B 型肝炎帶原之肝細胞癌患者接受肝動脈栓塞治療後血液中 DNA 之
定量分析
Quantitative Analysis of Circulating Plasma DNA in HBV-Positive Patients Following Transcatheter Arterial Embolization (TAE) for Hepatocellular Carcinoma
中文摘要
在大部分的肝癌患者，要完全治癒的機率很低，幾乎所有病人都會面臨腫瘤復 發的問題，使得如何追蹤治療中的病人，找出具有殘存癌細胞的患者成為一個重 要課題。經導管栓塞治療(TAE)是常用的肝癌非手術治療之一，患者接受 TAE 治療後，若是在被栓塞的腫瘤看到lipiodol 分布很完全， 通常可以預測其所造
成的病理性壞死也相當不錯。但是要用電腦斷層追蹤lipiodol 殘存程度，最好
的評估時機必須在TAE 四週以後，而在不完全殘留的腫瘤中殘存的 lipiodol 不 一定與壞死相關，常常會使得醫師在評估腫瘤壞死及復發造成困擾。雖然有人嚐 試用其他方法來評估壞死的嚴重度，仍有其不足之處。
在文獻中已有人提出血漿中β-globin gene 之高低可作為外傷患者疾病嚴重
程度的指標，我們嘗試以接受血管栓塞治療的B 型肝炎表面抗原陽性之肝癌患
者作為組織受傷之模式之一，探討肝臟在缺氧之後血漿中DNA (β-globin
gene 及 HBV DNA ) 之變化及其是否能預測 TAE 效果之好壞。
在西元2001 年四月至六月間，我們事先徵得病人之書面同意之後收集 14 位 B
型肝炎帶原之肝癌患者進行測試。將取得的病人全血經離心、抽取plasma DNA
之後，以real-time quantitative PCR 分析檢體之絕對及相對定量，再與同步 之臨床資料作比較。我們所選用於PCR 之 DNA target 為 HBV DNA 及β -globulin gene. Real-time quantitative PCR 所選用的螢光染劑為 SYBR Green I dye。所有病人在 TAE 後每天抽一次血與未作 TAE 前的 baseline 作 比較。以絕對及相對定量兩種方式作分析。
實驗中HBV DNA 及β-globulin gene 平均的 CV 值 (coefficients of variance) 分別為 1.24% 及 1.19% 。Standard Curve 在 7 個 log 範圍 之 correlation coefficient 可達 0.975。 不管是 HBV DNA 或β-globulin gene，在 TAE 之後是增加的。在有限可評估之病人中，HBV DNA 屬於 delayed peaking 型態的病人，其 1-2 個月後所做 CT 上的 lipiodol retention 較 early peaking 組病人好(79.31±28.79% vs 18.43±10.61 % ，p value : 0.017, power 0.98， student t＇s test )，暗示觀察 TAE 後血漿中 DNA 變化，可
以提早掌握栓塞之效果，具有預測效果。腫瘤之大小與血漿中DNA 增加的模式
無關。
此研究結果可進一步掌握肝癌患者接受TAE 後的血漿中 DNA 濃度之變化情形。
我們希望能藉此發現，不只可以預測TAE 治療的效果，更可作為日後發展用輔 助性治療（如：評估angiogenesis inhibitor 治療效果）之實驗設計之參考。
英文摘要
Background: Most patients with hepatocellular carcinoma would experience recurrence even after curative resection. Transcatheter arterial embolization (TAE) was one of the most important non-surgical therapies for hepatoma. Complete lipiodol retention after TAE often correlated with good pathologic necrosis. But for those with incomplete lipiodol retention, the correlation was not satisfactory. The most
appropriate time for assessing lipiodol retention was 4 weeks after TAE. Sometimes the high attenuation caused by lipiodol on CT confused clinicians in making
distinction between necrosis or recurrence. Although some resolutions have been proposed, most of them are image-based and have limitation in early detection.
Circulating plasma β-globin gene has been proposed as a prognostic marker in trauma patients. We used HBsAg -positive hepatoma patients receiving TAE as a model of injury to follow the changes of circulating plasma DNA (β-globin gene and HBV DNA) after ischemic insults and tried to find whether they could predict the extent of necrosis of TAE at early stage.
Methods: During April and June in 2001, total 14 patients with positive HBs Ag and HCC were studied after written consents. Plasma HBV DNA and β-globin gene were measured by real-time quantitative PCR daily after TAE for 5 consecutive days.
SYBR Green I dye was chosen as double-strand DNA specific dye. Clinical data and concurrent serum α-fetoprotein were analyzed. The DNA levels were analyzed both in absolute and relative quantification.
Results: The mean intra-assay coefficients of variation (CV) were 1.24% and 1.19%
for HBV DNA and β-globin gene respectively. Upon optimization of assay conditions, we were able to obtain a standard curve with linear range (correlation coefficient=
0.975) across at least 7 logs of DNA concentration. Both HBV DNA and β-globin gene increased after TAE during our observation. Changes of HBV DNA after TAE could be divided into two groups: delayed peaking group ( whose HBV DNA level continued rising after the end of 5th day after TAE) and early peaking group (whose HBV DNA level peaked within 5 days after TAE) . The delayed peaking group had better lipiodol retention (79.31±28.79%) than the early peaking group( 18.43±10.61
%，p value : 0.017， power 0.98，by student t’s test) . Tumor size was not related to the pattern of increasing.
Conclusions: Levels of circulating DNA elevated after ischemic insults. Delayed peaking group patients correlated with better lipiodol retention in subsequent CT. The understanding of the pattern of helps us to design related studies ( such as applications
of monitoring plasma tumor-specific DNA in HCC patients receiving TAE or angiognesis inhibitor treatment) more properly.
