HPV-Associated Metastatic Squamous Cell Carcinoma of Cervical Lymph Node Mimicking Nasopharyngeal Carcinoma: Diagnostic Utility of p16 and EBER In Situ Hybridization.
Year: 2026
Type: article
Study design: Case Report
Abstract: Background: Metastatic squamous cell carcinoma presenting as an upper cervical neck mass (Level II) frequently poses a diagnostic challenge regarding the identification of the primary tumor site. Non-keratinizing morphologies with prominent lymphoid stroma strongly suggest Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma, though they can occasionally mask Human Papillomavirus (HPV)-related oropharyngeal primaries. Case Presentation: A 53-year-old male presented with an enlarging cervical neck mass for investigation. Initial core needle biopsy of a Level II cervical lymph node revealed lymphoid tissue extensively infiltrated by nests and syncytial sheets of atypical epithelial cells with vesicular nuclei, conspicuous nucleoli, and a prominent lymphoid background, initially favoring metastatic non-keratinizing squamous cell carcinoma with differential diagnostic consideration of nasopharyngeal carcinoma. Subsequent immunohistochemical and molecular characterization demonstrated tumor cells that were strongly positive for CK AE1/AE3, p40, and p16, while EBER in situ hybridization was negative, and CD3 and CD20 highlighted background lymphocytes. These findings confirmed a diagnosis of HPV-associated metastatic squamous cell carcinoma. Conclusion: HPV-related metastatic squamous cell carcinoma can closely mimic EBV-associated nasopharyngeal carcinoma morphologically due to syncytial growth and prominent lymphoid stroma. A panel including p16 immunohistochemistry and EBER in situ hybridization is essential for accurate primary site attribution and prognostic stratification in unknown primary cervical nodal metastases. Keywords HPV-associated Squamous Cell Carcinoma, Cervical Lymph Node Metastasis, p16, EBER in situ hybridization, Nasopharyngeal Carcinoma mimic, Head and Neck Pathology. Introduction Cervical lymphadenopathy secondary to metastatic squamous cell carcinoma (SCC) is a frequent clinical presentation in head and neck oncology. When the primary site is occult, pathologists must rely heavily on morphologic clues and immunohistochemical panels to narrow the differential diagnosis between nasopharyngeal, sinonasal, and oropharyngeal primaries. Non-keratinizing squamous cell carcinomas characterized by syncytial aggregates of atypical epithelial cells embedded within a brisk lymphoid background strongly point toward Epstein-Barr virus (EBV)-driven nasopharyngeal carcinoma. However, high-risk Human Papillomavirus (HPV)-associated oropharyngeal squamous cell carcinomas can exhibit basaloid or non-keratinizing features that overlap morphologically, creating potential diagnostic pitfalls. Utilizing p16 immunohistochemistry as a surrogate marker for high-risk HPV alongside EBER (Epstein-Barr virus-encoded RNA) in situ hybridization is critical to establishing the correct biological etiology. Case Presentation Clinical Presentation A 53-year-old male presented with a persistent, enlarging neck mass. Diagnostic workup included a core needle biopsy of a Level II cervical lymph node. Histopathology (Initial Core Biopsy) Microscopic examination of Hematoxylin and Eosin (H&E) stained sections from the core biopsy showed multiple fragments of lymph node parenchyma extensively infiltrated and partially replaced by nests, sheets, and syncytial aggregates of atypical epithelial cells. The neoplastic cells displayed enlarged vesicular nuclei, irregular nuclear contours, conspicuous nucleoli, and moderate amounts of eosinophilic cytoplasm (Figure 1, Figure 2). Overt squamous maturation and keratin pearl formation were absent. A prominent reactive background lymphoid population was present alongside brisk mitotic activity and apoptotic bodies. Given the Level II location and syncytial architecture, the initial morphological impression favored metastatic non-keratinizing squamous cell carcinoma, raising a strong differential diagnosis of differentiated or undifferentiated nasopharyngeal carcinoma. Advanced Immunohistochemical & Molecular Findings To definitively characterize the tumor lineage and investigate viral association, specialized immunohistochemistry and in situ hybridization were performed: p40: Strongly positive in neoplastic epithelial cells. Cytokeratin AE1/AE3: Diffusely positive. p16: Diffusely positive (nuclear and cytoplasmic staining pattern consistent with HPV association). EBER (In Situ Hybridization): Negative (ruling out EBV-driven nasopharyngeal carcinoma). CD3 & CD20: Highlighted normal background reactive lymphocytes. The combined immunophenotype (p40+, CK AE1/AE3+, p16+, EBER-) confirmed the definitive diagnosis of HPV-Associated Metastatic Squamous Cell Carcinoma. Discussion The distinction between HPV-mediated oropharyngeal SCC and EBV-driven nasopharyngeal carcinoma is of paramount clinical importance, as treatment paradigms, response to therapy, and overall prognosis differ significantly. Morphologically, non-keratinizing SCCs in upper cervical lymph nodes (Levels II and III) often present with basaloid features, syncytial growth, and extensive tumor-infiltrating lymphocytes. These features historically led pathologists to suspect nasopharyngeal primaries, particularly in patients presenting with occult primary lesions. However, the global rise of HPV-associated oropharyngeal squamous cell carcinoma has established it as a primary consideration in cervical nodal metastases. Immunohistochemistry provides reliable tools for resolving this differential diagnosis. Overexpression of p16 serves as a robust surrogate marker for transcriptionally active high-risk HPV infection. Conversely, EBER in situ hybridization remains the gold standard for confirming EBV-driven malignancies such as nasopharyngeal carcinoma. In the present case, the combination of strong p16 positivity and negative EBER definitively redirected the origin from the nasopharynx to an HPV-related head and neck primary site (frequently palatine or lingual tonsils), sparing the patient inappropriate nasopharyngeal-directed evaluations and optimizing oncological management. Conclusions Metastatic non-keratinizing squamous cell carcinoma with prominent lymphoid stroma in cervical lymph nodes can morphologically mimic nasopharyngeal carcinoma. p16 immunohistochemistry and EBER in situ hybridization are indispensable ancillary tests required to differentiate HPV-associated squamous cell carcinoma from EBV-associated nasopharyngeal carcinoma. Accurate viral and lineage characterization directly impacts clinical staging, therapeutic targeting, and prognostic counseling in patients presenting with cervical nodal metastases of unknown primary. Figure Captions Figure 1 (Microfotografía - H&E, Low Power): Histologic section of the cervical lymph node showing extensive architectural replacement by confluent nests and sheets of atypical squamous cells within a prominent lymphoid background (H&E, 10x). Figure 2 (Microfotografía - H&E, High Power): High-power view demonstrating cohesive sheets of malignant epithelial cells with enlarged vesicular nuclei, prominent nucleoli, and brisk apoptotic activity (H&E, 40x). References Westra WH. The changing face of head and neck cancer in the 21st century: the impact of HPV. Otolaryngol Head Neck Surg. 2010;142(5):7-13. Shanbaug S, et al. HPV-associated head and neck squamous cell carcinoma: diagnostic and therapeutic updates. Head Neck Pathol. 2019;13(1):31-42. StGuily JL, et al. The role of HPV in head and neck cancer: a review. Eur Arch Otorhinolaryngol. 2020;277(8):2161-2170. Lewis JS Jr, et al. Provisional guidelines for p16 testing in oropharyngeal squamous cell carcinoma. Head Neck Pathol. 2018;12(2):147-152.
Author: Milanés Sánchez, José Luis
Language: English
Topic: Head and Neck Cancer Studies
Subfield: Otorhinolaryngology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W7225413465