检测血液中肝癌

StephenW

Cell Research (2015) 25:1279–1280. doi:10.1038/cr.2015.136; published online 24 November 2015

        Detecting hepatocellular carcinoma in blood

Lulu Hu1,2 and Chuan He1,2

• 1Department of Chemistry, Department of Biochemistry and Molecular Biology, and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637, USA
• 2Howard Hughes Medical Institute, The University of Chicago, Chicago, IL 60637, USA
  

Correspondence: Chuan He, E-mail: [email protected]

Liquid biopsy is ideal for early diagnosis of cancer and for prognosis upon treatment. Wen et al. describe a methylated CpG tandems amplification and sequencing method to profile hypermethylated CpG islands genome-widely in cell-free DNA, and further identify high performance markers in blood for potential detection of early stage hepatocellular carcinoma.

Early diagnosis is key to cancer prevention and treatment. When physiological consequences of cancer are observed it could be too late for the optimal treatment and therapy1. Traditional biopsy has been widely used for diagnosis; however, it is difficult to frequently perform biopsy. In many cases it is impossible to perform biopsy of solid tumors grown in deep tissues. Cell-free nucleic acids (cfNAs) offer an alternative option. The presence of cfNAs in blood was described in 1948. However, cfNAs such as DNA, mRNA and microRNAs (miRNAs) were not recognized as potential disease biomarkers until recently because of the rapid advance of sequencing technologies2,3,4. The apoptosis and necrosis of tumor tissues can lead to release of cell-free DNAs (cfDNAs) into the circulating system5; these cfDNAs contain crucial genetic and epigenetic information for early diagnosis if sensitive and accurate methods can be developed.

Human hepatocellular cancer, one of the most lethal cancers, is characterized by progressive accumulation of epigenetic changes6, among which hypermethylation of cancer-associated DNA offers distinct markers for diagnosis. DNA methylation patterns could change throughout the cancer development stages. If the same DNA methylation changes could be monitored in cfDNA released by tumor one could trace the emergence of the cancer, monitor the progression, and predict effects of treatments. Despite these advantages, current cfDNA detection is significantly hampered by the lack of sensitivity because only a very small amount of cfDNA could be obtained from plasma and serum. cfDNA is also heavily fragmented (between 200~400 bp), adding additional challenges.

Faced with these challenges, Wen et al.7 invent methylated CpG tandems amplification and sequencing (MCTA-Seq), a method that takes advantage of the fact that CpG tandems are highly enriched in the CpG island-containing promoters of human genome. These CpGs are typically unmethylated but tend to gain hypermethylation in hepatocellular carcinoma (HCC)6. The cfDNAs released into circulation carry the same hypermethylation patterns, thereby providing accurate information of the presence of HCC in patients. In their new method, cfDNA is treated with bisulfite, during which non-methylated C (cytosine) is converted to U (uracil) while methylated C remains unaffected. They then use a pair of primers to specifically amplify DNA loci that contain hypermethylated CGCGCGG, a sequence frequently presented in CpG islands and tend to be methylated in cancer tissues. The focus on the CGCGCGG-containing loci may miss other potential markers; however, it offers the sensitivity required for methylation detection in cfDNA. Validation data of MCTA-Seq shows that it is highly reproducible and sensitive, with the detection limit down to as low as 7.5 pg (~2.5 haploid genome equivalents). Existing biomarkers that are frequently hypermethylated in human cancers8, such as VIM, SEPT9, NDRG2 and RASSF18, could be detected with high sensitivity by using MCTA-Seq. The method, although limited by the requirement of the CGCGCGG sequence content, is genome-wide and offers sufficient information about CpG island methylation changes in HCC.

Wen et al. applied the new method to detect tumor-specific CGI methylation with plasma samples from HCC patients, cirrhosis patients, and normal individuals. Two types of biomarkers have been identified for early stage HCC diagnosis (Figure 1). Type I markers possess significantly higher methylated CGIs than cancer-free individuals. Type II markers are tissue-specifically methylated CGIs, which tend to be restricted to liver cells under normal circumstances but are released into the blood when malignance occurs. Type II markers dominate in the cfDNA at early stage of HCCs, making them sensitive signs of tumor emergence.

Figure 1.Hypermethylated cfDNA released into the blood can be detected with a new method. Cell-free DNA-containing hypermethylated CpG islands (mCGIs) circulating in the blood of heptocellular carcinoma patients can be detected for early diagnosis. These marker DNAs are released by either tumor cells undergoing apoptosis or necrosis (type I) or adjacent non-cancerous cells affected by tumor growth (type II).
Full figure and legend (44K)

The new method and the use of marker combination shown by Wen et al. provide a new strategy for DNA methylation detection from cfDNA. It may have widely applicable potential not only in HCC but also a cohort of other cancer types.

