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Purpose <p>Plasma circulating tumor HPV DNA (ctHPVDNA) persistence after curative-intent treatment may identify patients with HPV-positive cancers at risk for recurrence. Technical validation is required for use as an integral biomarker in a prospective clinical trial.</p> Methods <p>Development and analytical validation of a digital&#xa0;droplet PCR assay for detection and quantification of 13 high-risk HPV types (i.e., Cell-Free 13) was performed with oligonucleotides/plasmids encoding type-specific E6/E7 coding regions. Clinical performance, determinants of detection/quantification, and associations of pre-treatment ctHPVDNA with progression-free survival (PFS) were also evaluated in a prospective cohort of 272 head and neck cancer patients.</p> Results <p>Limit of detection, limit of quantification, and linear range of quantification were 5, 16 and 16–200,000 virus copies for all 13 high-risk HPV types. No cross-reactivity was detected across all 13 HPV types. At 10,000 copies, inter-assay coefficients of variation ranged from 0.3 to 4.6%. Multiplexing, DNA purification method, input plasma volume, total input cell-free (&lt; 1800&#xa0;ng) or genomic (&lt; 700&#xa0;ng) DNA did not affect HPV detection or quantification. The assay had a sensitivity of 91.7% (95%CI 87.3–94.9%) and specificity of 97.7% (95%CI 87.7–99.9%) for ctHPVDNA detection in the setting of newly&#xa0;diagnosed HPV-positive oropharyngeal cancer. Tumor and nodal stage categories, tumor viral load (ρ = 0.41, <i>p</i> &lt; 0.05), and HPV integration status were associated with ctHPVDNA quantitative level. Pre-treatment ctHPVDNA greater than the median (231 copies/ml) was associated with worse PFS (HR = 2.14, 95%CI 1.16–3.97, <i>p</i> = 0.0156) in univariate analysis. However, this was no longer significant after adjustment for clinical covariates (HR<sub>adj</sub> = 1.81, 95%CI 0.97–3.37, <i>p</i> = 0.0635).</p> Conclusion <p>Cell-Free 13 demonstrated excellent analytical performance and clinical sensitivity/specificity in HPV-positive oropharyngeal cancer. Pre-treatment ctHPVDNA may be associated with oncologic outcomes.</p>

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Development and Validation of an Assay to Quantify Plasma Circulating Tumor Human Papillomavirus DNA for 13 High-Risk Types that Cause 98% of HPV-Positive Cancers

  • 期刊论文

  • 发表日期：

    2025年2月25日

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  • Michael T. Wotman,
  • Weihong Xiao,
  • Robyn R. Du,
  • Bo Jiang,
  • Keiko Akagi,
  • Suyu Liu,
  • Maura L. Gillison

摘要

Purpose

Plasma circulating tumor HPV DNA (ctHPVDNA) persistence after curative-intent treatment may identify patients with HPV-positive cancers at risk for recurrence. Technical validation is required for use as an integral biomarker in a prospective clinical trial.

Methods

Development and analytical validation of a digital droplet PCR assay for detection and quantification of 13 high-risk HPV types (i.e., Cell-Free 13) was performed with oligonucleotides/plasmids encoding type-specific E6/E7 coding regions. Clinical performance, determinants of detection/quantification, and associations of pre-treatment ctHPVDNA with progression-free survival (PFS) were also evaluated in a prospective cohort of 272 head and neck cancer patients.

Results

Limit of detection, limit of quantification, and linear range of quantification were 5, 16 and 16–200,000 virus copies for all 13 high-risk HPV types. No cross-reactivity was detected across all 13 HPV types. At 10,000 copies, inter-assay coefficients of variation ranged from 0.3 to 4.6%. Multiplexing, DNA purification method, input plasma volume, total input cell-free (< 1800 ng) or genomic (< 700 ng) DNA did not affect HPV detection or quantification. The assay had a sensitivity of 91.7% (95%CI 87.3–94.9%) and specificity of 97.7% (95%CI 87.7–99.9%) for ctHPVDNA detection in the setting of newly diagnosed HPV-positive oropharyngeal cancer. Tumor and nodal stage categories, tumor viral load (ρ = 0.41, p < 0.05), and HPV integration status were associated with ctHPVDNA quantitative level. Pre-treatment ctHPVDNA greater than the median (231 copies/ml) was associated with worse PFS (HR = 2.14, 95%CI 1.16–3.97, p = 0.0156) in univariate analysis. However, this was no longer significant after adjustment for clinical covariates (HRadj = 1.81, 95%CI 0.97–3.37, p = 0.0635).

Conclusion

Cell-Free 13 demonstrated excellent analytical performance and clinical sensitivity/specificity in HPV-positive oropharyngeal cancer. Pre-treatment ctHPVDNA may be associated with oncologic outcomes.