Human papillomavirus-encoded circular RNA circE7 promotes immune evasion in head and neck squamous cell carcinoma.

Immune evasion represents a crucial milestone in the progression of cancer and serves as the theoretical foundation for tumor immunotherapy. In this study, we reveal a negative association between Human Papillomavirus (HPV)-encoded circular RNA, circE7, and the infiltration of CD8+ T cells in head and neck squamous cell carcinoma (HNSCC). Both in vitro and in vivo experiments demonstrate that circE7 suppresses the function and activity of T cells by downregulating the transcription of LGALS9, which encodes the galectin-9 protein. The molecular mechanism involves circE7 binding to acetyl-CoA carboxylase 1 (ACC1), promoting its dephosphorylation and thereby activating ACC1. Activated ACC1 reduces H3K27 acetylation at the LGALS9 gene promoter, leading to decreased galectin-9 expression. Notably, galectin-9 interacts with immune checkpoint molecules TIM-3 and PD-1, inhibiting the secretion of cytotoxic cytokines by T cells and promoting T cell apoptosis. Here, we demonstrate a mechanism by which HPV promotes immune evasion in HNSCC through a circE7-driven epigenetic modification and propose a potential immunotherapy strategy for HNSCC that involves the combined use of anti-PD-1 and anti-TIM-3 inhibitors.

Clinical implications of activation of the LIMD1-VHL-HIF1α pathway during head-&-neck squamous cell carcinoma development.

Background & objectives Given the importance of the role of hypoxia induced pathway in different cancers including head-and-neck squamous cell carcinoma (HNSCC), this study delved into elucidating the molecular mechanism of hypoxia-inducible factor-1α (HIF1α) activation in HNSCC. Additionally, it analyzes the alterations of its regulatory genes [von Hippel-Lindau (VHL) and LIM domain containing 1 (LIMD1)] and target gene vascular endothelial growth factor (VEGF) in head-and-neck lesions at different clinical stages in relation with human papillomavirus (HPV) infection. Methods Global mRNA expression profiles of HIF1α, VHL, LIMD1 and VEGF were evaluated from public datasets of HNSCC, followed by validation of their expression (mRNA/protein) in an independent set of HPV+ve/-ve HNSCC samples of different clinical stages. Results A diverse expression pattern of the HIF1α pathway genes was observed, irrespective of HPV infection, in the datasets. In validation in an independent set of HNSCC samples, high mRNA expressions of HIF1α/VEGF were observed particularly in HPV positive samples. However, VHL/LIMD1 mRNA expression was low in tumours regardless of HPV infection status. In immunohistochemical analysis, high/medium (H/M) expression of HIF1α/VEGF was observed in basal/parabasal layers of normal epithelium, with significantly higher expression in tumours, especially in HPV-positive samples. Conversely, high cytoplasmic VHL expression in these layers gradually decreased with the progression of HNSCC, regardless of HPV infection. A similar trend was noted in LIMD1 expression (nuclear/cytoplasmic) during the disease development. The methylation pattern of VHL and LIMD1 promoters in the basal/parabasal layers of normal epithelium correlated with their expression, exhibiting a gradual increase with the progression of HNSCC. The H/M expression of HIF1α/VEGF proteins and reduced VHL expression was associated with poor clinical outcomes. Interpretation & conclusions The results of this study showed differential regulation of the LIMD1-VHL-HIF1α pathway in HPV positive and negative HNSCC samples, illustrating the molecular distinctiveness of these two groups.

Exosomal transcript cargo and functional correlation with HNSCC patients' survival.

BACKGROUND: HPV status in a subset of HNSCC is linked with distinct treatment outcomes. Present investigation aims to elucidate the distinct clinicopathological features of HPV-positive and HPV-negative HNSCC and investigate their association with the HNSCC patient survival. MATERIALS AND METHODS: The total RNA of exosomes from HPV-positive (93VU147T) and HPV-negative (OCT-1) HNSCC cells was isolated, and the transcripts were estimated using Illumina HiSeq X. The expression of altered transcripts and their clinical relevance were further analyzed using publicly available cancer transcriptome data from The Cancer Genome Atlas (TCGA). RESULTS: Transcriptomic analyses identified 3785 differentially exported transcripts (DETs) in HPV-positive exosomes compared to HPV-negative exosomes. DETs that regulate the protein machinery, cellular redox potential, and various neurological disorder-related pathways were over-represented in HPV-positive exosomes. TCGA database revealed the clinical relevance of altered transcripts. Among commonly exported abundant transcripts, SGK1 and MAD1L1 showed high expression, which has been correlated with poor survival in HNSCC patients. In the top 20 DETs of HPV-negative exosomes, high expression of FADS3, SGK3, and TESK2 correlated with poor survival of the HNSCC patients in the TCGA database. CONCLUSION: Overall, our study demonstrates that HPV-positive and HPV-negative cells' exosomes carried differential transcripts cargo that may be related to pathways associated with neurological disorders. Additionally, the altered transcripts identified have clinical relevance, correlating with patient survival in HNSCC, thereby highlighting their potential as biomarkers and as therapeutic targets.

