Multimodal assessment of high-risk human papillomavirus in sinonasal squamous cell carcinoma.

High-risk human papillomavirus (hrHPV) is an emerging risk factor for sinonasal squamous cell carcinoma (SNSCC). The goal of this study was to assess the prevalence of hrHPV and subtype distribution in SNSCC and correlation with patient and clinical characteristics. This retrospective cohort study included 43 cases diagnosed with incident primary SNSCC at the University of Cincinnati Medical Center from 2010 to 2015. The prevalence of hrHPV was interrogated using a multi-assay approach that included p16 immunohistochemistry (IHC), RNA in-situ hybridization (ISH), and hrHPV DNA sequencing. The association of hrHPV with 5-year overall survival (OS) and 2-year disease-free survival (DFS) was assessed. Fourteen cases (32.6 %) were classified as hrHPV positive, based on the a priori definition of having either a positive RNAScope™ ISH test or hrHPV DNA and p16-positive IHC; 9 cases (20.9 %) were positive for all three tests. All cases that arose from an inverted sinonasal papilloma (ex-ISP) were negative for hrHPV. HPV16 was the most common subtype among hrHPV positive cases (58.8 %), followed by HPV18 (17.6 %). No significant association was observed between hrHPV and OS or DFS after adjusting for potential confounding. hrHPV is prevalent in a sizable fraction of SNSCC. Additional studies are needed to better elucidate the relationship with patient survival outcomes and determine the optimal testing modality for prognostication.

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.

Impact of the COVID-19 pandemic on the severity of newly diagnosed cases of head and neck cancer in southern Brazil.

This observational study aimed retrospectively assess the impact of the COVID-19 pandemic on the head and neck squamous cell carcinoma (HNSCC) diagnosis and severity of the disease in southern Brazil. All new cases diagnosed with HNSCC from March 11, 2019 to March 10, 2020 (pre-COVID-19) and from March 11, 2020 to March 10, 2021 (COVID-19) were included. The data collected were: date of the histopathological diagnosis, sociodemographic data, place of residence, data related to the tumor (location of the primary tumor, lymph node involvement, distant metastasis and TNM clinical staging), time elapsed between the diagnosis and treatment initiation. There was no significant difference in the number of new diagnoses in the COVID-19 group (n=45) compared to the pre-COVID-19 group (n=47). There was also no statistical difference regarding patients' sociodemographic profile, time between diagnosis and treatment, and overall TNM staging. However, the clinical N classification was more severe in the COVID-19 group (p=0.021). Patients diagnosed during the COVID-19 pandemic were 4.05 times more likely to have the N-stage more advanced (95% CI:1.62 - 10.12). Although there was no reduction in the number of new diagnosis of HNSCC during COVID-19 pandemic, the diagnosed cases showed lymph node metastasis in more advanced stages.

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.

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.

Clinical Predictors of Cisplatin Chemoradiation-Induced Ototoxicity in HPV-Positive Oropharyngeal Squamous Cell Carcinoma: A Case-Control Study.

BACKGROUND: Cisplatin-based chemoradiation is a standard treatment for many patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC), an etiologically distinct subset of head and neck cancer. Although associated with good long-term survival, clinical risk factors for ototoxicity have been understudied in this population. This study aimed to evaluate clinical predictors associated with ototoxicity in HPV-positive OPSCC patients treated with cisplatin chemoradiation. METHODS: This retrospective case-control study included 201 adult patients (>18 years) with histologically confirmed HPV-positive OPSCC who received cisplatin chemoradiation as their primary treatment from 2001 and 2019 at a single tertiary cancer center. Ototoxicity was determined using baseline and follow-up audiometry and the Common Terminology Criteria for Adverse Events v5.0 grading criteria (Grade ≥2). Multivariable logistic regression [adjusted odds ratio (aOR)] identified significant predictors that increased the odds of ototoxicity. RESULTS: A total of 201 patients [165 males; median (IQR) age, 57 (11) years] were included in the study. The incidence of ototoxicity in the worst ear was 56.2%, with the greatest hearing loss occurring at high frequencies (4-8 kHz), resulting in a loss of 12.5 dB at 4 to 6 kHz and 20 dB at 6 to 8 kHz. High-dose cisplatin administration compared to weekly administration [aOR 4.93 (95% CI: 1.84-14.99), P = .003], a higher mean cochlear radiation dose [aOR 1.58 (95% CI: 1.12-2.30), P = .01], smoking history [aOR 2.89 (95% CI: 1.51-5.63), P = .001], and a 10 year increase in age [aOR 2.07 (95% CI: 1.25-3.52), P = .006] were each independently associated with increased odds of ototoxicity. CONCLUSIONS: Clinical predictors of ototoxicity in HPV-positive OPSCC patients treated with cisplatin-based chemoradiation include the use of a high-dose cisplatin regimen, higher cochlear radiation doses, a history of smoking, and older age. With the rising incidence of this malignancy in Western countries and overall improved survivorship, our research motivates future studies into risk stratification and earlier interventions to mitigate and reduce the risk of ototoxicity.

