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p16 Immunohistochemistry in Oropharyngeal Squamous Cell Carcinoma Using the E6H4 Antibody Clone: A Technical Method Study for Optimal Dilution

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Abstract

Routine testing for p16 immunohistochemistry (with selective HPV-specific test use) has been recommended for clinical practice in oropharyngeal squamous cell carcinoma (OPSCC). Data suggests that the E6H4 clone performs best for this purpose, yet no studies have evaluated the optimal antibody concentration for OPSCC testing. We evaluated three concentrations (undiluted, 1:5, and 1:10) of the primary antibody solution for E6H4 using tissue microarrays from a cohort of 199 OPSCC patients with a > 70% staining cutoff for positivity. Concordance was evaluated using percent agreement and Cohen’s kappa. The concentrations were evaluated for sensitivity and specificity using high risk HPV RNA in situ hybridization (RNA-ISH) and also correlated with Kaplan–Meier overall survival analysis. Inter-rater agreement was very high between p16 results at each concentration and also with RNA in situ hybridization (p < 0.0001 for all). Agreement between p16 undiluted and 1:5 dilution (agreement 98.2%; Kappa 0.943; p < 0.0001) was very high and between p16 undiluted and 1:10 dilution (agreement 79.2%; Kappa 0.512; p < 0.0001) much lower. Intensity of the staining did decrease with the 1:5 and 1:10 dilutions compared to undiluted, but not in a manner that obviously would change test interpretation or performance. Results suggest that the E6H4 antibody performs well at dilutions of up to 1:5 fold with a minor decrease in staining intensity, minimum loss of sensitivity, and no loss of specificity in OPSCC patients. This could result in reagent and cost savings.

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Acknowledgements

We would like to thank Donna M. Posey for her wonderful assistance with clerical support and spreadsheet data management for the various aspects of this study. We also acknowledge the Translational Pathology Shared Resource (TPSR) supported by NCI/NIH Cancer Center Support Grant 2P30 CA068485-14 and the Vanderbilt Mouse Metabolic Phenotyping Center Grant 5U24DK059637-13.

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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. & Jeremy Shelton

  2. Department of Otolaryngology, Vanderbilt University Medical Center, Nashville, TN, USA

    James S. Lewis Jr. & Krystle Lang Kuhs

  3. Division of Epidemiology, Vanderbilt University School of Medicine, Nashville, TN, USA

    Krystle Lang Kuhs

  4. Department of Biostatistics, Vanderbilt University Medical Center, Nashville, TN, USA

    Derek K. Smith

  5. Department of Oral and Maxillofacial Surgery, Vanderbilt University Medical Center, Nashville, TN, USA

    Derek K. Smith

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  1. James S. Lewis Jr.
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  2. Jeremy Shelton
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Correspondence to James S. Lewis Jr..

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Ethical Approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors.

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Use of participant data was exempt from individual consent (study waiver) under institutional review board approval.

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Lewis, J.S., Shelton, J., Kuhs, K.L. et al. p16 Immunohistochemistry in Oropharyngeal Squamous Cell Carcinoma Using the E6H4 Antibody Clone: A Technical Method Study for Optimal Dilution. Head and Neck Pathol 12, 440–447 (2018). https://doi.org/10.1007/s12105-017-0871-5

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  • DOI: https://doi.org/10.1007/s12105-017-0871-5

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