Probes for HPV

Abstract

Objectives

In the present study, we comprehensively analyzed the prevalence of transcriptionally active HPV in tissue samples of Indian patients with leukoplakia - predominantly hyperplastic lesions and HNSCC. In addition, saliva samples from patients with HNSCC were screened for HPV detection.

Study Design

p16 overexpression was analyzed by immunohistochemistry. Leukoplakia (n = 121) and HNSCC (n = 427) tissue samples and the saliva of patients with HNSCC (n = 215) were tested for HPV using nested PCR. Positive samples were sequenced for subtyping. The presence of HPV E6/E7 mRNA was confirmed by RNA in-situ hybridization.

Results

p16 expression and HPV DNA were not detected in any of the leukoplakia specimens. Of the 427 HNSCC tumors, 9 showed p16 overexpression and 7/427 cases were positive for HPV16 DNA, either in saliva and/or tissue. E6/E7 mRNA positivity was observed in eight HNSCC samples, primarily from patients with no habit of tobacco consumption. The prevalence of high-risk HPV was restricted to oropharynx and larynx with very little concordance between p16 overexpression and HPV positivity. All patients with HPV positive saliva samples had transcriptionally active HPV present in their tumors.

Conclusion

Presence of HPV-DNA does not necessarily reflect transcriptionally active virus in tumors; hence, it is important to consider this fact while categorizing HPV associated tumors.

The role of human papillomavirus (HPV) as an etiologic and transformational agent in inverted Schneiderian papilloma (ISP) is unclear. Indeed, reported detection rates of HPV in ISPs range from 0 to 100%. The true incidence has been confounded by a tendency to conflate high- and low-risk HPV types and by the inability to discern biologically relevant from irrelevant HPV infections. The recent development of RNA in situ hybridization for high-risk HPV E6/E7 mRNA now allows the direct visualization of transcriptionally active high-risk HPV in ISP, providing an opportunity to more definitively assess its role in the development and progression of ISPs. We performed p16 immunohistochemistry and high-risk HPV RNA in situ hybridization on 30 benign ISPs, 7 ISPs with dysplasia, 16 ISPs with carcinomatous transformation, and 7 non-keratinizing squamous cell carcinomas (SCCs) with inverted growth that were unassociated with ISP. Transcriptionally active HPV was not detected in any of the 52 ISPs including those that had undergone carcinomatous transformation, but it was detected in two of seven (29%) non-keratinizing SCCs that showed inverted growth. There was a strong correlation between high-risk HPV RNA in situ hybridization and p16 immunohistochemistry (97%; p < 0.01). These results indicate that transcriptionally active high-risk HPV does not play a common role in either the development of ISP or in its transformation into carcinoma.

The aim of this study is comparing two in situ hybridization (ISH) detection methods for human papilloma virus (HPV) 16 E6/E7 mRNA, i.e. the RNAscope™ 2.0 High Definition (HD) and the upgraded RNAscope™ 2.5 HD version. The RNAscope™ 2.5 HD has recently replaced the RNAscope™ 2.0 HD detection kit. Therefore, this investigation starts from the need to analytically validate the new mRNA ISH assay and, possibly, to refine the current algorithm for HPV detection in oropharyngeal squamous cell carcinoma (OSCC) with the final goal to apply it to daily laboratory practice. The study was based on HPV status and on generated data, interpreted by a scoring algorithm. The results highlighted that the compared RNAscope HPV tests had a good level of interchangeability and enabled to identify OSCC that are truly driven by high risk-HPV infection. This was also supported by the comparison of the RNAscope HPV test with HPV E6/E7 mRNA real time reverse transcriptase-polymerase chain reaction (RT-PCR), in a fraction of cases where material for HPV E6/E7 mRNA real time RT-PCR was available. Furthermore, the algorithm that associates p16 immunohistochemistry (IHC) with the identification of HPV mRNA by RNAscope was more effective than the one that associated p16 IHC with the identification of HPV DNA by ISH.

Human papillomavirus (HPV) is an independent risk factor for development of oral squamous cell carcinoma (OSCC). This study aimed to investigate the role of HPV infection and the trend in percentage of HPV-associated OSCC over a five-year period in northeastern Thailand. In this case-control study, 91 exfoliated oral cell samples and 80 lesion cell samples from OSCC cases and exfoliated oral cells from 100 age/gender-matched controls were collected. HPV infection was investigated by PCR using GP5+/GP6+ primers followed by HPV genotyping using reverse line blot hybridization. Quantitative RT-PCR was used to evaluate HPV oncogene transcription. Temporal trends of HPVinfection were evaluated in archived formalin-fixed paraffin-embedded (FFPE) OSCC tissues using in situ hybridization. HPV DNA was found in 17.5% (14/80) of lesion samples from OSCC cases and 29.7% (27/91) of exfoliated oral cell samples from the same cases. These values were significantly higher than in exfoliated oral cell samples from controls (13%, 13/100). HPV-16 was the genotype most frequently found in OSCC cases (92.8%, 13/14 infected cases). Interestingly, HPV oncogene mRNA expression was detected and correlated with OSCC cases (P < 0.005). Of 146 archived FFPE OSCC samples, 82 (56.2%) were positive for high-risk HPV DNA and 64 (43.8%) cases were positive for HPV E6/E7 mRNA expression. There was a trend of increasing percentage of HPV-associated OSCC from 2005 to 2010. This was especially so for females with well-differentiated tumors in specific tongue sub-sites. We suggest that HPV infection plays an important role in oral carcinogenesis in northeastern Thailand.