Highly-multiplex detection of plasma cell-free human papillomavirus-16 DNA in oropharyngeal carcinoma

Background: Plasma cell-free Human Papillomavirus DNA (cfHPVDNA) is a biomarker for oropharyngeal carcinoma. Existing diagnostics may be limited by inadequate sensitivity or high cost/complexity for longitudinal monitoring.

Objectives: We hypothesized that sensitive and specific plasma cfHPVDNA detection may be achieved via a highly-multiplex qPCR method.

Study design: We designed and validated a single-tube one-step genotype-specific qPCR assay for detection of cfHPV16DNA in human plasma using >8,000 genomes spanning 18 genotypes. Amplicons were optimized for cfHPVDNA fragment size.

Results: The cfHPV16DNA qPCR amplicons spanned 16 % of the HPV16 genome. Amplicons were conserved in a median of 99.0 % of 3,944 genomes in silico. The 95 % lower limit of detection was 0.35 genome copies/reaction and the limit of blank was 0. Multiplexing achieved a tenfold improvement in sensitivity compared with single amplicons using in silico simulations of cfHPVDNA fragmentation, which was in close agreement with experimental observations. An assay was replicated for HPV18 with similar observations. Among 36 patients with head/neck mucosal carcinomas (26 HPV-positive, 12 HPV-negative), there was 100 % concordance with tissue HPV status and with NavDx digital PCR. Pre-treatment specimens with sub-genomic cfHPVDNA concentration were detected. False negatives were observed with single amplicons but not with this multiplexed method. Among 17 patients with post-treatment landmark specimens, there was 100 % PPV and 100 % NPV for recurrence.

Conclusions: This assay is specific for plasma cfHPVDNA detection and prognostic for recurrence. Sub-genomic sensitivity was in close agreement with in silico simulations. The format might be more accessible than dPCR or NGS for longitudinal testing.