肺癌之性別差異與預後因子之研究

• Main Authors: 陳冠宇, Chen, Kuan-Yu
• Other Authors: 楊泮池, 張富雄, 臺灣大學：
• Format: Text
• Language: Chinese; English
• Published: 2006
• Subjects: 肺癌; 性別; 預後; lung cancer; gender; prognosis; New Zealand; TNM; Iceland
• Online Access: http://ntur.lib.ntu.edu.tw/handle/246246/55464; http://ntur.lib.ntu.edu.tw/bitstream/246246/55464/1/ntu-95-D88421005-1.pdf

肺癌目前已經成為全球性的健康問題。肺癌在世界上，包括許多歐美已開發國家，都是發生率最高，死亡率也最高的疾病。在全世界所增加的癌症病患中，有12%是來自於肺癌。在未開發國家中，肺癌佔癌症死亡比率的14.6%；而在已開發國家中，肺癌佔癌症死亡的比率則高達22%。肺癌細胞的形成需要一個相當複雜的基因變化過程。經過階段性的基因變異累積下來，形成了具有不死特性的腫瘤細胞。目前為止有許多環境因子，包括吸煙或是空氣污染，可以是造成支氣管上皮或是肺泡細胞產生基因變異的起始者(initiator)。 在全球，平均男與女的比例大約為2.7:1。在男性，發生率及死亡率最高的地區是歐洲(特別是東歐)、北美、澳洲/紐西蘭、和南美洲。其次是中國、日本以及東南亞。發生率最低的地域則主要是在南亞（印度、巴基斯坦）和撒哈拉沙漠以下的非洲各國。在女性，肺癌的地域分佈則大約反映過去吸煙比率的分佈。發生率最高的地區是在北美和西北歐（英國、冰島、丹麥），其次則是澳洲/紐西蘭和中國。 在美國，男女癌症死亡原因的首位都是肺癌。在過去五十年來，肺癌一直是男性癌症死因的首位。而從1987年以來，肺癌也超越了乳癌成為女性癌症死因的首位。在歐洲，關於肺癌的發生率及死亡率在不同的區域有明顯的不同。以男性而言，東歐的肺癌發生率要比其他區域要高出許多。而以女性而言，肺癌的發生率則以北歐為最高，幾乎是西歐地區的兩倍。肺癌死亡率的分佈也呈現相似的情況。預測在2010年，大部分的歐洲國家，將出現男性肺癌死亡率的下降和女性肺癌死亡率的上升。在亞洲，女性的肺癌死亡率通常是小於西方國家的。然而，在過去數十年當中，亞洲女性的肺癌死亡率則有顯著的增加。在台灣，男性癌症死亡病因中，肝癌居於首位，每十萬人口44.92人。而肺癌則位居其次，每十萬人口43.03人。至於女性方面，肺癌早已躍居女性癌症死亡率的第一位，每十萬人口19.71人。 研究疾病的預後因子，有助於醫師針對病患個人給予建議，決定治療方法，以及對疾病本身的瞭解。關於肺癌的預後因子研究，不同的組織型態的肺癌，也有不同的發現。在小細胞肺癌，關於預後因子的研究並不多。臨床上公認體能狀況和疾病範圍是影響病人預後的重要因子。在實驗室測量的因子中，血清乳酸脫氫酶是較常被報告具有預後價值的。相對的，非小細胞肺癌的預後因子已經被大幅地研究。大致可分為兩類：腫瘤相關因子和宿主相關因子。其中，隨著基因體和蛋白質體技術的進步，分子生物學上的預後因子急速增加而被廣泛地研究。但根據TNM系統來判定的臨床和病理分期，目前仍然是非小細胞肺癌最有力的預後因子。 儘管有越來越多關於非小細胞肺癌病人預後的研究成果，在分析上仍然有些值得注意的地方：大部分的研究都是回溯性的；從這些統計資料中，辨認出的預後因子仍是相當有限；而在這當中，除了少數預後因子在不同的研究中持續穩定被確認，其他因子的研究結果常常出現相衝突的結論，而無法確定其預後價值。 在過去的研究中，肺癌的病人在不同性別與不同年齡層，其臨床特性也不一樣。基於肺癌，尤其是在女性肺癌方面的流行病學研究上發現的地域種族差異，在本研究中收集台大醫院的肺癌病人的臨床資料，透過統計學方法加以分析，試圖呈現台灣地區肺癌病人整體的臨床表徵，以及存活上的性別差異和年齡層間的的不同。在另一方面，針對肺癌的預後因子，希望能結合分子生物學以及臨床資料統計分析，就現有的肺癌預後因子作進一步的探討與釐清，同時透過生物資訊的方法與傳統生物技術的整合，建立一良好的研究模式，以發現更多新的預後因子。 本研究包含三個部分：肺癌的性別差異研究；肺癌已知的預後因子研究；和尋找新的肺癌預後因子。茲分述如下： 肺癌的性別差異 肺癌的性別差異以及在不同年齡層差別的釐清是非常重要的。因為這些差異對於臨床研究的設計非常重要，包括預防癌症藥物的發展、早期診斷的篩檢方法、以分子標記為基礎的分期方法、以及個人化的治療方式。因此本研究就肺癌病人不同的性別及年齡層，在組織學型態、分期、治療、及存活方面進行深入的分析。並且嘗試以多變數分析來探討台灣地區肺癌病患的臨床預後因子。 我們使用台大醫院的癌症登錄系統，將1991年1月到1999年12月所有診斷為肺癌的病人搜尋出來。我們回顧病人的臨床記錄；包括診斷時的年齡、性別、癌症家族史、吸煙史、腫瘤期別、組織學型態、治療方法、以及存活期間；並加以分析。從1991年到1999年，台大醫院每年的新診斷為肺癌的人數呈現增加的趨勢。在這段期間，肺癌病人男性：女性的比率呈現穩定的2:1。除了年齡層小於40歲的病人以外，增加的趨勢普遍地存在其他所有的年齡層的病人。而這個肺癌病人增加的趨勢，主要是來自肺腺癌病人數的增加。 腺癌在男性和女性的病人都是最常見的組織型態。相較於其他其他種類的肺癌而言，肺腺癌已成為一個越來越重要的問題。女性肺腺癌的比率 (73.5%)要比男性 (42.1%) 要顯著地來得高 (p<0.001)。在不同年齡層的組織型態分佈比例方面，隨著年齡的增加，腺癌的比例呈現減少的趨勢而鱗狀細胞癌呈現增加的趨勢。透過Cox 前進迴歸模型進行多變數分析，我們發現肺腺癌的患者也較其他組織型態的肺癌患者有較佳的預後。此外，女性肺癌病人較男性有更好的存活率。這樣的差異存在於50-69歲的這個年齡層。我們推測存在於這個年齡層的生理變化，如停經導致的性荷爾蒙變化，對女性肺癌預後的影響可能有其重要性。以社區人口為基礎，有關肺癌的性別與年齡差異研究，將有助於加強本研究中的結論。 肺癌已知的預後因子研究 要決定個別的肺癌病患預後並不容易，主要是由於病人在臨床上的表現相當的多樣化。而整個臨床病程又可以牽涉到許多致病機轉。各式各樣的臨床表現和進展過程，可以是原發腫瘤、轉移病灶、和腫瘤伴隨症候群的種種腫瘤細胞行為交織所致。因此要進行完整而精確的分析相當困難。 人體端粒酶(telomerase)是一個可以在染色體的端粒(telomere)加上重複的核酸序列TTAGGG的核糖蛋白質。端粒酶活性(telomerase activity)可以在幾乎所有的小細胞肺癌及80%的非小細胞肺癌偵測出來。相對地，在正常的人類體細胞中，端粒酶活性則無法被偵測出來。端粒酶活性被認為是非小細胞肺癌諸多重要的預後因子之一。既然絕大部分的肺癌病人的腫瘤都可以偵測到端粒酶活性，如果只是根據端粒酶活性的有無來預測肺癌病人的預後，實際上沒有太大用途。關於端粒酶活性的定量分析和病人預後的相關性之前很少被研究。我們假設端粒酶活性的定量，而非存在與否，可以預測肺癌病人的預後。我們使用與以往研究不同的非放射性定量方法，來研究端粒酶活性的高低和非小細胞肺癌病人臨床特徵和預後的相關性。 我們納入因非小細胞肺癌而在台大醫院接受手術切除的68位病人成為研究對象。切除的肺癌檢體及鄰近正常組織的端粒酶活性，使用TeloTAGGG Telomerase PCR ELISAplus kit (Roche molecular Biomedical, Mannheim, Germany)來加以測定。並且與臨床上和病理學上的諸多參數、如年齡、性別、組織型態、腫瘤的侵犯程度及淋巴結轉移程度做相關分析。高端粒酶活性定義為：所測量到的相對端粒酶活性值大於中位數。如果病人檢體的端粒酶活性值小於中位數、那麼這位病人則被歸類在低端粒酶活性這一組。 共有58 (85.3%)位病人的腫瘤組織及21 (30.9%)個鄰近非腫瘤組織檢體可偵測到端粒酶活性。腫瘤有端粒酶活性和沒有端粒酶活性的不同病人群，在年齡、性別、腫瘤侵犯程度、淋巴結轉移、組織型態以及病理分期方面，並沒有明顯的差異。在定量方面，非腫瘤組織的相對端粒酶活性值中位數為3.95，而腫瘤組織的相對端粒酶活性值中位數為536.95。不同年齡、性別、以及組織型態的病人，相對端粒酶活性值並沒有顯著的差異。然而，隨著病理分期、腫瘤侵犯程度、淋巴結轉移程度的增加，相對端粒酶活性值也隨著呈現增加的趨勢。 針對第I期的肺癌病人，低端粒酶活性的病人較高端粒酶活性的病人有更好的存活，並且有統計學上的差異。以平均無疾病存活的期間而言，低端粒酶活性這一組的病人要比高端粒酶活性的病人來的長。較高的端粒酶活性會導致較高的再發風險。對於第II期、第III期以及第IV期的病患，在端粒酶活性的高低兩組病人之間的總存活或是無疾病存活差異，並沒有統計學上的顯著意義。