James S. Lewis

The uses for circulating tumor high risk human papillomavirus DNA (ctHPVDNA) in the diagnostic work-up and treatment of HPV-associated oropharyngeal squamous cell carcinoma (HPV( +)OPSCC) patients are actively being investigated. We evaluated the concordance of ctHPVDNA with tissue-based HPV testing (p16 immunohistochemistry (IHC), DNA in situ hybridization (ISH), and/or RNA ISH) to assess its performance as a primary diagnostic test for patients' HPV status. We conducted a retrospective cross-sectional study of patients evaluated at a tertiary care multidisciplinary oropharyngeal cancer clinic with suspected HPV( +)OPSCC who underwent pre-treatment ctHPVDNA testing (HPV types 16, 18, 31, 33, 35) via the NavDx assay. Of 236 patients with OPSCC, 190 (81%) had a positive ctHPVDNA result. 41 (17%) were negative, and 3 (1%) were indeterminate. Of the 190 patients with a positive ctHPVDNA result, 190 (100%) had positive tissue-based HPV testing. Of the 41 patients with a negative result, 34 (83%) had positive tissue-based HPV testing and 7 (17%) were negative. ctHPVDNA demonstrated a sensitivity of 85% (95% CI 79-89%), specificity of 100% (95% CI 59-100%), NPV of 17% (95% CI: 7-32%), and PPV of 100% (95% CI 98-100%). The high specificity and PPV of ctHPVDNA indicate that in the setting of a positive result and high clinical suspicion, it may not be necessary for pathologists to test subsequent cytology or tissue specimens that are diagnostic for SCC for p16 or HPV. In the setting of a negative ctHPVDNA result, however, our findings indicate that HPV testing of a pathology specimen must still be performed.

Current spatial T cell receptor (TCR) profiling approaches lack the resolution needed to link clonal identity, transcriptional state, and spatial positioning of individual T cells in the tumor microenvironment. Here, we introduce a spatial TCR profiling strategy that resolves individual T cell clones together with their transcriptional states at single-cell resolution and applied the method to human head and neck squamous cell carcinoma. Presumed tumor-specific T cells were broadly dispersed throughout the tumor microenvironment, and cells of the same clone occupied distinct transcriptional states in different locations: Immune-rich regions contained more plastic or progenitor cells, whereas tumor-dense regions were enriched for exhausted states. Patients exhibited notably different spatial architectures of antitumor T cell responses, revealing variation that was not captured by high-resolution, spatially agnostic methods such as spectral flow cytometry and single-cell RNA sequencing. These results provide a blueprint for dissecting antigen-specific T cell states in human tumors and reveal how T cell states are spatially coordinated with local cues across the tumor microenvironment.

Squamous differentiation in head and neck tumors is so common that we sometimes don’t think about the diversity of lesions that can show it and how to tease them apart. Accurate molecular subclassification of squamous tumors now directly informs patient diagnosis, prognosis, and treatment. Viral-associated carcinomas remain the largest and most clinically important type. Human papillomavirus (HPV)–associated oropharyngeal squamous cell carcinoma (SCC) and Epstein–Barr virus (EBV)–associated nasopharyngeal carcinoma are defined by distinct oncogenic mechanisms, morphologic features, and clinical behavior, with routine use of p16 and EBER in situ hybridization now standard practice. Testing for viral association also aids in the work-up of metastatic squamous carcinoma of unknown primary. Translocation-associated carcinomas, once considered a non-sequitur, are now recognized as clinically distinct entities in head and neck pathology. DEK::AFF2 fusion nonkeratinizing SCC is typically a papillary sinonasal tumor with deceptively bland morphology while NUT carcinoma represents the opposite extreme, presenting as an aggressive, often midline, poorly differentiated carcinoma, a small subset of which can even be cytokeratin negative. Both can be diagnosed by fluorescence in situ hybridization (FISH) or by fusion specific immunohistochemistry. Finally, hyalinizing clear cell carcinoma (HCCC) is a bland, nested oral cavity and pharyngeal submucosal tumor with squamous immunophenotype and illustrates how EWSR1 fusions blur the boundaries between squamous and salivary neoplasms, with ancillary FISH sometimes needed to confirm the diagnosis. Viral and targeted molecular testing are critical to classification of these tumors. "So it's squamous" is no longer the end of the work up—it is the beginning of a more refined classification.

