專利名稱：：評估結(jié)腸直腸癌的制作方法評估結(jié)腸飾癌本申請是以下申請的分案申請申請日2003年3月29日；申請?zhí)?3131205.5;發(fā)明名稱同上。背景本申請要求2002年3月29日申請的序列號為60/368,798的美國臨時申請的利益。本發(fā)明涉及基于生物樣本基因表達分布圖的結(jié)腸飾癌的診斷和預測。結(jié)腸飾癌是一種鄉(xiāng)疾病，由據(jù)認為^131H種主要肝機制形成的腫瘤組成1)與染色體不穩(wěn)定性相結(jié)合的多發(fā)性結(jié)腸腺癌(APC)基因，或e-連環(huán)蛋白基因的突變，2)與M星序列不穩(wěn)定性相關的DNA錯配修復基因如MLH1,MSH2^PMS2和MSH6的突變，和包含縫餅列基因的突變，以及3)由腫瘤抑制基因啟動子區(qū)的過甲基化誘導的基因沉默。結(jié)腸直腸癌個體的遺傳互補可肖抱括遺傳不穩(wěn)定性、特異突變和基因沉默的不同結(jié)合。染色軒穩(wěn)定性(CIN)—般是癌癥的普遍特征。它意其中所有的或大部分的染色體丟失或增加了的非整倍體表型。伴隨短重復序列突變率的增加，在二倍體腫瘤中發(fā)現(xiàn)了微粒,穩(wěn)定性(MIN)。這兩種遺傳不穩(wěn)定性的形式在結(jié)腸直腸癌中很普遍。因此結(jié)腸直腸癌具有復雜的起源并且包括不同生物通路間大量的相互作用。用于協(xié)助提供診斷、預觀蜮治療監(jiān)測結(jié)果的血清標記、組織學和細胞學檢查通常不具有所期望的可信度。同時，4柳制蟲的遺傳標記(例如Ht縫因的表達增加)可能是有益的，癌癥的多樣性^t傳標記的組合可能成為最好的途徑。本發(fā)明是評估結(jié)腸OT癌存在或不存在，或者被認為患有結(jié)腸直腸癌的患者其可能狀態(tài)的方法。在本方法中，通過分析患者樣本的基因,分布圖確定患者是否得了結(jié)腸直腸癌，患者是否未得結(jié)腸直腸癌，患者是否可能要得結(jié)腸3M癌，或正在接受治療的結(jié)腸直腸癌患者對治療的反應。本發(fā)明的一個方面涉及用于實施該方法的制品。這樣的制品包括基因就分布圖或固定于機器可讀媒介如計算機可讀媒介的基因皿分布圖的表現(xiàn)物。.用來鑒別基因表達分布圖的制品也可以包括用于捕捉和/或顯示基因表達存在與否，戯達，號的基片或平面，如微陣列。發(fā)明詳述很少發(fā)現(xiàn)組織樣本中單純的特定核苷酸序列的存在或缺失具有診斷或預測價值。另一方面，關于多種蛋白、肽或mRNA,的信息正ffl^越，瞎視。具有駄蛋白、肽或mRNA的潛在可能的特定核苷,列(這樣的序列稱為"基因")僅在給定細胞的基因組中存在這一事實本身，并不能確定該蛋白、肽或mRNA是否會在給定的細胞中表達。能駄蛋白、肽或mRNA的給定基因是否表達以及表達到何種程度取決于多種復合因子。不考慮理解和評估這些因子的困難程度，檢觀瞎因表達可以為如腫瘤發(fā)生、轉(zhuǎn)移、細胞凋亡以及其他臨床有關王1^等重要事件的發(fā)生提^W用的信息。通,因表達分布圖可以發(fā)現(xiàn)基因激活或失活程度的相對指標。本發(fā)明的基因表達分布圖可用于結(jié)腸直腸癌患者的診斷和治療。樣品制備需要收集患者樣本。本發(fā)明4頓的病例樣本是那些疑為含有患病細胞的樣本，如取自結(jié)腸樣本或外科切除樣本的上皮細胞。獲得可疑樣本的一種有用技術(shù)是激光捕1￡微切片技術(shù)(LCM)。激光1^微切片K^提供了一種選擇需研究細胞的方法，并最小化由細胞種類的不均一性造成的易變性。因此，正常細胞和癌細胞之間的基因表達中度或細小的變化都可以被輕易檢測到。在優(yōu)選的方法中，樣本包括從外周血中提取的循環(huán)上皮細胞。這可以按照多種方法獲得，但最,的方法是Immunivest公司的美國專利6,136,182所描述的磁性分離技術(shù)，該專利在此引入作為參考。一旦得到含有所關注細胞的樣本，就提取其RNA，擴增，并獲得基因表達分布圖，優(yōu)逸的通過微陣列獲得合適框架(portfolio)中基因的表達分布圖。確立基因,分布圖的，方,括，確定一個倉嫩編碼蛋白質(zhì)或肽的基因所產(chǎn)生的RNA的量。這可M5I録酶PCR(RT-PCR)，靜性RT-PCR，實時RT-PCR，差異顯示RT-PCR，Northern雜交分析和其他相關實驗實現(xiàn)。盡管可育調(diào)單個PCR反應實施這些皮術(shù)，但最好擴增來自mRNA的互補DNA(cDNA)或互補RNA(cRNA)并用微陣歹樹其分析。對本領域的技術(shù)人員來說大量不同的陣列構(gòu)型及其生產(chǎn)方法是已知的，并且也在如下的美國專利中有描述5,445,934;5,532,128;5,556,752;5,242,974;5,384,261;5,405,783;5,412,087;5,424,186;5,429,807;5,436,327;5,472,672;5,527,681;5,529,756;5,545,531;5,554,501;5,561,071;5,571,639;5,593,839;5,599,695;5,624,711;5,658,734;和5,700,637;這些專利披露的技術(shù)在此弓l入作為參考。微陣列技術(shù)允許同時檢測數(shù)千基因的穩(wěn)定狀態(tài)的mRNA水平，因此它為檢測非控制的細胞增殖^m，如啟動、抑制或調(diào)節(jié)等提供了一個強有力的工具?，F(xiàn)在兩種微陣列技術(shù)正被廣泛鵬。第一種是cDNA陣列，第二種驗核苷酸陣列。盡管這些芯片在構(gòu)造上存在差異，但其下游數(shù)據(jù)的分析和輸出實質(zhì)上是相同的。這些分析的結(jié)果典型地是對由標記探針獲得的信號強度的測定，所述的探針用于檢測與陣列上己知位點的核,列雜交的、來自樣品的cDNA序列。典型地，所述的信號3驢與cDNA的含量，或^i兌樣本細胞中所表達的mRNA的量成比例。有許多這樣的技術(shù)可以獲得并且是有用的。tt^的檢測基因表達的方法可以在美國專利中找到，如Linsley等人的專利6,271002;Friend等人的專利6,218,122;Peck等人的專利6,218,114;Wang等人的專利6,004,755，其所公開的內(nèi)容均員引入作為參考。基因表ii^平的分析通過比較強^t行。最好^131制備一種待測樣品中基因皿強度相對于對照樣品中基因表達強度的比值矩陣進行。例如，可以將疾病組織中基因表達的強度與相同類型正常組織中的表達5M進行比較(例如患病的結(jié)腸組織樣本對正常的結(jié)腸組織樣本)。這個,強度的比11M示出被測樣本和對照樣本中基因表達的f，變化?；虮磉_分布圖也可以ttt很多方法顯示。最普遍的方法是將原始的熒光^販離陣系數(shù)處理綱狀圖，其中列標被觀脾品，行標基因。經(jīng)這樣的處理后那些具有相^達分布的基因就會相互鄰近。^基因的表達比率可用一種顏色顯示。例如，小于一的比率(表示下調(diào))可以出現(xiàn)在圖譜的藍色部分，而大于一的比率(表示上調(diào))可以出現(xiàn)在圖譜的紅色部分。商業(yè)上可購買到的計^t幾軟件禾Mmra合顯^S樣的數(shù)據(jù)，包括SiliconGenetics有限公司的"GENESPRING"，Partek有限公司的"DISCOVERY"和"INFER"軟件。用于本發(fā)明的方法的受調(diào)節(jié)基因如表1所示。差異表達的基因表現(xiàn)為在患病細胞中上調(diào)或下調(diào)。上調(diào)和下調(diào)是相對的術(shù)語，表示可檢測到的相對于某種基線的基因表達的量的差異(超出檢測系統(tǒng)的噪音影響)。在這種情況中，基線是正常細胞測得的基因表達。再使用同樣的觀懂方法確定患病細胞中所關注的基因相對于基線水平是上調(diào)或下調(diào)。上下文中，患病的，是指伴隨細胞非控制增殖而出現(xiàn)的機體狀態(tài)的改變，此種改變中斷、擾亂或潛在的打亂機體功能的適當彭見。當某人基因型或表型的一些方面一^^現(xiàn)出存在特定疾病時，他就被診斷為患有該種,。