Rapid and ultrasensitive detection of circulating human papillomavirus E7 cell-free DNA as a cervical cancer biomarker

Abstract

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% ( κ = 0.67) for HPV16 E7 and 100% ( κ = 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.

The Diagnostic Value of Circulating Cell-Free HPV DNA in Plasma from Cervical Cancer Patients

“…Several issues may explain these variable fin-dings. Firstly, there is a great variation in the methods used to detect ccfHPV DNA [ 15 , 31 , 32 , 34 , 35 , 36 , 37 , 39 , 40 , 41 , 42 , 43 , 53 ]. Since ddPCR offers a very high sensitivity and enables an absolute target quantification of the molecular target [ 52 ], this method is the obvious choice to determine the diagnostic performance of ccfHPV DNA for early detection of cervical cancer.…”

“…So far, there has been limited success in detecting ccfHPV DNA in plasma or serum, which has mainly been suggested to be caused by a lack of sensitivity of the me-thods used [ 32 , 33 , 34 ]. Recent studies have examined the use of digital droplet PCR (ddPCR) to measure ccfHPV DNA [ 31 , 35 , 36 , 37 ]. This method is based on partitioning of the sample in many reaction chambers or droplets [ 38 ], and it offers better sensitivity and quantification of poorly abundant nucleic acids, including ccfHPV DNA [ 17 , 31 , 32 , 39 , 40 , 41 , 42 , 43 , 44 ].…”

“…This method is based on partitioning of the sample in many reaction chambers or droplets [ 38 ], and it offers better sensitivity and quantification of poorly abundant nucleic acids, including ccfHPV DNA [ 17 , 31 , 32 , 39 , 40 , 41 , 42 , 43 , 44 ]. In these studies, detection rate ranges between 31% and 100% [ 31 , 35 , 36 , 37 ], underlining that findings on ccfHPV DNA in cervical cancer patients and the exact relationship between both its presence and quantity and other clinical parameters are still unclear.…”

Accurate determination of meat mass fractions using DNA measurements for quantifying meat adulteration by digital PCR

“…The results showed that our methods can detect pork DNA down to 2–5 copies µL –1 with 100% detection rate (data not shown). This result was in agreement with other publications for the LOD of ddPCR (Hindson et al ., 2011; Carolina et al ., 2020; Rungkamoltip et al ., 2021; Vishnuraj et al ., 2021).…”

Liquid Biopsy to Detect Minimal Residual Disease: Methodology and Impact

“…It can also be detected either by assaying for circulating viral antigens or antibodies directed against the virus [ 102 ] or the presence of the viral genome in the bloodstream [ 103 ]. Studies have mainly focused on detecting specific viral oncogenes in the plasma using PCR-derived methods, for example, quantitative PCR (qPCR) [ 103 ], digital PCR (dPCR) [ 104 , 105 ], and droplet digital PCR (ddPCR) [ 106 , 107 ]. Detection of the viral genome to assess MRD has shown interesting results, especially in Human Papilloma Virus (HPV)-derived and Epstein–Barr (EBV)-derived cancers.…”