使用Cox迴歸分析，我們發現每增加100單位的端粒酶活性，將使得死亡的風險比率增加13%。在控制其他可能影響死亡的變因如：年齡、性別、期別之後，端粒酶活性增加對死亡風險的影響並未有所改變。而就第I期的病患而言，每增加100單位的端粒酶活性將使得死亡的風險比率甚至增加高達33%。第I期的病患而言，每增加100單位的端粒酶活性將使得術後再發的風險比率增加13%。在控制其他可能影響死亡的變因如：年齡、性別之後，每增加100單位的端粒酶活性將使得術後再發的風險比率甚至增加高達16%。 我們的研究結果支持這樣的假說：端粒酶活性的高低，而非端粒酶活性的有無，才是真正的非小細胞肺癌的真正預後因子。這樣的發現，將有助於應用在臨床上預測非小細胞肺癌病人的存活。我們所使用的方法是一個非放射線活性的PCR-ELISA方法，而且進行了更詳細的分析。這些結果顯示端粒酶活性的高低，也許可以成為反映腫瘤侵犯程度的指標。同時也可以用來預測該腫瘤是否已經有了淋巴結轉移。然而，需要更大型的研究來確認是否端粒酶活性可以隨著腫瘤的擴展，淋巴結的擴散，以及病理分期的增加而增加。 尋找新的肺癌預後因子 以整個基因體為目標來進行的微陣列(microarray)分析，目前已經廣泛地被用來顯示癌細胞的整個基因體表現變化，同時也可以發現在不同環境條件下，癌細胞中不同的表現基因。研究者常常必須面對這樣的困難：必須設法在微陣列研究所產生大量的資訊中能夠做有效的運用；在原始的微陣列資料中做有效的轉譯，來塑造一個可行的模式，以便就所篩選出的研究目標基因群辨認其優先順序。 我們試圖建立一個方法學模式，從微陣列資料庫中擷取有用的資訊，以便來找尋肺癌的預後因子。我們假設在微陣列資料庫中，具有相似表現模式的基因，可能會隸屬於同範疇的生物功能，同時也可能對病人的臨床預後產生相同的影響。我們在Affymetrix Human Genome U133A 微陣列晶片上進行肺腺癌的微陣列分析。運用一個群聚/分類的程序，針對一個已知的非小細胞肺癌的預後因子，survivin (BIRC5)，利用肺腺癌所建立的微陣列資料庫，來搜尋表現相似的基因。其中，trophinin(TRO)是表現模式最相似的基因之一。從trophinin在胚胎著床的過程中囊胞侵犯到子宮內膜時所扮演的角色推測，trophinin可能也參與了癌細胞的侵襲過程。因此我們做了免疫切片染色來檢驗肺癌腫瘤檢體中trophinin的表現。然後和病人的臨床特徵及存活進行相關分析。我們也透過增加細胞中trophinin表現，以及使用小型干擾RNA(siRNA)抑制trophinin的表現，試圖來釐清trophinin在癌細胞侵襲過程中所扮演的角色。 總共有141位病患（41位女性和100位男性）的腫瘤蠟塊切片被用來進行免疫組織分析。在這群肺癌病人中發現，在女性的患者(p=0.003)以及年齡小於65歲的病人(p=0.027)有較高比率的的trophinin 表現。腫瘤有trophinin表現和沒有trophinin表現的不同病人群，在組織型態、病理分期、和淋巴結轉移方面，並沒有統計學上的差異。對肺腺癌的病人而言，在女性的患者(p=0.028)以及T1-2期的病人(p=0.021)有較高比率的trophinin 表現。而對鱗狀細胞癌的病人而言，腫瘤有trophinin表現和沒有trophinin表現的不同病人群，年齡、性別、病理分期、腫瘤侵襲、和淋巴結轉移方面，並沒有統計學上的差異。 我們發現在第I期的病患中，沒有trophinin表現的肺癌病人，他們的中位數存活期要比有trophinin表現的肺癌病人顯著地來的長(80.4 vs. 50.2 個月, p=0.042)。我們也使用了Cox 前進迴歸模型來針對第I期病患的年齡、性別、不同細胞型態、術式和trophinin 表現之有無進行多變數分析。當我們調整過其他預後因子之後，仍然顯示有trophinin表現的肺癌病患有較差的預後。在不同的組織型態的病人群中，我們發現：對肺腺癌的病人而言，trophinin表現的有無導致的存活差異較為顯著。在第I期的肺腺癌病患中，沒有trophinin表現的肺癌病人，他們的中位數存活期以及中位數無疾病存活期，要比有trophinin表現的肺癌病人顯著地來的長。而在鱗狀細胞癌的病患，trophinin表現的有無，並未發現有存活的差異。 為了瞭解trophinin 在癌細胞侵襲過程中所扮演的角色，我們採用了兩種不同的實驗方法。我們使用MDCK細胞株做為實驗的系統模型。這些轉殖後穩定表現trophinin的細胞株將被用來進行侵襲分析(invasion assay)。進行侵襲分析後的結果，跟母代未接受轉殖的MDCK細胞或是只接受載體轉殖的MDCK細胞比較起來，兩株接受轉殖後trophinin表現增加的細胞株，它們的侵襲能力大約增加為兩倍。其次，我們在具有高度侵襲能力的肺腺癌細胞株，CL1-5，使用三條不同的化學合成siRNA，分別來抑制trophinin的表現。在trophinin siRNAs的作用之下，內生的trophinin RNA 被抑制達80%-90%。CL1-5 細胞的內生性trophinin被抑制之後導致了侵襲能力的下降。我們推論：trophinin 可以促進細胞的侵襲。 Survivin 和trophinin在肺腺癌的微陣列資料庫中有相似的表現模式，這些觀察結果暗示著著survivin 和trophinin在表現的調控機轉上，可能有某部分的相同特性。Survivin是一個抗細胞凋亡的基因。然而，trophionin在功能上是否也有抗細胞凋亡的效果不得而知。不過在我們的研究中呈現出，trophinin可以促進癌細胞的侵襲/轉移。因此，我們認為這兩個基因雖然功能不同，卻一樣導致非小細胞肺癌的不良預後。更進一步來研究調控trophinin表現的機轉，然後將研究結果跟調控survivin的表現機轉的研究結果作比較，將可以得到更多新的發現，也有助於我們瞭解肺癌的進展過程。 透過臨床上的觀察以及流行病學的統計，可以發現肺癌在發生的病因學、治療的反應、以及死亡率和預後的表現，都存在有相當大的性別差異。有越來越多的文獻，為這樣的差異提供了生物學上的解釋基礎。然而，這些基礎仍然相當的薄弱。最主要是由於大部分的大型研究都是回溯性的。這樣的觀察性研究無可避免的會有許多病案篩選的偏見和研究方法的歧異。使用前瞻性的研究設計來進行大規模的篩檢登錄，可以更精準的評估性別的差異。在台灣，超過90%以上的女性肺癌患者都是不吸煙的。因此，吸煙以外的因子在台灣的肺癌研究變成相當的重要。目前關於肺癌發生的重要課題是：為什麼女性比男性容易得到肺癌？烹飪習慣、二手煙、基因多型性、荷爾蒙因素乃至於環境污染因素，都需要被小心謹慎的評估。對台灣女性肺癌如此高比率的腺癌，完整的流行病學資料將有助於提供可能的解釋以及深入研究的方向。特別是針對遺傳因子與環境因子互動，這樣的研究需要一個多中心的研究團隊來整合肺癌病人的多元性，並且使用分子生物技術來進行大規模的篩檢、藉以找出更多重要的危險因子和預後因子。 目前已知的肺癌預後因子的研究對象，大都是針對可接受手術切除腫瘤的病患。針對超過80%都是無法開刀的的非小細胞肺癌病患，以及100%接受化學治療或合併放射治療的小細胞肺癌病患而言，現有的預後因子，特別是分子生物學上的預後因子，仍然有相當大的研究空間。在少量細胞或是微量組織檢體中，得到分子生物學上預後因子的相關資訊，比較能夠符合實際臨床上評估病人預後的需要。在病人的體液中，包括血清、肋膜積液、或是甚至尿液，分析某些反映預後的特定基因產物的變化，更有助於臨床醫師對病患結果的預測。我們的實驗結果支持了預後因子定量分析的重要性。隨著越來越多的分子生物學上的預後因子被發現。其中，較能被確認其預後價值的因子，有必要針對檢體中表現量的高低與臨床資料相配合進行分析。 未來在尋找新的肺癌預後因子時，如何從龐大的基因體或蛋白質體資料庫中，找尋可能成為預後因子的基因或基因產物，將成為重要的任務。我們提出此一研究模式：使用已知的預後因子為基準，在基因體或蛋白質體資料庫找出的相似表現模式的基因，然後在病人腫瘤檢體中檢視其臨床價值。具有預後價值的因子再以分子生物實驗方法來確認其癌細胞轉移侵襲的相關角色功能。以這樣的方法來搜尋，將可以更迅速而且更能找到新的肺癌預後因子。新的預後因子在細胞生理所扮演的角色，可能和原有作為搜尋基礎的已知預後因子的功能不同。藉由分子生物技術的實驗方法，甚至可能開啟兩個不同細胞訊息傳遞途徑之間的連結。這樣的研究模式不只有助於發現新的預後因子，同時也有助於對癌細胞轉移侵襲機轉的進一步瞭解。 