然而，作出診斷或預測的行為包括確定疾病表象狀態(tài)的情況，如治療監(jiān)測。在治療監(jiān)測中，比較經(jīng)過一段時間治療后的基因表達確定基因表達分布圖是否有變化，或其變化與正常組織的模式更為一致來考慮療程的媒從而作出臨床判定。雌的，上升或下降調(diào)節(jié)的水平是以雜交的微陣列探針觀懂弓雖的成餘變化來區(qū)別的。2倍差異或p值小于0.5對于作出這樣的辨別^im的。即當認為與在正常細胞中相比一個基因在患病細胞中差異表達時，患病細胞中的強度至少是正常細胞的2倍或1/2。f，差異越大，該基因就M^用作診斷。本發(fā)明選擇用作分析表達分布圖的基因具有產(chǎn)生使該基因區(qū)另盱正?；蚧蚍钦{(diào)節(jié)基因的信號的表達水平，該信號翻出i頓臨床實驗室檢測設備的背景量。禾,統(tǒng)計值可用于確定地使調(diào)節(jié)基因與非調(diào)節(jié)基因和噪音區(qū)別幵。mm計^t驗發(fā)現(xiàn)在不同樣本組之間有最顯著差異的基因。Student'sT1^驗就是一^h發(fā)現(xiàn)兩組之間顯著差異的有力的統(tǒng)計檢驗的例子。pHM小，不同組之間基因顯示差異的證據(jù)就越有i劃艮力。然而既然微陣列可以同時檢測多個基因，那么就需要同時進行萬次的統(tǒng)計檢驗。因此，就存在偶爾看到極小p值的可能，可以利用Sidak校正以及隨機俗排列實^a行調(diào)整。T檢驗中p值小于.05就證明基因錢異顯著的。更有說服力的證據(jù)是加入Sidak校正因子后p值小于.05。對于每組中的大量樣品，經(jīng)過隨機俗排列檢紘p值小于.05錢異顯著的最有說服力的證據(jù)。另一個可用來選擇產(chǎn)生強于非調(diào)節(jié)基因和噪音信號的基因的參數(shù)是絕對信號差異測量值。優(yōu)選的，調(diào)節(jié)基因的表達形成的信號與正?；蚧蚍钦{(diào)節(jié)基因(以絕對值為基礎)至少有20%的差異。更雌的，調(diào)節(jié)基因形成的^ii模式與正?；蚧蚍钦{(diào)節(jié)基因至少有30%的差異。將基因分組以便于M獲得一組基因中一套基因的信息為診斷、預測或治療選擇等相關臨床判斷提供可靠的基礎。這些套基因構(gòu)成了本發(fā)明的框架(portfolios)o此例中，由該框架支持的判斷包括結(jié)腸鵬癌。基因表達分布圖的框架包含實施例3中描述的基因f^組合。對于大多數(shù)診斷標記物來說，最好^ffi盡量少的標記物就足以充分作出一個正確的醫(yī)學判斷。這就避免了^ffl那些懸而未決需作進一步分析的治療措施帶來的延誤，同時又避免了時間和資源的不合理l頓。此例中，這樣的最小量的框架包含實施例4中的基因組合。優(yōu)選的，構(gòu)^S樣的框架以保證該框架中的基因組合相對于單個基因或隨豐;i^擇的基因組合顯示出更好的靈敏度和特異性。在本發(fā)明中，框架的靈tt反映在一個基因在,狀態(tài)下相對于，正常狀態(tài)下所表現(xiàn)出的基因表達的成倍差異中。特異性可以反映在基因^ii信號與所關注,相關性的統(tǒng)計領懂中。例如，標準差就是這樣一個有用的度量。當考慮將一組基因摻AJ煤一框架中時，表達澳懂值的標準差越小其特異性越大。其他檢測變量，如相關系數(shù)也包括在使用范圍內(nèi)。最^tfe，ffiz:基因表達框架的方法^i用優(yōu)化算法，如在確定股票投資中廣泛使用的平均方差算法的。這一方法在同日期的TimJatkoe等人標題為"投資組合選擇(PortfolioSelection)"的同時待審的專利申請中有詳細泰逸。該方法實質(zhì)上要求一套輸入值(金,用中的股票，這里指艦S販觀啶的表達)，其可優(yōu)化收到的回報率(例如形成的信號)以進行應用，同時最小化回報率的可變性。許多商業(yè)軟件禾M^適^tfi^樣的操作。^說明書中稱為"Wagner軟件"的"Wagner聯(lián)合均值-方差優(yōu)化應用程序"是優(yōu)選的。這個軟件使用"Wagner^均值-方差優(yōu)化庫"中的功能，來確定有效邊界，皿Maikowitz意義上的優(yōu)化框架。由于該軟件用于金融分析目的，因此^ffi這種類型的軟件需要糊敫陣列數(shù)據(jù)進行轉(zhuǎn)換，使數(shù)據(jù)以股票回報值和風險測量值的方式輸入處理。例如，當Wagner軟件與微陣列§破測定協(xié)同應用時，要運用以下的繊轉(zhuǎn)換方法。首先il31確定那些其表超少顯示出某種微小差異水平的基因，對基因進fiil^擇。，的預選擇過程如下所述。選擇一個基線組。典型的基線組包含來自未患有所述疾病的群體的基因。例如，錢擇所用于診斷乳腺癌的基因框架時，可以將未患乳腺癌患者的樣本用作制備基線組。一旦選擇了基線組，就可以計算出基線組樣本中齡基因的表達指標的算術(shù)平均值和標準差。典型的，這個指標是微陣列記錄的熒光強度。隨后將通過計算得到的統(tǒng)計學數(shù)據(jù)用于計算旨基因的基線值(X4示準^+均值)。這就是基因的基線記錄，其它所有樣本都要與其相比較。X是由制定框架的旭擇出來的嚴l^錢。X的數(shù)鵬大京^格。iM的X數(shù)值范圍是0.5-3，更,的是2—3，最，的是3。隨后計算每個實驗樣本(那些表現(xiàn)出所關注疾病的樣本)與基線記錄的比值。再將這些比值轉(zhuǎn)換成易于軟件進行繊處理的底數(shù)為10的對數(shù)值。該方法使下調(diào)基因顯示為負值，這是{頓Wagner軟件并按照Maikman均值-方差算法進行優(yōu)化所必需的。將包含這些轉(zhuǎn)換比值的預處理數(shù)據(jù)作為輸入，替代用于金融分析目的的Wagner軟件中通常使用的資產(chǎn)回報值。一旦制定出有效邊界，對于一^合定的輸入水TO(回報率)^應于邊界上的一個點的方差，就可以選擇出一個優(yōu)化框架。這些輸入值或方差是制定框架的人預先確定的標準數(shù)集。換句話說，尋求最適框架的人決定一個可接受的輸A/K平(表明靈tt)或者一^t合定的方差水平(表明特異性)，選擇位于MH入7K平或方差相應的有效邊界的基因。當選定了輸入水平或方差，Wagner軟件就能夠選擇出這樣的基因。Wagner軟件可以像對股票投資組合中每支股票所做的那樣，分配所述框架中旨基因一個皿。將患者樣;^i架中的基因的表達，與用于構(gòu)建所述框架的差異^iiS因的計算值相比較，就可以確定該樣本是否患有該框架所診斷的疾病。優(yōu)選的，首先;將在選擇框架的過程中纟^^基因分配的權(quán)重乘以框架中*基因的強度值，力卩和得到框架值。然后用(基線組中框架的Y"示準M+均值)計算邊界值，其中Y是一個嚴格數(shù)值，具有與戰(zhàn)X相同的含義。一個樣本具有的框架值大于基線組的框架值，則此樣本就被歸為患有該病。需要時，可以根據(jù)提高可信度水平的己知統(tǒng)計學方法重mJ:^MI呈。任選的，可以重復進行J^lfffl到獲得預期的最高準確度?？蚣苓x擇的戰(zhàn)呈和未知量的表tBl程概括如下1.選擇基線組2.計算基線組樣本中*基因的均值和標準差3.計算旨基因的(X^示準^f均值)。這就是基線記錄，其它所有的樣本都要與這個基線記錄相比較。x是一嚴格變量，x數(shù)HM大m^格。4.計算齡實驗樣本對根據(jù)步驟3得到的基線記錄的比值。5.轉(zhuǎn)換比值，使值小于l的比值成為負數(shù)(例如〗柳底數(shù)為10的對數(shù))。(現(xiàn)在下調(diào)的基因有正確的負值以符合MV優(yōu)化的需要)。6.將這些轉(zhuǎn)換的比值作為輸入值，替換軟件應用中通常使用的資產(chǎn)回報值。7.該軟件將標繪出有效的邊界線，并且返回一組沿此有效邊界線上任意一點的優(yōu)化框架。8.在此有效邊界線±^擇一個所需的回報率或者方差。9.il31將在選擇框架的算法中所給定的權(quán)重乘以每個基因的強度值，加和計算每個樣本的框架值。10.將基線組的框架值的均働B上Y與基線組的框架值的標縫的乘積，計算出邊界值。大于該邊界值的數(shù)值應被歸為實驗組。11.<趟的，可以重復進行戰(zhàn)過程，直到獲得預期的最佳準確度?？