Early-stage lung cancers radiologically manifested as ground-glass opacities (GGOs) have been increasingly identified, among which pure GGO (pGGO) has a good prognosis after local resection. However, the optimal surgical margin is still under debate. Precancerous lesions exist in tumor-adjacent tissues beyond the histological margin. However, potential precancerous epigenetic variation patterns beyond the histological margin of pGGO are yet to be discovered and described. A genome-wide high-resolution DNA methylation analysis was performed on samples collected from 15 pGGO at tumor core (TC), tumor edge (TE), para-tumor tissues at the 5 mm, 10 mm, 15 mm, 20 mm beyond the tumor, and peripheral normal (PN) tissue. TC and TE were tested with the same genetic alterations, which were also observed in histologically normal tissue at 5 mm in two patients with lower mutation allele frequency. According to the difference of methylation profiles between PN samples, 2284 methylation haplotype blocks (MHBs), 1657 differentially methylated CpG sites (DMCs), and 713 differentially methylated regions (DMRs) were identified using reduced representation bisulfite sequencing (RRBS). Two different patterns of methylation markers were observed: Steep (S) markers sharply changed at 5 mm beyond the histological margin, and Gradual (G) markers changed gradually from TC to PN. S markers composed 86.2% of the tumor-related methylation markers, and G markers composed the other 13.8%. S-marker-associated genes enriched in GO terms that were related to the hallmarks of cancer, and G-markers-associated genes enriched in pathways of stem cell pluripotency and transcriptional misregulation in cancer. Significant difference in DNA methylation score was observed between peripheral normal tissue and tumor-adjacent tissues 5 mm further from the histological margin (p < 0.001 in MHB markers). DNA methylation score at and beyond 10 mm from histological margin is not significantly different from peripheral normal tissues (p > 0.05 in all markers). According to the methylation pattern observed in our study, it was implied that methylation alterations were not significantly different between tissues at or beyond P10 and distal normal tissues. This finding explained for the excellent prognosis from radical resections with surgical margins of more than 15 mm. The inclusion of epigenetic characteristics into surgical margin analysis may yield a more sensitive and accurate assessment of remnant cancerous and precancerous cells in the surgical margins.

中文翻译：

---

早期浸润性肺腺癌组织学边缘及其以外的 DNA 甲基化模式在放射学上表现为纯磨玻璃样混浊

影像学表现为磨玻璃样影（GGOs）的早期肺癌越来越多，其中纯GGO（pGGO）在局部切除后预后良好。然而，最佳手术切缘仍在争论中。癌前病变存在于组织学边缘以外的肿瘤邻近组织中。然而，尚未发现和描述超出 pGGO 组织学边缘的潜在癌前表观遗传变异模式。对从 15 pGGO 在肿瘤核心 (TC)、肿瘤边缘 (TE)、肿瘤旁 5 mm、10 mm、15 mm、20 mm 处收集的样本进行全基因组高分辨率 DNA 甲基化分析肿瘤和外周正常（PN）组织。TC 和 TE 用相同的基因改变进行了测试，在两名突变等位基因频率较低的患者中，在 5 mm 处的组织学正常组织中也观察到了这种情况。根据 PN 样本之间甲基化谱的差异，使用还原代表性亚硫酸氢盐测序 (RRBS) 鉴定了 2284 个甲基化单倍型块 (MHB)、1657 个差异甲基化 CpG 位点 (DMC) 和 713 个差异甲基化区域 (DMR)。观察到两种不同模式的甲基化标记：陡峭 (S) 标记在超出组织学边缘 5 mm 处急剧变化，渐变 (G) 标记从 TC 逐渐变为 PN。S 标志物占肿瘤相关甲基化标志物的 86.2%，G 标志物占其他 13.8%。S标记相关基因富含与癌症标志相关的GO术语，和 G 标记相关基因富含干细胞多能性和癌症中的转录失调途径。在距组织学边缘 5 mm 远的外周正常组织和肿瘤邻近组织之间观察到 DNA 甲基化评分的显着差异（MHB 标记 p < 0.001）。距离组织学边缘 10 mm 及以上的 DNA 甲基化评分与外周正常组织没有显着差异（所有标记中 p > 0.05）。根据我们研究中观察到的甲基化模式，暗示在 P10 处或以上的组织与远端正常组织之间的甲基化改变没有显着差异。这一发现解释了手术切缘超过 15 毫米的根治性切除术的良好预后。