Lung cancer has become a global health problem, accounting for 12% of new cases of cancer worldwide, followed by cancers of the breast and colo-rectum. Lung cancer is the leading cause of death from cancer among men and women in many Western countries, including the U.S. and countries in Europe. It is relatively more important in developed rather than developing countries, as it accounts for 22% versus 14.6% of cancer deaths. Lung cancer results from complex, genetic and epigenetic changes characterized by stepwise malignant progression of cancer cells in association with accumulation of genetic alteration. This process, referred to as multistep carcinogenesis, develops through the clonal evolution of initiated lung cells. Initiation consists in the acquisition of defined genetic alterations in a small number of genes that confer a proliferative advantage that facilitates progression towards invasive carcinoma. Many environmental factors, including smoking and air pollution, could act as an initiator of carcinogenesis in bronchial or bronchiolar-alveolar epithelial cells. The male to female ratio of lung cancer patients throughout the world is 2.7:1. In men, the areas with the highest incidence and mortality are Europe (especially Eastern Europe), North America, Australia/New Zealand, and South America. The rates in China, Japan, and South East Asia are moderately high, while the lowest rates are found in southern Asia (India, Pakistan), and sub-Saharan Africa. In women, the geographic pattern reflects different historical patterns of tobacco smoking. The highest incidence rates are observed in North America and North Western Europe (U.K., Iceland, Denmark) with moderate incidence rates in Australia, New Zealand, and China. In the United States, lung cancer is the most common cause of death from cancer in both males and females. For the past 50 years, lung cancer is the leading cause of death from cancer for males. Since 1987, lung cancer has become the most common cause of cancer death for females. There are substantial differences in incidence and mortality in the different regions and populations within Europe. In men, the incidence is highest in Eastern Europe. In women, the highest incidence of lung cancer is in Northern Europe, which is almost twice as high as Western Europe. There is a similar pattern in the distribution of mortality. Predictions for the year 2010 show that most European countries carry a decline in mortality for males, but an increase in females. In Asia, the mortality rates of lung cancer in women are generally lower than those in western countries. However, there has been a significant increase in the mortality rate from lung cancer in Asian women over the past few decades. In Taiwan, the mortality rates of lung cancer are high in both genders. For males, lung cancer is the second most common cause of death from cancer, accounting for 43.03 deaths per 100, 000 people. For females, lung cancer has been the leading cause of death from cancer, accounting for 19.71 deaths per 100,000 people. The research into prognostic factors helps clinicians to give personal advice, to choose treatment modalities, and to understand the pathogenesis of the disease. The prognostic factors might be different among the various histologic types of lung cancer. For small cell lung cancer, studies on prognostic factors are limited. It is well known that performance status and extent of disease are important prognostic factors. For laboratory parameters, serum lactate dehydrogenase is frequently reported as a prognostic factor. In contrast, the prognostic factors of non-small cell lung cancer have been widely studied. Those factors could be divided into two categories: tumor-related factors and host-related factors. Among these factors, molecular biologic factors increased rapidly with the progression of genomic and proteomic technologies. However, clinical and pathologic staging according to the TNM system