蛇x擇的，首先M31確定那些其就顯示出某種微小差異水平的基因，對基因進行i^擇。在這個可選擇的方法中，予i^擇im的以(^一A)給出的閾(CT,+(7),基礎，其中m是已知患有該疾病或病癥的樣本子集的均值，^是正常樣本子集的均值，at+on表示聯(lián)合標準差。3131根據(jù)如(戶-M4X)的關系式對的預選擇，也可在其中使用信噪比截止值。這就確?；诓町愓{(diào)節(jié)進衍頁選擇的基因具有臨床上的顯著差異。即超過了適于測定各種臨床參數(shù)的儀器產(chǎn)生的噪音7]C平。對于根據(jù)這些標準預選的*標記物^一個矩陣，其中列^^樣本，行表示標記物，并且齡元件都是根據(jù)對該標記物的^yj行標準化后所得到的3艘測量值，其中14撥弓賊測量值。也可以M體附加的邊賂件來定義優(yōu)化框架。例如，框架的大小可限定為固定的值域或ina物的數(shù)目。這或者可以s^制定更為嚴格的ut標準(例如以"-M4X)代替M4AT,))^i^頓禾M^特征如P艮制組合大小來實現(xiàn)。(CT+(X)例如可以^S邊^(qū)fH^有效邊界線從最優(yōu)化的IO個基因中選出。也可以^ffl所有的預選基因確定有效邊界線后再限定所選擇基因的數(shù)目(例如不超過10選雜架的戰(zhàn)呈也包括應用探索式的規(guī)則。雌的，這樣的規(guī)則是基于生物學和對用于產(chǎn)生臨床結(jié)果的技舞解的基礎上制定的。更雌的是，它們應用到優(yōu)化方法的輸出數(shù)據(jù)中。例如，選雜架的均方差法可以應用于乳腺癌樣本中大量差異表達基因的微陣列數(shù)據(jù)。這種方法得到的輸出數(shù)據(jù)將會是一套優(yōu)化的基因，包括既砂卜周血中表達也在患病孚L腺組織中表達的一些基因。如果這種檢測方法用到的樣本來自外周血，并且某些基因既在乳腺癌個例也在外周血中差異表達，那么就可以應用探索式的規(guī)則，從排除了那些在外周血中差異表達的基因而得到的有效邊界線上選擇框架。當然，這個規(guī)則可以在形成有效邊界線前應用，例如，將謝見則應用至iJ娜的預爐擇中?？梢詰闷渌c所考慮的生物學問題^i、然聯(lián)系的探索^見則。例如，可以應用只有給定百分比的框架可用特定的基因表示的規(guī)則。商業(yè)上可購買的軟件如Wagner軟件就提供了這類型的探索式規(guī)則。這是非常有用的，例如，除了正確度和精密度等，其他因素(例如，預期許可費)也會影響包括一個或多個基因的可用性。本發(fā)明的一個方飽括如上所述比較多種基因(離架)的基因表達分布圖以進行診斷。將構(gòu)成框架的旨基因的基因表達分布圖固定于如計算機可讀的媒倫上。這可以采取很多形式。例如，可以粒一個輸A^示疾病的信號范圍(例如強度測定值)的表格?？梢詫⒒颊叩膶嶋H數(shù)據(jù)與表格中的值進行比較以確定該患者的樣品屬于是正常的或患病的。在一個改進的具體實例中，表達信號的模式(例如熒光強度)以數(shù)字或圖形方式記錄。再將與患者樣本相結(jié)合使用的基因框架中的基因表達模式與上述表達模式比較。然后利用模式比較軟件確定該患者本是否具有^所討論的疾病的模式。當然這些比較也可用于確定該患者的結(jié)果是否正常。再將樣本的表達分布圖與正常m照細胞的框架相比較。如果樣本表達模式與結(jié)腸直腸癌的表達模式一致，那么(無抵消性的醫(yī)學理由)該患者就被診斷為結(jié)腸直腸癌陽性。如果樣本表達模式與正常^t照細胞的表達模式一致，那么該患者就被診斷為結(jié)腸1B癌陰性。許多公知的識另鵬式的方法是適用的。下列參考麟了一些例子加權(quán)閾值WeightedVoting:Golub,TR.,Sclonim,DK"TamayaJ>.，Huard5C,Gaasenbeek5M.5Mesirov,JP,Coller3JLoh,L.J)owning,JR.,CaligiuriMA31oomfieUCD.^Lander,ES.癌癥的^P分類利用基因表達檢觀啲分數(shù)現(xiàn)和分類預測?？茖WScience286:531-537，1999支持^fi機器SupportVectorMachine:Si^AL,WelslUB.，SapinosoJJVUen^SG^imitrov^RJLapp凡Schultz^G^Powell,SM.^Moskaluk,CA.friersoiOIF.Jr.^Hampton,GM.利用基因表達信號的人類癌癥的肝分類。癌癥研究CancerResearch61:7388-93,2001Ramaswamy,S.,Tamayo5R5Rifkin5R.5Mukheijee,S.,Yeang,GH.5Angelo5M.JLadd5C.JleichjVl.;atulippe3JVIesirov,JR^oggio,T.，Gerald^W.山odaJvL山ander,ES.,Gould,TR.禾,腫瘤基因表達信號的多種類癌癥診斷。美國國家科學院院刊ProceedingsoftheNationalAcademyofSciencesoftheUSA98:15149誦15154,2001K-最鄰近值K畫nearestNeighbors:TR.禾,腫瘤基因,信號的多種類瞎癥診斷。美國國家科學院院刊ProceedingsoftheNationalAcademyofSciencesoftheUSA98:15149-15154,2001相關系數(shù)CorrelationCoefficients:van，tVeerLJJDaiH,vandeVijverMJ，HeYD^HartAA>laoHPeterseHL,vanderKooyKJVIartonMJ,WitteveenAT,SchreiberGJ^KerkhovenRMJRobertsQLinsleyPS^emardsR^FriendSH.基因表達分布圖預測乳腺癌的臨床結(jié)果。自然Nature.2002Jan31:415(6871):530~6本發(fā)明的基因表達分布圖也可以與其他在癌癥診斷、預測、或治療監(jiān)測中所4頓的非遺傳的診斷方法齢4頓。例如，在一些瞎況中，將如上所述的基于基因表達診斷能力的方法與從如血清蛋白標記(例如癌胚性抗原)的傳統(tǒng)標記物中得到的數(shù)據(jù)結(jié)合使用是很有益的。存在許多這樣的標記物，包括如CA19-9,CA125,CK-BB和鳥苷酸環(huán)化酶C的分析物。在這樣的一個方法中，對ii治療患者周期性采血，然后將血樣作J^血清標iat—的酶聯(lián)免疫檢測。當標記的濃度暗示了腫瘤復發(fā)或治療失敗時，可以隨后對樣本源作基因表達分析。如果，所懷疑的腫塊，取細針吸取物并如上所述對取自腫塊的細胞進行基因表達分布圖分析。可選擇的，組織樣本可以取自先前摘取了腫瘤的組織的鄰近區(qū)域。當其他檢測結(jié)果不確定時，這種方式特別有用。當其它診斷的可靠性值得懷疑時，齢〗頓遺傳新己和其他診斷是最雌的。例如，^^周知CEA血清水平在相當禾M^t受到與患者癌癥狀錢無關系的因子的影響。當一個患者ltt測到在接受結(jié)腸癌治療后顯満規(guī)CEA檢測高水平時，那齢進行基因表ii/CEA檢測是有益的。本發(fā)明的制品包括用于治療、診斷、預測和其它,ifl古的基因表達分布圖的^物。這些分布圖表示物簡化為可被機器自動可讀的媒介物，如計，可讀媒介(磁性的，光學的及類似的)。制品也包括在這種媒介中Wf古基因毅分布圖的指令說明。例如，制品可倉抱括具有用于比較上述基因框架的基因表達分布圖的計算機指令的CDROM。制品可能也含有基因駄分布圖的數(shù)字記錄以用于與患者樣本的基因表達數(shù)據(jù)進行比較?？蛇x擇的，所述的分布圖可以不同的表現(xiàn)格式記錄。圖形記錄就是這樣的一種格式。上文提到的"GENESPRING"和"DISCOVER"計算^lfi^中^的聚類算法就可很好的協(xié)助這些娜形象化。根據(jù)本發(fā)明生產(chǎn)制造的不同，的物品是指用于揭示基因表達分布圖的媒介物或格式化檢測。這些可以包括，例如微陣列，其中將序列互補物或探針固定于基片上，它與^^所關注基因的序列^^￡一個有關在的易讀行列式。