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Tissue Fixation Conditions for p16 Immunohistochemistry and Human Papillomavirus RNA In Situ Hybridization in Oropharyngeal Squamous Cell Carcinoma

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Abstract

Human papillomavirus (HPV) has become a critical prognostic biomarker in oropharyngeal squamous cell carcinoma (OPSCC). While retrospective studies suggest that p16 immunohistochemistry and even HPV RNA in situ hybridization work well on tissues and tumors from a variety of labs and various fixation conditions, no formal study of fixation conditions has been performed to date. We took surgically resected specimens from three p16 and HPV RNA in situ hybridization positive OPSCC patients, divided their fresh tumors into small pieces, and varied the time to formalin fixation as 1, 3, 6, 24, and 48 h. Tumors were either held moistened at room temperature or were refrigerated. After fixation and processing, routine hematoxylin and eosin slides were generated and p16 immunohistochemistry and RNA in situ hybridization performed. All three tumors were nonkeratinizing and had strong and diffuse p16 expression at immediate fixation, which, surprisingly, remained positive for all fixation times and conditions and despite significant degeneration at the later points for two of the patients while for one, the nuclear signal dropped out of most cells at early and mid time points, particularly at room temperature, causing false negatives. HPV RNA in situ hybridization stayed positive in all specimens up to 48 h of cold ischemic time refrigerated and even at room temperature, except for overtly autolyzed tumor regions. These findings help to establish that, at least for standard nonkeratinizing, p16 and HPV RNA strongly positive OPSCC patients, and using the most common tests in clinical practice, relatively lenient time to fixation may be acceptable if it cannot be avoided. However, for some patients, p16 immunohistochemistry may be sensitive to signal loss with autolysis. HPV RNA in situ hybridization, in particular, seems remarkably resistant to pretest cold ischemic times.

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Acknowledgements

We would like to thank Natasha Jones, Mary Abbuhl, and Craig Self from the Vanderbilt University Hospital Anatomic Pathology histology laboratory for their help with performing the RNA ISH studies.

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Authors and Affiliations

  1. Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, 3020D Vanderbilt University Hospital, Nashville, TN, 37232-7415, USA

    James S. Lewis Jr., Shira B. Barnett & Mitra Mehrad

  2. Department of Otolaryngology, Vanderbilt University Medical Center, 3020D Vanderbilt University Hospital, Nashville, TN, 37232-7415, USA

    James S. Lewis Jr., Kyle Mannion & Mitra Mehrad

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  1. James S. Lewis Jr.
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  2. Shira B. Barnett
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Correspondence to James S. Lewis Jr..

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Lewis, J.S., Barnett, S.B., Mannion, K. et al. Tissue Fixation Conditions for p16 Immunohistochemistry and Human Papillomavirus RNA In Situ Hybridization in Oropharyngeal Squamous Cell Carcinoma. Head and Neck Pathol 14, 637–644 (2020). https://doi.org/10.1007/s12105-019-01090-6

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  • DOI: https://doi.org/10.1007/s12105-019-01090-6

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