is still the most powerful prognostic factor for non-small cell lung cancer. Although more and more prognosis research data for non-small cell lung cancer have been published, there are still some important issues that should be addressed: most studies are retrospective; the number of significant prognostic factors identified is still small; and study results of most prognostic factors identified were controversial or contradictory. In the previous literature, lung cancer patients with various ages and genders demonstrated different clinical characteristics. Epidemiologic studies of lung cancer have shown geographic and ethnic differences, especially for female lung cancer patients. Therefore, we reviewed and analyzed the clinical data of lung cancer patients at National Taiwan University Hospital in order to characterize lung cancer patients of different age groups and genders in regards to the distribution of histologic types, clinical stages, treatments, and survival. In addition, we tried to combine the molecular biological experiment and clinical data analysis to clarify the value of the known prognostic factor of lung cancer. Through the integration of bioinformatics method and conventional biotechnology, we also try to establish a new research model to find more novel prognostic factors. Our study consists of three sections: gender differences in lung cancer, the research of known prognostic factors for lung cancer, and identification of novel prognostic factors for lung cancer. Gender differences in lung cancer The importance of gender and age differences in lung cancer needs to be elucidated because of its implication in the design of experimental protocols for targeted chemoprevention, early disease screening, molecular markers-based staging, and individualized treatment. This study was designed to characterize lung cancer patients of different age groups and genders in regard to the distribution of histologic types, clinical stages, treatments, and survival. A multivariate analysis was also performed to identify the clinical prognostic factors for lung cancer patients in Taiwan. Patients with a diagnosis of lung cancer admitted to National Taiwan University Hospital between January 1991 and December 1999 were identified from a computer registration database through the ICD-9 coding system. The clinical records of the patients included were reviewed and analyzed for age at diagnosis, gender, family history of malignancy, history of smoking, stages of cancer, histology, treatment modalities, and survival. This study demonstrated the trend evolution of the distribution in histologic cell types and stages of lung cancer patients in Taiwan with respect to different genders and age groups. The annual number of lung cancer patients had a trend of increase from 1991 to 1999. The male to female ratio was constantly 2:1 during this period. The trend of increase was demonstrated in all age groups except for those patients at ages < 40 years old. The trend of increase in lung cancer patients was mainly due to the annually increase of patients with adenocarcinoma. Adenocarcinoma has been shown to be a predominant histologic type in both male and female lung cancer patients. It has become an emerging problem more than the other cell types of lung cancer. Females (73.5%) had a significantly higher percentage of adenocarcinoma than males (42.1%) (p<0.001). For the histological distribution among different age groups, a trend of decrease in adenocarcinoma and a trend of increase in squamous cell carcinoma were demonstrated, as the age increased. A multivariate analysis using Cox forward regression model was conducted for gender, age group, histology, stage, and treatment modalities. The adenocarcinoma group had better survival than other