Evaluation of ultrasensitive ddPCR and NGS for circulating tumor HPV DNA in minimal residual disease monitoring and lymph-node metastasis in cervical cancer: A prospective study.

Abstract

e17511 Background: Circulating tumor HPV DNA (ctHPV) is emerging as a promising biomarker for minimal residual disease (MRD) in cervical cancer. Prospective studies investigating the association of ctHPV at diagnosis with primary tumor size and lymph node metastasis (LNM), as well as its potential for longitudinal MRD monitoring, are limited. Establishing a rapid and clinically feasible method for ctHPV quantification could improve early risk stratification and guide precision management strategies. Methods: Newly diagnosed HPV16/18-positive cervical cancer patients are being prospectively enrolled, with a planned cohort of 83 patients. 76 patients have been analyzed to date. ctHPV levels at diagnosis were quantified using droplet digital PCR (ddPCR) and next-generation sequencing (NGS). The concordance between ddPCR and NGS was evaluated using correlation analysis and Bland–Altman plots. Associations of ctHPV with LNM status and trends according to primary tumor size were analyzed. Post-treatment ctHPV measurements are ongoing, and patients will continue longitudinal follow-up to evaluate ctHPV dynamics and their potential association with treatment response and prognosis. Results: Analyses presented here include 76 patients from the ongoing prospective cohort. ctHPV was detectable at diagnosis in all patients. ddPCR showed high concordance with NGS, with Bland–Altman analysis demonstrating minimal bias, supporting its use as a rapid and clinically feasible quantification method. Patients with LNM exhibited higher ctHPV levels at diagnosis compared with those without LNM, and ctHPV levels increased with greater primary tumor size. These findings highlight the potential of ctHPV to reflect tumor burden and LNM status at diagnosis. Conclusions: ctHPV at diagnosis is associated with primary tumor size and lymph node metastasis in cervical cancer. ddPCR provides a rapid and reliable method for ctHPV quantification. Ongoing and future longitudinal analyses will explore ctHPV dynamics during treatment and follow-up as a marker for MRD and prognosis, supporting its potential clinical utility in prospective patient management. Circulating tumor HPV DNA (ctHPV) levels measured by ddPCR and NGS in the prospective cohort, showing stratified by lymph node metastasis and primary tumor size. Analysis/cohort N ddPCR log10 (ctHPV + 1) (copies/ml) NGS log10 (ctHPV + 1) (copies/ml) Mean difference (95% LoA) P Value (ddPCR) P Value (NGS) ddPCR/NGS concordance 76 2.091 ± 1.19 2.27 ± 1.29 -0.18 (-0.72 – 0.36) # - - LNM positive 37 2.58 ± 1.24 2.80 ± 1.41 - 0.0003 0.0003 LNM negative 39 1.63 ± 0.94 1.77 ± 0.93 - Tumor <= 2 cm 9 1.25 ± 0.91 1.46 ± 0.89 - 0.0002 * 0.0005 * Tumor 2 – 4 cm 21 1.57 ± 0.86 1.71 ± 0.79 - Tumor >= 4 cm 46 2.49 ± 1.20 2.69 ± 1.38 - #: Bland–Altman analysis; *: Linear trend was evaluated by polynomial contrasts in one-way ANOVA.