Danese Elisa, Minicozzi Anna Maria, Benati Marco, Montagnana Martina, Paviati Elisa, Salvagno Gian Luca, Lima-Oliveira Gabriel, Gusella Milena, Pasini Felice, Lippi Giuseppe, Guidi Gian Cesare
Laboratory of Clinical Biochemistry, Department of Life and Reproduction Sciences, University Hospital of Verona, Verona, Italy.
National Centre for Bowel Research and Surgical Innovation (NCBRSI), Academic Surgical Unit, Barts and The London NHS Trust, Queen Mary University of London, London, United Kingdom.
PLoS One. 2015 May 6;10(5):e0126417. doi: 10.1371/journal.pone.0126417. eCollection 2015.
Although recent advances in circulating DNA analysis allow the prediction of tumor genomes by noninvasive means, some challenges remain, which limit the widespread introduction of cfDNA in cancer diagnostics. We analyzed the status of the two best characterized colorectal cancer (CRC) genetic and epigenetic alterations in a cohort of CRC patients, and then compared the degree to which the two patterns move from tissue to plasma in order to improve our understanding of biology modulating the concordance between tissues and plasma methylation and mutation profiles.
Plasma and tumor tissues were collected from 85 patients (69±14 years, 56 males). KRAS and SEPT9 status was assessed by allele refractory mutation system quantitative PCR and quantitative methylation-specific PCR, respectively. Six of the most common point mutations at codon 12 and 13 were investigated for KRAS analysis.
KRAS mutations and SEPT9 promoter methylation were present in 34% (29/85) and in 82% (70/85) of primary tumor tissue samples. Both genetic and epigenetic analyses of cfDNA revealed a high overall concordance and specificity compared with tumor-tissue analyses. Patients presenting with both genetic and epigenetic alterations in tissue specimens (31.8%, 27/85) were considered for further analyses. The median methylation rates in tumour tissues and plasma samples were 64.5% (12.2-99.8%) and 14.5% (0-45.5%), respectively. The median KRAS mutation load (for matched mutations) was 33.6% (1.8-86.3%) in tissues and 2.9% (0-17.3) in plasma samples. The plasma/tissue (p/t) ratio of SEPT9 methylation rate was significantly higher than the p/t ratio of KRAS mutation load, especially in early stage cancers (p=0.0108).
The results of this study show a discrepant rate of epigenetic vs. genetic alterations moving from tissue to plasma. Many factors could affect mutation cfDNA analysis, including both presence of tumor clonal heterogeneity and strict compartmentalization of KRAS mutation profile. The present study highlights the importance of considering the nature of the alteration when analyzing tumor-derived cfDNA.
尽管循环DNA分析的最新进展使得通过非侵入性手段预测肿瘤基因组成为可能，但仍存在一些挑战，这限制了游离DNA（cfDNA）在癌症诊断中的广泛应用。我们分析了一组结直肠癌（CRC）患者中两种特征最明显的CRC基因和表观遗传改变的状况，然后比较了这两种模式从组织转移到血浆中的程度，以加深我们对调节组织与血浆甲基化及突变谱一致性的生物学机制的理解。
收集了85例患者（年龄69±14岁，男性56例）的血浆和肿瘤组织。分别采用等位基因特异性突变系统定量PCR和定量甲基化特异性PCR评估KRAS和SEPT9的状态。对KRAS分析研究了密码子12和13处6种最常见的点突变。
原发性肿瘤组织样本中KRAS突变和SEPT9启动子甲基化的发生率分别为34%（29/85）和82%（70/85）。与肿瘤组织分析相比，cfDNA的基因和表观遗传分析均显示出较高的总体一致性和特异性。对组织标本中同时存在基因和表观遗传改变的患者（31.8%，27/85）进行了进一步分析。肿瘤组织和血浆样本中的甲基化率中位数分别为64.5%（12.2 - 99.8%）和14.5%（0 - 45.5%）。组织中KRAS突变负荷（匹配突变）的中位数为33.6%（1.8 - 86.3%），血浆样本中为2.9%（0 - 17.3）。SEPT9甲基化率的血浆/组织（p/t）比值显著高于KRAS突变负荷的p/t比值，尤其是在早期癌症中（p = 0.0108）。
本研究结果显示了从组织到血浆的表观遗传与基因改变的差异率。许多因素可能影响cfDNA突变分析，包括肿瘤克隆异质性以及KRAS突变谱的严格分区化。本研究强调了在分析肿瘤来源的cfDNA时考虑改变性质的重要性。
