p16(INK4A) immunohistochemical staining may be helpful in distinguishing branchial cleft cysts from cystic squamous cell carcinomas originating in the oropharynx

Abstract

Background: We investigated p16(INK4A) expression in branchial cleft cysts and its utility in distinguishing branchial cleft cysts from metastatic head and neck squamous cell carcinomas (SCCs) in fine-needle aspiration biopsies (FNABs).

Methods: A study set comprising 41 resections (15 SCC and 26 branchial cleft cysts) and a test set of 15 FNABs (11 SCC and 4 branchial cleft cysts) were analyzed with p16(INK4A) immunohistochemistry and human papillomavirus (HPV) polymerase chain reaction (PCR)/pyrosequencing. Cases with discrepant p16(INK4A) and PCR/pyrosequencing results were further evaluated with HPV in situ hybridization (ISH). SCCs were divided into keratinizing SCC and nonkeratinizing SCC groups and site of origin.

Results: Metastatic oropharyngeal nonkeratinizing SCC in the study set exhibited diffuse, strong p16(INK4A) (7 of 7) and HPV16 DNA positivity (6 of 6), while keratinizing SCC from the larynx and oral cavity was negative for p16(INK4A). p16(INK4A) reactivity in the branchial cleft cyst study set was characterized by focal, strong staining (6 of 21) involving the superficial squamous epithelium. HPV DNA was identified in 7 of 19 branchial cleft cyst study set cases by PCR/pyrosequencing, but these cases were negative by HPV ISH. In the test set, oropharyngeal nonkeratinizing SCC exhibited diffuse, strong p16(INK4A) (3 of 3) and HPV16 DNA (2 of 2), while metastatic keratinizing SCC was negative for p16(INK4A) and HPV DNA. All 4 FNABs of branchial cleft cysts were negative for p16(INK4A). Diffuse, strong p16(INK4A) correlated with oropharyngeal origin (P=.001) and nonkeratinizing morphology (P=.0001).

Conclusions: Branchial cleft cysts can exhibit focal strong reactivity limited to the superficial squamous epithelium and glandular epithelium. Although p16(INK4A) immunohistochemistry may be helpful in distinguishing oropharyngeal nonkeratinizing SCC from branchial cleft cysts in FNAB specimens, it is not helpful in cases of keratinizing SCC because these cases are typically negative for p16(INK4A).

FIGURE 1

(A) Metastatic cystic squamous cell carcinoma (SCC) from the larynx with a keratinizing morphology exhibiting keratin pearl formation and intercellular bridges between tumor cells. The cyst contains extensively keratinized tumors cells and debris. (B) Metastatic keratinizing SCC, seen in Figure 1A, showing negative staining with p16^INK4A immunohistochemistry; (C) fine-needle aspirate smear from a metastatic cystic SCC from the larynx showing extensive cytoplasmic keratinization, nuclear enlargement and hyperchromasia, and occasional single intact tumor cells in a background of histiocytes and degenerating cells; (D) cell block section from a metastatic keratinizing SCC from the larynx with (E) accompanying negative p16^INK4A immunohistochemical stain. (A: H&E stain, original magnification ×100; B: original magnification ×100; C: Papanicolaou stain, original magnification ×200; D: H&E stain, original magnification ×200; E: original magnification ×200).

FIGURE 2

(A) Metastatic cystic squamous cell carcinoma (SCC) from the oropharynx with a nonkeratizinating morphology exhibiting a monomorphic population of ovoid cells with indistinct cell borders and an extensive cystic component with degenerating cells. (B) Focal areas of keratinization in an otherwise nonkeratinizing SCC showing predominantly rounded nests of basaloid tumor cells with central comedo-like necrosis; (C) nonkeratinizing SCC, seen in Figures 2A–B, showing diffuse, strong full thickness p16^INK4A positive staining as well as staining of scattered tumor cells within the cyst contents; (D) fine-needle smear from a metastatic cystic SCC from the oropharynx showing a background of extensive degenerating cells with a rare crowded 3-dimensional cluster of tumor cells; (E) 3-dimensional clusters of tumor cells forming synctia without keratinization from a metastatic cystic SCC from the oropharynx; (F) cell block section from a metastatic nonkeratinizing oropharyngeal SCC with (G) accompanying p16^INK4A immunohistochemical stain showing diffuse, strong staining in the neoplastic cell clusters as well as scattered positive tumor cells dispersed singly (A,B: H&E stain, original magnification ×100; C: original magnification ×100; D: Diff-Quik, original magnification ×200; E: Papanicolaou stain, original magnification ×400; F: H&E, original magnification ×200; G: original magnification ×200).

FIGURE 3

(A) Branchial cleft cyst characterized by cytologically bland squamous lining cells without nuclear enlargement or mitotic activity; (B) branchial cleft cyst, seen in Figure 3A, showing focal, strong p16^INK4A immunohistochemical staining involving the superficial squamous cells lining the cyst without full-thickness staining; (C) fine-needle smear of branchial cleft cyst exhibiting bland squamous cells and anucleate squamous forming occasional clusters; (D) cell block section from the branchial cleft cyst with (E) accompanying negative p16^INK4A immunohistochemical stain (A: H&E, original magnification ×200; B: original magnification ×200; C: Papanicolaou stain, original magnification ×200; D: H&E, original magnification ×200; E: original magnification ×200).