Rungkamoltip Phetploy, Temisak Sasithon, Piboonprai Kitiya, Japrung Deanpen, Thangsunan Pattanapong, Chanpanitkitchot Saranya, Chaowawanit Woraphot, Chandeying Nutthaporn, Tangjitgamol Siriwan, Iempridee Tawin
National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency, Pathum Thani 12120, Thailand.
Chemical Metrology and Biometry Department, National Institute of Metrology (NIMT), Pathum Thani 12120, Thailand.
Exp Biol Med (Maywood). 2021 Mar;246(6):654-666. doi: 10.1177/1535370220978899. Epub 2020 Dec 13.
Circulating cell-free DNA (cfDNA) has attracted attention as a non-invasive biomarker for diagnosing and monitoring various cancers. Given that human papillomavirus (HPV) DNA integration and overexpression of E6/E7 oncogenes are pivotal events for carcinogenesis, we sought to determine if HPV E7 cfDNA could serve as a specific biomarker for cervical cancer detection. We applied droplet digital PCR (ddPCR) to quantify HPV16/18 E7 cfDNA from the serum of patients with cervical cancer, cervical intraepithelial neoplasia, and controls. HPV16/18 E7 cfDNA was highly specific for cervical cancer, displaying 30.77% sensitivity, 100% specificity, and an area under the curve of 0.65. Furthermore, we developed a sensitive isothermal detection of HPV16/18 E7 and the PIK3CA WT reference gene based on recombinase polymerase amplification combined with a lateral flow strip (RPA-LF). The assay took less than 30 min and the detection limit was 5-10 copies. RPA-LF exhibited 100% sensitivity and 88.24% specificity towards HPV16/18 E7 cfDNA in clinical samples. The agreement between RPA-LF and ddPCR was 83.33% ( = 0.67) for HPV16 E7 and 100% ( = 1.0) for HPV18 E7, indicating a good correlation between both tests. Therefore, we conclude that HPV E7 cfDNA represents a potential tumor marker with excellent specificity and moderate sensitivity for minimally invasive cervical cancer monitoring. Moreover, the RPA-LF assay provides an affordable, rapid, and ultrasensitive tool for detecting HPV cfDNA in resource-limited settings.
循环游离DNA（cfDNA）作为一种用于诊断和监测各种癌症的非侵入性生物标志物已受到关注。鉴于人乳头瘤病毒（HPV）DNA整合以及E6/E7癌基因的过表达是致癌作用的关键事件，我们试图确定HPV E7 cfDNA是否可作为宫颈癌检测的特异性生物标志物。我们应用液滴数字PCR（ddPCR）对宫颈癌、宫颈上皮内瘤变患者及对照者血清中的HPV16/18 E7 cfDNA进行定量。HPV16/18 E7 cfDNA对宫颈癌具有高度特异性，灵敏度为30.77%，特异性为100%，曲线下面积为0.65。此外，我们基于重组酶聚合酶扩增结合侧流条（RPA-LF）开发了一种灵敏的HPV16/18 E7和PIK3CA野生型参考基因等温检测方法。该检测方法耗时不到30分钟，检测限为5至10拷贝。RPA-LF对临床样本中HPV16/18 E7 cfDNA的灵敏度为100%，特异性为88.24%。RPA-LF与ddPCR对HPV16 E7的一致性为83.33%（κ=0.67），对HPV18 E7的一致性为100%（κ=1.0），表明两种检测方法之间具有良好的相关性。因此，我们得出结论，HPV E7 cfDNA是一种潜在的肿瘤标志物，对微创宫颈癌监测具有优异的特异性和中等灵敏度。此外，RPA-LF检测方法为在资源有限的环境中检测HPV cfDNA提供了一种经济、快速且超灵敏的工具。
