TWI390204B - Biomarker of bladder cancer and its detection method - Google Patents

Biomarker of bladder cancer and its detection method Download PDF

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TWI390204B
TWI390204B TW099104400A TW99104400A TWI390204B TW I390204 B TWI390204 B TW I390204B TW 099104400 A TW099104400 A TW 099104400A TW 99104400 A TW99104400 A TW 99104400A TW I390204 B TWI390204 B TW I390204B
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bladder cancer
biomarker
protein
urine
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Description

膀胱癌之生物標記及其檢測方法
本發明係有關一種生物標記及其檢測方法，特別是指一種膀胱癌之生物標記及其檢測方法。
人體在排尿前，腎臟所分泌之尿液是收集在膀胱中。膀胱癌在泌尿道癌症中是最常見，因此其相關的腫瘤標記發展是迫切需要的。2009年美國癌症學會對膀胱癌最近一期的判斷指出大約有70980個新的病例與並且每年會有14330人死於膀胱癌。男性罹患膀胱癌的機會是27人中就有一人會罹患膀胱癌，而女性罹患膀胱癌的機會是85人中就有一人罹患膀胱癌，且罹患膀胱癌的人將近90%是超過55歲。癌症是早期發現早期治療才能夠獲得較好的治癒成果。TNM系統是膀胱腫瘤分期中最常使用的，依據1997年之AJCC/UICC TNM分期系統包含有原發腫瘤分期相關描述(T分期)，淋巴結狀態(N分期)與遠端轉移(M分期)。組織學的分級是標示為低度(G1)、中度(G2)或者高度(G3)階段。目前這個分級已經被取代為低度或高度分級，其區別與癌細胞行為有關。非侵襲式腫瘤在組織學分級呈現多樣性，然大部分的侵襲式贅瘤是屬於高度分級。
大約70%膀胱癌呈現的是淺表性損傷(superficial lesions)。淺表性癌(Ta、Tis、T1)主要藉由腫瘤經尿道切除手術(TRU)來治療。膀胱內灌注免疫療法(例如卡介苗)或者化學療法(例如亞德利亞黴素(doxorubicin))可以作為殘餘病灶的結合療法或者作為預防工具。淺表性機能損傷的再發生率是50%～80%。大約15%的低度分級腫瘤會復發為高度分級腫瘤且傾向為肌肉層侵犯的機能損傷。對於具有肌肉層侵犯的膀胱癌病人而言，膀胱根除手術是治療的選擇方式之一，但這樣的方式會導致性功能的損傷與需要尿路重建。
雖然細胞學具有低敏感性與明確性，特別是對低度分級腫瘤。但是準確的非侵入式生物標記可以減少高成本與侵入式之膀胱鏡每一年的使用次數。因此，相較於細胞學，具有較高敏感性與明確性之生物標記可以大幅度改進對膀胱癌的處理。已經發現數個膀胱癌候選腫瘤生物標記存在尿液或膀胱癌細胞株中，可以作為初步診斷、偵測復發與評估治療反應。然而，目前的這些候選標記並沒有在敏感性與特異性兩者上具有較優良的特性，或者並沒有對大量的病人進行臨床上的使用。因此迫切需求能更有效偵測膀胱癌之生物標記。
因為尿液直接與膀胱內膀胱癌上皮細胞接觸，因此尿液樣品中富含有由膀胱癌細胞所分泌的蛋白質，可視為發現膀胱癌生物標記的可使用初始材料。最有希望搜尋出有用的膀胱癌生物標記的方式是在病灶發生時研究尿液中的蛋白質體(urine proteome)，並進而研究尿液蛋白質之整體圖譜變化。在基於質譜儀分析與資料庫搜尋的協助下所產生的已定義蛋白質的蛋白質含量可以大略的由胜肽光譜數(peptide spectral counts)或者emPAI(exponentially modified protein abundance index)系統判斷。然而，在尿蛋白質體分析上使用這些新的質譜數據分析策略仍是十分有限的。
有鑑於此，本發明遂針對上述習知技術之缺失，提出一種膀胱癌之生物標記及其檢測方法，以有效克服上述之該等問題。
本發明之主要目的在提供一種膀胱癌之生物標記及其檢測方法，其係用以協助診斷膀胱癌及區分癌細胞入侵程度，以幫助了解膀胱癌患者的病情，從而對症下藥，並提高治療成效。
