1887

Abstract

Cervical cancer is caused by human papillomavirus (HPV), but some cases may test HPV-negative. We previously tested 2850 Swedish cases and found that 394/2850 (13.8 %) cases tested HPV DNA-negative by PCR. Sequencing is the most thorough method to assess HPV status.

. We wished to assess whether deep sequencing might detect HPV sequences among these HPV-negative cervical cancer specimens, and to increase the likelihood of detecting transcriptionally active infections.

Out of the 2850 cancer cases, we sequenced a random sample of 92 HPV PCR-negative cervical cancers and 34 HPV PCR-positive cervical cancers. Four pools of blank blocks were sequenced as negative controls. To enrich for mRNA – a hallmark of active viral infection – the samples were extracted, reverse-transcribed, rRNA-depleted and then sequenced using the NovaSeq 6000 system (Illumina, USA). High-quality reads were aligned to the human genome and non-human reads were queried against HPV proteins.

We obtained a median of 23 million paired reads per sample. HPV was detected in 31/34 HPV PCR-positive cases. Among cases negative for HPV by PCR, 48/92 (52.2 %) contained HPV sequences, with HPV33 being the most commonly detected type among these (14/48 cases, 29.2 %). Comparison of the ratio of exon and intron sequences found that the sequenced material contained both DNA and RNA. Splice junctions were detected in 12 cases.

Apparently, some cervical cancers contain HPV that is difficult to detect by PCR. Sequencing may be a helpful tool for additional quality assurance for HPV testing methods.

Funding
This study was supported by the:
  • Joakim Dillner , Swedish Foundation for Strategic Research , (Award RB13-0011)
  • Joakim Dillner , Cancerfonden , (Award CAN2017/459)
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/content/journal/jgv/10.1099/jgv.0.001374
2019-12-20
2020-11-24
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