建立癌症蛋白基因體策略應用於非小細胞肺癌之多重表皮生長因子受體突變定量分析

Abstract

亞洲肺腺癌患者具有較高的表皮生長因子受體(EGFR)活化性突變發生率(主要為L858R點突變和19外顯子鹼基缺失)，含有此突變基因的肺腺癌患者對於標靶藥物酪胺酸激酶抑制劑具有較佳的藥物反應性。目前基因檢測方法已廣泛使用於鑑定肺腺癌患者EGFR突變以進行標靶藥物治療，然而，肺腺癌患者的藥物治療效果具有差異性；且經治療6到12個月後，患者都對標靶藥物產生抗藥性，其中多數肺腺癌患者已被鑑定出具有EGFR T790M抗藥性突變。為進一步了解基因突變鑑定是否已能因應於標靶藥物抑制EGFR突變蛋白質之效力關係，本研究發展一個以質譜技術平台為基礎之蛋白基因體策略，針對野生型與突變型的EGFR蛋白進行鑑定與定量分析。透過突變蛋白質氨基酸序列之生物資訊分析比對，篩選出相對應之蛋白質水解酶，並結合親和性純化EGFR蛋白質方法，以膠體輔助方式進行多重酵素平行水解作用產生適當的野生型及突變型胜肽，再以液相層析串聯質譜儀結合平行反應監測法進行胜肽分析。 本論文針對EGFR活化性突變，如點突變L858R、G719A、外顯子19鹼基缺失 (E746-A750del)、次要突變T790M、與一無突變位點的野生型序列，設計合成胜肽以應用於後續方法開發。透過資料庫比對胜肽所產生的特定碎片離子並於錯誤發現率小於百分之一條件下，可在具有基因外顯子19鹼基缺失的PC9肺癌細胞中，同時準確鑑定出具有突變胜肽與其相對應之野生型。此外亦可於具有L858R突變的H3255及H1975細胞，及有G719A突變的CL97細胞當中，分別鑑定到L858R突變胜肽。為進一步能定量突變型及野生型EGFR的表現量，我們利用含有同位素標定之合成胜肽分別建置其相對應的標準曲線，進行絕對定量質譜分析。根據各突變蛋白質與其相對應之野生型蛋白質質譜分析結果，其偵測極限值為0.06 到0.34 奈克，可定量極限值為 0.2到1奈克之間。我們將此策略結果應用在非小細胞肺癌的細胞株與人源化腫瘤異種移殖的老鼠腫瘤，突變型與野生型EGFR具有伴隨性異質表現情形。在具有外顯子19鹼基缺失的PC9與HCC827，和帶有L858R突變的H3255細胞，以及具有抗藥性突變的CL68 (外顯子19鹼基缺失/T790M) 和CL97 (G719A/T790M) 細胞中，突變型EGFR皆有高度表現，而野生型EGFR有低表現量。相較於前述細胞株，攜有抗酪胺酸激酶抑制劑T790M突變基因的H1975細胞中，突變型與野生型EGFR蛋白質有相同量但是較低的表現量。在源於PC9及CL68的腫瘤中，亦偵測到具有高度表現的突變型EGFR，同時伴隨存在著極少量的野生型態;其中於帶有CL68腫瘤的個別老鼠當中，突變型EGFR表現量有高度差異 (於1毫克蛋白質中含有27到108皮克突變蛋白質)。比對H3255細胞定量結果，源於H3255的老鼠腫瘤中並未偵測到高度表現的EGFR，且突變型與野生型的蛋白質比例也從細胞中測到3倍降至1.5倍。值得注意的是，源於具有抗藥性H1975的腫瘤中偵測到的野生型EGFR具有高度異質性表現。同時，透過此分析策略可於具有L858R基因突變之肺癌病患血漿中鑑定到無突變位點及L858兩種野生型胜肽，亦顯示出此策略方法可能作為非侵入性檢测EGFR蛋白質的潛力。綜合上述實驗結果，此研究所建立的癌症蛋白基因體策略，可準確於細胞與腫瘤中同時鑑定與定量多種EGFR突變蛋白質之表現，未來可進一步評估EGFR突變蛋白質表現量與酪胺酸激酶抑制劑治療適應性之相關性。

Epithelial growth factor receptor (EGFR) activating mutations have been discovered with high frequency in lung adenocarcinoma patients in East Asia. The lung cancer patients harboring an activating mutation, including L858R and exon19 deletion (E746-A750del, Del-19) in EGFR will be beneficial with responsiveness to EGFR tyrosine kinase inhibitors (TKI). Though EGFR gene test is widely used to select patient, however, the efficacy of TKI varied between TKI-treated patients. Furthermore, all patients develop resistance to TKI within 6 months to one year mainly due to acquired secondary mutation (T790M). To study whether gene test is satisfactory to access the efficacy of TKI targeting on EGFR protein level, we developed a mass spectrometry (MS)-based proteogenomics strategy for confident identification and absolute quantitation of wild-type and mutant EGFR protein. Specifically, the strategy integrated in silico analysis of mutant protein sequences for protease selection, affinity purification of EGFR, parallel enzymatic digestions, and liquid chromatography–parallel reaction monitoring–mass spectrometry (LC-PRM/MS) analysis. Synthetic peptides designed from EGFR activating mutations, including L858R, G719A and Del-19 and secondary mutation (T790M) as well as a shared peptide from non-mutation region were prepared for methodology development. The E746-A750 peptide and the corresponding wild type (WT) peptide were confidently identified (FDR<1%) in PC9 cell line which bearing Del-19. The L858R was unambiguously identified in H3255 and H1975 cells, and G719A was identified in CL97 cells. The standard curves for each mutant and WT peptide were constructed using isotopic-labeled peptides for quantification of endogenous peptides. For different mutant EGFR proteins and corresponding wild-type, the limit of detection ranges from 0.06 to 0.34 ng and the limit of quantitation is from 0.2 to 1 ng in LC/MS analysis. By using this strategy, our results revealed concomitant and heterogeneous expressions of mutant and wild-type EGFR proteins in NSCLC cell lines and xenograft tumors. The mutant EGFR proteins were highly expressed in PC9 and HCC827 (Del-19), H3255 (L858R), CL68(Del-19/T790M) and CL97 (G719A/T790M), while equal amount of wild-type and mutant EGFR were detected in the resistant H1975 cells (L858R/T790M). In PC9 and CL68 cell line-derived xenograft tumors, mutant EGFR is the primary form accompanied by a trace amount of wild-type EGFR. In addition, the CL68 derived tumors from different mice had different mutant EGFR expression (27 to 108 pg per mg lysate). Interestingly, though EGFR protein has high expression in H3255 cells, its expression is much lower in corresponding xenograft tumors and the ratio of L858R to WT expression decreased from 3-fold in H3255 cell to 1.5-fold in tumors. Notably, EGFR WT has extremely heterogeneous expression in the tumor derived from the TKI resistant H1975 cells. In the plasma, we detected the L858 wild-type peptide from one EGFR-L858R lung adenocarcinoma patient, indicating the potential of our approach for non-invasive detection of EGFR proteins. Our developed oncoproteogenomics strategy can precisely determine the multiple EGFR somatic mutations at protein level in cell lines and tumors. Further study is required to evaluate the association between expression levels of mutant EGFR proteins and the adaption to EGFR-TKIs treatments.