cell type groups. Female lung cancer patients had a better prognosis than males, which occurred mainly at the age of 50-69 years. We considered that physiological change in middle ages, such as change of sex hormone status due to menopause, might influence the prognosis. Further population-based studies on gender and age differences in lung cancer patients will help to reinforce these conclusions. Research of known prognostic factors for lung cancer Determining the prognosis for an individual patient with non-small cell lung cancer is difficult, in part because of the marked clinical heterogeneity of patients. Most patients presenting with one or more potential constellations in progression are, in turn, due to multiple potential manifestations of the primary tumor of involved metastatic sites and of paraneoplastic syndromes. Human telomerase is a ribonucleoprotein that adds repeated units of TTAGGG to the ends of telomeres. Telomerase activity was detected in almost all cases of small cell lung cancer and in 80% of non-small cell lung cancer cases. In contrast, in normal somatic cells, telomerase activity is usually undetectable. Telomerase activity is one of the most important prognostic factors in patients with non-small cell lung cancer. The detection of telomerase might be of little use in predicting the prognosis of lung cancer in patients since most of the patients have positive telomerase activity. Little has been studied regarding the quantitative analysis of telomerase activity and subsequent outcomes. We hypothesized that the quantification of telomerase activity, rather than the detection, would predict the prognosis of lung cancer. Thus, we used a non-radioactive quantitative method to investigate the correlation between the level of telomerase activity and the clinical features in patients with non-small cell lung cancer. Patients who underwent surgery for non-small-cell lung cancer at National Taiwan University Hospital were included as potential subjects. The level of telomerase activity for lung cancer tissue and adjacent non-cancerous tissue obtained at surgery was accessed according to the telomeric repeat amplification protocol (TRAP) method with modifications using a TeloTAGGG Telomerase PCR ELISAplus kit. Clinical and pathologic parameters, including age, gender, histology, pathological staging, tumor invasion, and lymph node metastasis, were evaluated with respect to the level of telomerase activity. The high relative telomerase activity (RTA) group was defined as a RTA level above the median value. Otherwise, patients were categorized in the low RTA group. Prominent telomerase-mediated 6-bp ladders using modified TRAP assay were detected in 58 (85.3%) lung cancer tissues and 21 (30.9%) adjacent non-neoplastic tissues. There was no difference between patient groups of positive and negative telomerase activity with respect to age, gender, tumor invasion, lymph node metastasis, histological type, and pathological staging. The median of the RTA value in non-cancerous tissues was 3.95, whereas that of cancer specimens was 536.95. There was no difference between the groups with respect to age, gender, and histological type of malignancy. However, there was a trend of increasing RTA in relation to the advance of pathological staging, depth of tumor invasion and lymph node metastasis. The difference was statistically significant for patients with stage I disease. The mean disease-free period was longer in the low RTA group than in patients with high RTA in patients with stage I disease. A high level of telomerase activity tended to correlate with a higher risk of recurrence. For patients with stage II, III and IV disease, no statistical significance of overall and disease-free survival difference between the low and high