支气管镜检查是疑似肺癌(LC)患者常用的初始诊断程序。在支气管镜检查期间获得的支气管冲洗(BW)样品的细胞学检查通常会产生有关LC诊断的不确定结果。本研究旨在确定分子生物标志物作为LC诊断的非侵入性方法。异常DNA甲基化用作LC的有用生物标志物。因此,13个患者匹配的I-III期肿瘤组织的基于微阵列的甲基化谱分析与进行非肿瘤组织,并鉴定了一组高度差异甲基化的基因。随后使用亚硫酸氢盐焦磷酸测序对其他组织和细胞系进行分析,发现了六个甲基化基因[具有血小板反应蛋白1型基序20的ADAM金属肽酶,叉头框C2(间充质叉头1),NK2相关转录因子,基因座5(果蝇),与LC相关的少突胶质细胞转录因子3、原钙粘蛋白γ亚家族A12(PCDHGA12)和配对相关同源异型盒1(PRRX1)]。接下来,一种高度灵敏和准确的检测方法,在单个封闭管中线性靶富集-定量甲基化特异性PCR,使用来自LC患者(n=68)和良性疾病患者(n=33)的BW样品进行临床验证。PCDHGA12和PRRX1甲基化被鉴定为检测LC的最佳表现的生物标志物。双标记组合的敏感性为82.4%,特异性为87.9%,曲线下面积为0.891。值得注意的是,小细胞LC的灵敏度为100%。双标记组合的阳性预测值为93.3%,阴性预测值为70.7%。敏感性高于细胞学,只有50%的灵敏度。两种标记组合的甲基化状态显示与性别无关,年龄或阶段,但与肿瘤位置和组织学有关。总之,本研究表明,PCDHGA12和PRRX1的调控区在LC中高度甲基化,可用于检测BW标本中的LC,作为临床细胞学诊断的辅助手段.
Bronchoscopy is a frequently used initial diagnostic procedure for patients with suspected lung cancer (LC). Cytological examinations of bronchial washing (BW) samples obtained during bronchoscopy often yield inconclusive results regarding LC diagnosis. The present study aimed to identify molecular biomarkers as a non-invasive method for LC diagnosis. Aberrant DNA methylation is used as a useful biomarker for LC. Therefore, microarray-based methylation profiling analyses on 13 patient-matched tumor tissues at stages I-III vs. non-tumor tissues were performed, and a group of highly differentially methylated genes was identified. A subsequent analysis using bisulfite-pyrosequencing with additional tissues and cell lines revealed six methylated genes [ADAM metallopeptidase with thrombospondin type 1 motif 20, forkhead box C2 (mesenchyme forkhead 1), NK2 transcription factor related, locus 5 (Drosophila), oligodendrocyte transcription factor 3, protocadherin γ subfamily A 12 (PCDHGA12) and paired related homeobox 1 (
PRRX1)] associated with LC. Next, a highly sensitive and accurate detection method, linear target enrichment-quantitative methylation-specific PCR in a single closed tube, was applied for clinical validation using BW samples from patients with LC (n=68) and individuals with benign diseases (n=33). PCDHGA12 and
PRRX1 methylation were identified as the best-performing biomarkers to detect LC. The two-marker combination showed a sensitivity of 82.4% and a specificity of 87.9%, with an area under the curve of 0.891. Notably, the sensitivity for small cell LC was 100%. The two-marker combination had a positive predictive value of 93.3% and a negative predictive value of 70.7%. The sensitivity was higher than that of cytology, which only had a sensitivity of 50%. The methylation status of the two-marker combination showed no association with sex, age or stage, but was associated with tumor location and histology. In conclusion, the present study showed that the regulatory regions of PCDHGA12 and
PRRX1 are highly methylated in LC and can be used to detect LC in BW specimens as a diagnostic adjunct to cytology in clinical practice.