Epithelial­derived head and neck squamous tumourigenesis (Review).

Head and neck squamous cell carcinomas (HNSCCs), a heterogeneous group of cancers that arise from the mucosal epithelia cells in the head and neck areas, present great challenges in diagnosis, treatment and prognosis due to their complex aetiology and various clinical manifestations. Several factors, including smoking, alcohol consumption, oncogenic genes, growth factors, Epstein­Barr virus and human papillomavirus infections can contribute to HNSCC development. The unpredictable tumour microenvironment adds to the complexity of managing HNSCC. Despite significant advances in therapies, the prediction of outcome after treatment for patients with HNSCC remains poor, and the 5­year overall survival rate is low due to late diagnosis. Early detection greatly increases the chances of successful treatment. The present review aimed to bring together the latest findings related to the molecular mechanisms of HNSCC carcinogenesis and progression. Comprehensive genomic, transcriptomic, metabolomic, microbiome and proteomic analyses allow researchers to identify important biological markers such as genetic alterations, gene expression signatures and protein markers that drive HNSCC tumours. These biomarkers associated with the stages of initiation, progression and metastasis of cancer are useful in the management of patients with cancer in order to improve their life expectancy and quality of life.

Smoking and alcohol by HPV status in head and neck cancer: a Mendelian randomization study.

HPV-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) are recognized as distinct entities. There remains uncertainty surrounding the causal effects of smoking and alcohol on the development of these two cancer types. Here we perform multivariable Mendelian randomization (MR) to evaluate the causal effects of smoking and alcohol on the risk of HPV-positive and HPV-negative HNSCC in 3431 cases and 3469 controls. Lifetime smoking exposure, as measured by the Comprehensive Smoking Index (CSI), is associated with increased risk of both HPV-negative HNSCC (OR = 3.03, 95%CI:1.75-5.24, P = 7.00E-05) and HPV-positive HNSCC (OR = 2.73, 95%CI:1.39-5.36, P = 0.003). Drinks Per Week is also linked with increased risk of both HPV-negative HNSCC (OR = 7.72, 95%CI:3.63-16.4, P = 1.00E-07) and HPV-positive HNSCC (OR = 2.66, 95%CI:1.06-6.68, P = 0.038). Smoking and alcohol independently increase the risk of both HPV-positive and HPV-negative HNSCC. These findings have important implications for understanding the modifying risk factors between HNSCC subtypes.

Associations of head and neck cancers with herpes zoster in the preceding five years.

This population-based study investigated the risk of having had prior herpes zoster within five years preceding a diagnosis of head and neck cancer. We conducted a case-control study that included 9,191 patients with a diagnosis of head and neck cancer in Taiwan's Longitudinal Health Insurance Database 2010 and 36,764 matched controls. We assessed the odds of patients with head and neck cancer having had a diagnosis of herpes zoster during the five years preceding head and neck cancer using multiple logistic regression analysis. The prevalence of prior herpes zoster among the total sample was 4.6%, 7.9% and 3.8% among patients with and without head and neck cancer, respectively (p < 0.001). The odds ratio of herpes zoster among the head and neck cancer- versus control group was 2.198 (95% CI = 2.001 ~ 2.415) after adjusting for sociodemographic characteristics and hypertension, diabetes, hyperlipidemia, tobacco use disorder, HPV infection, and alcohol dependence syndrome. Statistically significant excess odds were observed for all specific subtypes of head and neck cancer except for sinonasal cancer. Herpes zoster infection within the 5 years preceding a diagnosis of head and neck cancer may be a harbinger of developing head and neck cancer.

The Next Chapter in Cancer Diagnostics: Advances in HPV-Positive Head and Neck Cancer.

