Simple SummaryOver 95% of cervical cancers are caused by the human papilloma virus (HPV). Locally advanced HPV-related cancers release tiny bits of tumour containing HPV-DNA into the blood (cHPV-DNA). The presence of cHPV-DNA in the blood can serve as a potential marker of cancer and should be absent in pre-cancerous conditions (cervical intraepithelial neoplasia (CIN). We have developed an ultra-sensitive and specific test to measure blood cHPV-DNA levels. We performed a feasibility study to confirm our expectation that cHPV-DNA is not found in the blood of patients with CIN but is found in the blood of patients with very early cancers. In this study, we have confirmed that cHPV-DNA is absent in the plasma of women with CIN. In early cervical tumours, there was a low detection rate of cHPV-DNA. Therefore, more sensitive tests are required before cHPV-DNA can be used for the detection of very early cervical cancers.AbstractBackground: High-risk HPV infection is responsible for >99% of cervix cancers (CC). In persistent infections that lead to cancer, the tumour breaches the basement membrane, releasing HPV-DNA into the bloodstream (cHPV-DNA). A next-generation sequencing assay (NGS) for detection of plasma HPV circulating DNA (cHPV-DNA) has demonstrated high sensitivity and specificity in patients with locally advanced cervix cancers. We hypothesised that cHPV-DNA is detectable in early invasive cervical cancers but not in pre-invasive lesions (CIN). Methods: Blood samples were collected from patients with CIN (n= 52) and FIGO stage 1A-1B CC (n= 12) prior to treatment and at follow-up. DNA extraction from plasma, followed by NGS, was used for the detection of cHPV-DNA. Results: None of the patients with pre-invasive lesions were positive for CHPV-DNA. In invasive tumours, plasma from one patient (10%) reached the threshold of positivity for cHPV-DNA in plasma. Conclusion: Low detection of cHPV-DNA in early CC may be explained by small tumour size, poorer access to lymphatics and circulation, and therefore little shedding of cHPV-DNA in plasma at detectable levels. The detection rate of cHPV-DNA in patients with early invasive cervix cancer using even the most sensitive of currently available technologies lacks adequate sensitivity for clinical utility.