HPV Sequencing Facilitates Ultrasensitive Detection of HPV Circulating Tumor DNA

Abstract

◥Purpose: Human papillomavirus (HPV) DNA offers a convenient circulating tumor DNA (ctDNA) marker for HPV-associated malignancies, but current methods, such as digital PCR (dPCR), provide insufficient accuracy for clinical applications in patients with low disease burden. We asked whether a next-generation sequencing approach, HPV sequencing (HPV-seq), could provide quantitative and qualitative assessment of HPV ctDNA in low disease burden settings.Experimental Design: We conducted preclinical technical validation studies on HPV-seq and applied it retrospectively to a prospective multicenter cohort of patients with locally advanced cervix cancer (NCT02388698) and a cohort of patients with oropharynx cancer. HPV-seq results were compared with dPCR. The primary outcome was progression-free survival (PFS) according to end-of-treatment HPV ctDNA detectability.Results: HPV-seq achieved reproducible detection of HPV DNA at levels less than 0.6 copies in cell line data. HPV-seq and dPCR results for patients were highly correlated (R 2 ¼ 0.95, P ¼ 1.9 Â 10 -29 ) with HPV-seq detecting ctDNA at levels down to 0.03 copies/mL plasma in dPCR-negative posttreatment samples. Detectable HPV ctDNA at end-of-treatment was associated with inferior PFS with 100% sensitivity and 67% specificity for recurrence. Accurate HPV genotyping was successful from 100% of pretreatment samples. HPV ctDNA fragment sizes were consistently shorter than non-cancer-derived cell-free DNA (cfDNA) fragments, and stereotyped cfDNA fragmentomic patterns were observed across HPV genomes.Conclusions: HPV-seq is a quantitative method for ctDNA detection that outperforms dPCR and reveals qualitative information about ctDNA. Our findings in this proof-of-principle study could have implications for treatment monitoring of disease burden in HPV-related cancers. Future prospective studies are needed to confirm that patients with undetectable HPV ctDNA following chemoradiotherapy have exceptionally high cure rates.

Monitoring and adapting cancer treatment using circulating tumor DNA kinetics: Current research, opportunities, and challenges

“…The median half-life of this decline has been estimated at 3 to 8 days ( 89 , 90 ). Most of the patients clear ctDNA completely, and residual ctDNA at the end of treatment is a poor prognostic marker ( 52 , 53 , 91 , 92 ). However, even some patients without detectable end-of-treatment ctDNA nevertheless recur.…”

Circulating Human Papillomavirus DNA in Head and Neck Squamous Cell Carcinoma: Possible Applications and Future Directions

“…In a small study of 17 patients with HPV-related carcinoma of the cervix, Leung et al tested the utility of a novel detection method called HPVseq, which utilizes 150 bp long “bait” probes to detect E6 and E7, as well as full-length HPV 16, 18, 33, 45, 31, 33, and 35. They reported a 100% sensitivity, as all patients had detectable ctDNA down to 0.03 copies/mL [ 35 ].…”

Role of Epstein-Barr Virus and Human Papilloma Virus in the Development of Oropharyngeal Cancer: A Literature Review

“…The results demonstrated that HPV-seq outperformed digital PCR (dPCR) in detecting ctDNA and by providing quantitative and qualitative information. Therefore, HPV-seq represents a promising and sensitive method for ctDNA detection and analysis in HPV-associated malignancies [ 26 ]. As to other detection methods and HPV activity, further studies need to be conducted.…”