利用次世代定序法探討腸癌病人治療之抗藥性機轉

Abstract

結腸直腸癌(CRC)在台灣是最常見的癌症之一，預估每年有14000例的新增病和5000位死亡個案。早期的結腸直腸癌(第一和二期)藉由外科手術就有很高的治癒率，而在第三期只接受外科手術其復發率大約有50%，大部分的病患因癌症的轉移而死亡。EGFR抑制劑是標的藥物而且被限定使用在野生型KRAS的基因型。KRAS的突變在轉移性結腸直腸癌病患的發生率大約為35%~45%，而且導致EGFR下游路徑的活化。分子診斷來檢測KRAS基因的變異是現今最被使用的，而且腫瘤組織是由不同成分組成包含腫瘤細胞、非癌化細胞、發炎細胞以及間質細胞，因此我們在正常參考病患中使用次世代定序法來偵測突變序列當作背景值，並用Basic Local Alignment Search Tool (BLAST) 和Bowti2的序列比對工具進行資料分析。結果指出Bowti2結合Integrative Genomics Viewer (IGV)在資料分析上是一個比BLAST快速、靈敏以及準確的組合。大腸癌的100個病患中我們實驗結果是有29位病患找到密碼子12突變(61.70%)，有17位病患找到密碼子13突變(36.17%)然而有1位病患找到密碼子14突變(2.13%)。這也顯示出突變後被Aspartate取代的最多。在分子層面，KRAS突變導致GTPase本身的活性下降，進而促使KRAS本身和下游訊號路徑永久性的活化，因此傳達惡性的形成。這些單一胺基酸的替換是位在密碼子12以及13，然而KRAS突變也少頻率的發生在密碼子61和146。因此KRAS突變(突變的密碼子12、13、61和146)是一個對EGFR治療產生抗性的預測指標。藉由高通量定序法的策略已經可以依照各個腫瘤的特性為結腸直腸癌的病患訂定客製化的治療。臨床醫師可以從晚期病患的腫瘤活檢中獲得對治療產生抗藥性的臨床相關訊息，我們利用次世代定序法鑑定有潛能的生物指標就如同為結腸直腸癌的患者提供正確的治療方法。

Colorectal cancer (CRC) is one of the most common cancers in Taiwan with an estimated 14,000 new cases and 5,000 deaths each year. While early stage CRC (stage I and II) has a high cure rate after surgery, the recurrence rate is about 50% for stage III CRC after surgery alone and most patients with metastatic disease will ultimately succumb to their cancer. EGFR inhibitors represent “targeted agents” and their useis limited to have wild type KRAS genotype. KRAS mutations occur in approximately 35%-45% of mCRC patients, and lead to the constitutive activation of EGFR downstream pathways. Molecular biological methods for detecting variations of the KRAS gene are now used. Solid tumors are very heterogeneous, containing tumor cells, nonneoplastic cells, inflammatory cells, and mesenchymal cells. Therefore, we use next generation sequencing (NGS) to detect somatic variants at low frequencies in normal reference patients, and regard results as background. We also compare alignment tools(Basic Local Alignment Search Tool (BLAST) and Bowtie2) to analysis NGS data. Our comparison shows that Bowtie 2 combines Integrative Genomics Viewer (IGV) to achieve a combination of high speed, sensitivity and accuracy than BLAST. our result showed 46% KRAS mutation rate in 100 colorectal cancer, and mutations are detected in codons 12 (63.70%), 13 (36.17%) and 14(2.13%)On the molecular level, KRAS mutations result in reduced intrinsic GTPase activity, which in turn leads to permanent activation of KRAS itself and downstream signalling pathways, thereby mediating malignant transformation. These single amino acid substitutions typically affect hotspots at codons 12 and 13. However, KRAS mutations also occur in codons 61, and 146 at low frequencies. KRAS mutations (mutationat codons 12 ,13, 61 or 146) are predictive of treatment resistance to EGFR therapy. The mechanism of the sensitivity of oxaliplatin was through down-regulation of excision repair cross-complementation group 1 (ERCC1) which was a predictor of oxaliplatin resistance. Personalized CRC patient treatment based on characterizing the individual tumors by high throughput sequencing strategies has been attempted. we utilized next generation sequencing technologies (NGS) to identify potential biomarkers so as to identify treatment options for patients with mCRC.