可選擇的，根據(jù)本發(fā)明的制品可以被制作鵬于進行雜交、擴增，以及形^^所關注的基因^ii7K平的信號以用來檢測結(jié)腸飾癌的試劑盒。根據(jù)本發(fā)明制備的試劑盒包括確定基因鋭分布圖的格式化檢測。逸就包括了進行檢測所需的如試劑和說明指令等所有或部分材料。本發(fā)明進一步用下列非限定性的實施例說明。實施例根據(jù)本發(fā)明分析的基因^I過參考GenBank數(shù)據(jù)庫中的基因ID號鑒定的。它們通常與編碼產(chǎn)生蛋白質(zhì)或肽的全長核苷酸序列相關。本領域的技術(shù)人員公認錄核苷,列的鑒定不是一個分析觀點所必需的。即可以根據(jù)公知的為評估相關基因的基因表達設計探針的原鵬行部分序歹蜮ESTs選擇。實施例l陽樣品鵬和LCM收集接受結(jié)腸M腫瘤外禾存術(shù)患者的27個,羊冷凍組織樣本。19個樣本是結(jié)腸:IM惡性腫瘤樣本，8個樣本是正常的結(jié)腸粘膜。組織在獲得后的20-30併中內(nèi)快速液氮冷凍，并隨后貯存于-80。C。為了激光,將樣本切片(6um)，一切片封固于玻璃載片上，另一切片封固于已被固定于玻璃載片(MicroSlidesColorfrostVWRScientific,Media^PA)上的膜(PAL.M.)上封固于玻璃載片上的切片在冷的丙酮中固定，并用Mayer's蘇木精(Sigma^StLouis,MO)染色.病理學家為診斷和定級分析樣本。^fflDukes分級，由附帶的夕卜禾理學和臨麻艮告進行臨床階段評估。封固于膜上的切片在100%乙醇中固定5併中，在曙&7100%乙醇(100yg曙紅溶于lOOmJ脫水乙醇)中錢1併中，f3^I在100%乙醇中浸泡一次以除去未結(jié)合染料，空氣中千燥10iH中。結(jié)腸m腺癌中的兩個為等級l，10個為等級2，5個為等級3。一個惡性腫瘤樣本是盲腸的類癌瘤，另一個是轉(zhuǎn)移的黑色素瘤病變。兩個腺癌樣本表現(xiàn)為粘蛋白M3^，一個為章形細M型。腺癌樣本按Dukes分級劃分如下:DukesA:2,DukesB:5，DukesC:7，DukesD:3。六個腺癌樣本在手術(shù)l^文療過。在LCM中使用前，膜(LPC-MEMBERANEPENFOIL1.35umNo.8100^人L.M.GmbHMikrolaserTechnologje,Bernrie4Germany)和載片經(jīng)過預處理以除去RNA酶，并且增強組織樣品上的吸附。簡要的，載片用DEP恥洗滌，膜用RNaseAWAY(MolecularByproducts,Inc.,S加Diego,CA)洗滌并用DEPH20沖洗。膜粘附到玻璃載片上后，載片于12(TC烘烤8小時，用TI-SAD(DiagnosticProductsCorporation^LosAngdes,CA^以1:50溶于DEPH20,用脫脂棉過濾)，+37"孵育30^1中。臨4頓前，將10ul小份的RNA酶抑制劑溶液(RNA酶蛋白質(zhì)抑制劑2500U=33U/ulN211A^PromegaGmbH,Mannheim^Germany,0.5溶于400ul冷的含有0.15molNaCl,lOmmolTrispH8.0,0.25mmol二硫蘇糖醇的溶液)鋪展到待封固組織樣本的膜上。封固于膜上的組織切片用于LCM。利用PALM自動微光束技術(shù)(P.A.L.M.MicrolaserTechnologje,CarlZeiss,Inc.,Thomwo。4NY)Mfe約2000個上皮細胞樣品，并連接到ZeissAxiovert135顯微鏡中(CarlZeissJenaGmbH,Je叫Geraiany)。正常粘膜的外周基質(zhì)和癌癥樣本中偶然的干擾基質(zhì)成分都包括在內(nèi)。捕捉到的細胞置于100%乙醇的小管中于-80卩保存。實施例2-RNA提取和擴增利用ZymO"Spin柱(ZymoResearch,Qrange,CA92867)提取MJlLCM捕捉的細胞的總RNA。將約2ng總RNA重懸于10P17k并且利用T7RNA聚合酶進行兩輪擴增，擴增的RNA產(chǎn)量約為50ug。實施例3-cDNA微陣列雜交和定量利用一套由約20，000個人cDNA克隆組成的cDNA微陣列檢測樣本。將約30個植物基因印跡到微陣列上作為非特異雜頓照。利用LCM捕捉細胞的5ugaRNA合成Cy3標記的cDNA探針。探針用Qiagen's核苷酸去除柱(NucleotideRemovalColumns)純化然后經(jīng)14-16小時雜交到微陣列上。載片在掃描前經(jīng)過洗滌并于空中干燥。掃描微陣列的Cy3熒光并用ImaGene軟件(Biodiscoveiy，LosAngeles,CA)定量。對于旨cDNA克隆，進行重復點和重復陣列的四次測量并將所得^E值平均。禾廿用第三代微陣列點樣儀(MolecularDynamics)將cDNAs印跡到M^硅烷涂層的載片上(Coniing)。cDNAs經(jīng)PCR擴增，純化(QiagenPCR純化i式劑盒)，并與10MNaSCN印跡緩沖液1:1混合。雜交前微陣列在異丙醇中室溫孵育10併中。探針于95'C孵育2併巾，室溫孵育5，巾，然后加到三個平行測定的載片上。用DPX(Fluka)將蓋片密封到載片上并于42。C孵育過夜。然后將載片在1XSSC/0.2°/。SDS和0.1XSSC/0.2%SDS中于55。C洗滌5辦中，0.1XSSC中浸潤并干燥后用第三代微陣列掃描儀(MolecularDynamics)掃描。用AUTOGENE軟件(Biodiscoveiy,LosAngeles)分析齡點的熒光3賊。對芯片強度進行線性標準化使得每個芯片的強度記錄75&個百分點相當于值100。芯片上的旨基因通過將該基因的強度記錄除以所有樣本的基因表達值記錄的中間值標準化。在聚類前，那些至少在一個樣本中不具有100#大的弓艘記錄的基因被濾掉了以限制對相似性雖的背景影響。選#~套6255個基因作聚類分析。禾U用作為相似性觀懂的相關度進行分級聚類，4頓有表現(xiàn)正變化同時不考慮其負變化的基因的樣品聚類到一起(SiliconGenetic,Sunnyville,CA)。系統(tǒng)圖的每一個主要節(jié)點可被認為是樣本的一個亞組。通過將每個腫瘤亞組與正常組進行比較鑒定差異就的基因。篩選是基于"-A)給出的信噪(cr'+cr)比觀糧閾值，其中yt劍中瘤雅的平均數(shù)，Un敲常樣本賴的平繊，CJt+Ong結(jié)合標準差。組內(nèi)一個基因的強度記錄的變異系數(shù)必須低于0.33，才可將基因包括在成對方式比較之中。腫瘤組的中間值與正常組的中間值相比大于2或等于2時為上調(diào)，小于或等于0.5倍時為下調(diào)。如果一個基因符合所有標準則被選擇出來。在所有的比較中均被篩選出來的基因即被認為是在結(jié)腸M癌中穩(wěn)定調(diào)節(jié)異常的。利用T檢驗假定不等方差計算統(tǒng)計學顯著性p值。用于聚類的一套基因也用軟#^(PartedStLouis,MO)進行主成分分析(PCA)。然后將數(shù)據(jù)投射到簡化的三維空間。正常的和結(jié)腸1B腫瘤的樣本以映射表達水平表現(xiàn)出來。建立有很大上調(diào)差異的基因列表以用來區(qū)別腫瘤和正常樣本。利用皿)作為信噪比中止點先扭謎出123個基因。以等于駄于1.5的比率作為上調(diào)的劇氏標準。也包括(^'-產(chǎn)")的基因。已經(jīng)粒了一個四基因的框架，(cr,+cr")#^基因劍中瘤和正常細胞間都有至少3倍的表達差異。結(jié)腸飾癌中差異敏的基因。與正常結(jié)腸粘膜相比，39個基因^^f有的腫瘤樣本中差異魏。除了一個異常值，37個基因在所有的腫瘤中顯著下調(diào)。2個上調(diào)?；虻纳砟犨^微陣列上cDNA克隆的測序驗證。結(jié)果如表l所示。<table>tableseeoriginaldocumentpage16</column></row><table><table>tableseeoriginaldocumentpage17</column></row><table>^Mii結(jié)腸翻中瘤的優(yōu)化框架禾u用方差優(yōu)化算法生成多重基因基礎的標記，其中所包括的基因可結(jié)合使用以區(qū)別正常和腫瘤樣本。