RTA groups could be demonstrated. Using a Cox regression analysis, we found that every 100 units of increase in telomerase activity cause the hazard ratio of death to increase by 13%. The effect did not change even after controlling for other variables such as age, gender, and stage. For the subset of patients with stage I disease, an increase of every 100 units in telomerase activity causes an even higher increase of 33% in the hazard ratio of death. For every 100 units of increase in telomerase activity, there was a 13% increase in the hazard ratio of recurrence for patients with stage I disease. After controlling for age and gender, for every 100-unit increase in telomerase activity, there was a 16% increase in the hazard of disease recurrence. Our findings support the conclusion that the quantification of telomerase activity, rather than its presence, predicts the prognosis of lung cancer patients. This finding is helpful for application of telomerase activity to predict patient outcomes. Our data demonstrated the same trend of increase in telomerase activity as previously reported with the advance of tumor invasion, node metastasis, and pathological staging. These results showed a more detailed increase when using a non-radioactive method. These results indicated that the level of telomerase activity can be a more useful marker for tumor aggressiveness and may distinguish between primary tumors with potential for lymphatic spread and limited local tumors. Further study with a larger case number is warranted to clarify the trend of increase along with the advance of tumor invasion, nodal metastasis and pathological staging. Identification of novel prognostic factors for lung cancer Genome-wide microarray technologies, which are widely used to monitor global gene expression in cancer, have identified numerous differentially expressed genes suggesting that microarray has promised to shed light on the identification of disease markers for clinical use. However, researchers often faced the dilemma of effective utilization of the vast information source gathered through these microarray studies. The unmet needs include how to efficiently translate existing raw microarray data into a workable model to aid in the greater understanding of target prioritization. The combination of in silico biology and empirical methods may circumvent the bottlenecks from microarray profiling to target identifications. In this study, a methodology was first set up to extract useful information from a microarray dataset for identification of new prognostic factors of lung cancer. We hypothesized that those genes showing similar expression patterns in a microarray database, which might belong to the same categories of biological functions, may also have the same impact on clinical outcomes. Microarray profiling of lung adenocarcinoma was performed on Affymetrix Human Genome U133A microarray chips. Since survivin (BIRC5) is a poor prognostic factor for non-small cell lung cancer, we hypothesized those genes exhibit expression profiling similar to survivin might be of clinical prognostic importance for lung cancer. Trophinin (TRO) is one of the best-correlated genes. Considering its role in embryonic implantation with blastocyst invading the endometrium, trophinin may also mediate the invasion process of cancer cells. Immunohistochemical staining of trophinin in lung cancer specimens was also performed. The results were correlated with clinical characteristics and outcomes. Ectopic expression of trophinin and inhibition by small interfering RNA (siRNA), which were monitored for cell invasion abilities, were also attempted. Paraffin sections of lung cancer tissue from 141 patients (41 female and 100 male) were subjected to immunohistological assessment. The