Cytologic evaluation of p16 staining in head and neck squamous cell carcinoma in CytoLyt versus formalin‐fixed material

BACKGROUND: The management of high-risk human papillomavirus (HR-HPV)-related oropharyngeal head and neck squamous cell carcinomas (HNSCCs) are distinct from HNSCC linked to smoking and alcohol use. HR-HPV-positive HNSCC frequently presents as a cervical lymph node metastasis. Because fine-needle aspiration (FNA) is often the initial diagnostic procedure, evaluating HR-HPV status in cytology specimens is important. The overexpression of p16 is a surrogate for HR-HPV; however, the evaluation of p16 in FNAs remains controversial. METHODS: From September 2015 to December 2016, cytopathologists performed 25 FNAs of neck lymph nodes that were suspicious for HR-HPV-positive HNSCC. Initial passes produced smears for on-site evaluation and CytoLyt material. Additional passes were formalin-fixed. A CytoLyt cell block (CCB) and a formalin-fixed cell block (FFCB) were prepared, and p16 immunocytochemistry was performed. RESULTS: In 24 of 25 cases, the FFCB had diffuse (>/=70% of cells), strong nuclear/cytoplasmic p16 staining. In all 24 of these cases, HR-HPV was detected by in situ hybridization. The corresponding CCB had weak-to-moderate p16 staining in <70% of cells (range, 5%-60% of cells) in 17 cases, 4 had weak-to-moderate diffuse staining, and 4 were acellular. The percentage of p16-positive cells was significantly higher with FFCB than with CCB (formalin: 94% +/- 2%; CytoLyt, 38% +/- 7%; 2-tailed, paired Student t test; P < .001; Fisher exact test, P < .001). CONCLUSIONS: The fixative used had a drastic impact on p16 staining, which explained the staining variability reported in the literature. FFCBs show a diffuse staining pattern, which correlates with HR-HPV status, whereas CCBs show a weaker and inconsistent staining pattern, which is more difficult to interpret.