Third-Generation Nanopore HPV Integration Site for Precise Prediction of Cervical Cancer Prognosis

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

Cervical cancer is a significant health threat to women, with over 500,000 new cases and approximately 342,000 deaths worldwide annually, and about 107,000 new cases in China with 51,000 fatalities. Current screening methods include HPV testing, cytology, colposcopy, and biopsy, but none can detect HPV genome integration in persistently positive patients.

Our team has analyzed over 200 cervical cell samples using third-generation nanopore technology, focusing on HPV integration sites. We've developed a proprietary long-fragment capture and sequencing method (reads averaging 2-5kb) that identifies precise HPV insertion points and detects the complete Human-Virus-Human (H-V-H) viral insertion sequence. We found overlaps in HPV integration genes across different stages of cervical intraepithelial neoplasia (CIN) and in cancer patients with recurrence and metastasis, suggesting potential biomarkers for tumor progression and poor prognosis.

We also analyzed HPV sequence proportions and gene insertion numbers in samples from patients before and after radical radiochemotherapy, providing insights into treatment efficacy and prognosis. Our study aims to use a domestic nanopore sequencing platform and probes tailored to Chinese HPV infection patterns to detect integration sites in late-stage cervical cancer and post-treatment recurrence/metastasis patients. We aim to optimize our method to complete the entire detection process within eight hours, expanding the technology's application in point-of-care diagnostics and decentralization.

Study Overview

• Status: Completed
• Conditions: Cervical Cancers; HPV Testing; Nanopore Sequencing; HPV Integration Site Detection; Rapid Diagnosis
• Intervention / Treatment: Diagnostic test: HPV integration

Detailed Description

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.

• Study Type: Observational
• Enrollment (Actual): 200

Contacts and Locations

Study Locations

• China
  • Beijing
    • Peking, Beijing, China, 100730
      • Peking Union Medical College Hospital

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Older Adult
• Accepts Healthy Volunteers: No

• Sampling Method: Non-Probability Sample

Study Population

Patients enrolled in the study are those with advanced-stage cervical cancer (Stage IIIB and beyond), with TCT procedures performed by radiation oncologists. During the procedure, sterile physiological cotton balls are used to clean away external cervical secretions. A cytobrush is inserted into the cervical lesion or the squamocolumnar junction within the cervical canal to collect samples, rotated 3-4 times, left in place for several seconds, and then placed into a cell preservation fluid, sealed, and sent for testing. As a therapeutic department, the radiation oncology department is heavily involved in the entire process of diagnosis, treatment, and follow-up for cervical cancer patients. During treatment, patients undergo invasive brachytherapy, which has a higher level of sterility requirements and operational difficulty than TCT procedures, hence they are capable of performing TCT specimen collection.

Description

Inclusion Criteria:

• A. Undergoing radical radiochemotherapy at our institution or a collaborating institution; B. Pathologically confirmed as cervical cancer; C. Diagnosed as an advanced-stage patient (FIGO staging IIIB, IIIC, IVA, IVB), or a patient who has experienced local recurrence or distant metastasis after radical radiochemotherapy; D. Capable of regular follow-up visits; E. The patient or their family can understand the research protocol and are willing to participate in this study, providing written informed consent.

Exclusion Criteria:

• A. Confirmed by pathology as neuroendocrine carcinoma of the cervix; B. Have not undergone standardized radical treatment (for the cohort of stage IIIB and later cervical cancer patients, those who have not undergone standardized radical radiochemotherapy must be excluded. For the cohort of patients who have local recurrence or distant metastasis after radical radiochemotherapy, those who have only had cervical cancer surgery and recurrence, and those who have not undergone standardized radiochemotherapy after cervical cancer surgery must be excluded); C. HPV testing is negative, and there is no clear integration site of HPV in the genome; D. The patient or family members are unable to cooperate with the study or cannot provide written informed consent.

Study Plan

How is the study designed?

Number of groups / cohorts

1

Cohorts and Interventions

Group / Cohort

Intervention / Treatment

Advanced-stage cervical cancer patients group (Stage IIIB and beyond)

Diagnostic test: HPV integration; Collect late-stage cervical cancer (Stage IIIB and beyond) and post-treatment recurrence and metastasis specimens (TCT, tissue) associated with HPV infection. Utilize third-generation nanopore technology to detect the integration sites of HPV DNA based on the principle of probe hybridization capture. Obtain information on HPV integration and hot spot genes in the samples. By analyzing the integration of HPV in primary and recurrent/metastatic tissues, identify insertions and gene alterations associated with recurrence/metastasis, and develop a predictive formula.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Disease-free survival	Disease-free survival was defined as the period from the diagnosis of cervical cancer to events that included death or disease progression at local, regional, or distant sites or until the date of the last follow-up.	5 years

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Overall survival	Overall survival was defined as the period from the diagnosis of cervical cancer to the date of death or last follow-up.	5 years

Collaborators and Investigators

• Sponsor: Peking Union Medical College Hospital

Study record dates

Study Major Dates

• Study Start (Actual): September 19, 2019
• Primary Completion (Actual): August 31, 2024
• Study Completion (Actual): August 31, 2024

Study Registration Dates

• First Submitted: October 15, 2024
• First Submitted That Met QC Criteria: October 15, 2024
• First Posted (Actual): October 16, 2024

Study Record Updates

• Last Update Posted (Actual): October 16, 2024
• Last Update Submitted That Met QC Criteria: October 15, 2024
• Last Verified: September 1, 2019

More Information

Terms related to this study

• Additional Relevant MeSH Terms: Neoplasms; Urogenital Neoplasms; Neoplasms by Site; Uterine Neoplasms; Genital Neoplasms, Female; Uterine Cervical Diseases; Uterine Diseases; Female Urogenital Diseases; Female Urogenital Diseases and Pregnancy Complications; Urogenital Diseases; Genital Diseases; Genital Diseases, Female; Uterine Cervical Neoplasms
• Other Study ID Numbers: CHINTIGER

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO
• IPD Plan Description: To protect the privacy of the patients

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No