Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, Japan.
Cancer Med. 2021 Mar;10(6):2003-2012. doi: 10.1002/cam4.3755. Epub 2021 Feb 28.
Circulating tumor DNA (ctDNA) detected before surgery disappears after complete surgical resection of the cancer. Residual ctDNA indicates minimal residual disease (MRD), which is a cause of recurrence. The presence of long-fragment circulating cell-free DNA (cfDNA) or methylated cfDNA also implies the presence of cancer. In this study, we evaluated the prognostic value of cfDNA methylation and long-fragment cfDNA concentration in gastric cancer patients undergoing curative surgery METHODS: Ninety-nine gastric cancer patients were included. Peripheral blood samples were collected before and 1 month after surgery. In patients administered chemotherapy, samples were collected before starting chemotherapy. qPCR was performed to detect long- and short-fragment LINE-1. A plasma HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay to determine the concentration of HpaII small fragments was performed using ligation-mediated PCR and HpaII was quantified as the HpaII:MspI ratio to detect methylation levels of cfDNA.
Overall survival (OS) of patients with low methylation levels before starting treatment was significantly worse than that of patients with high methylation levels (P = 0.006). In the 90 patients who underwent curative surgery, recurrence-free survival (RFS) and OS of patients with low methylation levels before surgery were worse than those with high methylation levels (P=0.08 and P = 0.11, respectively). RFS and OS of patients with high concentrations of long-fragment LINE-1 after surgery were significantly worse than those with low concentrations of long-fragment LINE-1 (P = 0.009, P = 0.04).
Pre-surgical low methylation levels of LINE-1 are a negative prognostic factor. Post-surgical high concentrations of long-fragment LINE-1 indicate MRD and a high risk of recurrence.
手术前检测到的循环肿瘤 DNA（ctDNA）在癌症完全切除后消失。残留的 ctDNA 表示微小残留病（MRD），这是复发的原因。长片段游离细胞 DNA（cfDNA）或甲基化 cfDNA 的存在也意味着癌症的存在。在这项研究中，我们评估了 cfDNA 甲基化和长片段 cfDNA 浓度在接受根治性手术的胃癌患者中的预后价值。
纳入 99 例胃癌患者。在手术前和手术后 1 个月采集外周血样本。在接受化疗的患者中，在开始化疗前采集样本。通过 qPCR 检测长片段和短片段 LINE-1。通过连接介导的 PCR 进行血浆 HELP（HpaII 小片段富集通过连接介导的 PCR）检测，以确定 HpaII 小片段的浓度，使用连接介导的 PCR 并将 HpaII 量化为 HpaII：MspI 比来检测 cfDNA 的甲基化水平。
治疗前低甲基化水平患者的总生存率（OS）明显差于高甲基化水平患者（P=0.006）。在 90 例接受根治性手术的患者中，手术前低甲基化水平患者的无复发生存率（RFS）和 OS 差于高甲基化水平患者（P=0.08 和 P=0.11）。手术后长片段 LINE-1 高浓度患者的 RFS 和 OS 明显差于低浓度患者（P=0.009，P=0.04）。
术前低甲基化水平的 LINE-1 是一个负预后因素。手术后长片段 LINE-1 浓度升高表明 MRD 且复发风险高。
