發展甲基化快篩檢測工具用於肝癌之偵測：試驗性研究

Developing a fast-screening DNA methylation test for detection of hepatocellular carcinoma: A pilot study

朱育萱(Yu-Hsuan Chu)

指導教授 ： 于明暉

國立臺灣大學/公共衛生學院/流行病學與預防醫學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201602549

摘要

研究背景：肝癌目前仍是全球高致死率的癌症之一，且約有75％肝癌可歸因於慢性肝炎病毒感染所致，因此針對B型肝炎病毒帶原者發展出一套早期篩檢工具是肝癌防治上重要的目標。MethyLight是一套高敏感度且可快速大量偵測檢體的甲基化定量檢測技術，被認為是具有發展成常規臨床篩檢工具的技術之一。因此，本研究的目標是以一個肝癌特異性的甲基化生物標記基因結合MethyLight檢測技術的原理，發展一套甲基化快篩檢測工具的藍本用於肝癌之偵測。
材料與方法：研究選用的甲基化候選基因是根據先前研究團隊在全基因體肝癌分析中所找尋到且已應用焦磷酸定序法驗證成功的周邊白血球甲基化基因群裡挑選。利用即時定量PCR來偵測經亞硫酸鹽轉換過後的DNA甲基化狀態，並以十倍序列稀釋實驗將DNA從25ng稀釋至2.5pg評估MethyLight的擴增效率與敏感度；同時選出24對肝癌病例手足配對的檢體來測試MethyLight實際應用之表現，並計算出每個樣本的甲基化百分比，再將此結果與先前研究中焦磷酸定序法的結果進行斯皮爾曼等級相關係數分析。
研究結果：在研究中挑選出的合適引子對與螢光探針，依甲基化位點不同，擴增效率介於93%∼99%。此檢測工具可偵測到的甲基化DNA極限為0.025 ng，且可正確辨別甲基化與未甲基化片段，而Ct值在組間與組內的變異係數分別介於0.08%∼0.46%和0.03%∼1.3%之間；此外MethyLight所計算出的甲基化百分比與焦磷酸定序法測得的甲基化程度相比具有顯著性相關(斯皮爾曼等級相關係數分析： 0.43~0.49)。
結論：MethyLight是可能可做為定量偵測甲基化異常生物標記的臨床快篩檢測工具，然而此研究建立出的肝癌甲基化快篩檢測工具僅為雛形，未來仍需更多後續研究以評估臨床上應用的可行性。

關鍵字

肝癌 ； DNA甲基化生物標記 ； 甲基化檢測技術

並列摘要

Background Aim：Hepatocellular carcinoma (HCC) remained a highly fatal malignancy, and chronic hepatitis B virus (HBV) infection accounts for approximately 75% of the liver cancer deaths worldwide. It is thus urgent to develop a screening test for early detection of HCC among HBV carriers. MethyLight is a sensitive, high-throughput assay for quantitation of DNA methylation, which has the potential to be applied in routine clinical testing. In this study, we aimed to conduct a pilot study on the development of a fast-screening DNA methylation test based on the MethyLight technology for detection of HCC.
Materials Methods：A candidate DNA methylation biomarker was selected according to an epigenome-wide association analysis of HCC on peripheral leukocyte DNA with the Infinium HumanMethylation 450 BeadChip array and experimentally validated by pyrosequencing. We used real-time polymerase chain reaction (PCR) on bisulfite-treated genomic DNA to detect methylation status on selected methylation sites. A ten-fold serial dilution experiment from 2.5 pg to 25 ng was conducted to assess the efficiency and detection limit of the assay. Percent methylated reference (PMR) was calculated for each sample as the index of methylation degree. In samples from 24 case-sibling pairs, to evaluate the performance of our assay we compared the results between MethyLight and pyrosequencing, which was used as a reference method for the validation of the newly developed method.
Results：On the basis of the selected optimal primer sets and probe, the PCR efficiencies ranged from 93%~99%, depending on targeted CpG loci. This assay was capable of detecting methylation degrees at 0.025 ng DNA and discriminating between methylated and unmethylated DNA. The inter-assay and intra-assay coefficients of variance of the Ct values ranged from 0.08% to 0.46% and 0.03% to 1.3%, respectively. There was a significant correlation between PMR values and the methylation degrees derived from pyrosequencing (spearman correlation coefficients: 0.43~0.49).
Conclusions：The MethyLight may be a useful tool as a fast-screening test for quantification of clinically relevant aberrant methylation. Our developed method needs further evaluation before application.

並列關鍵字

Hepatocellular carcinoma ； DNA methylation biomarker ； MethyLight

參考文獻

1. Torre, L.A., et al., Global cancer statistics, 2012. CA Cancer J Clin, 2015. 65(2): p. 87-108.

2. Ferlay, J., et al., Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer, 2015. 136(5): p. E359-86.

3. Perz, J.F., et al., The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J Hepatol, 2006. 45(4): p. 529-38.

4. Chen, C.H., et al., Estimation of seroprevalence of hepatitis B virus and hepatitis C virus in Taiwan from a large-scale survey of free hepatitis screening participants. J Formos Med Assoc, 2007. 106(2): p. 148-55.

5. Nguyen, V.T., M.G. Law, and G.J. Dore, Hepatitis B-related hepatocellular carcinoma: epidemiological characteristics and disease burden. J Viral Hepat, 2009. 16(7): p. 453-63.