禾倆樣本和如實施例1-3所描述的微陣列進行纟破檢測。首先，基于腫瘤和正常樣本間的預定5倍差異，對待分析的娜進ffiP員選。根據(jù)這個標準預選出來的基因的表達數(shù)據(jù)隨后作如下使用。以一瞎移性樣本作為基線，計算出每個基因的強度測量值的平均數(shù)和標準差。然后對于每個基線基因計算鑒別值X"示準M+平均匍(X值指定為3)。這個iiffl來確保結(jié)果框架是有說服力的。再計算每個轉(zhuǎn)移性樣本的鑒別值與基線值的比率。這個比率隨后被轉(zhuǎn)換成常用對數(shù)。然后這個數(shù)據(jù)被輸入Wagner軟件，產(chǎn)生一個有效邊界，從中選擇出一個4基因框架。這套基因包括一未知序列，前膠原I型，核糖體蛋白L21的大亞基，和纖連蛋白。這些基因確定為Seq.ID.No.42，Seq.ID.No.43，Seq.ID.No.44和Seq.ID.No.45的序列。換句話說，形成框架的基因組合可以為關于結(jié)腸直腸癌的臨床判定提供有用的診斷信息。這在將選自該框架的基因組合與附加標記(遺傳或非遺傳的)相結(jié)合時特別有益。優(yōu)化的基因框架>giI1264443IgbIN92134.1IN92134za23fD9.rlSoaresfetalliverspleenINFLSHomosapienscDNAcloneMAGE:2934175，similartogbIM87908IHUMALNE32Humancarcinomacell-derivedAluKNAtranscriptj(rRNA);gbX57025一malINSULIN-LKEGROWTHFACTORIAPRECURSOR(HUMAN)>giI2221047IgbIAA490172.1IAA490172ab06b08.slStratagenefetalretina937202HomosapienscDNAcloneMAGE:8399913，similartogb:J03464PROCOLLAGENALPHA2①CHAINPRECURSOR(HUMAN)>giI2188912IgbIAA464034.1IAA464034zx86跳rlSoaresovarytumorNbHOTHomosapienscDNAcloneMAGE:8106175,similartoSW:RL21一HUMANP4677860SRBOSOMALPROTEINL21>giI834491IgbIR62612.1IR62612yil2d01.slSoaresplacentaNb2HPHomosapienscDNAcloneMAGE:1390093,similartogb週761"CdslFIBRONECTINPRECURSOR(HUMAN);4頓不同的標準但相同的方法，艦軟件選出了又一個四基因框架。這些是Seq.ID.No.46，Seq.ID.No.47，Seq.lD.No.48和Seq.ID.No.49的序列。兩個基因與第一個四基因框架重疊。兩4^化框架也可以結(jié)合形成一個六基因框架。優(yōu)化的基因框架>giI2114953IgbIAA431245.1IAA431245.1zw78d06,rlSoares—testis一NHTHomosapienscDNAcloneMAGE:7823155'similartowp:F36H1.2CE05814ANKYR1NLIKE〉giI2156172IgbIAA443497.1IAA443497zw34d03.rlSoaresovarytumorNbHOTHomosapienscDNAcloneMAGE:771173>giI2221047IgbI2IAA490172ab06跳slStmtagenefetalretina937202HomosapienscDNAcloneMAGE:8399913'similartogb:J03464PROCOLLAGENALPHA2①CHAINPRECURSOR(HUMAN)>giI1264443IgbIN92134.1IN92134za23f09.rlSoaresfetalliverspleenINFLSHomos叩ienscDNAcloneMAGE:2934175，similartogbIM87908IHUMALNE32Humancarcinomacell-derivedAluRNAtranscriptj(rRNA);gbX57025一malINSULIN-LKEGROWTHFACTORIAPRECURSOR(HUMAN);<110>鵬G,Y腦<120>刑鵬腸iraS<歸CDS267IBNP<歸孤<141>2003-03-21<150>60/368,798<151>2002普29<160>49<170>Patentlnversion3.1<210〉1<211>1500<212>IM<213>人<柳>1atggcttcgaccaccacctgcaccaggttcacggacgagtatcagcttttcgaggagctt60ggaaagggggcattctcagtggtgagaagatgtatgaaaattcctactggacaaggatat120gctgccaaaattatcaacaccaaaaagctttctgctagggatcatcagaaactagaaaga180gaagctagaatctgccgtcttttgaagcaccctaatattgtgcgacttcatgatagcata240tcagaagagggctttcactacttggtgtttgatttagttactggqggtgagctgtttgaa300gacatagtggcaagagaatactacagtgaagctgatgccagtcattgtatacagcagatt360ctagaaagtgttaatcattgtcacctaaatggcatagttcacagggacctgaagcctgag420aatttgcttttagctagcaaatccaagggagcagctgtgaaattggcagactttggctta480gccatagaagttcaaggggaccagcaggcgtggtttggttttgctggcacacctggatat540cUtctccagaagttttacgtaaagatccttatggaaagccagtggatatgtgggcatgt600ggtgtcattctctatattctacttgtggggtatccacccttctgggatgaagaccaacac腳agactctatcagcagatcaaggctggagcttatgattttccatcaccagaatgggacacg720gtgactcctgaagccaaagacctcatcaataaaatgcttactatcaaccctgccaaacgc780atcacagcctcagaggcactgaagcacccatggatctgtcaacgttctactgttgcttcc840atgatgcacagacaggagactgtagactgcttgaagaaatttaatgctagaagaaaacta900aagggtgccatcttgacaactatgctggctacaaggaatttctcagcagccaagagtttg960ttgaagaaaccagatggagtaaaggagtcaactgagagttcaaatacaacaattgaggat1020gaagatgtgaaagcacgaaagcaagagattatcaaagtcactgaacaactgatcgaagct1080stcaacaatg鵬actttgaagcctacacaaaaatctgtgacccaggccttactgctttt1140gaacctgaagcttt鵬taatttagtggaagggatggattttcaccgattctactttgaa■aatgctttgtccaaaagcaataaaccaatccacactattattctaaaccctcatgtacat體ctggtaggggatgatgccgcctgcatagcatatattaggctcacacagtacatggatggc1320agtggaatgccaaagacaatgcagtcagaagagactcgtgtgtggcaccgccgggatgga1380