201250245 .. 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種肺腺癌之診斷指標，特別是關於一 種肺腺癌預後之曱基化分子指標’以及將該肺腺癌預後之 曱基化分子指標應用於肺腺癌分子診斷之用途。 【先前技術】 肺癌乃工業國家惡性腫瘤死亡率之首位，五年存活率 僅10至15%’其向死亡率之主因在於肺癌之高復發率及習 用偵測技術之不足。 肺腺癌（lung adenocarcinoma)屬於非小細胞癌，係肺 癌的主要種類，佔所有肺癌之40-50%，其比例逐年增加。 不同於鱗狀細胞肺癌，肺腺癌主要好發於肺部周邊位置， 如外側肺葉，且容易侵犯女性及非吸煙患者，肺腺癌的致 病原因尚未明朗，並且初期症狀不明顯，因此在其早期診 斷及預後分析上更顯困難。 習用肺癌篩檢之方法包含胸部χ光、痰液細胞學檢查 以及靈敏度較高之電腦斷層及正子攝影，其中，該胸部χ 光及痰液細胞學檢查雖為臨床慣用之肺癌診斷手段，卻具 有專-性及靈敏度不佳等問題，無法善盡早期偵測以有效 =善肺癌高致死率n而電職層及正子攝影雖係現 今腫2部位及腫瘤分期判斷的重要指標，惟價格昂貴，非 般常人所能負擔，因此在臨床肺癌偵測之應用上往往受 限0 按，美國公開第2010/0167296號專利案，揭示一種非 201250245 小細胞肺炎之預後偵測方法’係以上皮細胞生長因子接受 體(epithelial growth factor receptor; EGFR)、巨嗤細胞聚落 刺激因子(macrophage colony stimulating factor; CSF-1)及 碳酸軒酶抑制劑(carbonic anhydraselX; CAIX)等生物標記 之表現值作為非小細胞肺癌預後狀況之判斷依據。 又’美國公開第 2010/0317002 號、第 2010/00068207 號、第2009/0214536 5虎及第2009/0297507號專利案則分別 揭示與肺癌診斷相關之蛋白質標記，包含CACNA1E、 ADAM10、DDR2 及 ADAMTS-7 等。 然而，前述各前案所示之分子診斷方法皆係利用偵測 各生物標記於RNA或蛋白質層面之表現值，作為肺癌診斷 的手段’而RNA及蛋白質皆屬不易保存且極不穩定之分子 層次’不僅在檢體處理上較為複雜、困難，更容易因該RNA 及蛋白質檢體之不穩定性而衍生偽陽性等問題，以致前述 各檢測方法之專一性與靈敏度無法有效提升，無法適任於 臨床肺癌檢測之使用。 據此可知，習用技術缺乏一具有高專一性、靈敏度， 並且可廣泛應用於臨床肺腺癌早期診斷及預後分析之工 具，因此’有必要針對習用技術加以改進，以有效預測肺 腺癌之預後狀況而改善其高致死率。 基因體的甲基化為當代生醫研究的重點之一，據研究 才曰出基因啟動子上CpG島群(cpG island)的過度甲基化 與癌症的形成與轉移息息相關。抑癌基因(tum〇r suppressor genes)啟動子的過度甲基化可導致許多參與調控的轉錄因 子及聚合雜素無法正常結合至啟料位置，造成該抑癌 201250245 基因不表現，料致染色體料穩定及錢，因而衍生癌 症的產生因此’特疋基因的曱基化可作為腫瘤形成的分 子才曰心’ $ ’現今仍未有與肺腺癌相關之曱基化分子 的開發及應用。 ” 【發明内容】 本心月之主要目的係提供一種肺腺癌預後之曱基化 分子指標，係針對肺腺癌之早期彳貞測及預後判斷具有高度 專一性及靈敏度。 本發明之次一目的係提供一種肺腺癌預後之甲基化 分子指標，係直接針對檢體之DNA層面進行檢測，可避 免習用RNA或蛋白質分子指標衍生偽陽性之疑慮。 本發明之再一目的係提供一種肺腺癌預後之曱基化 刀子才曰&，係可以應用於臨床肺腺癌分子診斷，可兼具有 高診斷率及低成本之效益。 本發明之再一目的係提供一種肺腺癌預後之甲基化 分子指標於臨床肺腺癌分子診斷之用途，以建立一肺腺癌 早期诊斷及預後分析的工具，進而有效改善肺腺癌的高致 死率。 為達到前述發明目的，本發明所運用之技術内容包含 有： 一種肺腺癌預後之甲基化分子指標，包含C16orf25、 RALGPS2、SPOCD 卜 KIAA0746、PDE10A、KIAA0649、 IER5、SCARF2、KCNJ8、FAM3B、PHLDA3、SORCS 卜 AGTR卜 PAX7、ZNF496、WNK4、RAB7U、HISTIH4D、 201250245 SYT2、CTSE、CDKLl、NFAMl、PAQR6、TMEM129、 SLAMF8、EFNA2、NOTCH4、OR1A2、AGTRL1、BDKRB1、 SEMA4A、KRT4、NCOR2、GNS、PNPLA2、ANKRD9、 ALDH1A3、BLVRA之其一或其組合；其中，該上述之％ 個甲基化分子指標包含37個基因及一個NCBI序列代碼為 C16orf25之序列，皆與肺腺癌轉移與致死率相關。 其中，該肺腺癌包含第一期至第四期之肺腺癌。 其中，該肺腺癌致死率係指預後存活3年以下。 如申請專利範圍第1項所述之肺腺癌預後之甲基化分 子指標應用於肺腺癌分子診斷之用途，包含偵測檢體基因 的曱基化程度，對照正常檢體；以及依據該檢體基因中該 甲基化分子指標的T基化程度，作為肺腺癌預後之判斷依 據。 其中，該檢體係癌組織、血液、血漿或帶有游離核酸 之體液。 其中’该檢體基因的甲基化程度係由焦填酸測序或基 因體曱基化晶片之方法進行分析。 其中，該肺腺癌包含第一期至第四期之肺腺癌。 【實施方式】 ▲為讓本發明之上述及其他目的、特徵及優點能更明顯 易懂，下域舉本發明之較佳實施例，並配合所附圖式， 作詳細說明如下： ―本毛明所述之「4檢族群」，係指自台北地區榮民總 醫Ik的39名初期肺腺癌患者’包含第—期及第二期的肺腺 201250245 癌患者’其中’ 27名肺腺癌患者的預後狀況較佳，存活率 達3年以上，12名肺腺癌患者的預後狀況則較差，存活率 僅3年以下。該「筛檢族群」係用於本發明之甲基化分子 標記的韩選。 本發明所述之「驗證族群」，係彳旨自台北地區榮民總 醫院内與該「篩檢族群」完全獨立的另外3G名肺腺癌患 者’包含第-期及第二義肺腺癌病患，其巾26名肺膝癌 患者的預後狀況較佳，存活率達3年以上，4名肺腺癌患 者的預後狀關㈣，存活補3年町，制於本發明 甲基化分子標s己之診斷性的驗證。 本發明所述之「臨床病理資料」，係依據該「筛檢族 群」或「驗證族群」的臨床預後資料所建立，包含各患者 的存活率、_轉移程度及其他職狀況等。 〜 本發明所述之「基因㈣基化變異圖譜」，乃由肺腺 癌組織檢體進行基因體甲基化分析所得之結果，經比對本 ::月，「臨床病理資料」而建立，係具有與肺腺癌預後較 差有關之甲基化基因。 睛蒼照第1圖所示，本發明係利用基因甲基化分才 法，綜合本發明之臨床病理#料，進而建立—基因體甲^ =異圖譜，該基因體曱基化變異圖譜包含與肺腺癌制 ^差:度相關之甲基化分子標記。該甲基化分子標記御 對料肺腺癌患者之預後狀況具有高度指標性及靈敏度 2 ’可進-步應用於臨床肺腺癌分子診斷之開發，以去 升肺腺癌之診斷率，並且改善肺腺癌之高致死率。 請再參照第1圖所示，本發明之甲基化分子標記乃〇 201250245 經由以下各步狀操作顿得，包含 S1; 一筛選步驟S1;以及-驗證步驟S3。斤^驟 該甲基化分析步驟82則係先採取該筛 織檢體基因甲基化分析法偵測各檢體基因的^基 化私度，㈣配合該篩檢族群的臨床病理資料，歸納並選 取各檢體基財’與肺腺癌預後較差具有關連性的甲基化 基因。本實施例係细-商職因體甲基化晶>Mllumina hum_ethylation27 bead 啊（祖㈤叫 ％ usa)分析各 檢體中約14,000個基因的甲基化狀態，同時配合各檢體之 臨床病理資料，自該14，_個基財擷選與肺腺癌預後差 相關’如存活率低於3年以下，之甲基化探針約27,578個。 本實施例中’各基因之甲基化狀態縣經計算而換得-甲 基化數值(β-value) ’再依據該曱基化數值之高低挑選與肺 腺癌預後差有關的27,578個探針。 该篩選步驟S2則係由該甲基化分析步驟S1所獲得之 甲基化基因中，進一步挑選具有特異性且專一性地與肺腺 癌之存活率低及轉移相關之基因。本發明之篩選步驟S2 較佳係包含二階段，第一筛選階段S21 ;以及第二筛選階 段S22。其中’該第一篩選階段係先於該甲基化基因中去 除區隔性較低或者係具有干擾性的甲基化基因，包含具有 基因多型性（single-nucleotide polymorphism)、重複片段 (repeated sequences)以及甲基化數值（p-vaiue)過高或過低 之基因’如β-value大於0.9或者小於〇.1，此外，為避免 X染色體上之基因所衍生的劑量補償作用（d〇sage compensation)產生干擾，位於該X染色體上之基因同步於 201250245 §亥第一篩選階段S21中剔除。該第二篩選階段S22係利用 統°十刀析的方式，如c〇x迴歸分析(cox regressi〇n anaiysis) 或貝氏迴歸分析(bayesian logistic regression software)等， 再進一步於該第一筛選階段S21所選取之基因中’挑選與 肺腺癌之存活率、預後狀況較差有關的基因，如存活率低 於3年或產生轉移情形者。 