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.

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.

Definitive radio(chemo)therapy versus upfront surgery in the treatment of HPV-related localized or locally advanced oropharyngeal squamous cell carcinoma.

BACKGROUND: The treatment of stage I-III HPV+ oropharyngeal squamous cell carcinoma (HPV-OPSCC) is based on either surgery ± adjuvant therapy or exclusive radio±chemotherapy. We sought to compare these two therapeutic strategies in terms of efficacy, tolerance and quality of life (QoL). METHODS: Patients treated for stage I-III HPV-OPSCC from 2010 to 2021 in 3 academic centers were included and sorted according to the treatment strategy: surgery or exclusive radio±chemotherapy. Efficacy and tolerance were retrospectively assessed, and a transversal exploratory QoL assessment was performed using QoL instruments. RESULTS: A total of 83 patients were included, with 21 undergoing non-minimally invasive surgery and 62 receiving definitive radio-±chemotherapy. 2-year progression-free survival (PFS) and overall survival (OS) were respectively 80% and 86% in the surgical group and 92% and 95% in the non-surgical group, with no significant difference. At the end of treatment, 64.5% of patients presented with a grade III toxicity, without significant difference between the two groups. No patient had late grade III toxicity at 24 months. Forty-five patients (11 in the surgical group, 34 in the non-surgical group) participated in an exploratory quality-of-life analysis. Patients reported significantly more fatigue and loss of appetite after surgery, whereas patients in the radio±chemotherapy group described significantly more salivary and oral problems and difficulty swallowing, but the median time between treatment completion and the response to the questionnaires. CONCLUSION: There was no significant difference in efficacy, physician-reported toxicity and overall patient-reported quality of life was found between non-minimally invasive surgery and radio±chemotherapy in the treatment of stage I-III HPV-OPSCC.

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.

Type I conventional dendritic cells and CD8+ T cells predict favorable clinical outcome of head and neck squamous cell carcinoma patients.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common tumor entities worldwide, with human papillomavirus (HPV) infection contributing to cancer development. Conventional therapies achieve only limited efficiency, especially in recurrent or metastatic HNSCC. As the immune landscape decisively impacts the survival of patients and treatment efficacy, this study comprehensively investigated the immunological tumor microenvironment (TME) and its association with patient outcome, with special focus on several dendritic cell (DC) and T lymphocyte subpopulations. Therefore, formalin-fixed paraffin-embedded tumor samples of 56 HNSCC patients, who have undergone resection and adjuvant radiotherapy, were analyzed by multiplex immunohistochemistry focusing on the detailed phenotypic characterization and spatial distribution of DCs, CD8+ T cells, and T-helper cell subsets in different tumor compartments. Immune cell densities and proportions were correlated with clinical characteristics of the whole HNSCC cohort and different HPV- or hypoxia-associated subcohorts. Tumor stroma was highly infiltrated by plasmacytoid DCs and T lymphocytes. Among the T-helper cells and CD8+ T cells, stromal regulatory T cells and intraepithelial exhausted CD8+ T cells expressing programmed cell death protein-1 (PD-1+) and/or lymphocyte-activation gene-3 (LAG-3+) were the predominant phenotypes, indicating an immunosuppressive TME. HPV-associated tumors showed significantly higher infiltration of type I and type II conventional DCs (cDC1, cDC2) as well as several CD8+ T cell phenotypes including exhausted, activated, and proliferating T cells. On the contrary, tumors with hypoxia-associated gene signatures exhibited reduced infiltration for these immune cells. By multivariate Cox regression, immune-related prognostic factors were identified. Patient clusters defined by high infiltration of DCs and T lymphocytes combined with HPV positivity or low hypoxia showed significantly prolonged survival. Thereby, cDC1 and CD8+ T cells emerged as independent prognostic factors for local and distant recurrence. These results might contribute to the implementation of an immune cell infiltration score predicting HNSCC patients' survival and such patient stratification might improve the design of future individualized radiochemo-(immuno)therapies.