proportion of positive trophinin expression was significantly higher for female patients (p=0.003) and patients with an age less than 65 years (p=0.027). No statistically significant correlations were found between trophinin expression and histological type, pathological staging, or lymph node metastasis. For patients with lung adenocarcinoma, the proportion of positive trophinin expression was also significantly higher for female patients (p=0.028) and patients with T1-2 disease (p=0.021). For patients with squamous cell carcinoma, no statistically significant correlations were found between trophinin expression and age, gender, pathological staging, tumor invasion, or lymph node metastasis. We found that among patients with stage I disease, the survival period for individuals without trophinin expression was significantly longer (80.4 vs. 50.2 months, p=0.042). A multivariate analysis, using Cox forward regression model, was conducted for gender, age, histology, type of surgery, together with trophinin expression for patients with stage I disease. The patients with positive trophinin expression had a poor survival after adjusting for other prognostic variables. As for the various histologic types of lung cancer, the difference of survival between patients with and without trophinin expression was statistically significant for adenocarcinoma. The difference for overall survival and disease-free survival period between patients with and without trophinin expression was even more significant in stage I lung adenocarcinoma (overall survival: 78.8 vs. 39.6 months; p=0.006, Figure 3, disease-free survival period: 69.8 vs. 34.5 months; p=0.03, Figure 4). For squamous cell carcinoma, there was no difference between patients with and without trophinin expression. To provide a mechanistic basis for trophinin as a novel poor prognostic factor and to delineate the biological effects of trophinin in cellular invasion, we took two approaches. First, we used MDCK renal epithelial cells as our model system, which is suitable for the cell invasion assay, to establish various trophinin stable clones. These stable clones were used in the invasion assay. Overexpression of trophinin in two different MDCK cell clones resulted in a two-fold increase in invasion ability compared with those of vector control (vehicle) or parental MDCK cells. Second, we employed three different chemical synthesized siRNA to knock down the expression of trophinin in a highly invasive lung adenocarcinoma cell line, CL1-5. Endogenous trophinin RNA was knocked down by 80-90% in all three trophinin siRNAs. Knock down of endogenous trophinin in CL1-5 led to a decrease in invasion ability as compared to the vehicle transfected parental CL1-5 cells. Together, these data are in agreement with the idea that trophinin may promote cell invasion. Survivin and trophinin have similar expression patterns in lung adenocarcinoma microarray dataset, which suggests trophinin and survivin might have some similar characteristics of expression regulation in common. Survivin is an anti-apoptosis gene; however, whether trophinin has a role in anti-apoptosis remains to be determined. Interestingly, trophinin might be an enhancer of cancer invasion/metastasis, suggesting these two genes of different functions leading to the same prognosis of non-small cell lung cancer. As such, further investigation is warranted to clarify the regulation mechanism of trophinin during cancer metastasis as compared with the findings of studies concerning regulation of survivin expression. Through clinical observation and epidemiological analysis, we found significant gender differences in carcinogenesis, treatment response, mortality rates and prognosis. Emerging reports in the literature have provided the biological