# Evaluation of targeted next‐generation sequencing for detection of HPV genotypes and sublineages in cervical liquid‐based cytology SurePath samples from the Danish screening program

## TL;DR

This study shows how next-generation sequencing can detect HPV sublineages in cervical screening samples, which could help improve risk assessment for cervical cancer.

## Contribution

A validated NGS protocol was developed to identify HPV sublineages in SurePath LBC samples, enabling future risk stratification studies.

## Key findings

- NGS successfully identified HPV sublineages in 99.1% of HPV-positive SurePath LBC samples.
- Sublineages from the A lineage were most common for most HPV genotypes, while B-type sublineages were more frequent for HPV45, 56, and 66.
- HPV31 showed the most diverse sublineage distribution across A, B, and C lineages.

## Abstract

The carcinogenicity of HPV genotypes is well established. However, HPV genotypes have sublineages with individual risk profiles, and these are much less described with respect to carcinogenicity. Research to characterize HPV sublineages by next‐generation sequencing (NGS) on screening‐derived liquid‐based cytology (LBC) samples is limited because of the technical and quality assurance challenging nature of sublineage analysis. This study aimed to evaluate the feasibility of detecting HPV sublineages from 14 HPV genotypes in SurePath LBC samples from Danish cervical cancer screening. We included 41 HPV plasmids (the Global HPV LabNet DNA Genotyping Proficiency Panel 2023) to quality assure the NGS approach and 120 SurePath LBC samples from the screening program for proof of concept. Our results of the HPV plasmids showed the correct sublineage for all included HPV genotypes except for HPV68b, where the coverage was inadequate for sublineage analysis. The NGS analysis enabled HPV sublineage analysis in 99.1% (112/113) of HPV‐positive SurePath LBC samples. Sublineages belonging to the A lineage were most frequent for HPV16, 18, 31, 33, 35, 39, 51, 52, 58, 59, and 68, while B‐type sublineages showed the highest frequency in HPV45, 56, and 66. The most diverse sublineage data was obtained for HPV31 with sublineages from the A, B, and C lineages. In conclusion, our method enables the identification of HPV sublineages in SurePath LBC screening samples. This information can be used in future studies to determine the usefulness of HPV sublineage analysis in screening settings for risk stratification and clinical management of HPV‐positive women.

What's new?

Human papillomavirus (HPV) assays currently used in cervical cancer screening detect genotypes without providing information on viral lineages and sublineages. However, genotype‐specific sublineages may have distinct risk profiles, which could be harnessed to further improve risk stratification. In this proof‐of‐concept study, a next‐generation sequencing panel and analytical protocol were developed specifically for cervical SurePath liquid‐based cytology samples to identify HPV sublineages. The validated protocol could be applied in future studies to determine the usefulness of HPV sublineages in risk stratification and clinical management.