Recently, there have been significant increases in oropharyngeal cancers (OPC) infected with the sexually transmitted human papillomavirus (HPV), known for driving cancer progression in cervical carcinomas. Over 85% of these patients are infected with the high-risk strain, HPV16. Recent research has shown that HPV+ OPC patients represent a distinct entity, with discrete molecular characteristics, treatment responsiveness and survival. Traditional detection methods are inadequate in addressing these variabilities as they are DNA-based and consequently do not confirm a transcriptionally active virus. Furthermore, these tests are invasive and costly. A biologically accurate, non-invasive high-risk HPV test is crucial for clinical decision making and treatment planning for OPC patients.
We are developing a robust diagnostic assay to non-invasively detect HPV16 mRNA oncogenes from blood and saliva of OPC patients.
HPV16 encodes for several viral oncogenes such as E6/E7. We have developed and optimised an RT-qPCR multiplexing assay to detect these two mRNA oncogenes in patient specimens.
Our assay detected E6/E7 in HPV+ cell lines, SiHa and Caski, with no significant difference in Ct values or PCR efficiencies to a traditional singleplex reaction (P<0.05). We then carried out serial dilutions of RNA input and determined that the LOD of our assay was 100pg. Next, we assessed our method in OPC patient tissue, which accurately differentiated between HPV+/- specimens with no significant difference in the PCR efficiency between the methods (P<0.05). Finally, we evaluated our assay in serum and saliva and obtained equivalent results.
Development of a biologically relevant test to precisely measure HPV is needed for clinical decision making and treatment planning for OPC patients. We have shown that HPV16 E6/E7 mRNA can be detected using hydrolysis probe RT-qPCR in cell lines and patient specimens. Our method reduces sample input, reagent costs, and is amenable to POC approaches. Our assay is different from other commercial or diagnostic kits as we measure the RNA, which is confirmation of active oncogene transcription and viral replication.