Clinical cancer research : an official journal of the American Association for Cancer Research | 2021
HPV sequencing facilitates ultrasensitive detection of HPV circulating tumor DNA.
Abstract
PURPOSE\nHuman papillomavirus (HPV) DNA offers a convenient ctDNA marker for HPV-associated malignancies, but current methods such as digital (d)PCR provide insufficient accuracy for clinical applications in patients with low-disease burden. We asked whether a next-generation sequencing approach (HPV-seq) could provide quantitative and qualitative assessment of HPV ctDNA in low-disease-burden settings.\n\n\nEXPERIMENTAL DESIGN\nWe conducted preclinical technical validation studies on HPV-seq and applied it retrospectively to a prospective multicentre cohort of locally-advanced cervix cancer patients (NCT02388698) and a cohort of oropharynx cancer patients. HPV-seq results were compared with dPCR. The primary outcome was progression-free survival (PFS) according to end-of-treatment HPV ctDNA detectability.\n\n\nRESULTS\nHPV-seq achieved reproducible detection of HPV DNA at levels <0.6 genome equivalent in cell line data. HPV-seq and dPCR results for patients were highly correlated (R2=0.95, p=1.9x10-29) with HPV-seq detecting ctDNA at levels down to 0.03 copy/mL plasma in dPCR-negative post-treatment 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 pre-treatment samples. HPV ctDNA fragment sizes were consistently shorter than non-cancer-derived cfDNA fragments, and stereotyped cfDNA fragmentomic patterns were observed across HPV genomes.\n\n\nCONCLUSIONS\nHPV-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.