Foundation Health Research Institute-Fundación Jiménez Díaz University Hospital, Madrid 28040, Spain.
Department of Surgery, School of Medicine, Universidad Autónoma de Madrid, Madrid 28029, Spain.
World J Gastroenterol. 2017 Oct 21;23(39):7087-7097. doi: 10.3748/wjg.v23.i39.7087.
To assess G12D mutation detection by droplet digital PCR (ddPCR) in stool-derived DNA from colorectal cancer (CRC) patients.
In this study, tumor tissue and stool samples were collected from 70 patients with stage I-IV CRC diagnosed by preoperative biopsy. mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffin-embedded (FFPE) tumor tissues. The G12D mutation was then analyzed by ddPCR in FFPE tumors and stool-derived DNA from patients with this point mutation. Wild-type (WT) tumors, as determined by pyrosequencing, were included as controls; analysis of FFPE tissue and stool-derived DNA by ddPCR was performed for these patients as well.
Among the total 70 patients included, mutations were detected by pyrosequencing in 32 (45.71%), whereas 38 (54.29%) had WT tumors. The frequency of mutations was higher in left-sided tumors (11 located in the right colon, 15 in the left, and 6 in the rectum). The predominant point mutation was G12D (14.29%, = 10), which was more frequent in early-stage tumors (I-IIA, = 7). In agreement with pyrosequencing results, the G12D mutation was detected by ddPCR in FFPE tumor-derived DNA, and only a residual number of mutated copies was found in WT controls. The G12D mutation was also detected in stool-derived DNA in 80% of all fecal samples from CRC patients with this point mutation.
ddPCR is a reliable and sensitive method to analyze G12D mutation in stool-derived DNA from CRC patients, especially at early stages. This non-invasive approach is potentially applicable to other relevant biomarkers for CRC management.
评估粪便来源的结直肠癌（CRC）患者 DNA 中 G12D 突变的数字液滴 PCR（ddPCR）检测。
本研究共纳入 70 例经术前活检诊断为 I-IV 期 CRC 的患者，收集肿瘤组织和粪便样本。通过对福尔马林固定石蜡包埋（FFPE）肿瘤组织获得的 DNA 进行焦磷酸测序分析，确定突变状态。然后，通过 ddPCR 分析这些患者点突变的 FFPE 肿瘤和粪便来源的 DNA 中 G12D 突变。野生型（WT）肿瘤通过焦磷酸测序确定，作为对照；对这些患者的 FFPE 组织和粪便来源的 DNA 也进行了 ddPCR 分析。
在总共纳入的 70 例患者中，32 例（45.71%）通过焦磷酸测序检测到 突变，而 38 例（54.29%）WT 肿瘤。左侧肿瘤的 突变频率较高（右结肠 11 例，左结肠 15 例，直肠 6 例）。主要的点突变是 G12D（14.29%，n=10），在早期肿瘤（I-IIA）中更为常见（n=7）。与焦磷酸测序结果一致，ddPCR 检测到 FFPE 肿瘤衍生 DNA 中的 G12D 突变，WT 对照中仅检测到少量突变拷贝。在所有携带该点突变的 CRC 患者粪便样本中，80%的样本中均检测到粪便来源的 G12D 突变。
ddPCR 是一种可靠且敏感的方法，可分析 CRC 患者粪便来源的 DNA 中的 G12D 突变，特别是在早期阶段。这种非侵入性方法可能适用于 CRC 管理的其他相关生物标志物。
