Targeted Next Generation Sequencing for Human Papillomavirus Genotyping in Cervical Liquid-Based Cytology Samples

Abstract

At present, human papillomavirus (HPV) testing is replacing morphology-based cytology as the primary tool for cervical cancer screening in several countries. However, the HPV assays approved for screening lack detection for all but one of the possibly carcinogenic HPV types and do not genotype all included HPV types. This study demonstrates the use of a targeted HPV next generation sequencing (NGS) panel to detect and genotype all 25 carcinogenic, probably carcinogenic, and possibly carcinogenic HPV types as well as the low-risk types HPV6 and HPV11. The panel was validated using a cohort of 93 paired liquid-based cytology samples (general practitioner (GP)-collected cervical samples and cervico-vaginal self-samples (SS)). Overall, the targeted panel had a sensitivity (GP = 97.7%, SS = 92.1%) and specificity (GP = 98.0%, SS = 96.4%) similar to the commercial HPV assays, Cobas® 4800 HPV DNA test (Roche) and CLART® HPV4S assay (GENOMICA). Interestingly, of the samples that tested positive with the NGS panel, three (6.4%) of the GP-collected samples and four (9.1%) of the self-samples tested positive exclusively for HPV types only included in the NGS panel. Thus, targeted HPV sequencing has great potential to improve the HPV screening programs since, as shown here, it can identify additional HPV positive cases, cases with HPV integration, variants in the HPV genome, and which HPV type is dominant in multi-infected cases.

Whole-genome analysis of human papillomavirus 67 isolated from Japanese women with cervical lesions

“…On the other hand, using an NGS panel that targets all carcinogenic and probably/possibly carcinogenic HPV types, Andersen et al . demonstrated that 5 out of 93 cases (5.4%) diagnosed as atypical squamous cells of undetermined significance were positive for HPV67 in cervical-vaginal self-collected samples [ 28 ]. These estimates suggest that the rate of HPV67 infection in the general population might be higher than previously thought.…”

Circulating cell‐free HPV DNA is a strong marker for disease severity in cervical cancer

“…The designing of the NGS HPV genotyping panel is described in detail in Andersen et al [29]. This panel includes the 25 IARC-classified carcinogenic (HPV16, 18,31,33,35,39,45,51,52,56,58,59), probably carcinogenic (HPV68), and possibly carcinogenic (HPV26, 30,34,53,66,67,69,70,73,82,85,97) HPV genotypes and the two most common low-risk HPV genotypes HPV6 and HPV11 [42].…”

“…In two previous studies published by our group, we validated a targeted next‐generation sequencing (NGS) panel for both HPV detection and genotyping, as well as evaluation of HPV integration status [ 28 , 29 ], which has been shown to reflect viral load [ 30 , 31 ] and prognosis [ 30 , 31 , 32 , 33 ] in CC. Integration of HPV DNA was previously considered a critical event in the process of cervical carcinogenesis [ 34 , 35 , 36 ].…”

Review of the Standard and Advanced Screening, Staging Systems and Treatment Modalities for Cervical Cancer

“…Furthermore, the next-generation sequencing (NGS) can be explicitly used to improve the specificity and sensitivity of HPV detection. The NGS panel can effectively detect the presence of all HPV genotypes present in archival formalin-fixed, paraffin-embedded (FFPE), liquid-based cytology (LBC), and plasma samples [ 60 , 61 ]. Thus, the NGS technology lowers the chance of false-negative results, compared with the traditional PCR-based assays, and provides more accurate screening results for the subsequent diagnosis and treatment planning.…”