PO.CL01.23 · 临床研究
基于DNA甲基化的模型预测可切除NSCLC患者的隐匿性淋巴结转移
DNA methylation-based model predicts occult lymph nodal metastasis for resectable NSCLC patients
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摘要 Abstract
中文摘要
背景：携带病理性淋巴结转移（LNDM）（pN1-2）的非小细胞肺癌（NSCLC）患者复发风险显著更高。然而，10%~15%经术前CT分期为cN0的患者经病理评估证实存在淋巴结受累。支气管内超声引导下经支气管针吸活检（EBUS-TBNA）可改善术前淋巴结分期，但对多个站点的广泛采样会增加操作风险和复杂性。因此，需要探索LNDM生物标志物。本研究旨在开发一种基于肿瘤DNA甲基化的模型来预测胸内淋巴结转移，从而更精确地界定接受EBUS-TBNA评估的患者人群。
方法：回顾性收集68例可切除NSCLC患者的原发肿瘤组织及配对的术前血浆（pN0=24，pN1=24，pN2=20，所有pN2均为纵隔淋巴结转移）。提取肿瘤DNA并进行酶转化和甲基化测序（EM-Seq）。通过比较pN0与pN1/2（Wald检验）识别与淋巴结受累相关的差异甲基化CpG位点。使用LASSO回归构建淋巴结转移甲基化评分，并通过ROC分析评估其性能。对定位于启动子的差异甲基化位点进行基因本体（GO）和通路富集分析。该模型在术前血浆游离DNA上的进一步验证正在进行中。
结果：揭示了pN0与pN1/2肿瘤之间6,745个差异甲基化CpG位点（p<0.05）。一个8-CpG甲基化评分以0.962的AUC区分有无淋巴结转移的患者（截断值2.0；敏感性1.0；特异性0.841）。甲基化评分随病理淋巴结分期升高而增加（中位数pN0=0.76，pN1=2.74，pN2=3.30，pN1对比pN0 p<0.01，pN2对比pN0 p<0.01，pN2对比pN1 p=0.26）。未发现与原发肿瘤大小的相关性（r=-0.08，p=0.505），使其仅与LNDM相关。在差异甲基化位点中，28%（1,872个位点）位于启动子区域。在淋巴结阳性肿瘤中，富集的GO条目包括上皮细胞极性和细胞-细胞黏附，而淋巴结阴性肿瘤则表现出苯丙氨酸、酪氨酸以及甘氨酸/丝氨酸代谢的富集。
结论：为可切除的cN0 NSCLC开发了一种与胸内淋巴结受累的存在和范围强相关的DNA甲基化模型。该模型有潜力在术前识别最需要接受EBUS-TBNA的患者。其在治疗前血浆中的预测性能正在验证中，基于血浆的结果将在AACR上呈现。
查看英文原文 English abstract
Background: Patients with non-small cell lung cancer (NSCLC) harboring pathologic lymph nodal metastases (LNDM) (pN1-2) have substantially higher recurrence risks. However, 10~15% of patients staged as cN0 via preoperative CT have lymph node involvement confirmed by pathologic evaluation. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) improves preoperative nodal staging, but extensive sampling of multiple stations increases procedural risk and complexity. Thus, exploring LNDM biomarkers are needed. This was aimed to develop a tumor DNA methylation-based model to predict intrathoracic nodal metastases indicating more precised patient population receiving EBUS-TBNA evaluation.
Methods: Primary tumor tissue and paired preoperative plasma from 68 patients with resectable NSCLC was retrospectively collected (pN0=24, pN1=24, pN2=20, all pN2 were mediastinal nodal metastases). Tumor DNA was extracted and subjected for enzyme conversion and methylation sequencing (EM-Seq). Differentially methylated CpG sites associated with nodal involvement were identified by comparing pN0 vs pN1/2 (Wald test). A lymph-node-metastasis methylation score was constructed using LASSO regression and its performance assessed by ROC analysis. Promoter-localized differentially methylated sites were subjected to Gene Ontology (GO) and pathway enrichment analyses. Further validation of this model on preoperative plasma cell-free DNA is undergoing.
Results: Differentially methylated 6,745 CpG sites between pN0 and pN1/2 tumors (p<0.05) were disclosed. An 8-CpG methylation score discriminated patients with and without nodal metastases with an AUC of 0.962 (cut-off 2.0; sensitivity 1.0; specificity 0.841). The methylation score increased with pathologic nodal stage (median pN0=0.76, pN1=2.74, pN2=3.30, pN1 vs pN0 p<0.01, pN2 vs pN0 p<0.01, pN2 vs pN1 p=0.26). No correlation to primary tumor size (r=-0.08, p=0.505) was found, making it only LNDM relevant. Among differentially methylated loci, 28% (1,872 sites) were located in promoter regions. In node-positive tumors, enriched GO terms included epithelial cell polarity and cell-cell adhesion, whereas node-negative tumors showed enrichment of phenylalanine, tyrosine, and glycine/serine metabolism.
Conclusions: A DNA methylation model strongly associated with the presence and extent of intrathoracic nodal involvement was developed for resectable, cN0 NSCLC. This model has the potential to preoperatively identify patients who most warrant EBUS-TBNA. Its predictive performance in pre-treatment plasma is being validated, and plasma-based results will be presented at the AACR.
利益披露 Disclosure
L. Jiang, None..
X. Li, None..
Y. Chen, None..
C. Zhu, None..
Z. Li, None.