Development of a high-performance multi-probe droplet digital PCR assay for high-sensitivity detection of human papillomavirus circulating tumor DNA from plasma

Objectives: To develop a high-performance droplet digital PCR (ddPCR) assay capable of enhancing the detection of human papillomavirus (HPV) circulating tumor DNA (ctDNA) in plasma from patients with HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC). Materials and Methods: Plasma samples from subjects with HPV+ OPSCC were collected. We developed a high-performance ddPCR assay designed to simultaneously target nine regions of the HPV16 genome. Results: The new assay termed 'ctDNA HPV16 Assessment using Multiple Probes' (CHAMP-16) yielded signifi-cantly higher HPV16 counts compared to our previously validated 'Single-Probe' (SP) assay and a commercially available NavDx (R) assay. Analytical validation demonstrated that the CHAMP-16 assay had a limit of detection (LoD) of 4.1 copies per reaction, corresponding to < 1 genome equivalent (GE) of HPV16. When tested on plasma ctDNA from 21 patients with early-stage HPV+ OPSCC and known HPV16 ctDNA using the SP assay, all patients were positive for HPV16 ctDNA in both assays and the CHAMP-16 assay displayed 6.6-fold higher HPV16 signal on average. Finally, in a longitudinal analysis of samples from a patient with recurrent disease, the CHAMP-16 assay detected HPV16 ctDNA signal similar to 20 months prior to the conventional SP assay. Conclusion: Increased HPV16 signal detection using the CHAMP-16 assay suggests the potential for detection of recurrences significantly earlier than with conventional ddPCR assays in patients with HPV16+ OPSCC. Criti-cally, this multi-probe approach maintains the cost-benefit advantage of ddPCR over next generation sequencing (NGS) approaches, supporting the cost-effectiveness of this assay for both large population screening and routine post-treatment surveillance.

Automated summary

A high-performance ddPCR assay called CHAMP-16 was developed to enhance the detection of HPV ctDNA in plasma from patients with HPV+ OPSCC. The assay targets nine regions of the HPV16 genome and showed significantly higher HPV16 counts compared to previous assays. The CHAMP-16 assay has the potential to detect recurrences earlier and is cost-effective for population screening and post-treatment surveillance.