aagtggcagaatgttcatttteatcgctcggggtcaccaacagtacccatcaagccaccc1440tgtattccaaatgggaaagaaaacttctcaggaggcacctctttgtggcaaaacatetga1500<210>2<211>5761<212>鵬<213>人<400〉2cacaccgcagtatgcggtgccctttactctgagctgcgcagccggccggccggcgctggt60tgaacagactgccgctgtact鵬gtggcctggagggactcagcaaattctcctgccttc120aacttggcaacagttgcctggggtagctctacacaactctgtccagcccacagcaatgat180tccagaggccatggggagtggacagcagctagctgactggaggaatgcccactctcatgg240caaccagtacagcactatcatgcagcagccatccttgctgactaaccatgtgacattggc300cactgctcagcctctgaatgttggtgttgcccatgttgtcagacaacaacaatccagttc360cctcccttcgaagaagaataagcagtcagctccagtctcttccaagtcctctctagatgt420tctgccttcccaagtctattctctggttgggagcagtcccctccgcaccacatcttctta480taattccttggtccctgtccaagatcagcatcagcccatcatcattccagatactcccag540ccctcctgtgagtgtcatcactatccgaagtgacactgatgaggaagaggacaacaaata600caagcccagtagctctggactgaagccaaggtctaatgtcatcagttatgtcactgtcaa660tgattctccagactctgactcttctttgagcagcccttattccactgataccctgagtgc720tctccgaggc幼tagtggatccgttttggaggggcctggcagagttgtggcagatggcac780tggcacccgcactatcattgtgcctccactgaaaactcagcttggtgactgcactgtagc840aacccaggcctcaggtctcctgagcaataagactaagccagtcgcttcagtgagtgggca900gtcatctggatgctgtatcacccccacagggtatcgagctcaacgcggggggaccagtgc960agcacaaccactcaatcttagccagaaccagcagtcatcggcggctccaacctcacagga1020gagaagcagcaacccagccccccgcaggcagcaggcgtttgtggcccctctetcccaagc1080cccctacaccttccagcatggcagcccgctacactcgacagggcacccacaccttgcccc1140ggcccctgctcacctgccaagccaggctcatctgtatacgtatgctgccccgacttctgc1200tgctgcactgggctcaaccagctccattgctcatcttttctccccaca^gttcctcaag■gcatgctgcagcctataccactcaccctagcactttggtgcaccaggtccctgtcagtgt1320tgggcccagcctcctcacttctgccagcgtggcccctgctcagtaccaacaccagtttgc1380cacccaatcctacattgggtcttcccgaggctcaacaatttacactggatacccgctgag1440tcctaccaagatcagccagtattcctacttatagttggtgagcatgagggaggaggaatc1500atggctaccttctcctggccctgcgttcttaatattgggctatggagagatcctccttta腳ccctcttgaaatttcttagccagcaacttgttctgcaggggcccactgaagcagaaggtt1620tttctct鵬ggaacctgtctcagtgttgactgcattgttgtagtcttcccaaagtttgc1680cctatttttaaattcattatttttgtgacagtaattttggtacttggaagagttcagatg1740cccatcttctgcagttaccaaggaagagagattgttctgaagttaccctctgaaaaatat扁tttgtctctctgacttgatttctataaatgcttttaaaaacaagtgaagcccctctttat1860ttcattttgtgttattgtgattgctggtcaggaaaaatgctgatagaaggagttgaaatc腦tgatgacaaattactttttgtttgtttataaactcagacttgcctatttt1980attttaaaagcggcttacacaatctcccttttgtttattggacatttaaactt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gccttgtgcaggcaatttgacagagtgttggatgtgtc1260atgtcatcatgtcaaacccaaatatttgacctaagggatcctttattctgcccagtggct1320aacUtaacaacatccctaatacaactgtttattcaaatgcacggtggtccctgttagag1380ttagacctctagactcacctgttctcacgccctgttttaatttaacccagctat鵬atg1440ccagataacagaattgctgcctacgagctgaacagggaggagtttgtgcagttgctgaca1500cttcttgttgcacataaataaatacagtgggtactatagagactcagttgcaaaaattaa1560caaatatgctgcttgattaaaatgg^taggcttctcatgtggctcattctttaatctatt腳ctcttttatttggtttggttcatggggtctctgcctatggatcatacttcaaactctt從1680tgtgatcctcctgattgtcacaatattagttaccctggtgtgctgtattctctaaaacct1740ttaaatgtttgcatgcagccattcgtcaaatgtcaaatattctctctttggctggaatga1800caaaaactca幼taaatgtatgattaggaggacatcataacctatgaatgatggaagtcc1860aaaatgatggtaactgacagtagtgttaatgccttatgtttagtcaaactctcatttagg1920tgacagcctggtgactccagaatggagccagtcatgctaaatgccatatactcacactga腦aacatgaggaagcaggtagatcccagaacagacaaaactttcctaaaaacatgagagtcc2040aggctgtctgagtcagcacagtaagaaagtcctttctgctttaactcttagaaaaa^ta2100atatgaagtattctgaaattaaccaatcagtttatttaaatcaatttatttatattcttc2160tgttcctggattcccattttacaaaacccactgttctactgttgtattgcccagtaggag2220ctatcactatattttgcagaatggaaactgccctgactcttgaatcacaaataaaagcca2280attgtatctgtt2292<220〉<221>其欲征<222>(212)..(212)<223>儲鵬<220><221〉其挪征<222>(451)..