The rapid rise of head and neck squamous cell carcinoma is due, in part, to the increased incidence of HPV-mediated oropharyngeal squamous cell carcinoma (OPSCC). This is a viral-mediated disease and as such likely co-exists with the virus in two distinct states: episomal, or outside the host genome, and integrated into the genome itself. Understanding the integration status of the virus in OPSCC could aid in determining future treatment paradigms, but first a rapid, low-cost assay for detecting integration is needed.
Methods: RNA from formalin-fixed, paraffin embedded tissues (FFPE) from 123 known OPSCC tumors was extracted and assayed on an nCounter MAX Analysis System with the Human PanCancer IO 360 with an additional eight HPV genes included in the target codeset following the manufacturer’s protocol. As suggested in the protocol, total RNA input was adjusted to 100 ng based on DV300 values from TapeStation 4200 profiles: (100/percent of sample > 300 nt) x 100 ng. A panel standard was run on each cartridge to allow for normalization. HPV E6 and E5 levels were constructed as a ratio to determine integration status. For verification runs, RNAseq was performed on libraries generated from the same samples and human-viral reads were used to identify integrated tumor samples. Sequencing was performed on a NovaSeq 6000 system with a 2x50 bp paired-end configuration following the manufacturer’s protocol. The pools were designed to target about 105 million clusters per library on average on either a S2 flow cell (n=40) or a S4 flow cell (n=93).
Results: Use of the E6:E5 expression ratio was highly sensitive and specific (92.6% and 95.1%, respectively) for detection of integrated HPV16 in OPSCC samples. This was confirmed by RNAseq-based human-viral reads and subsequent E6/E7:E2/E5 reads as determined by viral genome RNAseq analysis. Additionally, HPV16 detection by this method was also both highly sensitive (100%) and highly specific (89.1%).
Conclusions: We have developed a method to rapidly determine HPV presence and integration status using RNA signatures in OPSCC tumors. This technology could be used to identify HPV-positive tumors with integration status and could aid in selection of appropriate treatment modalities.