肝癌自體抗原之鑑定與生物標記開發

Abstract

肝癌 (hepatocellular carcinoma, HCC) 是台灣最常見的惡性腫瘤之一，位居台灣十大癌症死亡原因的前二位。目前肝癌血液檢查的診斷方式是偵測甲型胎兒蛋白 (alpha-fetoprotein, AFP) 的含量，若 AFP 的量超過 20 ng/mL 則可能罹患肝癌，然而在慢性肝炎病人中肝細胞再生時 AFP 也會增高表特異性不佳，此外腫瘤小於兩公分的肝癌病人 AFP 的靈敏度只有 43 %，顯示 AFP 在肝癌早期無法成為有效的檢測工具，因此尋找有效的生物標記作為診斷肝癌早期的指標是非常重要的。腫瘤細胞異常表現的蛋白質會引發身體的免疫反應產生自體抗體 (autoantibody)，這些引起免疫反應的蛋白質稱為腫瘤相關抗原 (tumor-associated antigen, TAA)，過去利用血清蛋白質體分析 (serological proteome analysis, SERPA) 鑑定到數個可能為肝癌 TAA 的蛋白質，挑選肝癌血清反應較高的蛋白質 hnRNP L、DEAD、lamin A/C 與 hnRNP B1 作確認。由一維電泳免疫轉染法發現肝癌病人血清與 hnRNP L 及 DEAD 反應頻率較高，分別為 64.5 % 和 67.7 %，與正常人、B型肝炎帶原者及肝硬化病人血清比較都有顯著差異，p value 小於 0.01。肝癌病人血清與 hnRNP L 或 DEAD 有反應的頻率是 85.5 % 特異性為 45 %。肝癌病人血清與 hnRNP L 和 DEAD 同時有反應的頻率是 46.8 % 特異性為 95 %。利用酵素連結免疫吸附分析 (enzyme-linked immunosorbent assay, ELISA) 發現肝癌病人血清對具有抗原決定部位 (epitope) 之短肽 hnRNP L 反應頻率較好，與其他三組血清比較 p value 小於 0.01。為了實行多重生物標記偵測，初步利用 Illumina Veracode Carboxyl Beads 作為 multiplexed protein assay，發現肝癌病人血清與短肽 hnRNP L 反應的頻率是 67.0 %，與其他三組血清比較 p value 小於 0.01。這些結果顯示 hnRNP L 與 DEAD 可為肝癌的 TAA，且短肽 hnRNP L 較適合作為肝癌生物標記。

Hepatocellular carcinoma (HCC), one of malignant tumors in Taiwan, is the top two causes of cancer death. Serum alpha-fetoprotein (AFP), at a cutoff value of 20 ng/mL, is currently the most widely used tumor marker for diagnosis of HCC. However, some patients with cirrhosis or hepatic inflammation can have an elevated AFP. Furthermore, sensitivity of AFP decreases to 43 % when tumor diameter is less than 2 cm. It shows that AFP can’t be an effective early detection tool for HCC so it’s important to find valid biomarkers as early indicators of HCC. Abnormal proteins, also known as tumor-associated antigens (TAAs), expressed by tumor cells would trigger the immune response to generate autoantibodies. Many TAAs were identified by serological proteome analysis (SERPA) and four proteins, hnRNP L, DEAD, lamin A/C, and hnRNP B1, were selected to do further research. Using 1D immunoblot, hnRNP L and DEAD were identified as HCC-related antigens, showing a higher seropositivity, 64.5 % and 67.7 %, in HCC patients than in controls, HBV-carrier, or cirrhosis patients. In addition, combination of hnRNP L or DEAD showed a high seropositivity, 85.5 %, in HCC patients, and specificity is 45 %. Combination of hnRNP L and DEAD showed a high specificity, 95 %, and sensitivity is 46.8 %. It also showed that the peptide epitope of hnRNP L had a higher seropositivity in HCC patients than other three groups by enzyme linked immunosorbent assay (ELISA). For detecing multiple biomarkers, Illumina Veracode Carboxyl Beads as multiplexed protein assay was used to screen the peptide of hnRNP L, and results indicated that the seropositivity is 67 % in HCC patients. In brief, hnRNP L and DEAD may be HCC-related antigens, and the epitope of hnRNP L is better to be a biomarker for early diagnosis of HCC.