Abstract
BackgroundHuman papillomavirus (HPV) is a necessary cause of cervical cancer, although some invasive cervical cancers may test negative by HPV PCR. We previously requested all invasive cervical cancers in Sweden during 10 years and subjected them to PCR. We also optimised methods for deep sequencing of formalin-fixed paraffin-embedded samples.MethodsUsing Novaseq 6000, we simultaneously sequenced total DNA and cDNA from 392 HPV PCR-negative cervical cancers. Non-human reads were queried against all known HPVs. The complete database now contains PCR and/or deep sequencing data on 2850 invasive cervical cancers.ResultsHPV sequences were detected in 169/392 of HPV PCR-negative cervical cancers. Overall, 30 different HPV types were detected, but only 5 types were present in proportions above 3% of cancers. More than 92% of tumours were HPV-positive in PCR and/or sequencing (95% confidence interval: 91.1–93.1%). Exploring possible reasons for failure to previously detect HPV suggest that more sensitive type-specific PCRs for HPV 31, 33, 45 and 73 targeting retained regions of HPV would have detected most of these (117/392).ConclusionsUnbiased deep sequencing provides comprehensive data on HPV types in cervical cancers and appears to be an important tool for quality assurance of HPV screening.
Highlights
Human papillomavirus (HPV) is a necessary cause of cervical cancer, some invasive cervical cancers may test negative by human papillomavirus (HPV) PCR
Sample collection HPV genotyping was previously performed based on L1 amplification and consequent probe hybridisation on formalin-fixed paraffin-embedded (FFPE) blocks from all cervical cancers occurring during a 10-year period (2002–2011) in Sweden.[13]
If HPV negative (n = 483), the FFPE blocks were subjected to HPV 16 and 18 real-time PCR (rt-PCR) targeting E6 and E7 genes of the HPV genome
Summary
Human papillomavirus (HPV) is a necessary cause of cervical cancer, some invasive cervical cancers may test negative by HPV PCR. An HPV genotype that diverges in its genomic sequence from the sequences designed for primers/probe may escape amplification and/or hybridisation, and remain undetected.[8,9,10] HPV detection failure can be readily circumvented by subjecting specimens to unbiased high throughput sequencing of the total nucleic acids of the sample, as HPV can be detected without prior knowledge/assumptions of which genotype-specific sequences may be present.[8,11,12] if cDNA is sequenced, the data will show if there is viral transcriptional activity, which is typically essential for both initiation and maintenance of the malignant phenotype
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