Development of a Third-Generation Nanopore HPV Integration Site Detection Kit for Precise Prediction of Cervical Cancer Prognosis

Cervical cancer is the most common malignant tumor of the reproductive tract, posing a serious threat to women's health. According to data from the World Health Organization (WHO), there are over 500,000 new cases of cervical cancer globally each year, with approximately 342,000 deaths. In China, there are about 107,000 new cases annually, with around 51,000 patients dying from cervical cancer. The market for detection is vast and continues to grow. The main methods of examination after cervical cancer screening and treatment include HPV testing, cytological screening, colposcopy, and cervical biopsy. All HPV tests on the market are qualitative and semi-quantitative, and there is currently no product on the market that can detect whether HPV integrates into the genome in patients with persistent HPV positivity.

Our team, in previous research, enrolled over 200 cervical cell samples, including those from patients with high-risk HPV infection without intraepithelial neoplasia, cervical intraepithelial neoplasia (CIN) I, CIN II, CIN III, and samples from locally advanced cervical cancer before and after radiochemotherapy at multiple time points. Using third-generation nanopore technology, we have completed experiments and analysis on 93 cervical cell samples. Based on the probe hybridization capture principle, we enriched and sequenced the integration sites of HPV viral DNA. Preliminary conclusions are as follows:

1. Through our self-developed experimental technology process, we achieved long-fragment virus capture and sequencing (average read length 2-5kb), identifying HPV insertion sites and more precise genomic localization. Moreover, nanopore long-fragment sequencing can detect the complete Human-Virus-Human (H-V-H) viral insertion structure sequence.
2. There is a certain proportion of overlap between the HPV insertion genes in the inflammation group, CIN I, CIN II, CIN III groups, and the HPV insertion genes in cancer patients with recurrence and metastasis, which can further assist in screening for molecular markers related to tumor progression and poor prognosis.
3. The proportion of HPV sequences and the number of inserted genes in patient samples before and after radical radiochemotherapy can further assess the effectiveness of treatment and prognosis, providing support data for pre-treatment intervention and risk warning.

The goal of this study is to collaborate with a domestically produced nanopore sequencing platform and self-designed probes targeting the characteristics of HPV infection in the Chinese population. We aim to conduct insertion site detection on cohorts of late-stage cervical cancer (stage IIIB and beyond) and patients with recurrence and metastasis after radical cervical cancer radiochemotherapy. We will further establish and optimize the long-fragment virus enrichment method and nanopore sequencing experimental process to complete the entire detection process within one working day (8 hours). This will also expand the application of the technology in bedside diagnosis and decentralized directions.