Inverted papilloma (IP) is a benign neoplasm of the nasal cavity and paranasal sinuses, known for its variable risk of recurrence and potential for developing carcinoma. Emerging evidence has shown high rates of activating EGFR mutation, and a smaller subset is associated with low-risk human papillomavirus (lrHPV). While certain morphologic features, including an inverted growth pattern, are well-established, the presence of condylomatous features, such as large fungating lesions with thick undulating surface epithelium, hyperkeratosis, cytoplasmic clearing, raisinoid nuclei, and binucleation (koilocytic changes) in low-risk HPV-associated IP suggests that these tumors may be a distinct subtype of sinonasal papilloma (SP) with features similar to low-risk HPV-associated anogenital condylomas. This study presents a series of SP with condylomatous morphology and explores the association with lrHPV, the clinicopathologic features, and the rates of carcinoma development. In total, 17 cases of SP exhibiting condylomatous morphology were retrospectively identified. We performed lrHPV and high-risk HPV (hrHPV) RNA in situ hybridization and p16 immunohistochemistry and gathered detailed clinical and pathologic data along with treatment, disease follow-up, and outcomes. These condylomatous papillomas almost all developed in active smokers, were large, were primarily located in the nasal cavity (47%), and showed frequent transformation to invasive squamous cell carcinoma (29%). This malignant transformation rate is much higher than what has been reported for inverted, exophytic, and oncocytic papillomas. The tumors were almost uniformly associated with transcriptionally-active lrHPV (94%) and were consistently negative for p16 and hrHPV. This study shows that a subgroup of IPs with condylomatous morphology have a predilection for the nasal cavity, strong association with lrHPV, and high rates of carcinoma. These findings support the concept that these tumors are a distinct (fourth) type of SP with a higher risk of malignant transformation.

DEK::AFF2 fusion nonkeratinizing squamous cell carcinoma (NKSCC) is an emerging entity in the sinonasal tract, temporal bone, and skull base. However, the clinical behavior of these tumors has not been well studied. Here, we report the largest cohort of DEK::AFF2 carcinomas to determine if morphology, mitotic rate, and/or Ki-67 IHC are associated with patient outcomes, including a comparison with high-risk human papillomavirus (HPV)-associated and independent patients. We solicited cases of molecularly or AFF2 immunohistochemistry (IHC) proven DEK::AFF2 SCC from surgical pathologists to collect patient demographic, clinical, and outcome data. Using representative H&E slides, we characterized the morphology and counted mitoses. Ki-67 immunohistochemistry was performed. We also compared the DEK::AFF2 survival rates to those in a cohort of AFF2 IHC-negative HPV-associated and HPV-independent SCC. DEK::AFF2 carcinomas most commonly arose in the nasal cavity (13/30, 43%), and the average number of recurrences was 1.8 (range: 0 to 10). At the last follow-up, most patients were disease free (19/30, 63%) or were alive with disease (9/30, 30%). There was an average mitotic rate of 2 per 2 mm² (range: 0 to 9) and Ki-67 proliferation rate of 26% (range: 3% to 60%). Local recurrence was common, but morphology, mitotic activity, and Ki-67 index were not associated with recurrence or survival. On Kaplan-Meier survival analysis, DEK::AFF2 patients had lower disease-free survival but otherwise had similar outcomes to conventional SCC patients. Our multi-institutional study shows that local recurrence is common in DEK::AFF2 fusion nonkeratinizing SCC patients, but patients have survival rates similar to conventional SCC. Despite showing a range of different features and proliferation rates, traditional grading by morphology, mitotic rate, and/or Ki-67 activity does not seem to be predictive of outcome.

Human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HPV-positive HNSCC) has distinct biological characteristics from HPV-negative HNSCC. Using an AI-based analytical platform on meta cohorts, we profiled expression patterns of viral transcripts and HPV viral genome integration, and classified the tumor microenvironment (TME). Unsupervised clustering analysis revealed five distinct and novel TME subtypes across patients (immune-enriched, highly immune and B-cell enriched, fibrotic, immune-desert, and immune-enriched luminal). These TME subtypes were highly correlated with patient prognosis. In order to understand specific factors associated with prognosis, we used the unsupervised clustering of an HPV-positive HNSCC cohort from The Cancer Genome Atlas (TCGA) (n = 53) based on HPV transcript expression, and identified four HPV-related subtypes (E2/E5, E6/E7, E1/E4 and L1/L2). Utilizing both viral transcript and TME subtypes, we found that the E2/E5 HPV subtype was associated with an immune-enriched TME and had a higher overall survival rate compared to other subtypes. The E2/E5 subtype was also enriched for samples without HPV-genome integration, suggesting that the episomal HPV status and E2/E5 expression pattern may be associated with an inflamed microenvironment and improved prognosis. In contrast, E6/E7 subtype samples were associated with the fibrotic and immune-desert TME subtypes, with lower values of T-cell and B-cell gene expression signatures and lower overall survival. Both E1/E4 and L1/L2 subtypes were associated with the immune-enriched luminal subtype. Our results suggest that HPV-transcript expression patterns may drive the modulation of the TME, and thereby impact prognosis.