Objectives Circulating tumor DNA assays have the potential to facilitate early detection of cancer recurrence as has been demonstrated in breast, bladder, colorectal, and most recently HPV-associated oropharyngeal cancer. We sought to develop a droplet digital PCR (ddPCR) assay for the quantification of circulating tumor HPV DNA in cervical cancer patients.
Methods Primers were designed to specifically detect an amplicon within the E7 gene encoded by high-risk HPV 16 and 18. Each reaction assay contained 2x ddPCR EvaGreen Supermix (10 µL), respective primers (4 µL), target DNA (1 µL), and DNAase free water (5 µL). Optimal annealing temperature and primer concentration were determined by running temperature and concentration titrations of PBS spiked with target gene fragments of HPV 16. Primers targeting the E7 gene of HPV 16 and 18 were combined into a single assay and HPV DNA quantification performed in control plasma samples.
Results Titration analysis demonstrated good correlation between expected HPV DNA copies and detected copies by ddPCR (R2 = 0.9763). The HPV 16 and 18 assays tested individually and in combination were specific for the HPV strain of interest with no cross-reactivity to the other HPV strain.
Conclusions We developed a highly sensitive and specific ddPCR assay to detect the two dominant high-risk HPV subtypes responsible for to cervical cancer. We plan to perform a prospective pilot study to validate our assay and its clinical utility in detecting minimal residual disease and treatment response.
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