HPV16 Phylogenetic Variants in Anogenital and Head and Neck Cancers: State of the Art and Perspectives.

HPV16 is responsible for approximately 60% and 90% of global HPV-induced cervical and oropharyngeal cancers, respectively. HPV16 intratype variants have been identified by HPV genome sequencing and classified into four phylogenetic lineages (A-D). Our understanding of HPV16 variants mostly derives from epidemiological studies on cervical cancer (CC) in which HPV16 B, C, and D lineages (previously named "non-European" variants) were mainly associated with high-grade cervical lesions and cancer. Although a predominance of HPV16 lineage A (previously named "European variants") has been observed in head and neck squamous cell carcinoma (HNSCC), epidemiological and in vitro biological studies are still limited for this tumor site. Next Generation Sequencing (NGS) of the entire HPV genome has deepened our knowledge of the prevalence and distribution of HPV variants in CC and HNSCC. Research on cervical cancer has shown that certain HPV16 sublineages, such as D2, D3, A3, and A4, are associated with an increased risk of cervical cancer, and sublineages A4, D2, and D3 are linked to a higher risk of developing adenocarcinomas. Additionally, lineage C and sublineages D2 or D3 of HPV16 show an elevated risk of developing premalignant cervical lesions. However, it is still crucial to conduct large-scale studies on HPV16 variants in different HPV-related tumor sites to deeply evaluate their association with disease development and outcomes. This review discusses the current knowledge and updates on HPV16 phylogenetic variants distribution in HPV-driven anogenital and head and neck cancers.

Advances in human papillomavirus detection and molecular understanding in head and neck cancers: Implications for clinical management.

Head and neck cancers (HNCs), primarily head and neck squamous cell carcinoma (HNSCC), are associated with high-risk human papillomavirus (HR HPV), notably HPV16 and HPV18. HPV status guides treatment and predicts outcomes, with distinct molecular pathways in HPV-driven HNSCC influencing survival rates. HNC incidence is rising globally, with regional variations reflecting diverse risk factors, including tobacco, alcohol, and HPV infection. Oropharyngeal cancers attributed to HPV have significantly increased, particularly in regions like the United States. The HPV16 genome, characterized by oncoproteins E6 and E7, disrupts crucial cell cycle regulators, including tumor protein p53 (TP53) and retinoblastoma (Rb), contributing to HNSCC pathogenesis. P16 immunohistochemistry (IHC) is a reliable surrogate marker for HPV16 positivity, while in situ hybridization and polymerase chain reaction (PCR) techniques, notably reverse transcription-quantitative PCR (RT-qPCR), offer sensitive HPV detection. Liquid-based RT-qPCR, especially in saliva, shows promise for noninvasive HPV detection, offering simplicity, cost-effectiveness, and patient compliance. These molecular advancements enhance diagnostic accuracy, guide treatment decisions, and improve patient outcomes in HNC management. In conclusion, advances in HPV detection and molecular understanding have significant clinical management implications. Integrating these advancements into routine practice could ultimately improve patient outcomes.

LRG1 and SDR16C5 protein expressions differ according to HPV status in oropharyngeal squamous cell carcinoma.

The increasing incidence of oropharyngeal squamous cell carcinoma (OPSCC) is primarily due to human papillomavirus, and understanding the tumor biology caused by the virus is crucial. Our goal was to investigate the proteins present in the serum of patients with OPSCC, which were not previously studied in OPSCC tissue. We examined the difference in expression of these proteins between HPV-positive and -negative tumors and their correlation with clinicopathological parameters and patient survival. The study included 157 formalin-fixed, paraffin-embedded tissue samples and clinicopathological data. Based on the protein levels in the sera of OPSCC patients, we selected 12 proteins and studied their expression in HPV-negative and HPV-positive OPSCC cell lines. LRG1, SDR16C5, PIP4K2C and MVD proteins were selected for immunohistochemical analysis in HPV-positive and -negative OPSCC tissue samples. These protein´s expression levels were compared with clinicopathological parameters and patient survival to investigate their clinical relevance. LRG1 expression was strong in HPV-negative whereas SDR16C5 expression was strong in HPV-positive tumors. Correlation was observed between LRG1, SDR16C5, and PIP4K2C expression and patient survival. High expression of PIP4K2C was found to be an independent prognostic factor for overall survival and expression correlated with HPV-positive tumor status. The data suggest the possible role of LRG1, SDR16C5 and PIP4K2C in OPSCC biology.

