On the path of developing a gold standard method for molecular diagnostics of HPV-related oropharyngeal cancers

Oropharyngeal cancer refers to cancer of the tonsil, the back third of the tongue, soft palate, and side and back walls of the throat. In 2015, there were an expected > 13,000 new cases of oropharyngeal cancer in the United States and it is becoming more prevalent every year. Like most head and neck cancers, oropharyngeal cancer is more common among older men with a mean age of 63 and the male: female incidence ratio is 2.7:1. However, recently, oropharyngeal cancer patients have become younger and more commonly, female as human papillomavirus (HPV) infection has emerged as an etiology. Strikingly, the risk of developing oropharyngeal cancer is 16 times higher in HPV-positive patients. In Europe and North America, HPV infection accounts for about 70 to 80 % of all cases. Over 95 % of all oropharyngeal cancers diagnosed are of the squamous cell carcinoma type. These so-called oropharyngeal squamous cell carcinoma (OPSCC) develop from the squamous cells, which are flat, scale-like cells that normally form the lining of the mouth and throat.

HPV is a group of more than 150 related viruses named for the warts (otherwise known as papillomas) that some HPVs can cause. Each strain of HPV virus is given a number, which is known as its HPV type. HPV is the most common sexually transmitted infection (STI); in fact, it is so common that nearly all sexually active men and women get it at some point in their lives. About 79 million Americans are currently infected with HPV and about 14 million people become newly infected each year. Though most HPV infections go away on their own within a couple of years, some HPV infections do persist. HPV infections that do not go away can cause changes in the cells in the infected area, which can lead to genital warts or cancer lesions. As a result, approximately 17,600 women and 9,300 men in the USA are affected every year by cancers caused by HPV – the equivalent of a new case every 20 minutes. Accordingly, monitoring HPV status has become a powerful prognostic marker and routine testing of all OPSCCs for the presence of HPV is advocated today by several influential consensus groups including the National Comprehensive Cancer Network, the College of American Pathologists and the Cancer Care Ontario. For example, overexpression of the cellular protein p16 by immunohistochemistry (IHC) is widely used as a surrogate marker of active HPV infection, but p16 overexpression can also be induced by non-viral mechanisms, most likely resulting in an overestimation of the true prevalence of HPV-related cancer cases. Indeed, most studies show that about 20% of OPSCCs are p16-positive by IHC, but HPV-negative by DNA in situ hybridization (DISH).

In order to get a better understanding and a more reliable classification of HPV-related OPSCCs, Lisa M. Rooper and colleagues from the Johns Hopkins Medical Institutions, Baltimore, USA, analyzed eighty-two head and neck squamous cell carcinoma cases that at time of diagnosis had already undergone p16 IHC and HPV DISH using the broad-spectrum Ventana Inform HPV III Family 16 probe. In this recent study, they compared these initial findings with HPV analysis by RNA in situ hybridization (RISH) using probes complementary to E6/E7 mRNA for direct visualization of the viral transcripts of these oncogenes. In addition, they also compared these results to HPV detection by DISH utilizing Enzo’s PATHO-GENE® HPV type 16/18/31/33/51 probe. Their investigations showed that out of 42 originally HPV-discordant cases, i.e., cases that had been positive for p16 in IHC but negative in DISH using the Ventana Inform probe, seven could now be detected with Enzo’s PATHO-GENE® HPV probe. On the other hand, in almost 90% of these discordant cases, transcriptional HPV activity could be detected by (RISH) using probes complementary to E6/E7 mRNA. This evidence underscores that transcriptional activation of the viral oncoproteins E6 and E7 might become the gold standard method for detection of clinically relevant HPV infection in oropharyngeal cancers. With the recent launch of the FLOWSCRIPT® HPV E6/E7 assay kit, Enzo offers a cost-effective and reliable method that is perfectly suited for molecular diagnostics of E6/E7 mRNA in such potentially HPV-related cancer cases.

From our superior labeling technology for microarrays to labeling systems, probes and detection assays for in situ hybridization, Enzo Life Sciences provides a complete set of molecular diagnostic tools for the study of HPV, some of which are described below.

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