本發明之另一目的再提供一種膀胱癌之生物標記及其檢測方法，其可與現有方法，例如潛血、NMP22分子檢驗、膀胱鏡與細胞學檢驗等合併，以利於膀胱癌檢測與(或)癌組織侵入或惡性的判斷。
為達上述之目的，本發明提供一種用以檢測膀胱癌的生物標記，其係選自於載脂蛋白質A1(APOA1)、載脂蛋白質A2(APOA2)、PRDX2、HCII及SAA4其中之一或一個以上，且該生物標記存在於一受檢測者之尿液檢體中。
本發明提供一種用以檢測膀胱癌的生物標記，其係選自下列69個蛋白質其中之一或一個以上，且此生物標記存在於一受檢測者之尿液檢體，此69個蛋白質為為為Protein S100-P(S100P)、Cerulopasmin precursor(SP)、Serum amyloid A-4 protein precursor(SAA4)、Isoform 1 of Complement factor B precursor(Fragment)(CFB)、Afamin precursor(AFM)、載脂蛋白質A1(Apolipoprotein A-I precursor,APOA1)、載脂蛋白質A2(Apolipoprotein A-Π precursor,APOA2)、Isoform 1 of Fibrinogen alpha chain precursor(FGA)、Isoform Gamma-B of Fibrinogen alpha chain precursor(FGG)、Apolipoprotein B-100 precursor(APOB)、Alpha-1-acid glycolprotein 1 precursor(ORM1)、Transthyretin precursor(TTR)、ALB protein(ALB)、Serotransferrin precursor(TF)、Hemopexin precursor(HPX)、Antithrombin Ⅲ variant(SERPINC 1)、Angiotensinogen precursor(AGT)、187kDa protein(C3)、FLJ00385 protein(Fragment)(IGHM)、Glutathione S-transferase P(GSTP1)、Fibrinogen beta chain precursor(FGB)、Beta-2-glycoprotein 1 precursor(APOH)、Complement C2 precursor(Fragment)(C2)、Apolipoprotein A-IV precursor(APOA4)、ENO1P protein(EDARAD)、Hemoglobin subunit alpha(HBA1；HBA2)、Peptidyl-prolyl cis-transisomerase A(PPIA；LOC 654188；LOC 653214)、Hemoglobin subunit delta(HBD；HBB)、Alpha-2-macroglobulin precursor(A2M)、Alpha-1-antitrypsin precursor(SERPINA1)、Vitamin D-binding protein precursor(GC)、Immunglobulin heavy chain variable region、Myosin-reactive immunoglobulin heavy chain variable region(Fragment)、Heparin cofactor 2 precursor(SERPIND1(HCII))、Peroxiredoxin-2(PRDX2)、heterogeneous nuclear ribonucleoprotein D-like(HNRPDL)、Keratin-8-like protein 1、Isoform 2 of Apolipoprotein-L1 precursor(APOL1)、Ig heavy chain V-I region EU、Protein S100-A6(S100A6)、Fetuin-B precursor(FETUB)、Factor VII active site mutant immunoconjugate(F7)、RcTPI1(Fragment) (LOC729708)、Isoform a 1 of Acyl-CoA-binding protein(DBI)、IGHA1 protein(IGHA1)、Ig heavy chain V-III region GAL、Macrophage migration inhibitory factor(MIF)、14-3-3 proteln theta (YWHAQ)、Ig