Moderate SCRIB Expression Levels Correlate with Worse Prognosis in OPSCC Patients Regardless of HPV Status.

Head and neck cancers rank as the sixth most prevalent cancers globally. In addition to traditional risk factors such as smoking and alcohol use, human papillomavirus (HPV) infections are becoming a significant causative agent of head and neck cancers, particularly among Western populations. Although HPV offers a significant survival benefit, the search for better biomarkers is still ongoing. In the current study, our objective was to investigate whether the expression levels of three PDZ-domain-containing proteins (SCRIB, NHERF2, and DLG1), known HPV E6 cellular substrates, influence the survival of HNSCC patients treated by primary surgery (n = 48). Samples were derived from oropharyngeal and oral cancers, and HPV presence was confirmed by PCR and p16 staining. Clinical and follow-up information was obtained from the hospital database and the Croatian Cancer registry up to November 2023. Survival was evaluated using the Kaplan-Meier method and Cox proportional hazard regression. The results were corroborated through the reanalysis of a comparable subset of TCGA cancer patients (n = 391). In conclusion, of the three targets studied, only SCRIB levels were found to be an independent predictor of survival in the Cox regression analysis, along with tumor stage. Further studies in a more typical Western population setting are needed since smoking and alcohol consumption are still prominent in the Croatian population, while the strongest association between survival and SCRIB levels was seen in HPV-negative cases.

Context-Dependent Regulation of Peripheral Nerve Abundance by the PI3K Pathway in the Tumor Microenvironment of Head and Neck Squamous Cell Carcinoma.

Recent studies have highlighted neurons and their associated Schwann cells (SCs) as key regulators of cancer development. However, the mode of their interaction with tumor cells or other components of the tumor microenvironment (TME) remains elusive. We established an SC-related 43-gene set as a surrogate for peripheral nerves in the TME. Head and neck squamous cell carcinoma (HNSCC) from The Cancer Genome Atlas (TCGA) were classified into low, intermediate and high SC score groups based on the expression of this gene set. Perineural invasion (PNI) and TGF-ß signaling were hallmarks of SChigh tumors, whereas SClow tumors were enriched for HPV16-positive OPSCC and higher PI3K-MTOR activity. The latter activity was partially explained by a higher frequency of PTEN mutation and PIK3CA copy number gain. The inverse association between PI3K-MTOR activity and peripheral nerve abundance was context-dependent and influenced by the TP53 mutation status. An in silico drug screening approach highlighted the potential vulnerabilities of HNSCC with variable SC scores and predicted a higher sensitivity of SClow tumors to DNA topoisomerase inhibitors. In conclusion, we have established a tool for assessing peripheral nerve abundance in the TME and provided new clinical and biological insights into their regulation. This knowledge may pave the way for new therapeutic strategies and impart proof of concept in appropriate preclinical models.

Enhancement of Tumor Antigen-specific T-cell Responses by Immune Checkpoint Blockade in Human Papillomavirus-related Head and Neck Squamous Cell Carcinoma.

BACKGROUND/AIM: Human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) is clinically and immunologically distinct from HPV-negative HNSCC. Herein, we investigated the presence of tumor antigens HPV E6/E7 and wild-type p53-specific T-cell responses, and the impact of immune checkpoint blockade in patients with HPV-positive HNSCC. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) from patients with HPV-positive HNSCC were stimulated with HPV E6/E7 or wild-type p53-derived peptide mixture and evaluated using the interferon-γ enzyme-linked immunosorbent spot assay. Flow cytometry was performed to analyze the proportion of T-cell subsets and T cells expressing immune checkpoint molecules. RESULTS: HPV E6/E7-specific T cells were detected in 22 (95.7%) of 23 patients, whereas wild-type p53-specific T cells were detected in 3 (15.0%) of 20 patients. Seven (43.8%) of 16 patients exhibited wild-type p53-specific T-cell responses, as determined using whole proteins instead of peptides. Immune checkpoint blockade enhanced wild-type p53-specific T-cell responses in 9 (45.0%) of 20 patients. Flow cytometric analysis of PBMCs revealed that responders exhibiting enhanced wild-type p53-specific T-cell responses following immune checkpoint blockade had a significantly higher proportion of Ki-67+CD4+ T cells, Ki-67+CD8+ T cells, regulatory T cells, PD-1+CD4+ T cells, and TIM-3+CD4+ T cells than non-responders. CONCLUSION: Our findings indicate that tumor antigen-specific T cells are present in the peripheral blood of patients with HPV-positive HNSCC. Blockade of checkpoint pathways can enhance T-cell responses in certain patients, probably via activated T cells, Tregs, and/or exhausted CD4+ T cells.