(451)<223>儲鵬備〉13gaaacaacaacagtgtaatctttaacagggatgttaaaggtaagaagtcaggaagat鵬60ccaaaatgattgagtatgataaagaattttgcatggcgattaaaatagaaaacctataaa120tgtagaaaaagcag&tctggacttagcaaagaaacaatatagtttggaga鄉(xiāng)catgaaa180taagttctutcatgttcactgctggtcacancataacagagagtgatgtggagagcttt240gggaaggtttcacgttgagttacatcagtggtcaaoaatggagcaacaagactccgtaga300ggatgccaccctgggagaattgcaagggaaaggaggctgaagcacaactggtaatagcct360tcagatatttaatggatatgcaaataaagctctgattaattgtattttcacttattatat420atcatctttggacctttctaaaagtgggacnctagaaaagatatactgaaactccaaaag480aatacttcagctcgagttgaatggattcaagatgttgtt519o12322224S2、155372222<<<<<400>14ggctcgcatccccatagtgctgggttacagtgaaggtacgccccgcgctctgctctggag60aggcagggtgggatagggaacgtctcgagtggcgcccgcagtcatggtggtgttcgttgg120ccgccgcctcccggcgctcctagggctgtttaagaagaagggctctgccaaggctgagaa180tgacaaacatctaagtgtagggcctggccaggggccagggtctgcagtggatgagcacca240ggacaacgtcttctttccca&tgggcgacccccccacctggaagagctgcacactcaggc300ccaggaggggctccgctccctacaacaccaagagaaacagaaactgaaca鄉(xiāng)gtggctg360ggaccatggagacacccagagtatccagtcctcccggacggggccggatgaagacaacat420ctccttctgcagtcagaccacatcctacgtggctgagagctccacagcagaggacgcgct柳ctccatccgctcggagatgatccagcgcaaaggctccaccttccgaccccatgactcatt540tcccaaatctggaaagtcagggcggcgtcggc鵬agcggcggagcactgtgct鵬act600cccgcagcatgtgcagaaggagcttggcctgaggaatgagcgtgaggcaccaggcacgcc腳ccgggctcctggtgcac鵬atgccgtacgcatccccacagtggacggccgcccccgagg720cacctcagggatgggggcccgggtgtccctgcaggcgctggaggcggaggcagaggctgg780cgctgagacagaggccatgctgcagcgccacattgaccgtgtctacc鵬atgacacctt840tgttggccggtccacgggtacccgggccccaccattgacccggcccatgtccctagcagt900gcctggattgacaggag鵬cagggcctgcagagcccctgagcccggccatgtccatctc960cccccaggccacctacctgtcgaagttgattccacatgctgtgctgccgcctacagtgga,cgtggtggccctaggccgctgcagcctgcgcacactaagccgctgcagcctgcactcggc1080cagcccagcctcagtccgctcgctggggcgcttctcctccgtctccagcccacagccccg1140cagccgccacccatcctcctccagtgacacctggagccactctcaatcctccgacaccat1200tgtgtctgacggttccaccctctcctctaagggtggctctgagggccagccggagagctc1260tacggctagcaatagcgtggtaccccctccccagggaggcagt鵬aggggctctcccag1320tgggggcagcactgctg鄉(xiāng)ccteagacacactcagcattcggagcagtgggcagttgtc1380tggccggagtgtgtccctgcgtaagctgaagcggcctccaccccctccccgccggaccca1440ctccctccatcagc鵬gcttagcagtgcctgatgggccattagggttgccccctaagcc1500tgagcgtaagcagcagccccagctgcctcggccacccaccactggtggctcagaaggggc1560gggggcagcaccctgtccacccaacccagccaacagctgggtacctggcttgtctcc鵬1620tggttcccggcgccccccacgfftccccagaacggacactttcgccctccagtggatactc1680gagccaaagtggtactcccaccctccctcccaagggcctggcaggtccccctgcttcccc薩aggcaaggcccagccccctaaaccagagcgtgtcacgtctcttcgctcccctggggcctc1800cgtctcctcttccctcacgtctttatgttcctcctcctctgacccagccccctcagaccg鵬ctctgggccacagatattgacccccctgggtgacaggtttgtcatacctcctcaccccaa腳ggtgcctgcccccttctccccacctccctccaagcccaggagccctaacccagctgccccl朋Otgctctagccgcccctgctgtggttcctgggcctgtttctaccactgacgccagtcctca2040gtcccctcccactccccagacaaccttgactccactgcaggagtctcctgtcatctccaa2100agaccagtcacccccaccttccccacccccatcttatcatccacccccaccacccactaa2160gaagccagagfftggttgtggaggcaccatctgcctcagagactgctgaggagcccctcca2220agatcccaactggccccctcccccaccccctgcccctgaggagcaggacctgtccatggc扁tgacttccccccaccagaggaggcttttttctctgtggccagccctgagcctgcaggccc2340ttcaggctecccagagcttgtcagctccccggctgcttcgtcctcctcagctactgcttt2400gcagattcagcccccgggtagcccagaccctcctccagct9cgccagccccagctcctgc2460tagttccgccccagggcatgtggccaagctccctcagaaggaaceggtgggctgtagcaa2520gggtggt鵬cctcccagggaggacgtaggtgcgcccctggtcacgccctcgctcctgca2580gatggtgcggctgcgctccgt鵬tgctccaggaggggctcccaccccagcact鵬gcc2640atcggccccccagaaaccactgcgaagggccctgtcagggcgggccagcccagtgcctgc2700cccctcctca鵬ctccatgctgcggtccgactcaaggcctgcagcctggccgccagtga2760aggcctctcaagtgctcagcccaacggaccgcctgaggcagagccacggcctccccagtc2820ccctgcctcaacggccagtttcatcttctccaagggctctaggaagctgcagctggagcg2880gcccgtgtcccctgagacccaggctgacctccagcggaatctggtggcagaactccggag2940catcteagagcagcggccaccccaggccccaaagaagtcacctaaggctcccccacctgt3000ggcccgcaagccgtctgtgggagtccccccacccgcctcccccagttaccctcgagct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ggatattegttataatactgttcttcactatgctgtttgtggtca480aagtttgtcattagttgaaaaactgcttgaatacgaagctgat523<220><221〉其撒征<222>(641)..