Human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC), particularly oropharyngeal squamous cell carcinoma (OPSCC), is an increasingly prevalent pathology worldwide, especially in developed countries. For diagnosing HPV in HNSCC, the combination of p16 immunohistochemistry (IHC) and polymerase chain reaction (PCR) offers high sensitivity and specificity, with p16 IHC being a reliable initial screen and PCR confirming HPV presence. Advanced techniques like next-generation sequencing (NGS) and RNA-based assays provide detailed insights but are primarily used in research settings. Regardless of HPV status, standard oncological treatments currently include surgery, radiation, and/or chemotherapy. This conventional approach does not account for the typically better prognosis of HPV-positive HNSCC patients, leading to increased chemo/radiation-induced secondary morbidities and reduced quality of life. Therefore, it is crucial to identify and detect HPV positivity and other molecular characteristics of HNSCC to personalize treatment strategies. This comprehensive review aims to summarize current knowledge on various HPV detection techniques and evaluate their advantages and disadvantages, with a focus on developing methodologies to identify new biomarkers in HPV-positive HNSCC. The review discusses direct and indirect HPV examination in tumor tissue, DNA- and RNA-based detection techniques, protein-based markers, liquid biopsy potentials, immune-related markers, epigenetic markers, novel biomarkers, and emerging technologies, providing an overall insight into the current state of knowledge.

Expression and prognosis of DSG-2, CXADR, CD46 in head and neck squamous cell carcinoma.

OBJECTIVES: Investigating the expression and prognostic significance of adenovirus receptors DSG-2, CXADR and CD46 in head and neck cancer. METHODS: 104 patients with HNSCC (77 OPSCC, 27 LSCC) were retrospectively included in the study. Immunohistochemical staining was performed on all selected slides to detect the expression of DSG-2, CXADR, CD46 and the immunoreactive score (IRS) was determined from the number of positively stained tumor cells and their staining intensity. Furthermore, the respective HPV status was determined by immunohistochemical staining against p16 and HPV-PCR. RESULTS: 81.7 % of the tumors showed DSG-2, 34.6 % of the tumors showed CXADR and 57.7 % of the tumors showed CD46 expression. A high DSG-2 IRS correlated significantly with an advanced tumor size (p= 0.003), increased grading (p=0.012) and positive HPV status (p=0.024) in OPSCC. A high CXADR IRS was significantly associated with a positive lymph node status (p= 0.041) in LSCC and an advanced AJCC stage (p= 0.012) and a positive HPV status (p= 0.009) in OPSCC. No significant correlation could be shown regarding CD46 expression and clinical tumor data. There was no effect of DSG-2, CXADR, and CD46 expression on 5-year overall and on 5-year disease-free survival. CONCLUSION: No prognostic significance of the expression of DSG-2, CXADR or CD46 in HNSCC was seen. DSG-2, CXADR and CD46 are expressed in HNSCC, so that optimization of oncotherapy with adenoviral vectors appears promising. Due to the significantly increased expression of DSG-2 and CXADR in advanced OPSCC tumors, there is potential for optimizing oncotherapy here in particular.

Simultaneous p53 and p16 Immunostaining for Molecular Subclassification of Head and Neck Squamous Cell Carcinomas.

PURPOSE: Our aim was to assess the ability of simultaneous immunohistochemical staining (IHC) for p16 and p53 to accurately subclassify head and neck squamous cell carcinomas (HNSCC) as HPV-associated (HPV-A) versus HPV-independent (HPV-I) and compare p53 IHC staining patterns to TP53 mutation status, p16 IHC positivity and HPV status. METHODS: We stained 31 HNSCCs for p53 and p16, and performed next-generation sequencing (FoundationOne©CDx) on all cases and HPV in-situ hybridization (ISH) when sufficient tissue was available (n = 23). p53 IHC staining patterns were assessed as wildtype (wt) or abnormal (abn) patterns i.e. overexpression, null or cytoplasmic staining. RESULTS: In a majority of cases (28/31) interpretation of p16 and p53 IHC was straightforward; 10 were considered HPV-A (p16+/p53wt) and 18 cases were HPV-I (p16-/p53abn). In the remaining three tumours the unusual immunophenotype was resolved by molecular testing, specifically (i) subclonal p16 staining and wild type p53 staining in a tumour positive for HPV and with no TP53 mutation (HPV-A), (ii) negative p16 and wild type p53 staining with a TP53 mutation and negative for HPV (HPV-I), and (iii) equivocally increased p16 staining with mutant pattern p53 expression, negative HPV ISH and with a TP53 mutation (HPV-I). CONCLUSION: Performing p16 and p53 IHC staining simultaneously allows classification of most HNSCC as HPV-A (p16 +, p53 wild type (especially basal sparing or null-like HPV associated staining patterns, which were completely specific for HPV-A SCC) or HPV-I (p16 -, p53 mutant pattern expression), with the potential for limiting additional molecular HPV or mutational testing to selected cases only.

