Cytological interpretation of p16 immunohistochemistry in head and neck carcinomas: does the choice of fixative matter?

Fine-needle aspiration (FNA) of nodal metastases plays a key role in the diagnosis of oropharyngeal squamous cell carcinoma (OPSCC). Because of significant clinical implications of human papillomavirus (HPV)-related OPSCC, immunohistochemistry for p16 as a surrogate marker for high-risk HPV is an important ancillary test. After our laboratory switched from CytoLyt to formalin fixative for FNA needle rinses generating cell block (CB) material, we investigated the impact of this protocol change on the accuracy of p16 results. FNA specimens of head and neck lesions with p16 staining performed on CB, from 1 year before and after the implementation of formalin-fixed CB (FCB) were identified. Nuclear and cytoplasmic p16 expression was scored and compared to p16 status on corresponding surgical specimens. There were no false-positive results with either fixative. CytoLyt-fixed CB (CCB) had 47% (7 of 15) false-negative cases, whereas FCB had none, with 100% diagnostic accuracy for p16-negative (n = 6) and p16-positive (n = 15) results. False-negative CCB showed 0% to 10% nuclear and 0% to 65% weak cytoplasmic staining, whereas true-positive CCB showed 10% to 85% nuclear and 35% to 90% cytoplasmic staining. p16-negative FCB showed 0% nuclear and cytoplasmic staining, and p16-positive FCB showed 30% to 100% moderate-strong nuclear and cytoplasmic staining. Interobserver variability was greater with CCB. In our laboratory, formalin fixation of CB material improved the accuracy of p16 interpretation. Staining in FCB was also more robust than CCB, which showed weaker cytoplasmic and more focal nuclear staining. Therefore, we advocate formalin fixation for head and neck cytology specimens that may require p16 testing.

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