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

Abstract

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.

Lymphoepithelioma‐like carcinoma of the uterine cervix: Cytomorphologic features and diagnostic pitfalls by liquid‐based cytology

“…immunostaining is known to be weak or negative in alcohol-fixed specimens. 9 It was also important to exclude other poorly-differentiated or rare malignancies in the differential diagnosis, especially because these generally have a much worse prognosis than LELC. Glassy cell carcinoma, for example, affects a similarly middle-aged patient population, is also associated with high risk HPV subtypes, has a similar immunoprofile, and can exhibit prominent lymphoplasmacytic infiltrates, but portends an unfavorable prognosis.…”

“…Thus, we chose to classify the lesion as “atypical glandular cells,” which we felt would provide the necessary clinical guidance to sample for both glandular (endometrial) and squamous lesions per ASCCP guidelines. Preparation of a cell block was also not deemed helpful, especially given the fact that p16 immunostaining is known to be weak or negative in alcohol‐fixed specimens …”

Molecular Diagnostics in Human Papillomavirus-Related Head and Neck Squamous Cell Carcinoma

“…al. recently suggested that the variation in the optimal cut-off for FNAB specimens reported in the literature could be due to differences in fixation and preparation methods utilized, as FNAB specimen processing is not standardized across institutions [39]. In their study, they compared p16 IHC staining patterns between cell blocks prepared from specimens preserved in CytoLyt (an ethanol-based fixative) and cell blocks prepared from specimens fixed in formalin.…”

Acinar cell induced autolysis is a frequent occurrence in CytoLyt‐fixed pancreatic fine needle aspiration specimens: An analysis of 157 cytology samples

“…Indeed, it has been shown to have several advantages. However, it has more recently been shown to impair the immunoreactivity of some antibody clones including MIB1 and p16, among others 9‐11 . On the other hand, formalin has been found to be equally adept at preserving tissue both for diagnosis as well as immunocytochemical and molecular studies 1,8 …”