The role of herpes simplex virus infection in the etiology of head and neck cancer-a Mendelian randomization study.

Introduction: Head and neck cancer (HNC) is a complex disease, and multiple risk factors can lead to its progression. Observational studies indicated that herpes simplex virus (HSV) may be correlated with the risk of HNC. However, the causal effects and direction between them were still unclear. Methods: This study utilized a Mendelian randomization (MR) approach for causality assessment between HSV infection and Head and neck cancer based on the latest public health data and Genome-Wide Association Study (GWAS) data. The causal effects were estimated using IVW, weighted median, and MR-Egger. A reverse MR analysis was subsequently performed. Cochrans Q test, MR-Egger intercept test, leave one out analysis, and the funnel plot were all used in sensitivity analyses. Results: Genetically predicted higher level of HSV-1 IgG was causally related to HNC (OR=1.0019, 95%CI=1.0003-1.0036, p=0.0186, IVW) and oral and oropharyngeal cancer (OR=1.0018, 95%CI=1.0004-1.0033, p=0.0105, IVW). The reverse MR analysis did not demonstrate a reverse causal relationship between HSV and HNC. However, HSV-2 infection was not causally related to HNC data and oropharyngeal cancer data. Sensitivity analysis was performed and revealed no heterogeneity and horizontal pleiotropy. Conclusion: Collectively, a significant association was noted between HSV infection and increased risk of HNC, providing valuable insights into the etiology of this malignancy. Further in-depth study is needed to validate these findings and elucidate the underpinning mechanisms.

p16, p53 and EGFR expression in head and neck squamous cell carcinoma and their correlation with clinicopathological parameters.

INTRODUCTION: Oral squamous cell carcinoma is a major cause of death throughout the developed world. Human papillomavirus (HPV) type 16 has also been suggested to play a role in etiology of head and neck squamous cell carcinoma (HNSCC). p16 expression is now being used as a surrogate marker of HPV infection in squamous cell carcinoma. Dysfunction in the p53 tumor suppressor gene is implicated in many cancers, including head and neck cancer. Overexpression or mutation of EGFR is found in 80%-100% of the patients with HNSCC, and is associated with poor prognosis and decreased survival. MATERIALS AND METHODS: In this cross-sectional observation study, total of 100 cases of HNSCC were taken. p16, p53, and EGFR expression was determined by immunohistochemical staining and correlated with clinicopathological parameters. p16 expression was also correlated with expression of p53 and EGFR. The obtained results were analyzed and evaluated using Chi-square test, value of P < 0.05 was taken significant. RESULTS: p16, p53, and EGFR were positive in 60%, 44%, and 58% cases, respectively. A statistically significant association was observed between p16 with age, site of the tumor, abnormal sexual habits and lymph node involvement. Significant expression also seen between p53 with age and abnormal sexual habits and immunohistochemical expression of p16 with p53 and EGFR. CONCLUSION: Immunohistochemical expression of p16 can be used as a surrogate marker of HPV. Study of p16, p53, and EGFR expression may provide clinicians with more exact information in order to evaluate tumor aggressiveness and treatment modalities.

Potential Transcript-Based Biomarkers Predicting Clinical Outcomes of HPV-Positive Head and Neck Squamous Cell Carcinoma Patients.