(641)<223〉條鵬<400>48ttcttttggaaaaccaaacatgctttatttcatttttttcacaatttatttaaacatctc60acatatacaaaataggrtacaatttaatttttctgcttgcccaagaaacaaagcttctgtg120gaaccatggaagaagatgaaaatgagactggcaaagaacaaatgctgaatctgaagaaga180ggacaacUt鵬caaataatctgcatacttttaatt鵬aataagatggaaaatatgaa240tgctaaatcaaattttttaaaaaatacaccacacgatacaactcaatacaggagtatttc300ttctcaaattcttctagcaccatcaacattcttcaagtatctgaaatactattaattagc360acctttgtattatgaacaaaacaaaacaaggacctcagttcatctctgtctaggtcagca420cctaacaatgtggatcacactcatgggaaagtgttttgaggtagtttaaacctttggaag480tttgggttttaaacttccctctgtggaagatattcaaaagccacaagtggtgcaaatgtt540tat鵬ttttatttttcaatttttattttggttttcttacaaaggttgacatttttcata600acaggtgtaagagtgttgaaaaaaaaatttcaatttttggngggaac鵬ggaaggagtt660aatgaaact669<400>47tccaaggtcatggcaaaacatctgaagttcatcgccaggactgtgatggtacaggaaggg60aacgtggaaagcgcatacaggaccctaaacagaatcctcactatggatgggctcattgag120gacattaagcatcggcggtattatgagaagccatgccgccgcgacagagggaaagctatg180aaaggtgccggcggatctacaacatggaaatggctcgcaagatcaacttcttgatgcgaa240agaatcgggcagatccgtggcagggctgctgaggcctgtgggtgggacaccagtgcgaaa300ccctcatccagttttctctccatctcttttctttgtacaatcccatttcctattaccatt360ctctgcaataaactcaaatcacatgtctgc390>>>>o1232222<<<<43,o123<<<<9A.>>>>o1232222<<<<<220><221>其W征<222>柳..柳<223>條鵬<220><221>其游征<222>(347)..(347)<223>儲鵬<220〉<221〉其辦征<222>(349)..(349)<223>儲鵬<湯<221〉其嫩征<222>(361)..(361)<223>條鵬<220><221〉其抓征<222>(362)..(362)<223>條鵬<220><221〉其挪征<222>(363)..(363)〈223〉條鵬<220〉<221〉其嫩征<222>(401)..(401)<223>ffit鵬<220〉<221>其撒征<222>(428)..(428)<223>儲鵬<400>49ctttttatatttattttcatcgctacacaaacattttttaggagtttgattctacctcca60ttttggttagatatacaaactctaccccatgagggantgtatggtgtatttctagattta120gcaacaattttcttgaaaaatgtacaatactatagaaaaatgaagatagtaaataccagg180tataagttaataacagtgtttcttttgtteagtaataatgaactgtgtactagcactgaa240ctttaggccctcctatttgcgtattttctgtttgtatatttttaaatagaggaattgtga300ttataatattattattttggaatatcctaaatcataaattcaaaacntnatttagttttt360咖ttttttttttaagatggagtcccgctttgtcccaggcnggagtgcagtggcatgatc420tcagctcnctg43權(quán)利要求1.包括Seq.ID.No.46-49的基因組合在制備用于評估結(jié)腸直腸癌狀況方法的試劑中的應用，所述方法包括鑒定每個基因相對于正常種群的相同基因的表達的差異調(diào)節(jié)。2.權(quán)禾腰求l的應用，其中被調(diào)節(jié)的基因表達存在至少2倍差異。3.權(quán)利要求1的應用，其中表示差異調(diào)節(jié)的p值低于0.05。4.權(quán)利要求1的應用，進一步包括i頓非遺傳基礎的結(jié)腸艦癌診斷方法。5.權(quán)利要求4的應用，其中在非遺傳基礎的結(jié)腸m癌診斷方法中j頓的非遺傳基礎的癌癥豐斜己選自癌胚性抗原、CA19-9、CA125、CK-BB和鳥苷酸環(huán)化酶C。6.—種診斷框架，包括S叫ID.No.4649的基因組合的分離的核,列或其互補序列。7.權(quán)利要求6的診斷框架，其位于適合鑒定包含于其上的基因的差異,的基片上。8.權(quán)利要求7的診斷框架，其中所述的基片用于微陣列。9.權(quán)利要求8的診斷框架，其中所述的微陣列是cDNA微陣列。10.權(quán)利要求8的診斷框架，其中所述的微陣列驗核苷酸微陣列。11.一種用于診斷結(jié)腸艦癌的試劑盒，其包含包括Seq.ED.No.4649的基因組合的分離的核,列或其互補序列。12.權(quán)利要求ll的試劑盒，進一步包括進行微陣列分析的試劑。13.權(quán)利要求ll的試劑盒，進一步包括媒倫，所述的核,歹蜮其互補序列艦該媒介物進行檢測。14.包括Seq.ID.No.4649的基因組合在制備用于評估結(jié)腸娜癌療效方法的試劑中的應用，所述方法包括鑒定旨基因相對于正常種群的相同基因的表達的差異調(diào)節(jié)。15.權(quán)利要求14的應用，其中對療效的評估包括確定患者是否好轉(zhuǎn)，非好轉(zhuǎn)，皿，可能好轉(zhuǎn)或可能aS:。16.iff古結(jié)腸飾癌狀況的制品，其包含包括S叫ID.No.4649的基因組合中的分離的核,列或其互補序列。17.評估結(jié)腸1^癌狀況的制品，其包含包括Seq.ID.No.4649的基因組合的分離的核,列或其互補序列的恭，。全文摘要通過分析一組基因的表達對相信患有結(jié)腸直腸癌的患者的結(jié)腸直腸癌存在與否或其可能狀態(tài)進行評估的方法。本發(fā)明還涉及多種媒介物，如微陣列中基因表達分布圖，也包括含有基因表達分布圖的試劑盒。文檔編號G01N33/574GK101603092SQ20091014896公開日2009年12月16日申請日期2003年3月29日優(yōu)先權(quán)日2002年3月29日發(fā)明者Y·王申請人:奧索臨床診斷有限公司