本實施例之篩選步驟S3則係先自該27,578個探針中 删除含有基因多型性（SNP)、重複片段（repeated sequences)、X染色體基因以及不具區分性的探針，如筛選 族群樣本經分析獲得之甲基化數值皆大於0.9或者皆小於 〇.1的探針’再利用統計軟體Super PC analysis進行嚴謹的 計算’得出與肺腺癌預後狀況有關之曱基化探針約2〇〇 個。該Super PC analysis係為進一步分析基因與疾病預後 關係的統什軟體，為相關分析領域者所能了解並應用之統 計分析方式。 該驗證步驟S3係利用一定量曱基化分析方法再次檢 視該篩選步驟S2所榻選之基因’以建立一基因體曱基化變 異圖譜’該基因體曱基化變異圖譜包含與肺腺癌預後較差 高度相關之基因。本實施例之驗證步驟S3係採用焦磷酸測 序(pyrosequencing)之方式驗證該200個曱基化探針，藉由 統計分析的方法’挑選可同時於該IUumina humanmethylation27 bead chip及該焦碟酸測序之分析平台 中得到相同結果的探針，以獲得本發明與肺腺癌轉移及存 活率低於3年以下高度相關之基因體曱基化變異圖譜。 請參照第1表所示，本實施例之基因體甲基化變異圖 10 201250245 5普係具有38個與肺腺癌預後高度相關之探針，係分別對應 37個基因以及序列代碼為ci6orf25的序列[參考自美國國 家衛生院(Nation Institution of Health)，生物技術資訊中心 (National Center for Biotechnology Information)]。其中，該 第1表所示之R值係為相關數值(correlation coefficient)， 係指各基因曱基化程度在晶片筛選平台與焦鱗酸測序分析 平台間之相關性’該R值愈趨近於1則代表二者之相關性 愈好。 第1表：38個在篩選平台與焦填酸測序分析平台高度 相關之探針 探針ID 基因 R值 探針ID 基因 R價 1 C16orf25 0.815 20 CTSE 0.934 2 RALGPS2 0.874 21 CDKL1 6.843 3 SPOCD1 0.858 22 NFAM1 0.931 4 KIAA0746 0.927 23 PAQR6 0.91 5 PDE10A 0.907 24 TMEM129 0.942 6 KIAA0649 0.946 25 SLAMF8 0.984 7 IER5 0.824 26 EFNA2 0.972 8 SCARF2 0.835 27 NOTCH4 0.896 9 KCNJ8 0.97 28 OR1A2 0.926 10 FAM3B 0.95 29 AGTRL1 0.935 11 PHLDA3 0.975 30 BDKRB1 0.949 12 SORCS1 0.959 31 SEMA4A 0.954 13 AGTR1 0.985 32 KRT4 0.901 14 PAX7 0.933 33 NCOR2 0.825 15 ZNF496 0.962 L 34 GNS 0.873 16 WNK4 0.979 35 PNPLA2 0.899 17 RAB7L1 0.963 36 ANKRD9 0.8 18 fflSTIH4D 0.963 37 ALDH1A3 0.946^ 19 SYT2 0.946 38 BLVRA Γ〇845 此外’請參照第2圖所示，組合該38個與肺腺癌預 後高度相關之探針進行該篩檢族群之診斷試驗。本實施例 201250245 係利用焦峨g曼測序(pyrosequencing)^方式針對該筛檢族群 的癌組織檢體進行分析，計算各癌組織檢體基因中的甲基 化程度，再與該篩檢族群之臨床病理資料進行比對，以推 得該38個探針的操作者接受特徵曲線(recdver叩沉此叫 characteristic curve;又稱R〇c曲線）。據此可得知該％ 個探針對於肺腺癌的預後狀況具有良好的專一性及靈敏 度，具有應用於臨床肺腺癌檢測之潛力。 綜上所述，經由各步驟之篩選而獲得的38個探針， 係與肺腺癌預後較差相關，並且具有良好的專一性及靈敏 度，該38個探針即為本發明之曱基化分子標記，並且可進 一步應用於臨床肺腺癌檢測方法之開發，藉由偵測患者檢 體中，包含該38個探針序列之基因的甲基化程度，進一步 與正常檢體進行輯，並且作為肺制織航之判斷依 據。其中，該患者檢體可以係癌組織、血液或者其他可能 帶有游離核酸之體液，以方便自各患者取得檢體進行試 驗，而各基因的曱基化程度則可利用焦磷酸測序或基因體 曱基化晶片等分析方法加以計算，較佳係利用㈣酸測序 的方式，以便於臨床診斷之快速操作與進行，同時可降低 診斷成本。另外，該38個探針中包含少數具有低訊號，或 者高僞性的探針，如探針ID為8、18及38，較佳係於檢 測過程巾屏除，以提升該甲基化分子標記於臨床檢測上之 靈敏度與專一性。 為進一步證實本發明之曱基化分子標記確實針對肺 腺癌的預後狀況具有高度的專一性及靈敏度，本實施例係 組合该35個探針(屏除探針ID為8、18及38之探針），利 —12 — 201250245 測序的方式，針對韩檢族群及驗證族群的癌組 織4欢體進仃分析，並且分顺算各檢縣因的Μ化程度。 :參照第3、4圖及第2表所示，係該35個探針的甲 :化程度示賴，顯示該35個探針皆在肺腺癌患者中呈現 冋度的甲基化’並且該35個探針的甲基化與肺腺癌患者且 有明確的顯著性。 ”201250245 .. 6. Description of the Invention: [Technical Field] The present invention relates to a diagnostic index for lung adenocarcinoma, in particular to a thiolated molecular index of the prognosis of a lung adenocarcinoma and the prognosis of the lung adenocarcinoma The thiolated molecular index is applied to the molecular diagnosis of lung adenocarcinoma. [Prior Art] Lung cancer is the leading cause of malignant tumor mortality in industrial countries, with a five-year survival rate of only 10 to 15%. The main cause of mortality is the high recurrence rate of lung cancer and the lack of conventional detection techniques. Lung adenocarcinoma belongs to non-small cell carcinoma and is the main type of lung cancer, accounting for 40-50% of all lung cancers, and its proportion is increasing year by year. Unlike squamous cell lung cancer, lung adenocarcinoma mainly occurs in the peripheral part of the lung, such as the lateral lobes, and is easy to invade women and non-smokers. The cause of lung adenocarcinoma is not clear, and the initial symptoms are not obvious, so Its early diagnosis and prognosis analysis is more difficult. The method of screening for lung cancer includes chest sputum, sputum cytology, and computerized tomography and positron photography with high sensitivity. Among them, the chest sputum and sputum cytology are clinically used for lung cancer diagnosis, but have Specialized and poor sensitivity, etc., can not do the early detection to effectively = good lung cancer high fatality rate n while the electric occupation and positron