Next-Generation Sequencing-Based HPV Genotyping Assay Validated in Formalin-Fixed, Paraffin-Embedded Oropharyngeal and Cervical Cancer Specimens

Abstract

Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5 + /6 + (BSGP)5 + /6 + . To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost.

Recent Advances on the Molecular Mechanism of Cervical Carcinogenesis Based on Systems Biology Technologies

“…In addition to DNA microarray and methylation arrays, deep sequencing such as next-generation sequencing (NGS) has also been widely used to understand genetic changes in cervical cancer [62,63]. For example, one group validated 20 different HPV genotypes in 266 cervical cancer specimens using NGS approach [64]. One study described cervical cytology by deep sequencing to investigate and compare HPV metagenomes for correlation with disease states [65].…”

Current and Emerging Molecular Tests for Human Papillomavirus–Related Neoplasia in the Genomic Era

“…11 HPV typing is feasible in paraffin-embedded tissue specimens. 9,45,80 Progress in Developing Genomic Tests for HPV-Related Neoplasia…”

“…39 Genomic methods have recently been described. 9,10,45,80 Cell-free DNA assays for HPV DNA in plasma show promise as a noninvasive measure of HPV þ carcinoma burden, and work is underway to evaluate this approach to monitor treatment efficacy, to identify recurrence, and to enhance early cancer detection. 27,30 A combinatorial approach is envisioned in which plasma is analyzed for viral DNA and for mutations common in carcinoma (eg, in PIK3CA).…”

HPV Genotyping of Modified General Primer-Amplicons Is More Analytically Sensitive and Specific by Sequencing than by Hybridization

“…Similar studies reported at the time of planning this study, typically combined the HPV consensus primers MY09/11,PGMY or a modified PGMY, which amplify a 450 nt fragment, in combination with 454 sequencing technology [18–20]. A recent study utilized the Ion Torrent platform in combination with the HPV broad-spectrum general primers (BSGP5+/6+) on formalin-fixed, paraffin-embedded cancer specimens, showing high sensitivity and specificity [21]. The BSGP5+/6+ primer system was designed by Schmitt et al [22] and consists of a pool of 3 reverse primers and 9 forward primers targeting the L1 gene.…”