Nanoplate based digital PCR assay for effective quantification of plasma HPV circulating tumor DNA.

High risk Human papillomavirus (hrHPV) is a leading cause of cervical cancer. Numerous studies have demonstrated the utility of plasma HPV circulating tumor DNA (ctDNA) for disease monitoring in cervical cancer, nevertheless with diverse accuracies. We investigated whether higher sample and reaction volumes using nanoplate based digital PCR (dPCR) system offered enhanced sensitivity and reproducibility, crucial for detecting low-copies of HPV ctDNA. A multiplex dPCR assay was optimized for detecting hrHPV16, 18, and 31 plasma ctDNA from pre-treatment and follow-up plasma samples of 87 cervical cancer patients using Qiagen QIAcuity One dPCR. The assay demonstrated 98% sensitivity and 100% specificity for detecting HPV ctDNA from pre-treatment plasma cell-free DNA (cfDNA). Persistence of HPV ctDNA during follow up associated with disease relapse, while clearance of HPV ctDNA was observed in patients without recurrence. Higher sample and dPCR reaction volumes increased the sensitivity of the test, while HPV ctDNA monitoring effectively reflected disease burden. Trial registration: Clinical Trials Registry of India (CTRI/2020/01/022862); registered on 20 January 2020.