photography is an important indicator of the current swollen 2 sites and tumor staging, but the price is expensive, It is not affordable for ordinary people, so it is often limited in the application of clinical lung cancer detection. According to US Patent Publication No. 2010/0167296, a prognostic method for non-201250245 small cell pneumonia is disclosed. Expression of biomarkers such as epithelial growth factor receptor (EGFR), macrophage colony stimulating factor (CSF-1) and carbonic anhydrasel (CAIX) as non-small cells Judging basis for the prognosis of lung cancer. The US Patent Publication Nos. 2010/0317002, 2010/00068207, 2009/0214536 5 and 2009/0297507 respectively disclose protein markers related to the diagnosis of lung cancer, including CACNA1E, ADAM10, DDR2 and ADAMTS- 7 and so on. However, the molecular diagnostic methods shown in each of the foregoing cases use the detection of the expression value of each biomarker at the RNA or protein level as a means of diagnosis of lung cancer, and RNA and protein are difficult to preserve and extremely unstable molecular level. 'It is not only complicated and difficult to process, but also prone to false positives due to the instability of the RNA and protein samples. As a result, the specificity and sensitivity of the above detection methods cannot be effectively improved, and it is not suitable for clinical use. Use of lung cancer detection. According to this, the conventional technology lacks a tool with high specificity, sensitivity, and can be widely used in the early diagnosis and prognosis analysis of clinical lung adenocarcinoma. Therefore, it is necessary to improve the conventional technology to effectively predict the prognosis of lung adenocarcinoma. The situation improves its high mortality rate. The methylation of the genome is one of the focuses of contemporary biomedical research. According to the research, the hypermethylation of the CpG island group (cpG island) on the gene promoter is closely related to the formation and metastasis of cancer. Over-methylation of the promoter of tumor suppressor gene (tum〇r suppressor genes) can cause many transcription factors and polymeric impurities involved in regulation to fail to bind to the starting position, resulting in the non-expression of the cancer suppressor 201250245 gene. Stable and money, and thus the emergence of cancer, so the thiolation of the 'special gene can be used as a molecule for tumor formation. ' Now there is still no development and application of thiolated molecules related to lung adenocarcinoma. [Summary of the Invention] The main purpose of this heart month is to provide a molecular index of the prognosis of lung adenocarcinoma, which is highly specific and sensitive to the early detection and prognosis of lung adenocarcinoma. The objective of the present invention is to provide a methylation molecular index for prognosis of lung adenocarcinoma, which is directly detected for the DNA level of the sample, and can avoid the doubt that the conventional RNA or protein molecular index is derived from false positive. A further object of the present invention is to provide a lung. The prognosis of adenocarcinoma is based on the molecular diagnosis of clinical lung adenocarcinoma, which has the advantages of high diagnostic rate and low cost. A further object of the present invention is to provide a prognosis of lung adenocarcinoma. The methylation molecular index is used for molecular diagnosis of clinical lung adenocarcinoma to establish a tool for early diagnosis and prognosis analysis of lung adenocarcinoma, thereby effectively improving the high mortality rate of lung adenocarcinoma. To achieve the aforementioned object, the present invention The technical content of the application includes: A methylation molecular index for the prognosis of lung adenocarcinoma, including