Dissecting Crucial Gene Markers Involved in HPV-Associated Oropharyngeal Squamous Cell Carcinoma from RNA-Sequencing Data through Explainable Artificial Intelligence.

BACKGROUND: The incidence rate of oropharyngeal squamous cell carcinoma (OPSCC) worldwide is alarming. In the clinical community, there is a pressing necessity to comprehend the etiology of the OPSCC to facilitate the administration of effective treatments. METHODS: This study confers an integrative genomics approach for identifying key oncogenic drivers involved in the OPSCC pathogenesis. The dataset contains RNA-Sequencing (RNA-Seq) samples of 46 Human papillomavirus-positive head and neck squamous cell carcinoma and 25 normal Uvulopalatopharyngoplasty cases. The differential marker selection is performed between the groups with a log2FoldChange (FC) score of 2, adjusted p-value < 0.01, and screened 714 genes. The Particle Swarm Optimization (PSO) algorithm selects the candidate gene subset, reducing the size to 73. The state-of-the-art machine learning algorithms are trained with the differentially expressed genes and candidate subsets of PSO. RESULTS: The analysis of predictive models using Shapley Additive exPlanations revealed that seven genes significantly contribute to the model's performance. These include ECT2, LAMC2, and DSG2, which predominantly influence differentiating between sample groups. They were followed in importance by FAT1, PLOD2, COL1A1, and PLAU. The Random Forest and Bayes Net algorithms also achieved perfect validation scores when using PSO features. Furthermore, gene set enrichment analysis, protein-protein interactions, and disease ontology mining revealed a significant association between these genes and the target condition. As indicated by Shapley Additive exPlanations (SHAPs), the survival analysis of three key genes unveiled strong over-expression in the samples from "The Cancer Genome Atlas". CONCLUSIONS: Our findings elucidate critical oncogenic drivers in OPSCC, offering vital insights for developing targeted therapies and enhancing understanding its pathogenesis.

Explainable prediction model for the human papillomavirus status in patients with oropharyngeal squamous cell carcinoma using CNN on CT images.

Several studies have emphasised how positive and negative human papillomavirus (HPV+ and HPV-, respectively) oropharyngeal squamous cell carcinoma (OPSCC) has distinct molecular profiles, tumor characteristics, and disease outcomes. Different radiomics-based prediction models have been proposed, by also using innovative techniques such as Convolutional Neural Networks (CNNs). Although some of these models reached encouraging predictive performances, there evidence explaining the role of radiomic features in achieving a specific outcome is scarce. In this paper, we propose some preliminary results related to an explainable CNN-based model to predict HPV status in OPSCC patients. We extracted the Gross Tumor Volume (GTV) of pre-treatment CT images related to 499 patients (356 HPV+ and 143 HPV-) included into the OPC-Radiomics public dataset to train an end-to-end Inception-V3 CNN architecture. We also collected a multicentric dataset consisting of 92 patients (43 HPV+ , 49 HPV-), which was employed as an independent test set. Finally, we applied Gradient-weighted Class Activation Mapping (Grad-CAM) technique to highlight the most informative areas with respect to the predicted outcome. The proposed model reached an AUC value of 73.50% on the independent test. As a result of the Grad-CAM algorithm, the most informative areas related to the correctly classified HPV+ patients were located into the intratumoral area. Conversely, the most important areas referred to the tumor edges. Finally, since the proposed model provided additional information with respect to the accuracy of the classification given by the visualization of the areas of greatest interest for predictive purposes for each case examined, it could contribute to increase confidence in using computer-based predictive models in the actual clinical practice.