Assess Transcriptionally Active HPV Biomarkers in Head and Neck Cancer Biopsies

Detect HPV E6/E7 mRNA expression with tissue morphological context

• High Sensitivity and Specificity—single molecule detection with single-target specificity by proprietary probe design and detection technology
• Broadest Spectrum—with flexibility of individual genotyping, pooled high-risk and low-risk panels (HPV-HR7, HPV-HR18, HPV-LR6) and custom pooled panels
• Robust and Easy Methodology—scalable with automation for routine analysis

Detection of Gold-standard E6/E7 Oncogene Transcripts Using RNAscope® ISH

Evidence for transcriptional active of the viral oncogenes E6/E7 is regarded as the gold standard for presence of clinically relevant high-risk human papillomavirus (HPV), but detection of E6/E7 mRNA can be challenging using conventional techniques (Bishop et al., 2013). As a causal agent in head and neck squamous cell carcinoma (HNSCC), it is critical that the detection method enable pathologist review of tissue morphology and be of the highest specificity and sensitivity for accurate assessment of within the tissue microenvironment of FFPE specimens (Figure 1, 2). RNAscope HPV Biomarker Detection Reagents and its proprietary “double Z” oligonucleotide probes specific for each subtype E6/E7 mRNA enable high specificity detection of viral transcripts in routine FFPE tumor biopsies.

FIGURE 1. Schematic illustration of the Biology of HPV infection provides points of HPV Detection (Bishop et al). Testing E6/E7 transcripts by RNA ISH is desirable because it indicates the presence of transcriptionally active virus and enables visualization of viral transcripts directly in tumor cells in tissue sections, unlike RT-PCR (Upko et al, 2011).

FIGURE 2. High risk HPV E6/E7 mRNA expression in Head and Neck Squamous Cell Carcinoma (HNSCC). FFPE section of HNSCC hybridized with a pool of HPV 16, 18, 31, 33, 35, 52 and 58 genotype probes, showing cytoplasmic punctate dots only in the tumor cells (40X magnification).

Highest Sensitivity and Specificity

Current methodologies for HPV testing include PCR-based amplification and DNA in situ Hybridization (ISH). PCR amplification of HPV DNA is more sensitive, but it is less specific than DNA ISH. Published studies (Bishop et al., 2013, Upko et al., 2011, and Schache et al., 2013) indicate that RNAscope-based ISH assay is more sensitive than DNA ISH in detecting HPV in Oropharyngeal Squamous Cell Carcinoma (OSCC) and results correlate well with p16 immunohistochemistry (IHC) staining (Table 1). RNAscope ISH technology is an ideal platform giving the high sensitivity and specificity needed for detection of HPV biomarkers in Head and Neck Squamous Cell Carcinoma (HNSCC) samples.

TABLE 1. Using the current “gold-standard” qPCR as the reference method, RNAscope® Technology demonstrated the best sensitivity and specificity for HPV status determination than existing methods (Schache et al., 2013). Abbreviations: IHC=immunohistochemistry; ISH=in situ hybridization; NPV=negative predictive value; PPV=positive predictive value; qPCR=quantitative PCR

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