C16orf25, RALGPS2, SPOCD, KIAA0746, PDE10A, KIAA0649 , IER5, SCARF2, KCNJ8, FAM3B, PHLDA3, SORCS, AGTR, PAX7, ZNF496, WNK4, RAB7U, HISTIH4D, 201250245 SYT2, CTSE, CDKL1, NFAM1, PAQR6, TMEM129, SLAMF8, EFNA2, NOTCH4, OR1A2, AGTRL1, BDKRB1 One or a combination of SEMA4A, KRT4, NCOR2, GNS, PNPLA2, ANKRD9, ALDH1A3, BLVRA; wherein the above-mentioned % methylation molecular index comprises 37 genes and a sequence of NCBI sequence code C16orf25, both The lung adenocarcinoma metastasis is related to the mortality rate. The lung adenocarcinoma includes the first to fourth stages of lung adenocarcinoma. Among them, the lung adenocarcinoma mortality rate refers to the prognosis survival for less than 3 years. The methylation molecular index of the prognosis of lung adenocarcinoma is applied to the molecular diagnosis of lung adenocarcinoma, which comprises detecting the degree of thiolation of the test gene, comparing the normal sample; and according to the test gene The degree of T-based molecularization of molecular markers is used as a basis for judging the prognosis of lung adenocarcinoma. Among them, the cancer system, blood, plasma or body fluid with free nucleic acid. The degree of methylation is analyzed by a method of coke-loading acid sequencing or gene-based thiolated wafers, wherein the lung adenocarcinoma comprises lung adenocarcinomas of the first to fourth stages. [Embodiment] ▲ For the present invention The above and other objects, features, and advantages will be more apparent from the following description of the preferred embodiments of the invention. , refers to 39 patients with initial lung adenocarcinoma from the Rongmin Chief Medical Ik in Taipei area, including patients with stage- and second-stage lung gland 201250245 cancer patients. Among them, 27 patients with lung adenocarcinoma have better prognosis and survive. With a rate of more than 3 years, the prognosis of 12 patients with lung adenocarcinoma was poor, and the survival rate was only 3 years or less. This "screening population" is used for the selection of methylated molecular markers of the present invention. The "verification group" described in the present invention is a patient of the 3G lung adenocarcinoma that is completely independent of the "screening population" in the Taipei City Veterans General Hospital, and includes the first-stage and second-stage lung adenocarcinoma. In the patient, 26 patients with lung and knee cancer had better prognosis, and the survival rate was more than 3 years. The prognosis of 4 patients with lung adenocarcinoma (4), surviving 3 years, was made in the methylation molecule of the present invention. Diagnostic verification of the standard. The "clinical pathology data" described in the present invention is based on clinical prognostic data of the "screening population" or "validation group", and includes survival rate, degree of metastasis, and other occupational status of each patient. ~ The "gene (four)-based variation map" described in the present invention is obtained by analyzing the gene methylation of a lung adenocarcinoma tissue sample, and is established by comparing: "monthly, "clinical pathological data". It has a methylation gene associated with poor prognosis of lung adenocarcinoma. In the first aspect of the present invention, the present invention utilizes the gene methylation and fractionation method to synthesize the clinical pathology of the present invention, and further establishes a genome-like heteromorphic map, and the gene thiosylation variation map includes A molecular marker associated with lung adenocarcinoma: degree-related methylation. The methylation molecular marker has a high index and sensitivity for the prognosis of patients with lung adenocarcinoma. 