Feasibility Study of Blood Biomarker Identification in Cervical Cancer

Feasibility Study of Blood Biomarker Identification in Cervical Cancer: Performance of Cell-free DNA Versus Extracellular Vesicles

This study aims to explore whether a simple blood test can be used to help detect and monitor cervical cancer. The investigators are studying tiny fragments and particles found in blood, called cell-free DNA (cfDNA) and extracellular vesicles (EVs), to determine whether these can act as early warning signs (biomarkers) of cervical cancer.

Cell-free DNA (cfDNA) refers to tiny fragments of genetic material (DNA) that are naturally released from cells into the bloodstream when cells die or renew. Cell-free DNA circulates naturally in the blood. In people with cancer, some of the cfDNA originates from cancer cells; this component is called circulating tumour DNA (ctDNA). Analysis of cfDNA can detect signs of cancer, such as specific genetic changes or pieces of viral DNA, including human papillomavirus (HPV) DNA, without requiring removal of tissue from the cervix.

Extracellular vesicles (EVs) are very small "packages" released by cells into the blood and other body fluids. EVs carry messages between cells and contain important information such as proteins, fats, and genetic material, including RNA. In cancer, tumour cells release EVs that reflect tumour behaviour and activity. Analysis of EVs can provide information about cancer growth and response to treatment.

Together, cfDNA and EVs can act as tiny messengers or "fingerprints" of biological processes occurring inside the body. Analysis of cfDNA and EVs in blood samples may support the development of a simple, less invasive, more comfortable, and more accessible approach to detecting and monitoring cervical cancer compared with current screening and diagnostic methods. A blood test could also assist doctors in monitoring treatment response or detecting early signs of recurrence without requiring a surgical biopsy.

The expected outcomes of this study are to identify specific patterns or "signatures" in extracellular vesicles (EVs) and cell-free DNA (cfDNA) found in blood samples. These signatures could help to:

Detect cervical cancer earlier, before symptoms appear or the disease progresses.

Monitor treatment response in real time without the need for invasive tests. Predict whether cervical cancer might recur or spread after treatment. Support more personalised care by helping doctors select the most suitable treatment for each participant.

This study is an open-label, prospective, experimental study of patients with cervical cancer. The primary aim is to evaluate the feasibility, clinical utility, and analytical performance of extracellular vesicles (EVs) and cell-free DNA (cfDNA) as minimally invasive, blood-based biomarkers for the early detection, treatment monitoring, and post-treatment surveillance of cervical cancer, with the potential to inform future screening and therapeutic strategies.

Cervical cancer continues to impose significant physical, emotional, and financial burdens on women worldwide. Despite advances in prevention and treatment, a critical need remains for more effective, non-invasive tools to support early detection, monitor treatment response, and guide clinical decision-making. As the field of cervical cancer diagnosis evolves, interest is growing in the use of blood-based biomarkers, particularly extracellular vesicles (EVs) and cell-free DNA (cfDNA), to improve patient care.

This study aims to evaluate the potential of EVs and cfDNA as minimally invasive biomarkers in the management of cervical cancer. Analysis of EVs and cfDNA is intended to provide deeper insights into the roles of these biomarkers in tumour development, progression, and intercellular communication. The anticipated outcomes include the identification of EV- and cfDNA-based signatures that can:

Facilitate earlier detection of cervical cancer. Enable real-time monitoring of treatment response. Predict recurrence and disease progression. Support the development of personalised therapeutic strategies.

By generating preliminary comparative data on the diagnostic accuracy of EVs and cfDNA, this study will provide a critical foundation for future large-scale investigations. The study is intended to contribute to the development of a novel, blood-based screening and monitoring tool that could enable earlier intervention and improve long-term outcomes.

A reliable, non-invasive test could also provide a more accessible and acceptable alternative to current cervical screening methods, particularly for women who face cultural, systemic, or personal barriers to participation. This is especially important for under-screened populations, including Aboriginal and Torres Strait Islander women and culturally diverse communities.

Ultimately, this research is expected to advance understanding of the biological role of EVs and cfDNA in cervical cancer pathogenesis and progression. The research supports the development of clinically effective, personalised diagnostic tools that may improve outcomes, reduce healthcare costs, contribute to Australia's national strategy to eliminate cervical cancer as a public health issue, and address persistent inequities in access to care.

Study Intervention

This prospective cohort study will enrol 100 patients with stage IA to IVB cervical cancer who are undergoing either primary surgical treatment, radiotherapy (RT), or chemoradiotherapy (CRT).

The study intervention involves the collection of approximately 40 mL of blood at baseline, following the initial diagnosis of cervical cancer, for the analysis of cell-free DNA, extracellular vesicles, and germline DNA. Additional blood samples of the same volume will be collected at two subsequent time points: at 8 weeks following surgery or completion of chemoradiotherapy (CRT), and at 6 months post-baseline.

In addition, quality-of-life questionnaires will be completed by participants at the following time points:

First time point: Baseline: For participants undergoing primary surgery, baseline is the date of surgery. For participants undergoing definitive radiotherapy or chemoradiotherapy, baseline is the date of the first radiation dose. Baseline assessments can be performed up to 30 days before the date of surgery or up to 30 days before the first radiation dose.

Second time point: Immediately after completion of radiotherapy or chemoradiotherapy (+14 days), or 8 weeks after surgery (±14 days).

Third time point: 6 months post-baseline (±14 days). Follow-up (24 Months)

Participant disease status will be recorded at 24 months (±30 days) through review of participants' medical records.

In accordance with current European Society for Medical Oncology (ESMO) guidelines, patients diagnosed with early-stage invasive cervical cancer, such as FIGO stage IA to IB2 and selected IIA1, typically undergo primary surgical treatment, including radical hysterectomy or simple hysterectomy. For these patients, the second blood collection will occur at 8 weeks post-surgery (±14 days).

For patients with locally advanced cervical cancer (LACC), for whom primary surgery is not recommended because of poor outcomes or increased morbidity associated with combined treatments, such as FIGO stages IB3, IIA2, IIB, and IIIA-IIIB, the preferred or definitive treatment is concurrent chemoradiotherapy (CRT) followed by internal radiation (brachytherapy). In these cases, the second blood sample will be collected at the end of treatment (+14 days).

Inclusion Criteria:

• Females, over 18 years, with histologically confirmed squamous cell carcinoma or adenocarcinoma of the cervix (stage 1a to 4b)
• Undergo intensive primary surgical treatment, radiotherapy (RT) or chemoradiotherapy (CRT).
• Signed written informed consent

Exclusion Criteria:

• Unable to provide informed consent
• Serious concomitant systemic disorders incompatible with the study (at the discretion of the investigator)
• Prior malignancies <5 years before inclusion, except for successfully treated keratinocyte skin cancers, or ductal carcinoma in situ.