高度特异性液滴数字 PCR 检测子宫内膜癌中普遍存在的循环肿瘤 DNA 甲基化。

Highly Specific Droplet-Digital PCR Detection of Universally Methylated Circulating Tumor DNA in Endometrial Carcinoma.

机构信息

Centre de Recherche des Cordeliers, « Equipe labélisée Ligue Contre le Cancer », CNRS SNC 5096, Sorbonne Université, Université de Paris, INSERM, Paris, France.

Department of Medical Oncology, Hopital Cochin, Institut du Cancer Paris CARPEM, AP-HP, APHP Centre, Paris, France.

出版信息

Clin Chem. 2022 Jun 1;68(6):782-793. doi: 10.1093/clinchem/hvac020.

DOI:10.1093/clinchem/hvac020

PMID:35323926

Abstract

BACKGROUND

No circulating biomarker is available for endometrial carcinoma (EC). We aimed to identify DNA positions universally hypermethylated in EC, and to develop a digital droplet PCR (ddPCR) assay for detection of hypermethylated circulating tumor DNA (meth-ctDNA) in plasma from patients with EC.

METHODS

DNA positions hypermethylated in EC, and without unspecific hypermethylation in tissue/cell types releasing circulating cell-free DNA in plasma, were identified in silico from TCGA/Gene Expression Omnibus (GEO) data. A methylation-specific ddPCR (meth-ddPCR) assay following bisulfite conversion of DNA extracted from plasma was optimized for detection of meth-ctDNA according to dMIQE guidelines. Performances were validated on a retrospective cohort (n = 78 tumors, n = 30 tumor-adjacent tissues), a prospective pilot cohort (n = 33 stage I-IV patients), and 55 patients/donors without cancer.

RESULTS

Hypermethylation of zinc finger and SCAN domain containing 12 (ZSCAN12) and/or oxytocin (OXT) classified EC samples from multiple noncancer samples with high diagnostic specificity/sensitivity [>97%; area under the curve (AUC) = 0.99; TCGA/GEO tissues/blood samples]. These results were confirmed in the independent retrospective cohort (AUC = 0.99). Meth-ddPCR showed a high analytical specificity (limit of blank = 2) and sensitivity (absolute lower threshold of detection = 50 pgmethDNA/mLplasma). In the pilot cohort, meth-ctDNA was detected in pretreatment plasma samples from 9/11 and 5/20 patients with advanced and non-advanced EC, respectively. 2 of 9 patients had ctDNA detected after macroscopic complete surgery and experienced progression within 6 months. No healthy donors had any copy of hypermethylated DNA detected in plasma.

CONCLUSIONS

Meth-ddPCR of ZSCAN12/OXT allows a highly specific and sensitive detection of ctDNA in plasma from patients with EC and appears promising for personalized approaches for these patients.

摘要

背景

目前尚无用于子宫内膜癌（EC）的循环生物标志物。我们旨在鉴定 EC 中普遍存在高甲基化的 DNA 位置，并开发用于检测来自 EC 患者血浆中高甲基化循环肿瘤 DNA（meth-ctDNA）的数字液滴 PCR（ddPCR）检测方法。

方法

从 TCGA/Gene Expression Omnibus（GEO）数据中进行了计算机预测，以鉴定在 EC 中高甲基化且在释放血浆中游离细胞循环 DNA 的组织/细胞类型中无非特异性高甲基化的 DNA 位置。根据 dMIQE 指南，优化了从血浆中提取的 DNA 经亚硫酸氢盐转化后进行甲基化特异性 ddPCR（meth-ddPCR）检测的方法，以检测 meth-ctDNA。在回顾性队列（n=78 个肿瘤，n=30 个肿瘤相邻组织）、前瞻性试点队列（n=33 例 I-IV 期患者）和 55 例无癌症患者/供体中对性能进行了验证。

结果

锌指和 SCAN 结构域包含 12（ZSCAN12）和/或催产素（OXT）的高甲基化将来自多个非癌症样本的 EC 样本分类，具有高诊断特异性/敏感性[>97%；曲线下面积（AUC）=0.99；TCGA/GEO 组织/血液样本]。在独立的回顾性队列中也得到了证实（AUC=0.99）。Meth-ddPCR 显示出高分析特异性（空白限制=2）和灵敏度（绝对检测下限=50pgmethDNA/mLplasma）。在试点队列中，在高级和非高级 EC 患者的预处理血浆样本中分别检测到 11 名和 20 名患者中的 9/11 和 5/20 名患者的术前血浆中存在 meth-ctDNA。9 名患者中有 2 名在肉眼完全手术后检测到 ctDNA，并在 6 个月内发生进展。没有健康供体在血浆中检测到任何高甲基化 DNA 拷贝。