2 ' can be applied to the development of molecular diagnosis of clinical lung adenocarcinoma to improve the diagnosis rate of lung adenocarcinoma, and Improve the high mortality rate of lung adenocarcinoma. Referring again to Fig. 1, the methylated molecular marker of the present invention is obtained by the following steps in 201250245, including S1; a screening step S1; and - a verification step S3. The step of methylation analysis step 82 is to first detect the geneification of each sample gene by the methylation analysis method of the screening protein gene, and (4) cooperate with the clinicopathological data of the screening group to summarize And select the methylation genes of each specimen's basic financial 'associated with the poor prognosis of lung adenocarcinoma. In this embodiment, the methylation status of about 14,000 genes in each sample is analyzed by the fine-commercial methylation crystals >Mllumina hum_ethylation27 bead (the ancestor (five) is called % usa), and the clinical pathology of each sample is combined. According to the data, since the 14 _ a basic financial selection is poorly correlated with the prognosis of lung adenocarcinoma, if the survival rate is less than 3 years, the methylation probe is about 27, 578. In this example, 'the methylation status of each gene is calculated and exchanged for the -methylation value (β-value)'. Based on the value of the thiolation value, 27,578 probes related to the poor prognosis of lung adenocarcinoma are selected. needle. In the screening step S2, among the methylated genes obtained in the methylation analysis step S1, genes having specificity and specificity associated with low survival rate and metastasis of lung adenocarcinoma are further selected. The screening step S2 of the present invention preferably comprises two stages, a first screening stage S21; and a second screening stage S22. Wherein the first screening stage is preceded by the methylation gene to remove a lesser or interfering methylation gene, comprising a single-nucleotide polymorphism, a repeating fragment (repeated) Sequences and genes with too high or too low methylation values (p-vaiue) such as β-value greater than 0.9 or less than 〇.1, in addition, to avoid dose-compensation by genes on the X chromosome (d〇 Sage compensation produces interference, and the gene located on the X chromosome is eliminated in the first screening stage S21 of 201250245. The second screening stage S22 is performed by means of a method such as c〇x regression analysis (cox regressi〇n anaiysis) or Bayesian logistic regression software, etc., and further to the first screening. Among the genes selected in stage S21, 'select genes associated with poor survival rate and prognosis of lung adenocarcinoma, such as those with a survival rate of less than 3 years or a metastatic situation. The screening step S3 of the present embodiment first removes the gene polymorphism (SNP), the repeated sequences, the X chromosome gene, and the non-discriminating probe from the 27, 578 probes, such as the screening population sample. The thiolated probes related to the prognosis of lung adenocarcinoma were obtained by analyzing the probes with methylation values greater than 0.9 or less than 〇.1 'Reuse statistical software Super PC analysis'. One. The Super PC analysis is a statistical analysis method for further analysis of the relationship between genes and disease prognosis, and is a statistical analysis method that can be understood and applied by relevant analytical fields. The verification step S3 is to re-examine the gene selected by the screening step S2 by using a certain amount of thiolation analysis method to establish a gene thiosylation variation map. The gene thiosylation variation map contains the prognosis of the lung adenocarcinoma. Poorly highly related genes. The verification step S3 of the present embodiment verifies the 200 thiolated probes by means of pyrosequencing, and the method of statistical analysis 'selects both the IUumina