LBPO.CL02 · 临床研究 · Late-Breaking
基于血清游离DNA的双重MethyLight检测用于基于液体活检的上皮性卵巢癌检测
Serum cell free DNA based duplex methyLight assay for liquid biopsy based detection of epithelial ovarian cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：卵巢癌作为印度第三大常见的妇科癌症，因其非特异性的早期症状和晚期疾病表现，诊断和预后不佳。异常的CpG岛高甲基化代表了癌变的一个初始且关键的标志，能够使肿瘤抑制基因沉默，并作为在不同体液中用于癌症诊断的有前景的可检测生物标志物。肿瘤抑制基因RUNX3和ZNF154的异常甲基化在卵巢癌中频繁被观察到，作为循环游离DNA中微创生物标志物具有前景。
方法：RUNX3和ZNF154被鉴定为上皮性卵巢癌中潜在的高甲基化肿瘤抑制候选基因。进行了靶向NGS/BSAS以确认这些富含CpG的高甲基化区域在EOC中发生异常甲基化。基于这一初步数据，我们开发并验证了一种双重MethyLight检测（基于定量荧光的实时PCR），以在100例（肿瘤70例，正常30例）组织和70份配对血清样本（肿瘤50例，正常20例）中同时检测两个基因的高甲基化。本研究在组织和血清cfDNA队列中开发并验证了针对这些基因（以COL2A1作为内源性对照）的双重MethyLight检测，以评估其诊断性能。采用MethyLight检测计算得出的甲基化参考百分比值构建ROC曲线，以评价该检测的诊断性能。在组织队列中，ZNF154的敏感性和特异性分别报告为89%和90%，RUNX3分别为87%和83%。在血清cfDNA样本中，RUNX3的敏感性和特异性分别为84%和90%，ZNF154的敏感性和特异性分别为90%和92%。基于任一基因高甲基化的双重panel优于单重标志物，在血清队列中对早期EOC实现了92%的敏感性和94%的特异性，AUC为0.95。该双重panel表现出显著的临床病理学相关性，p<0.05。
结论：这项基于双重MethyLight的试点研究证明了在cfDNA中用于EOC诊断的稳健诊断性能，支持其作为微创筛查工具的潜力。此外，其临床转化尚需多中心验证。
查看英文原文 English abstract
Background: Ovarian cancer, being the third most common gynaecological cancer in India, exhibits poor diagnosis and prognosis due to its non-specific early symptoms and late-stage disease presentation. Aberrant CpG island hypermethylation represents an initial and key hallmark of carcinogenesis, enabling the silencing of tumour suppressor genes and serving as a promising detectable biomarker in different body fluids for cancer diagnosis. Aberrant methylation of tumour suppressor genes RUNX3 and ZNF154 is frequently observed in ovarian cancer, offering promise as minimally invasive biomarkers in circulating cell-free DNA.
Methods: RUNX3 and ZNF154 are identified as potential hypermethylated tumour suppressor candidates in epithelial ovarian cancer. Targeted NGS/BSAS was performed to confirm that these hypermethylated CpG-rich regions were aberrantly methylated in EOC. With this preliminary data, we developed and validated a duplex MethyLight assay (quantitative fluorescence-based real-time PCR) to simultaneously detect hypermethylation of both genes in n-100 (T-70, N-30) tissue and 70 matched serum samples (T-50, N-20). Our present study developed and validated a duplex MethyLight assay targeting these genes (with COL2A1 as endogenous control) in both tissue and serum cfDNA cohorts to assess their diagnostic performance. ROC curves were built employing calculated Percentage of Methylated Reference values from MethyLight assays to evaluate the diagnostic performance of the assay. In the tissue cohort, the sensitivity and specificity of ZNF154 are reported as 89% and 90%, respectively, with RUNX3 showing 87% and 83%, respectively. In serum cfDNA samples, RUNX3 demonstrated a sensitivity and specificity of 84% and 90%, respectively, and ZNF154 demonstrated a sensitivity and specificity of 90% and 92%, respectively. The duplex panel, which is based on either gene hypermethylation, outperformed singleplex markers, achieving 92% sensitivity and 94% specificity with an AUC of 0.95 for early-stage EOC in the serum cohort. The duplex panel demonstrated significant clinicopathological correlation with p<0.05.
Conclusion: This duplex MethyLight-based pilot study demonstrates robust diagnostic performance for EOC diagnosis in cfDNA, supporting its potential as minimally invasive screening tool. Furthermore, multi-centre validation is necessary for its clinical translation.
利益披露 Disclosure
D. Bisht, None..
M. Sachan, None.