2784 - Highly-Multiplex Detection and Quantitation of Plasma Cell-Free Human Papillomavirus-16 DNA in Oropharyngeal Carcinoma

P. E. Clark¹, N. Karasik², S. R. Campbell¹, N. M. Woody³, J. Ku⁴, N. Silver⁴, D. Bottalico⁴, B. Prendes⁴, E. Lamarre⁴, J. Scharpf⁴, T. Sussman², E. Yilmaz⁵, J. L. Geiger⁶, H. Wang⁷, T. A. Chan⁸, S. A. Koyfman³, and J. A. Miller¹; ¹Cleveland Clinic, Cleveland, OH, ²Department of Hematology/Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, ³Department of Radiation Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, ⁴Department of Otolaryngology, Head and Neck Institute, Cleveland Clinic, Cleveland, OH, ⁵Department of Medical Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, ⁶Department of Hematology and Medical Oncology, Taussig Cancer Center, Cleveland Clinic, Cleveland, OH, ⁷Department of Laboratory Medicine, Diagnostics Institute, Cleveland Clinic, Cleveland, OH, ⁸Department of Radiation Oncology, Cleveland Clinic Foundation, Cleveland, OH

Purpose/Objective(s): Plasma high-risk Human Papillomavirus (HPV) DNA is a biomarker for oropharyngeal carcinoma. Existing molecular diagnostics utilizing single-target real-time PCR (qPCR), multiplex digital PCR (dPCR), or hybrid-capture next-generation sequencing (NGS) may be limited by inadequate sensitivity, low genomic coverage, specialized instrumentation, or impractical cost/complexity for longitudinal monitoring and minimal residual disease assessment. We hypothesized that sensitive and specific plasma HPV16 DNA detection may be achieved via a highly-multiplex qPCR method. Materials/

Methods: We designed and validated a highly-multiplex single-tube one-step genotype-specific qPCR assay for detection of cell-free HPV16 DNA in human plasma. Recurrently-mutated or conserved regions within and across HPV genotypes were identified using more than 7,000 whole genome sequences spanning 18 genotypes. Amplicons were optimized for cell-free HPV16 DNA fragment size, genotype specificity, and maximal genomic coverage. Synthetic whole-genome HPV16 DNA served as a positive control for analytical validation. A human gene served as an endogenous internal control for extraction and amplification. Pre-treatment plasma specimens from patients with oropharyngeal carcinomas with HPV status confirmed by tissue/cytology served as clinical controls.

Results: The multiplex qPCR assay covered 16% of the HPV16 genome with median amplicon size 99 (range, 81-124). Oligonucleotides were conserved in a median of 99.0% (range, 95.2-99.7%) of 4,000 HPV16 genomes in silico. The 95% lower limit of detection was 0.35 HPV16 genome copies/reaction (95% CI 0.22-0.48) and the limit of blank was 0. In a cohort of 33 patients (18 HPV-positive, 15 HPV-negative), there was 100% concordance with tissue/cytology HPV status. The assay detected plasma HPV16 DNA below 0.5 genome copies/reaction in clinical samples.

Conclusion: A highly-multiplex qPCR assay was specific for detection of plasma HPV16 DNA among subjects with oropharyngeal carcinoma. Sub-genomic analytical sensitivity was achieved via increased genomic coverage relative to single- or dual-target assays. The one-step single-reaction qPCR format might be more accessible than dPCR or NGS due to low cost/complexity, rapid turnaround time, and widespread availability of qPCR instrumentation and trained personnel. Future studies are warranted to directly compare the performance of existing assays for minimal residual disease detection.