humanmethylation 27 bead chip and the pyrolysis acid sequencing The probes with the same results in the analysis platform were used to obtain the genome thiolation variation map of the present invention which is highly correlated with lung adenocarcinoma metastasis and survival rate below 3 years. Please refer to the first table, the genome methylation variation of the present embodiment is shown in Fig. 10 201250245. The general system has 38 probes highly correlated with the prognosis of lung adenocarcinoma, corresponding to 37 genes and the sequence code is ci6orf25. Sequence [reference from the National Institute for Health Information, National Center for Biotechnology Information]. Wherein, the R value shown in the first table is a correlation coefficient, which refers to the correlation between the degree of thiolation of each gene between the wafer screening platform and the pyromycin sequencing analysis platform. A value of nearly 1 indicates a better correlation between the two. Table 1: 38 probe probes highly correlated with the pyrosial acid sequencing analysis platform. Gene R value Probe ID Gene R price 1 C16orf25 0.815 20 CTSE 0.934 2 RALGPS2 0.874 21 CDKL1 6.843 3 SPOCD1 0.858 22 NFAM1 0.931 4 KIAA0746 0.927 23 PAQR6 0.91 5 PDE10A 0.907 24 TMEM129 0.942 6 KIAA0649 0.946 25 SLAMF8 0.984 7 IER5 0.824 26 EFNA2 0.972 8 SCARF2 0.835 27 NOTCH4 0.896 9 KCNJ8 0.97 28 OR1A2 0.926 10 FAM3B 0.95 29 AGTRL1 0.935 11 PHLDA3 0.975 30 BDKRB1 0.949 12 SORCS1 0.959 31 SEMA4A 0.954 13 AGTR1 0.985 32 KRT4 0.901 14 PAX7 0.933 33 NCOR2 0.825 15 ZNF496 0.962 L 34 GNS 0.873 16 WNK4 0.979 35 PNPLA2 0.899 17 RAB7L1 0.963 36 ANKRD9 0.8 18 fflSTIH4D 0.963 37 ALDH1A3 0.946^ 19 SYT2 0.946 38 BLVRA Γ〇 845 In addition, please refer to Figure 2 to combine the 38 probes highly correlated with the prognosis of lung adenocarcinoma for the diagnostic test of the screening population. In the present embodiment, 201250245 analyzes the cancer tissue samples of the screening group by using pyrosequencing method, calculates the degree of methylation in each cancer tissue sample gene, and then compares with the screening group. The clinical pathological data were compared to extract the operator of the 38 probes to accept the characteristic curve (recdver sinking this called a qualitative curve; also known as the R〇c curve). It can be seen that the % probes have good specificity and sensitivity for the prognosis of lung adenocarcinoma, and have the potential to be applied to the detection of clinical lung adenocarcinoma. In summary, the 38 probes obtained through the screening of each step are related to the poor prognosis of lung adenocarcinoma, and have good specificity and sensitivity. The 38 probes are the thiolated molecules of the present invention. Marked, and can be further applied to the development of clinical lung adenocarcinoma detection methods, by detecting the degree of methylation of the gene containing the 38 probe sequences in the patient's specimen, further collating with the normal specimen, and The basis for judging the lung weaving. Among them, the patient's specimen may be cancerous tissue, blood or other body fluids that may have free nucleic acids, so that it is convenient to obtain samples from each patient for testing, and the degree of thiolation of each gene can be pyrosequencing or genetic sputum. The calculation method such as the base wafer is calculated, and the method of (4) acid sequencing is preferably used to facilitate the rapid operation and progress of the clinical diagnosis, and at the same time, the diagnosis cost can be reduced. In addition, the 38 probes contain a small number of probes with low signal or high pseudo-symmetry, such as probe IDs 8, 18 and 38, preferably in the detection process screen to enhance the methylation molecular marker. Sensitivity and specificity in clinical testing. In