Heterogeneity of p16 immunohistochemistry and increased sensitivity of RNA in situ hybridization in cytology specimens of HPV-related head and neck squamous cell carcinoma

Background Many patients with human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HPV-HNSCC) initially present with cervical lymph node metastases. Although p16 immunohistochemistry (IHC) is the most commonly used surrogate marker for HPV, however criteria in cytologic material are not well established. The objective of this study was to better characterize p16 IHC in cell blocks of metastatic HPV-HNSCC, and to evaluate the performance of HPV RNA in situ hybridization (RNA ISH). Methods p16 IHC was performed on cell blocks from 97 metastatic HPV-HNSCC fine-needle aspiration specimens with HPV status confirmed by DNA or RNA ISH or polymerase chain reaction (PCR). Tumor cellularity (<100 cells, 100-500 cells, and >500 cells) and quality (presence of cell clusters, necrosis) were recorded. p16 staining intensity and extent (1%-9%, 10%-69%, and >= 70%) were scored. In addition, RNA ISH was performed on 38 PCR-positive cases. Results p16 IHC was positive in 90 of 97 cases (93%), demonstrating variable patterns. p16 staining was found to be moderate to strong in 69 cases, with 37 cases (38%) demonstrating positivity in >= 70% of tumor cells. Weak staining occurred in 21 cases (22%) and 7 cases (7%) were negative. Of the 60 cases with weak and/or absent expression or staining in <70% of cells, 30 cases (50%) had <100 tumor cells, 12 (20%) lacked cell clusters, and 19 cases (32%) had extensive necrosis. RNA ISH was positive in 37 of 38 cases (97%) that were HPV positive by PCR. Conclusions p16 is heterogeneous in cell blocks of metastatic HPV-HNSCC, suggesting that any p16 positivity should prompt confirmatory HPV studies. RNA ISH appears to demonstrate high sensitivity, and laboratories even may consider using RNA ISH as a first-line HPV test in cytologic specimens.