StephenW

细胞研究（2015）25：1279年至1280年。 DOI：10.1038 / cr.2015.136;网上公布的24月2015年
检测血液中肝癌

露露Hu1,2和川He1,2

    化学，生物化学与分子生物学系，研究所和生物物理动力学，芝加哥大学，芝加哥，伊利诺伊60637，美国教研室
    2Howard休斯医学研究所，芝加哥大学，芝加哥，伊利诺伊60637，USA

函授：传他，电子邮件：[email protected]

液体活检是理想的癌症早期诊断和治疗后预后。 Wen等。描述一个甲基化的CpG串联物扩增和测序方法来分析甲基化CpG岛基因组广泛在无细胞的DNA，并进一步确定在血液高性能标志物的早期肝细胞癌电位检测。

早期诊断是关键，癌症的预防和治疗。当癌症的生理后果的观察到它可能是太晚了最佳的治疗和therapy1。传统的活检已广泛用于诊断;然而，难以频繁执行活检。在许多情况下，它不可能执行在深部组织生长的实体肿瘤的活检。无细胞核酸（cfNAs）提供一种替代选择。 cfNAs在血液中存在于1948年。但是被描述，cfNAs如DNA，基因和微RNA（miRNA）没有被看作是潜在的疾病生物标志，因为测序technologies2,3,4的快速推进，直到最近。凋亡和肿瘤组织的坏死可以导致释放的无细胞的DNA（cfDNAs）到循环系统5;这些cfDNAs如果包含敏感和准确的方法，可开发的关键遗传和表观遗传信息的早期诊断。

人类肝细胞癌，最致命的癌症之一，其特点是后生changes6是逐渐蓄积，其中与癌症相关的DNA甲基化提供了独特的标记进行诊断。 DNA甲基化模式可能会改变整个癌症的发展阶段。如果相同的DNA甲基化的改变可能在cfDNA来监测释放肿瘤人们可以追踪癌症的出现，监测的进展，并预测治疗效果。尽管有这些优点，目前cfDNA检测显著由缺乏敏感性而受到阻碍，因为只有极少量的cfDNA可以从血浆和血清中获得。 cfDNA也严重碎片化（200〜400碱基对），增加了额外的挑战。

面对这些挑战，闻等[7]发明甲基化的CpG串联物扩增和测序（MCTA-SEQ），一种方法是利用这样的事实即CpG的串联物高度富集人类基因组的包含CpG岛的启动子优势。这些的CpG通常未甲基化的，但往往获得甲基化在肝细胞癌（HCC）6。释放到循环cfDNAs携带相同的甲基化模式，从而提供肝癌患者中存在的准确信息。在他们的新方法，cfDNA物用亚硫酸氢盐，在此期间，非甲基C（胞嘧啶）被转换为U（尿嘧啶）而甲基C保持不受影响。然后，他们使用一对引物特异性地扩增包含甲基化CGCGCGG，一个序列频繁出现在CpG岛，而且往往在癌组织中被甲基化DNA的位点。重点对含CGCGCGG位点可能会错过其他潜在的标记;然而，它提供了所需的甲基化检测中cfDNA的灵敏度。 MCTA-SEQ的验证数据表明，它是高度可再现和灵敏，与检测限向下到低至7.5皮克（〜2.5单倍体基因组当量）。现有的生物标志物经常甲基化在人类cancers8，如VIM，SEPT9，NDRG2和RASSF18，能高灵敏度使用交通局-SEQ进行检测。的方法中，虽然限制由CGCGCGG序列含量的要求，是全基因组，并提供关于在HCC CpG岛甲基化的变化的足够信息。

Wen等。应用新的方法来检测肿瘤特异性的CGI甲基化与来自HCC患者，肝硬化患者，和正常人血浆样品。两种类型的生物标志物已确定为早期肝癌诊断（图1）。 I型标志物具有显著高甲基化的CGI比无癌症的个体。 II型标志物是组织特异性甲基化的CGI，倾向于在正常情况下被限制在肝细胞但释放到血液中时发生恶性。第二类指标占主导地位的cfDNA在肝癌的早期阶段，使他们的肿瘤出现敏感症状。
图1。
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释放到血液中高甲基化cfDNA可以用一个新的方法来检测。无细胞含DNA的甲基化CpG岛（mCGIs）流通在肝细胞癌患者的血液可用于早期诊断来检测。这些标记DNA被释放或者肿瘤细胞经历凋亡或坏死（类型I）或受肿瘤生长（II型）相邻的非癌细胞。
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的新的方法和使用标记组合的由闻等人示出。提供DNA甲基化检测从cfDNA的新战略。它可以具有不仅在HCC，还包括其他的癌症类型的队列广泛适用的潜力。
Xìbāo yánjiū (2015)25:1279 N