膵臓嚢胞において進行した新生物を、新しいDNAメチル化マーカーを用いて正確に検出できることを実践する。
本実施例では、膵臓嚢胞において進行した新生物を、嚢胞液中の新しいDNAメチル化マーカーのアッセイによって検出できることを実証する。
Claims (6)
- 被検体から得られた試料における高度膵異形成をスクリーニングする方法であって、当該方法は、
上記試料における、BMP3、AK055957およびST8SIA1のマーカー、ならびにFRMD4AおよびZNF781のうちから選択される少なくとも1つの追加マーカーについてCpG部位のメチル化レベルを測定することを含み、
当該測定は、
上記試料におけるゲノムDNAを重亜硫酸塩を用いて処理すること、
上記重亜硫酸塩処理されたゲノムDNAを、BMP3、AK055957およびST8SIA1それぞれのマーカー用のプライマーの組、ならびにFRMD4Aおよび/またはZNF781用の少なくとも1つの追加プライマーの組、を用いて増幅すること、および
メチル化特異的PCR、定量的メチル化特異的PCR、メチル化感受性DNA制限酵素分析、定量的重亜硫酸塩パイロシークエンシング、または重亜硫酸塩ゲノムシークエンシングPCRによって、上記CpG部位のメチル化レベルを決定することを介して行われる、方法。 - 上記試料は膵臓組織および/または膵臓嚢胞液を含む、請求項1に記載の方法。
- 上記試料は便試料、血液試料、および/または血液分画試料を含む、請求項1に記載の方法。
- 上記選択される上記マーカー用のプライマーは、
配列番号45および46からなる、BMP3に対するプライマーの組、
配列番号19および20からなる、FRMD4Aに対するプライマーの組、
配列番号41および42からなる、ZNF781に対するプライマーの組、
配列番号3および4からなる、AK055957に対するプライマーの組、および
配列番号35および36からなる、ST8SIA1に対するプライマーの組、
から選択される請求項1に記載の方法。 - 上記CpG部位がコード領域または制御領域に存在する、請求項1に記載の方法。
- 上記マーカーについての上記CpG部位のメチル化レベルを、上記測定することが、上記CpG部位のメチル化スコアを決定すること、および上記CpG部位のメチル化頻度を決定することからなる群から選択される決定を含んでいる、請求項1に記載の方法。
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