Human papillomavirus (HPV)-positive Head and Neck Squamous Cell Carcinomas (HNSCC) comprise a particular cancer entity traditionally associated with better clinical outcomes. Around 25% of HNSCC are HPV positive, HPV16 being the most prevalent type. Nevertheless, close to 30% of the HPV-positive patients have an unfavorable prognosis, revealing that this type of tumor exhibits great heterogeneity leading to different clinical behaviors. Efforts have been made to identify RNA molecules with prognostic value associated with the clinical outcome of patients with HPV-positive HNSCC, with the aim of identifying patients at high risk of metastasis, disease recurrence, and poor survival, who would require closer clinical follow-up and timely intervention. Moreover, the molecular identification of those HPV-positive HNSCC patients with good prognosis will allow the implementation of de-escalating therapeutic strategies, aiming to reduce side effects, resulting in a better quality of life. This review compiles a series of recent studies addressing different methodological and conceptual approaches aimed at searching for potential gene expression-based biomarkers associated with the prognosis of patients with HPV-positive HNSCC.

Human papillomavirus-associated head and neck squamous cell carcinoma cells lose viability during triggered myocyte lineage differentiation.

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease, and death rates have remained at approximately 50% for decades. New tumor-targeting strategies are desperately needed, and a previous report indicated the triggered differentiation of HPV-negative HNSCC cells to confer therapeutic benefits. Using patient-derived tumor cells, we created a similar HNSCC differentiation model of HPV+ tumor cells from two patients. We observed a loss of malignant characteristics in differentiating cell culture conditions, including irregularly enlarged cell morphology, cell cycle arrest with downregulation of Ki67, and reduced cell viability. RNA-Seq showed myocyte-like differentiation with upregulation of markers of myofibril assembly. Immunofluorescence staining of differentiated and undifferentiated primary HPV+ HNSCC cells confirmed an upregulation of these markers and the formation of parallel actin fibers reminiscent of myoblast-lineage cells. Moreover, immunofluorescence of HPV+ tumor tissue revealed areas of cells co-expressing the identified markers of myofibril assembly, HPV surrogate marker p16, and stress-associated basal keratinocyte marker KRT17, indicating that the observed myocyte-like in vitro differentiation occurs in human tissue. We are the first to report that carcinoma cells can undergo a triggered myocyte-like differentiation, and our study suggests that the targeted differentiation of HPV+ HNSCCs might be therapeutically valuable.

Knowledge, and attitude of medical students about role of human papilloma virus, and vaccine in head and neck cancer.

BACKGROUND: Papilloma DNA virus which is most common sexually transmitted disease to both sexes. The infection either benign or malignant affecting head and neck region. AIM OF THE STUDY: Assess the level of knowledge, and attitude, of medical students about Human Papilloma virus, vaccine, and its role in head and neck cancer development. MATERIALS AND METHODS: A descriptive cross-sectional survey was conducted on 357 undergraduate medical students. Data were collected by online Google form researcher made questionnaires which was analyzed using SPSS 25. RESULTS: There are 357 medical undergraduate students from different educational stages participated in this study. This study was shown 176 (49.3 %) of medical students agreed that smoking Tobacco are the most common causes for development oral cancer followed by viruses 98 (27.5 %), that 57.4 % of medical students reported that HPV was the main viral cause. As for the questions concerning HPV mode of transmission, almost (85.7 %) stated sexual transmission, (79.8 %) skin to skin direct contact. Most of the participants (92.2 %) agreed that primary prevention can decrease the risk of infection with HPV, and 43.4 % strongly agreed that vaccination plays an important role in HPV prevention. CONCLUSIONS: There is a requirement within the existing curriculum and syllabus to include more education, seminars, and training courses on HPV, role in HNC, prevention, and vaccination, and mainly for students in the preclinical academic years. Application of a virtual classroom viral module or communicating workshop would likely improve knowledge and attitudes of the students for their future medical tasks.

Comment on, "Characterization of oral microbiota in HPV and non-HPV head and neck squamous cell carcinoma and its association with patient outcomes".

The article "Characterization of oral microbiota in HPV and non-HPV head and neck squamous cell carcinoma and its association with patient outcomes" by Chan et al. investigates the relationship between oral microbiota, HPV infection, and patient outcomes in head and neck squamous cell carcinoma (HNSCC). This comprehensive study, involving 166 Chinese adults, utilized advanced sequencing techniques to profile bacterial and HPV regions in paired tumor and control tissues. The findings highlight the complex interplay between microbiota dysbiosis, HPV infection, and HNSCC progression. Despite the robustness of the study, limitations include potential biases in DNA extraction and PCR amplification, and unaccounted environmental factors. Recommendations for future research include increasing sequencing depth, comparing DNA extraction methods, using multiple bioinformatics pipelines, and controlling for environmental variables. Longitudinal studies and microbiota-targeted interventions are suggested to further elucidate the role of oral microbiota in HNSCC and improve patient outcomes.

