A Highly Sensitive Real-Time RT-PCR Assay for Detection of HPV-16 E7

Lizhi Yu*, John Rassa, Yixin Wang and Sarah Hersey

Bristol Myers Squibb, Summit, New Jersey, USA,


Highly sensitive HPV-16 assays suitable for clinical settings are critical to screen patients for emerging HPV targeted therapies. In this light, we developed a novel TaqMan RNA PCR assay for qualitative detection of HPV-16 E7 gene transcript using formalin-fixed and paraffin-embedded (FFPE) samples. Furthermore, the assay was compared to another SYBR green RNA PCR assay, a DNA genotyping assay and a composite method consisting of p16 IHC and HPV DNA genotyping assay. The results of 82 clinical HNSCC samples demonstrated that the TaqMan RNA assay detected 19% of the negative samples by the SYBR green RNA assay, 27% by the DNA genotyping assay and 24% by the composite assay while capturing 100% of the positive samples detected by the SYBR green RNA assay or the composite method. Meanwhile, 100% of the negative samples by the TaqMan assay were also negative by at least two other assays. To interrogate the discordant samples that were positive by the TaqMan RNA assay but negative by the SYBR green RNA assay, the analytical sensitivity of the two assays was evaluated using a simulated HPV-16 positive RNA sample panel. The result illustrated that the TaqMan RNA assay has sensitivity 10-fold or greater than that of the SYBR green RNA assay. Our study indicates that 1) laboratory-developed HPV-16 RNA PCR assays may have significant variability in performance even with similar methodologies. 2) A notable portion of HPV-16 negative samples by the methods reported from HNSCC clinical studies could be positive for the viral transcripts. These findings highlight the urgency of developing more accurate and reliable HPV-16 assays to select patients that are most likely to benefit from treatment regimens of HPV-targeted therapies.

Keywords: HPV-16 Assays, RNA, Therapy