order to further confirm that the thiolated molecular marker of the present invention is highly specific and sensitive to the prognosis of lung adenocarcinoma, this embodiment combines the 35 probes (the screen removal probe IDs of 8, 18 and 38) Acupuncture), Li-12—201250245 The method of sequencing, for the analysis of the cancer tissue of the Korean and ethnic groups, and the degree of degeneration of each county. : Referring to Figures 3, 4 and 2, the degree of A of the 35 probes is shown, indicating that the 35 probes exhibit methylation in the lung adenocarcinoma patients and Methylation of the 35 probes was clearly associated with lung adenocarcinoma patients. ”
2表.35 ^與肺腺癌預德高度相 基因 基因 C16orf25 CTSE RALGPS2 CDKL1 SPOCD1 NFAM1 WNK4 PAQR6 RAB7L1 TMEM129 PDE10A SLAMF8 IQAA0649 EFNA2 IER5 NOTCH4 KCNJ8 OR1A2 KIAA0746 AGTRL1 PHLDA3 BDKRB1 ZNF496 SEMA4A PAX7 KRT4 AGTR1 NCOR2 SORCS1 ’ GNS FAM3B IPNPLA2 SYT2 ANKRD9 ALDH1A3 關之基因 本發明之曱基化分子標記於實際應用於臨床肺腺癌 之預後診斷分析’係先自篩檢患者中採得檢體，該檢體可 以為組織，較佳為0.5立方公分(〇.5cm3)，或者係血漿較佳 為500微升(5〇〇μ1) ’再分別針對各檢體進行基因體核酸或 游離核酸的萃取，所取得之核酸DNA經定量分析後，另 —13 — 201250245 取1微克(lgg)之核酸DNA進行亞硫酸鹽（sodium bisulfite) 處理。再將處理後之核酸DNA純化回收成20微升(20μ1) 之體積’擷取其中的1微升(Ιμΐ)並利用聚合酶連鎖反應 (PCR)進行獨立基因的放大，再將所獲得之產物（PCR products)加入特定的定序引子進行焦填酸測序，由各基因 所呈現的訊號以焦磷酸測序儀定量該基因的甲基化程度 (範圍：0〜100%) ’並且配合各基因統計之權重係數進一 步分析及判斷，如第3表所示，係列出本實施例之8個範 例基因的權重係數’該權重係數係代表各基因的重要程度。 本實施例係利用公式(A)計算而推得各基因所代表之 風險值，其中’各基因的風險值還須經加總而得一總值， 該總值係代表各篩檢患者的死亡風險值，並且依據各基因 權重係數及基因甲基化程度之不同，該總值可能有正負值 的差異’其中數值愈趨近於正值且數值愈大，代表高死亡 風險，也就是篩檢患者的預後較差。 (A):單一基因甲基化程度（％) X權重係數^風險值 第3表:與肺癌預後相關基因之權重係數 基因 權重係數 CTSE H 0.0845 NTFAM1 -0.0507 TMEM129 0.0247 EFNA2 -0.0134 AGTRL1 Γ -0.0466 BDKRB1 -0.0117 SEMA4A -0.0075 ALDH1A3 -0.0813 201250245 > :參照第5圖所示，係該35個探針的曱基化程度， 與°亥篩檢族群、驗證族群之臨床病理資料所推算而得之 曲線，由此可知，該35個探針，對於肺腺癌的預後 狀況^有良好的專一性及靈敏度，確實可應用於臨床肺腺 癌的診斷，作為肺腺癌預後狀況之判斷依據。 本發明之甲基化分子標記，乃係利用基因甲基化分析 ，，綜合本發明之臨床病理資料，經由嚴謹的統計方式計 异而獲得，包含38個與肺腺癌預後較差高度相關的探針， 對於臨床肺腺癌病患的預後具有指標性、專一性以及良好 的靈敏度。 本發明之甲基化分子標記，係屬DNA分子標記，可 直接就檢體之DNA層面進行偵測、分析，可避免檢體的 過度處理及製備，以及容易衍生偽陽性等缺點，具有提升 肺腺癌診斷之專一性及靈敏度的功效。 本發明之甲基化分子標記可應用於臨床肺腺癌新興 檢測方法的開發，作為肺腺癌預後的評估指標，可藉由偵 測患者檢體中，包含該38個探針序列之基因的曱基化程 度，進一步與正常檢體進行比對，以作為肺腺癌預後狀況 之判斷依據，並且建立一個具有高專一性、靈敏度，並且 可廣泛應用於臨床肺腺癌早期診斷及預後分析之工具，改 善肺腺癌之高致死率，為本發明之功效。 雖然本發明已利用上述較佳實施例揭示，然其並非用 以限定本發明，任何熟習此技藝者在不脫離本發明之精神 和範圍之内，相對上述實施例進行各種更動與修改仍屬本 發明所保濩之技術範臂，因此本發明之保護範圍當視後附 —15 — 201250245 之申請專利範圍所界定者為準。 【圖式簡單說明】 第1圖：本發明之甲基化分子指標之篩選流程圖。 第2圖：本發明之曱基化分子指標之診斷性示意圖。 第3圖：本發明之曱基化分子指標之曱基化程度示意圖。 第4圖：本發明之曱基化分子指標之曱基化程度示意圖。 第5圖：本發明之曱基化分子指標之診斷性示意圖。 【主要元件符號說明】 S1甲基化分析 52 篩選步驟 S21第一篩選階段 S22第二篩選階段 53 驗證步驟 16 —2 Table .35 ^Highly phased gene gene with lung adenocarcinoma C16orf25 CTSE RALGPS2 CDKL1 SPOCD1 NFAM1 WNK4 PAQR6 RAB7L1 TMEM129 PDE10A SLAMF8 IQAA0649 EFNA2 IER5 NOTCH4 KCNJ8 OR1A2 KIAA0746 AGTRL1 PHLDA3 BDKRB1 ZNF496 SEMA4A PAX7 KRT4 AGTR1 NCOR2 SORCS1 ' GNS FAM3B IPNPLA2 SYT2 ANKRD9 ALDH1A3 gene of the present invention The thiolated molecular marker of the present invention is applied to a prognostic diagnosis of clinical lung adenocarcinoma. The sample is obtained from a patient who first screens the test, and the sample may be a tissue, preferably 0.5 cubic centimeters. (〇.5cm3), or the plasma is preferably 500 μl (5〇〇μ1)' and then extract the genomic nucleic acid or free nucleic acid for each sample separately. After the quantitative analysis of the obtained nucleic acid DNA, another 13 — 201250245 Take 1 microgram (lgg) of nucleic acid DNA for sodium bisulfite treatment. The processed nucleic acid DNA is purified and recovered into a volume of 20 μl (20 μl), and 1 μl of the sample is taken and amplified by a polymerase chain reaction (PCR), and the obtained product is obtained. (PCR products) Adding specific sequencing primers for pyrolysis, and the signal presented by each gene quantifies the degree of methylation of the gene by pyrosequencing (range: 0~100%)' and cooperates with each gene. The weight coefficients are further analyzed and judged. As