5. The utility of HPV PCR and p16 immunohistochemistry in the differential diagnosis of second branchial cleft cysts and level 2 cystic metastatic squamous cell carcinomas by fine needle aspiration cytology

Background Branchial cleft cysts (brCC) can resemble level 2 nodal cystic metastatic squamous cell carcinomas (mSCC). mSCC in this location often arise from an HPV-associated oropharyngeal primary. P16 immunohistochemistry (IHC) is used as a surrogate marker for infection by oncogenic HPV in this setting and can be combined with HPV PCR to identify this subset of mSCC. Aim (1) Evaluate the usefulness of p16 IHC and HPV PCR in the separation of brCC and mSCC. (2) Assess how different PCR techniques perform in this setting. Methods 54 brCC and 35 mSCC with cell blocks were assessed. 15/35 (42.8%) mSCC were oropharyngeal in origin. P16 IHC was scored by three pathologists. HPV detection was carried out by multiplex nested PCR (nPCR) and a newer multiplex tandem PCR (tPCR) technique. Results No brCC had ≥20% p16 positive cells in contrast to 17/35 (48.6%) mSCC; 12/17 (70.6%) were oropharyngeal in origin. tPCR identified HPV DNA in 20/54 (37%) brCC and 24/35 (68.6%) mSCC; 15/24 (62.5%) were oropharyngeal in origin. nPCR identified HPV DNA in 1/54 (1.8%) brCC and 14/27 (51.8%) mSCC; 10/14 (71.4%) were oropharyngeal in origin. No HPV PCR+ brCC were p16+ (≥20% cells staining). P16+ was present in 17/24 (70.8%) tPCR HPV+ mSCC and 13/14 (92.8%) nPCR HPV+ mSCC. 10/13 (76.9%) p16+/nPCR HPV+ mSCC and 12/17 (70.6%) p16+/tPCR HPV+ mSCC were of oropharyngeal origin. Conclusions (1) tPCR in isolation is not useful in separating brCC and mSCC; (2) dual p16/nPCR HPV DNA+ has the highest predictive value for mSCC from the oropharynx; (3) more experience is required with tPCR in this clinical setting given the high rate of HPV DNA detection in brCC.