mu heavy chain disease protein、HP protein(HP)、Serum paraoxonase/arylesterase 1(PON1)、Complement component C9 precursor(C9)、Fructose-bisphosphate aldolase A(ALDOA)、Kallikrein B,plasma(Fletcher factor)1(KLKB1)、Inter-alpha-trypsin inhibitor heavy chain H2 precursor(ITIH2)、Protein S100-A4(S100A4)、Malate dehydrogenase,mitochondrial precurscr(MDH2)、Protein S100-A11(S100A11)、Extracellular matrix protein 1 precursor(ECM1)、Complement factor H-related protein 3 precursor(CFHR3)、Hemoglobin subunit beta(HBB)、101 kDa protein(NDST1)、Nucleoside diphosphate kinase A(NME1)、Apolipoprotein C-III precursor(APOC3)、Histone H4(HIST1H4)、Isoform 1 of Haptoglobin-related protein precursor(HPR)、TALDO1 protein(TALDO1)、IGHG4 protein(IGHG4)以及Myosin-reactive immunoglobulin heavy chain variable region(Fragment)。
本發明尚提供膀胱癌的檢測方法，其包含有下列步驟提供一受檢測者之尿液檢體；提供至少一生物標記；以及檢測此生物標記)在尿液檢體中含量的呈現程度；其中此生物標記係選自於或脂蛋白質A1(APOA1)、載脂蛋白質A2(APOA2)、PRDX2、HCII及SAA4其中之一或一個以上，或是上述的69個蛋白質。
底下藉由具體實施例詳加說明，當更容易瞭解本發明之目的、技術內容、特點及其所達成之功效。
本發明係揭示69個膀胱癌特異之尿液蛋白質(如表一所示)作為新的生物標記，尿液中具有本發明所述之69個膀胱癌各標記含量的呈現程度較高的病人表示該病人罹患膀胱癌之機率較高或者膀胱癌細胞侵入程度較嚴重。在該69個膀胱癌特異之尿液蛋白質中有5個是較受到注目的，其係各為載脂蛋白質A1(APOA1)、載脂蛋白質A2(APOA2)、PRDX2、HCII及SAA4。
請一併參閱第1圖，其係本發明之一種膀胱癌的偵測方法步驟流程圖。如圖所示，首先如步驟S1所述，提供一受檢測者之尿液檢體；接續，如步驟S2所述，提供至少一生物標記，其係由上述之69個膀胱癌特異之尿液蛋白質中選出；以及最後，如步驟S3所述，偵測此生物標記在此尿液檢體中含量的呈現程度，以判斷該受檢測者是否有膀胱癌或者膀胱癌惡化程度。
其中該呈現程度係與健康對象之尿液檢體中之該生物標記的含量相比對，以作為膀胱癌存在的指證。或者是與受檢測者先前之尿液檢體中之該生物標記的含量相比對，以作為為膀胱癌之癌細胞侵入程度的指證。
本發明所提出的膀胱癌檢測方法可與現有方法，例如潛血、NMP22分子檢驗、膀胱鏡與細胞學檢驗等合併，以利於膀胱癌檢測與(或)癌組織侵入或惡性的判斷。
在本發明中係利用iTRAQ定量技術於尿液檢體中發現具有明顯差異含量的蛋白質。並在個別樣品中進一步利用西方墨點法進行確認此發明的效果。由一個或者以上個不同表現之蛋白質結合所形成之多重生物標記組(marker panel)能夠早期偵測、診斷或區別出膀胱癌的侵入階段，從而對症下藥，並提高治療成效。
雖然本發明對該生物標記的含量是利用西方墨點法進行確認，但也可使用質譜法抗體偵測法、螢光、冷光、免疫法或層析等技術進行檢測。
實施例 iTRAQ標定與陽離子交換(SCX)液相層析分餾與鹼性相色層分析