basis of explanation for the gender differences. However, there are still limitations for the basis. The main reason is that most large-scale studies are retrospective. These studies have some case selection bias and discrepancy in methodology. Prospective studies with population-based screening are necessary for further investigation of gender differences. In Taiwan, more than 90% of female lung cancer patients are non-smokers. Thus, risk factors other than smoking are critical for female lung cancer patients in Taiwan. The important issue for lung cancer carcinogenesis is the following: Why are women more susceptible to lung cancer than men? Factors including cooking, passive smoking, gene polymorphism, hormone factors, and air pollution should be carefully investigated. Complete epidemiological data is helpful to provide insight toward the explanation for such a high proportion of female lung adenocarcinoma in Taiwan. The interaction between genetic risk factors and environmental ones should be investigated. A multiple-center research team would be able to integrate the variety of lung cancer patients, to perform large-scale screening by using biotechnologies, and to identify novel risk and prognostic factors. The major research subjects for prognostic factors of lung cancer are patients with resectable non-small cell lung cancer. For most patients with unresectable non-small cell lung cancer and small cell lung cancer patients who will not undergo operation, much is left to be studied regarding prognostic factors, especially for molecular biological factors. It would be more useful to develop methods for clinicians to obtain prognostic information from small tissue specimen via biopsy or from scanty cells via aspiration. Analysis of the changes of tumor-specific gene products in body fluids, including blood, pleural effusion, or urine, would be highly practical for clinicians in predicting patient outcomes. Our results support the importance of quantitative assay for prognostic factors. Through the emergence of identified prognostic factors, it will be necessary to perform quantitation and correlate with clinical data to confirm prognostic values. In the future, while searching for the novel prognostic factors for lung cancer, it will become a great task to screen candidate genes or gene products from the genomic or proteomic database. We established a research model, in which we used a known prognostic factor as a template, and then searched for genes or gene products with similar expression patterns. The expression of genes or gene products was examined in the tumor specimens. The factors with prognostic values are investigated by experimental methods to clarify the role in cancer invasion/metastasis. Through this methodology, we could identify the novel prognostic genes more efficiently. The prognostic factor identified might have a different function in cell biology from the template gene. We might be able to find the links between different cell signal transduction pathways by molecular biological approaches. The research model is helpful not only for identification of prognostic factors, but also for understanding the mechanism of cancer invasion/metastasis. 壹、中文摘要 --- 5 貳、緒論 --- 12 一、 研究動機及背景 --- 12 1. 肺癌的流行病學及危險因子 --- 12 2. 肺癌的分子生物學及遺傳變異 --- 15 3. 肺癌的診斷與治療 --- 19 二、 研究問題及重要性 --- 25 1. 肺癌的死亡率研究 --- 25 2. 肺癌的預後因子 --- 28 三、 研究的假說與特定目的 --- 33 肺癌的性別差異 --- 33 肺癌已知的預後因子研究 --- 33 尋找新的肺癌預後因子 --- 34 參、研究的方法與材料 --- 35 肺癌的性別差異研究 --- 35 肺癌的端粒酶活性定量研究 --- 36 尋找新的肺癌預後因子 --- 39 肆、結果 --- 43 肺癌的性別差異研究 --- 43 肺癌的端粒酶活性定量研究 --- 46 新的肺癌預後因子Trophinin --- 48 伍、討論 --- 51 肺癌的性別差異研究 --- 51 肺癌的端粒酶活性定量研究 --- 56 新的肺癌預後因子Trophinin --- 61 陸、展望 --- 65 肺癌的性別差異 --- 66 肺癌已知的預後因子研究 --- 68 尋找新的肺癌預後因子 --- 70 柒、論文英文簡述 --- 74 捌、參考文獻 --- 85 玖、圖表 --- 110 壹拾、附錄 --- 136