shown in the third table, the weighting coefficients of the eight example genes of the present embodiment are listed. The weighting coefficient represents the importance of each gene. In this embodiment, the risk value represented by each gene is calculated by using formula (A), wherein the risk value of each gene is also added to obtain a total value, which represents the death of each screening patient. The risk value, and depending on the weight coefficient of each gene and the degree of methylation of the gene, the total value may have a difference between positive and negative values. The value is closer to a positive value and the larger the value, which represents a high risk of death, that is, screening. The patient's prognosis is poor. (A): Degree of methylation of a single gene (%) X Weight coefficient ^ Risk value Table 3: Weight coefficient of genes associated with lung cancer prognosis Gene weight coefficient CTSE H 0.0845 NTFAM1 -0.0507 TMEM129 0.0247 EFNA2 -0.0134 AGTRL1 Γ -0.0466 BDKRB1 -0.0117 SEMA4A -0.0075 ALDH1A3 -0.0813 201250245 > : Refer to Figure 5 for the degree of thiolation of the 35 probes, and the curve derived from the clinical pathological data of the screening group and the verification population. From this, it can be seen that the 35 probes have good specificity and sensitivity for the prognosis of lung adenocarcinoma, and can be used for the diagnosis of clinical lung adenocarcinoma as a basis for judging the prognosis of lung adenocarcinoma. The methylation molecular marker of the present invention is obtained by using gene methylation analysis, synthesizing the clinical pathological data of the present invention, and obtaining it through rigorous statistical methods, including 38 probes which are highly correlated with the prognosis of lung adenocarcinoma. The needle has an index, specificity and good sensitivity for the prognosis of patients with clinical lung adenocarcinoma. The methylation molecular marker of the invention belongs to the DNA molecular marker, and can directly detect and analyze the DNA layer of the sample, can avoid the excessive treatment and preparation of the sample, and is easy to derive pseudo-positive defects, etc., and has the lungs improved. The specificity and sensitivity of adenocarcinoma diagnosis. The methylation molecular marker of the invention can be applied to the development of an emerging detection method for clinical lung adenocarcinoma, and can be used as an indicator for evaluating the prognosis of lung adenocarcinoma by detecting the gene of the 38 probe sequences in the patient sample. The degree of thiolation is further compared with normal samples to determine the prognosis of lung adenocarcinoma, and to establish a highly specific, sensitive, and widely used in early diagnosis and prognosis analysis of clinical lung adenocarcinoma. The tool improves the high mortality rate of lung adenocarcinoma and is the efficacy of the present invention. While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is the scope of the invention, and the scope of the invention is defined by the scope of the patent application of the above-mentioned application. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart showing the screening of methylation molecular indicators of the present invention. Figure 2: Diagnostic schematic of the thiolated molecular index of the present invention. Figure 3: Schematic diagram of the degree of thiolation of the thiolated molecular index of the present invention. Figure 4: Schematic diagram of the degree of thiolation of the thiolated molecular index of the present invention. Figure 5: Diagnostic schematic of the thiolated molecular index of the present invention. [Main component symbol description] S1 methylation analysis 52 Screening step S21 first screening stage S22 second screening stage 53 Verification step 16 —