將由符合年齡控制條件與相同組織學階段或者病理學階段之病人的尿蛋白質混合，以減少個別差異與強化訊號。在這個研究中，疝氣的病人是被選作為非癌症對照組(Non tumor，NT)次群組。依據four-plex iTARQ(Applied Biosystems,Foster City,CA)製造商的操作建議，對每一次群組之混合尿蛋白質(100微克(μg))進行處理。簡單來說，一單位的iTARQ試劑(定義為標示100微克蛋白質所需的試劑總量)溶解於70微公升(μL)酒精中，加入每一次群組的已混合尿蛋白質，經進行還原(reduce)、半胱胺酸鍵結(cysteine-blocked)與胰蛋白酵素水解反應。114、115、116與117 iTARQ試劑加入非癌症對照組(NT)、低分級/早期期別(LgEs)、高分級/早期期別(HgEs)與高分級/重度期別(HgAs)次群組的胜肽片段，在室溫下反應1小時。隨後混合四組樣品並利用真空離心方式乾燥。將臨床樣品1利用SCX色層分析分為數個部份；來自於臨床樣品部2則分別利用SCX與RP色層分析分為數個部份。充提後的部分(elution fractions)混合成42個部分並真空乾燥，以適合進行奈米ESI-LC-MS/MS分析。
利用LTQ-Orbitrap PQD進行LC-ESI MS/MS分析
每一個分離的胜肽混合物以緩衝液E(包含有0.1%甲酸之水溶液中)回溶，並且每部分的取2微克溶於緩衝液A內，以流速0.2微公升/分鐘(μL/min)裝載穿過一捕捉管柱(trap column)，並且在一具有15微米尖端(New Objective,Woburn,MA)之resolving 10-cm analyticalC18 PicoFritTM column(內直徑為75微米)中分離。LC setup線上與利用Xcalibur 2.0 software(Thermo Fisher)操作之LTQ-Orbitrap(linear ion trap-orbitrap)(Thermo Fisher,San Jose,CA,USA)結合。為了擷取串聯質譜數據，以PQD(pulsed Q dissociation)在LTQ中偵測胜肽離子碎片。每次一次質譜掃描伴隨著三個二次質譜掃瞄，MS掃瞄之m/z掃瞄範圍是350至2000Da。
蛋白質識別與利用連續資料庫搜尋進行定量
MS/MS圖譜利用MASCOT engine(Matrix Science,London,UK;version 2.2.04)比對來自於European Bioinformatics Institute(http://www.ebi.ac.uk/)之non-redundant International Protein Index(IPI)human sequence database v3.27(released at March 2007;67,528 sequences;28,353,548 residues)以進行蛋白質的身分鑑定，在蛋白質分子量比對允許10ppm的質量誤差，在PQD片段離子是0.5Da，容許兩個未完全胰蛋白消化之位點，氧化設為的可能的結構改變，iTRAQ(N terminal),iTRAQ(K)與MMTS(C)是固定的結構改變。胜肽的電荷狀態是設定為+2與+3。蛋白質識別與定量的確認是利用共享資源Trans-Proteomic Pipeline(TPP)software(Version 4.0)進行確認。由MASCOT搜尋產生DAT檔案。MS原始資料與DAT檔案隨後以TPP軟體理與分析。於此，使用PeptideProphet與ProteinProphet probability scores來確認誤測率低於0.7%。每一蛋白質比例的定量是利用Libra program來達成，用TPP內的定量套件來對MS/MS圖譜進行定量，定量離子的最小強度門檻是20。
西方墨點法分析
尿蛋白質經過去除鹽分與濃縮後，蛋白質總量利用DC protein assay量測，接著以西方墨點法分析的每一尿液樣品。來自於個別或混合樣品的尿蛋白質(100克)溶解於SDS膠體中並且電泳轉換至PVDF膜(Bio-Rad,Hercules CA,USA)。此膜的阻塞(block)是利用具有5%脫脂奶粉的trisbuffered saline(Bio-Rad,Hercules CA,USA)與0.1%Tween-20(Sigma,St Louis)(TBST)在室溫下進行1小時。西方墨點法分析以下列的抗體進行：anti-apolipoprotein A-I(anti-APOA1,1:500,ab58924,Abcam),anti-apolipoprotein A-II(anti-APOA2,1:250,ab54796,Abcam),anti-heparin cofactor 2 precursor(anti-HCII,1:2000,MAB0769,Abnova),anti-peroxiredoxin 2(anit-PRDX2,1:5000,AF3489,R&D systems),anti-s100A6(1:200,AF4584,R&D systems),與anti-s100A8(1:200,AF4570,R&D systems)。西方墨點法主要將抗體依照horseradish peroxidase-conjugated antibody進行，並且依據製造商的說明利用強化的化學發光偵測來建構。絕對蛋白質APOA1能階的定量，apolipoprotein A-I protein purified from human plasma(A0722,Sigma-Aldrich,USA)，是作為西方墨點分析法中的定量標準。