Differential tumor immune microenvironment coupled with tumor progression or tumor eradication in HPV-antigen expressing squamous cell carcinoma (SCC) models.

Human papilloma virus (HPV) is an etiological factor of head and neck squamous cell carcinoma (HNSCC). To investigate the role of HPV antigen in anti-tumor immunity, we established mouse models by expressing HPV16 E6 and E7 in a SCC tumor cell line. We obtained two HPV antigen-expressing clones (C-225 and C-100) transplantable into C57BL/6 recipients. We found that C-225 elicited complete eradication in C57BL/6 mice (eradicated), whereas C-100 grew progressively (growing). We examined immune tumor microenvironment (TME) using flow cytometry and found that eradicated or growing tumors exhibited differential immune profiles that may influence the outcome of anti-tumor immunity. Surprisingly, the percentage of CD8 and CD4 tumor-infiltrating lymphocytes (TILs) was much higher in growing (C-100) than eradicated (C-225) tumor. However, the TILs upregulated PD-1 and LAG-3 more potently and exhibited impaired effector functions in growing tumor compared to their counterparts in eradicated tumor. C-225 TME is highly enriched with myeloid cells, especially polymorphonuclear (PMN) myeloid-derived suppressor cells (MDSC), whereas the percentage of M-MDSC and tumor-associated macrophages (TAMs) was much higher in C-100 TME, especially M2-TAMs (CD206+). The complete eradication of C-225 depended on CD8 T cells and elicited anti-tumor memory responses upon secondary tumor challenge. We employed DNA sequencing to identify differences in the T cell receptor of peripheral blood lymphocytes pre- and post-secondary tumor challenge. Lastly, C-225 and C-100 tumor lines harbored different somatic mutations. Overall, we uncovered differential immune TME that may underlie the divergent outcomes of anti-tumor immunity by establishing two SCC tumor lines, both of which express HPV16 E6 and E7 antigens. Our experimental models may provide a platform for pinpointing tumor-intrinsic versus host-intrinsic differences in orchestrating an immunosuppressive TME in HNSCCs and for identifying new targets that render tumor cells vulnerable to immune attack.

Head and neck squamous cell carcinomas of unknown primary: Can ancillary studies help identify more primary tumor sites?

A subset of head and neck squamous cell carcinomas present solely as metastatic disease in the neck and are of unknown primary origin (SCCUP). Most primary tumors will ultimately be identified, usually in the oropharynx. In a minority of cases, the primary site remains elusive. Here, we examine the role of ancillary testing, including mutational signature analysis (MSA), to help identify likely primary sites in such cases. Twenty-two cases of SCCUP in the neck, collected over a 10-year period, were classified by morphology and viral status; including human papillomavirus (HPV) testing by p16 immunohistochemistry (IHC) and RT-qPCR, as well as Epstein-Barr virus (EBV) testing by EBER-ISH. CD5 and c-KIT (CD117) IHC was done to evaluate for possible thymic origin in all virus-negative cases. Whole exome sequencing, followed by MSA, was used to identify UV signature mutations indicative of cutaneous origin. HPV was identified in 12 of 22 tumors (54.5%), favoring an oropharyngeal origin, and closely associated with nonkeratinizing tumor morphology (Fisher's exact test; p = 0.0002). One tumor with indeterminant morphology had discordant HPV and p16 status (p16+/HPV-). All tumors were EBV-negative. Diffuse expression of CD5 and c-KIT was identified in 1 of 10 virus-negative SCCUPs (10%), suggesting a possible ectopic thymic origin rather than a metastasis. A UV mutational signature, indicating cutaneous origin, was identified in 1 of 10 (10%) virus-negative SCCUPs. A cutaneous auricular primary emerged 3 months after treatment in this patient. Primary tumors became clinically apparent in 2 others (1 hypopharynx, 1 hypopharynx/larynx). Thus, after follow-up, 6 tumors remained unclassifiable as to the possible site of origin (27%). Most SCCUPs of the neck in our series were HPV-associated and thus likely of oropharyngeal origin. UV signature mutation analysis and additional IHC for CD5 and c-KIT for possible thymic origin may aid in further classifying virus-negative unknown primaries. Close clinical inspection of hypopharyngeal mucosa may also be helpful, as a subset of primary tumors later emerged at this site.