Human Papillomavirus Negative High Grade Cervical Lesions and Cancers: Suggested Guidance for Hpv Testing Quality Assurance

Abstract

Background: Some high-grade cervical lesions and cervical cancers (HSIL+) test negative for human papillomavirus (HPV). The HPV-negative fraction varies between 0.03% and 15% between different laboratories. Monitoring and extended re-analysis of HPV-negative HSIL+ could thus be helpful to monitor performance of HPV testing services. We aimed to a) provide a real-life example of a quality assurance (QA) program based on re-analysis of HPV-negative HSIL+ and b) develop international guidance for QA of HPV testing services based on standardized identification of apparently HPV-negative HSIL+ and extended re-analysis, either by the primary laboratory or by a national HPV reference laboratory (NRL).

Methods: There were 116 initially HPV-negative cervical specimens (31 histopathology specimens and 85 liquid-based cytology samples) sent to the Swedish HPV Reference Laboratory for re-testing. Based on the results, an international QA guidance was developed through an iterative consensus process.

Result: Standard PCR testing detected HPV in 55.2% (64/116) of initially “HPV-negative” samples. Whole genome sequencing of PCR-negative samples identified HPV in an additional 7 samples (overall 61.2% HPV positivity). Reasons for failure to detect HPV in an HSIL+ lesion are listed and guidance to identify cases for extended re-testing, including which information should be included when referring samples to an NRL are presented.

Conclusion: Monitoring the proportion of and reasons for failure to detect HPV in HSIL+ will be helpful to promote high performance of HPV testing services. We encourage implementation of QA strategies based on re-analysis of “HPV negative” HSIL+ samples.

Note:
Funding declaration: This work was supported by the Bill and Melinda Gates Foundation (INV-021790)

Conflict of Interests: K Cuschieri’s institution has received research funding or gratis consumables to support research from the following commercial entities in the last 3 years: Cepheid, Euroimmun, GeneFirst, SelfScreen, Hiantis, Seegene, Roche, Abbott, Hologic and Vaccitech. M.P.’s institution received research funding, free-of-charge reagents, and consumables to support research in the last 3 years from Qiagen, Seegene, Abbott, and Roche, all paid to his employer. Sciensano the employer of M.A. received funding in the framework of Valgent and VALHUDES, which are 2 researcher induced protocols for evaluation of HPV tests on cervical and vaginal samples respectively (see Arbyn et J Clin Virol 2016 & 2018). M.A, did not receive any financial or material benefit from these projects

Ethical Approval: Ethical approval was granted by the Swedish National Ethical Review Agency (decision 2023- 02457-01). The work was classified as quality assurance and all re-testing used coded samples.

Keywords: human papillomavirus, HSIL, Cervical Cancer, quality assurance, HPV testing, screening