2013-01-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/646857摘要： 肺癌是癌症死因的第一名。近年來標靶治療的使用，以及對癌症致病基因的研究愈來愈透徹，使得肺癌個人化醫療愈受重視。肺癌表皮生長因子受體（EGFR）突變，與病人使用EGFR tyrosine kinase inhibitor (TKI) 的效果有相當好關聯性。另外，腫瘤有EML4-ALK轉位對crizotinib－ALK抑制劑有相當好的療效。這些突變的蛋白質會促成癌細胞產生、生長以及存活，如果這些突變的蛋白質被抑制後，癌細胞就會死亡。這觀念稱作「driver mutation and oncogene addiction」。除了上述兩種突變，肺癌細胞的致癌基因突變，仍可見於k-Ras, HER2, B-Raf, PIK3CA, AKT2, MEK1, …等。但這些突變可能有如EGFR突變一般，有人種的差異。台灣地區需要有一個廣泛性的在地研究。肺癌病患診斷時多為晚期不能開刀，臨床上診斷檢體的取得是靠小切片或細胞學診斷。必須能從微小的臨床診斷檢體，測出致癌基因的突變。但是腫瘤中通常混雜著癌細胞及各種基質（stroma）細胞或發炎細胞，造成DNA突變檢測率偏低。因為正常細胞表現致癌突變基因的RNA量極低，而癌細胞則大量表現，使用RNA做基因突變檢測，比起用DNA檢測，可將正常細胞的干擾降到最低，可增加檢測敏感度。所以本計劃以微小的臨床檢體，包括細針抽取或切片、支氣管切片或刷取及沖洗細胞、肋膜積液細胞等，利用RNA方法，對致癌基因的突變做一廣泛的檢測。目前收集了427個肺癌檢體，主要是肺腺癌。已經建立RT-PCR以及基因定序方法，包括exons18-21 of EGFR, exons 1-2 of K-Ras, exons18-21 of Her2, exons 11–15 of B-Raf, exon 9 and exon 20 of PIK3CA, MEK1 exon 2, and exons 9-12 of AKT2 以及EML4-ALK translocaion。完成定序的有400個檢體，其中部分RNA量不足，部分基因無法完成RT-PCR 及定序。定序結果mutation rates 分別為153/400, 17/400, 9/393, 13/389, 11/385, 2/385, 1/379 以及41/392。因為除EGFR外之突變發生率仍低，需要收集個多檢體繼續研究，了解台灣地區對上述致癌基因突變發生率，以及各致癌基因突變之肺癌病人之臨床特徵及預後。希望能為將來台灣肺癌個人化醫療，奠定基礎。<br> Abstract: Lung cancer is the leading cause of cancer-related death worldwide. Most patients have advanced disease at the time of diagnosis. The concept of personalized cancer therapy has attracted much attention. Recently, a strong association of somatic mutations in the tyrosine kinase domain of EGFR with clinical efficacy to EGFR tyrosine kinase inhibitors (TKIs) has been demonstrated. Another example is that crizotinib (PF-02341066), a dual MET/ALK tyrosine kinase inhibitor, has a favorable treatment response in EML4-ALK positive patients with advanced lung cancer.These mutations occur in genes that encode signaling proteins critical for cellular proliferation and survival. Mutant oncogenes drive tumor formation and maintenance. This concept is called driver mutation and oncogene addiction. In addition to EGFR and EML4-ALK, there are other driver mutations found in lung cancer, such as: K-Ras, HER2, B-Raf, PIK3CA, AKT2, MEK1...etc. Those driver mutations have important roles in lung cancer carcinogenesis of lung cancer and may have different distribution among different ethnicities. Little information is known about the mutation rates of various driver genes except of EGFR in lung cancer patients of Taiwan. Comprehensive and concurrent analysis of these known recurrent driver mutations in a large cohort of advanced lung cancer is necessary. However, even in prospectively conducted clinical trials, only about 30% of the patients had specimens available for gene testing. The reason for the difficulty of tissue procurement is that the majority of the lung cancer patients are in an advanced stage. Diagnostic minute tissue samples of lung cancer (such as needle biopsy, aspiration cytology or effusion cytology), which might be the only diagnostic material, are used for gene testing. Because the non-tumor cells do not express oncogenic proteins and RNAs, using RNA instead of genomic DNA as the source for sequencing could minimize the influence of nontumor cells. The use of RNA as template is an effective approach to improve sensitivity of mutations gene testing for comprehensive and concurrent analysis of driver oncogenes in heterogeneous and minute diagnostic samples.In this project, we use clinical diagnostic minute samples using multiplex RT-PCR and direct sequencing to detect the driver mutation and identify multiple molecularly distinct subsets of lung cancer in Taiwan. In the first part of this project, we have collected 427 lung cancer diagnostic samples. Most of them are adenocarcinoma. We have established the methods of detection the mutations of exons18-21 of EGFR, exons 1-2 of K-Ras, exons18-21 of Her2, exons 11–15 of B-Raf, exon 9 and exon 20 of PIK3CA, MEK1 exon 2, and exons 9-12 of AKT2 and EML4-ALK translocations. We have completed the sequencing of 400 samples. A tiny portion of RT-PCR failed due to the little amount of RNA. However, the mutation rates of those genes are 153/400, 17/400, 9/393, 13/389, 11/385, 2/385, 1/379 and 41/392 in the current status. We will continue the collections and genetic analysis of the samples and hopefully we can pave the way for personalized clinical trials in lung cancer.肺癌致癌基因突變臨床微小的檢體Lung cancerOncogeneMutationsMinute samples