統計分析
利用平均數、標準偏差、中位數與四分位差對對個別的西方墨點結果進行統計分析。APOA1、APOA2、PRDX2、HCII與SAA4在尿液及濃縮尿液蛋白質中的表現含量與癌症病患間的差異是利用nonparametric Mann-Whitney U test來執行。統計分析是利用SPSS軟體(version 13.0,SPSS Inc,Chicago,IL)實施。當0.05的雙尾p值(two-tailed p values)或者更少是被認為是具有統計意義的。Receiver operator characteristic(ROC)分析與曲線下面積(AUC)也被計算。
請參閱第2圖，其係利用西方墨點分析法(較低的控制板)對個別尿蛋白內五個候選蛋白質進行偵測。由個別尿液樣品製備的蛋白質均量利用SDS-PAGE分離，轉換至PVDF膜並且利用相關的抗體探測。(a)APOA1在100微克的尿蛋白質中之含量。(b)APOA2在100微克的尿蛋白質中之含量。(c)HCΠ在50微克的尿蛋白質中之含量。(d)PRDX2在100微克的尿蛋白質中之含量。(e)SAA4在50微克的尿蛋白質中之含量。依據全部尿蛋白質總額(左側徒刑)與等同的尿液體積(右側圖形)計算介於NT、LgEs、HgEs、HgAs與UTI/HU間的蛋白質相對表現量倍率(fold changes)。藉由與NT次群組比較，每一次群組的平均相對表現量標記在每一盒狀圖的頂端。在每一線道，盒狀圖的水平線指示數據點的第10、第25、第50、第75與第90百一分段值。
表一由iTRAQ結果所找到之膀胱癌病患尿液樣品內69個高度表現蛋白質之清單
表2是在膀胱癌與相較於NT群組之LgEs、HgEs、HgAs及UTI/血尿次群組之選擇的尿蛋白質的相對表現量。
表3(a)是在個別樣品的總尿蛋白質的固定總量下藉由西方墨點法的p值、ROC曲線的AUC值，與APOA1、APOA2、HCII與PRDX2表現的感度與特異性表現的總結。3(b)是在個別樣品在固定總尿液體積下藉由西方墨點法的p值、ROC曲線的AUC值，與APOA1、APOA2、HCII與PRDX2表現的敏感性與具體性表現的總結。
唯以上所述者，僅為本發明之較佳實施例而已，並非用來限定本發明實施之範圍。故即凡依本發明申請範圍所述之特徵及精神所為之均等變化或修飾，均應包括於本發明之申請專利範圍內。
第1圖係本發明之一種膀胱癌的偵測方法步驟流程圖。
第2圖a、b、c、d與e是分別在較低的控制板處是利用西方墨點分析法對個別尿蛋白內五個候選蛋白質進行偵測。左側控制板是依據固定全部尿蛋白質總量與在右側控制板處依據等同的尿液體積計算介於NT、LgEs、HgEs、HgAs與UTI/HU間的蛋白質相對表現量(fold changes)。

Claims (8)

  1. 一種用以檢測膀胱癌的生物標記，其係選自於載脂蛋白質A2(APOA2)、HCII及SAA4其中之一或一個以上，且該生物標記存在於一受檢測者之尿液檢體中。
  2. 如申請專利範圍第1項所述之生物標記，其係利用iTRAQ定量技術所測定。
  3. 一種膀胱癌的檢測方法，其包含有下列步驟：提供一受檢測者之尿液檢體；提供至少一生物標記；以及檢測該生物標記在該尿液檢體中含量的呈現程度；其中該生物標記係選自於載脂蛋白質A2(APOA2)、HCII及SAA4其中之一或一個以上。
  4. 如申請專利範圍第3項所述之膀胱癌的檢測方法，其中該呈現程度係與健康對象之尿液檢體中之該生物標記的含量相比對。
  5. 如申請專利範圍第3項所述之膀胱癌的檢測方法，其中該呈現程度係與該受檢測者先前之尿液檢體中之該生物標記的含量相比對。
  6. 如申請專利範圍第3項所述之膀胱癌的檢測方法，其中該呈現程度係為膀胱癌存在和/或癌組織侵入或惡性程度的指證。
  7. 如申請專利範圍第3項所述之膀胱癌的檢測方法，其中該生物標記的含量是利用西方墨點法、質譜法、抗體偵測法、螢光、冷光、免疫法或層析等技術檢測。
  8. 如申請專利範圍第3項所述之膀胱癌的檢測方法，其更可與潛血、NMP22分子檢驗、膀胱鏡與細胞學檢驗等合併，以利於膀胱癌檢測與/或癌組織侵入或惡性的判斷。
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