Quantification of Human Papillomavirus cell - free DNA from low volume blood plasma samples by digital PCR

The incidence rate of human papillomavirus-driven oropharyngeal cancer (HPV-OPC) is increasing in many countries with high human development index. HPV cell-free DNA (cfDNA) isolated from relatively large amounts of blood plasma (typically 3-4 ml) has been successfully used for therapy surveillance of HPV-OPC patients. A currently highly discussed application of HPV-cfDNA is early detection of HPV-OPC in symptom-free individuals. This requires particularly sensitive and specific cfDNA detection methods as viral copy numbers can be very low. To study the predictive power of pre-diagnostic HPV-cfDNA, archived samples from epidemiological cohorts with limited plasma volume are an important source. To establish a cfDNA detection workflow optimized for low plasma volumes we compared two cfDNA purification methods (MagNA Pure 96 and QIAamp ccfDNA/RNA) and two digital PCR systems (Biorad QX200 and QIAGEN QIAcuity One). Final assay validation included 65 low volume plasma samples from OPC patients with defined HPV status (34 HPV16+, 1 HPV33+, 2 HPV58+, 28 HPV-negative) stored for 2-9 years. MagNA Pure 96 yielded a 28% higher cfDNA isolation efficiency in comparison to the QIAamp ccfDNA/RNA kit. Both digital PCR systems showed comparable analytic sensitivity (limit of detection 6-17 copies for both HPV16 and HPV33), but the higher multiplexing capacity allowed the QIAcuity to detect both HPV types in the same assay. In the validation set, the assay had a sensitivity of 80% (n=28/35) for detecting HPV16 and HPV33, and a specificity of 97% (n=29/30). In samples with ≥750 µl plasma available, the sensitivity was 85% (n=17/20), while in samples with <750 µl plasma the sensitivity was 73% (n=11/15). Despite the expected drop in sensitivity with decreased plasma volume, the assay is sensitive and highly specific even in low volume samples, and thus suited for studies exploring HPV-cfDNA as an early HPV-OPC detection marker in low volume archival material.