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Automated versus manual detection of HPV in head and neck cancers

Human papillomavirus (HPV) is a DNA virus from the papillomavirus family that is capable of infecting human keratinocytes of the skin or mucous membrane. In a majority of cases, HPV infection will not cause any physical symptoms and may remain unnoticed. Unfortunately, the presence of HPV might also lead to the development of benign papillomas (e.g. warts) or malignant cancers of the cervix, head and neck, penis, rectum, and vagina. At least a dozen high-risk HPV strains have been identified. Two of them (HPV types 16 and 18) are believed to be linked with 70% of cervical cancers and are responsible for a unique type of head and neck cancer in the throat. HPV infection is the most common sexually-transmitted disease and it is estimated that there are 529,000 new cases of cervical cancer and 275,000 deaths per year worldwide. To stop these numbers from rising, two HPV vaccines shielding against HPV-induced cancers were created using the L1 protein of the viral capsid: Gardasil® (Merck & Co.) and Cervarix® (GlaxoSmithKline). The latter protects against HPV types 16 and 18 only while the former also protects against HPV types 6 and 11, strains responsible for 90% of genital warts.


In situ hybridization (ISH) is a technique relying on the use of a labeled complimentary DNA or RNA to quantify and localize a specific DNA or RNA sequence in a tissue section. Briefly, the probe hybridizes to the target sequence at elevated temperature while unbound or excess probe is washed away under stringent conditions. Salt/detergent concentrations as well as temperature of hybridization are taken into account when optimizing the complementary binding of the probe to the target sequence and preventing unspecific binding. Quantification and localization can then be carried out using a chromogen, a fluorochrome, or a radioactive compound. In situ hybridization is a method of choice for the clinical diagnosis of certain chromosomal abnormalities and infections, notably HPV infections. It was found to be especially useful for molecular testing of HPV infection in cervical lesions and is now also integrated into the pathologic assessment of head and neck squamous cell carcinomas (HNSCCs).

Back in 2010, Singhi and colleagues from the Johns Hopkins Medical Institutions demonstrated that a strategy incorporating the immunohistochemistry (IHC) of p16 (an inhibitor of cyclin-dependent kinases 4 and 6 and a biomarker for head and neck squamous cell carcinoma (HNSCC)) and the in situ hybridization of HPV allowed the detection of HPV in a high percentage of HNSCCs. With the ever increasing number of HPV-associated cancers being diagnosed, there is a need for a fast and reliable method to successfully detect HPV in tissue. To that effect, Dr. Fatima and coworkers from Emory University Hospital compared manual automated HPV ISH with manual HPV ISH and p16 IHC using tissue sections from metastatic HNSCCs. Using Enzo Life Sciences’ PATHO-GENE® HPV type 16/18 probe, they demonstrated that automated HPV ISH combined with careful evaluation under high magnification was a more sensitive approach in determining HPV status in tissue sections compared to manual HPV ISH and p16 IHC. In addition, automated HPV ISH lowered the overall experimental costs and reduced the time-to-diagnosis from seven hours to one hour. Altogether, this study highlights the relevance of using automated methods for the clinical analysis of HPV in HNSCCs and their potential application to other forms of HPV-associated cancers.

As a long time contributor to the HPV market, Enzo Life Sciences developed the first non-radioactive HPV probes for in situ hybridization detection in 1986. Today, Enzo Life Sciences continues to contribute to the field of HPV research, notably with a new flow cytometry-based platform, the FLOWSCRIPT® HPV E6/E7 Assay that allows the single cell detection of mRNAs for HPV oncoproteins E6 and E7. Some of the products offered by Enzo Life Sciences and used for the detection of HPV are listed below:

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References:

  1. A.D. Singhi, et al. Comparison of human papillomavirus in situ hybridization and p16 immunohistochemistry in the detection of human papillomavirus-associated head and neck cancer based on a prospective clinical experience. Cancer (2010) 116: 2166-73.
  2. N. Fatima, et al. Automated and manual human papilloma virus in situ hybridization and p16 immunohistochemistry: comparison in metastatic oropharyngeal carcinoma. Acta Cytol. (2013) 57: 633-40.

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