Prognostic value of circulating HPV cell-free DNA in cervical cancer using liquid biopsy

Abstract

Liquid biopsies, which analyze circulating tumor cells or cell-free circulating tumor DNA (ctDNA) from blood, have emerged as promising cancer detection and monitoring tools. Specifically, human papillomavirus (HPV) cell-free (cf) DNA is gaining recognition as a prognostic marker in high-risk HPV-related cancers. However, detecting circulating markers for cervical cancer (CC) requires highly sensitive techniques to quantify circulating HPV DNA. This study aimed to evaluate the use of droplet digital PCR (ddPCR), a highly sensitive technique, for detecting and quantifying circulating HPV DNA in cervical cancer patients, both at baseline (before chemo- or radiotherapy) and during follow-up, to assess its utility as a prognostic marker. Blood samples were collected from 60 cervical cancer patients (Stages I-IV) at AIIMS, New Delhi, at baseline and three months post-treatment. Samples from 10 healthy controls were also included. Plasma was separated and stored at - 80 °C, and cfDNA was extracted from 1 ml of plasma. The presence of high-risk HPV types, HPV16 and HPV18, in cfDNA from 35 patients was assessed using ddPCR. The median concentration of cfDNA in cervical cancer patients was 9.35 ng/µL at baseline, which decreased to 7 ng/µL after three months of treatment. In healthy controls, the median cfDNA concentration was 6.95 ng/µL. ddPCR screening showed that detection rates for HPV18 and HPV16 detection were 45.71% and 82.86%, respectively. A significant correlation was observed between cf HPV16 DNA levels and tumor size, suggesting its potential as biomarker for disease burden.

Keywords: Cervical cancer; Circulating cell-free DNA; Circulating tumor DNA; Digital droplet PCR; Human papillomavirus.

Fig. 1

Detection of Circulating Free DNA in Plasma Samples of Cervical Cancer Patients and Healthy Controls. (A) Quantification of circulating free DNA in plasma samples from cervical cancer patients (n = 60) at two different time points: baseline (prior to any treatment) and three months post-therapy. The results are compared with plasma samples from healthy controls (n = 10). (B) Comparative analysis of plasma cell free DNA levels between cervical cancer patients and healthy controls.

Fig. 2

Concentration of Circulating Cell Free HPV DNA (ccfHPV DNA) in Cervical Cancer Patients by Clinical Criteria (A) Concentration of circulating HPV DNA (copies mL, log scale) detected by ddPCR in cervical cancer patients, stratified by FIGO 2018 staging criteria. The stages are grouped into 2 categories: IB3, IIA, IIB patients and IIIB, IIIC1, IIIC2, IVA patients. This panel illustrates how the concentration of c-HPV DNA varies with disease progression, suggesting a correlation between higher c-HPV DNA levels and advanced disease stages. Statistical significance between groups was assessed using the t-test or Mann-Whitney U test, as appropriate. (B) Concentration of ccfHPV DNA (copies /mL, log scale) in cervical cancer patients categorized by age groups (in years). The figure explores whether patient age influences ccfHPV DNA levels, providing insights into the demographic factors that may affect viral load in cervical cancer patients. Statistical analysis was conducted to determine any significant differences between age groups. (C) Concentration of ccfHPV DNA (copies mL, log scale) based on histological classification of cervical cancer: Non-Keratinizing Squamous Cell Carcinoma (NKSCC), Squamous Cell Carcinoma (SCC), and Clear Cell Adenocarcinoma (CAA). This panel evaluates the variation in ccfHPV DNA levels across different histological subtypes of cervical cancer, highlighting the potential differences in viral DNA presence related to tumor type. Statistical comparisons were made to assess significance among these groups. (D) Concentration of ccfHPV DNA (copies/mL, log scale) correlated with tumor size (measured in centimeters). This figure demonstrates the relationship between tumor size and ccf HPV DNA levels, suggesting that larger tumors may release more viral DNA into the bloodstream. The data support the use of ccfHPV DNA quantification as a proxy for tumor burden, which could aid in monitoring disease progression and treatment efficacy. Statistical significance was determined using appropriate tests. These dots outside the box represent the outliers.

Fig. 3

Schematic representation of the steps involved in extraction, quantification and analysis of cfDNA from the whole blood of cervical cancer patients.