ABSTRACT Background Although the impact of increased time to treatment initiation (TTI) on outcomes for patients with oropharyngeal squamous cell carcinoma (OPSCC) has been well‐studied, a deeper understanding of the mechanisms underlying delay in patients with human papillomavirus (HPV)‐negative OPSCC is lacking in the current literature. Objective To assess differences in sociodemographic factors and treatment timelines between patients with HPV‐negative OPSCC with shorter versus. longer TTI. Methods Patients treated for HPV‐negative OPSCC at a single academic institution between 2013 and 2023 were retrospectively identified via chart review and dichotomized by the cohort median TTI (53.5 days; defined as the time from biopsy to first treatment initiation). Clinical timelines between delayed and nondelayed patients were compared using descriptive statistics and Mann–Whitney U testing. Independent predictors of delayed TTI (> 53.5 days) were evaluated using multivariate logistic regression modeling, with adjusted odds ratios (aORs) and 95% confidence intervals reported. Results Seventy‐six patients were identified. On multivariable analysis, male sex (aOR 3.28; 95% CI 1.02–10.49), unmarried status (aOR 5.96; 95% CI 1.36–26.07), primary chemoradiation versus surgery (aOR 0.25; 95% CI 0.07–0.85), and biopsy available before arrival (aOR 4.08; 95% CI 1.32–17.36) were independently and significantly (p< 0.05) associated with delayed treatment initiation. Treatment timeline analysis revealed that both the interval from biopsy to referral and the interval from PET scan to treatment initiation differed significantly between delayed and nondelayed patients (p< 0.05). Conclusion Primary nonsurgical treatment and lack of social support were found to be independently associated with treatment delay in patients with HPV‐negative OPSCC. These findings highlight opportunities for improving the care of HPV‐negative OPSCC at the specialty level.

This study analyzed contemporary trends in major salivary gland carcinoma (MSGC) incidence in the United States from 2000 to 2022 using the SEER 17 Registries. Age-adjusted incidence rates (IRs) per 1,000,000 persons and temporal trends were evaluated using annual percent change (APC) and average APC (AAPC). A total of 20,522 MSGC cases were identified, with an overall IR of 10.4 and a significant increasing trend over time (APC = 0.42%, P = 0.015). Incidence increased particularly among females (APC = 0.81%, P = 0.002), older age groups (60–79 years: APC = 0.40%, P = 0.036; ≥80 years: APC = 1.12%, P = 0.010), the Black population (APC = 0.80%, P = 0.029), and parotid gland primaries (APC = 0.40%, P = 0.023). In stage-specific analyses restricted to 2004–2022, localized disease also increased significantly (APC = 0.89%, P = 0.002). Among major histologic subtypes, adenocarcinoma, not otherwise specified, showed a significant decreasing trend (AAPC=–3.33%, P < 0.001), whereas salivary duct carcinoma (AAPC = 10.41%, P < 0.001) and carcinoma ex pleomorphic adenoma (APC = 2.20%, P = 0.022) showed increasing trends. Female-focused subgroup analyses demonstrated increasing incidence in both age groups: females aged ≥ 60 years (APC = 0.64%, P = 0.012) and those aged < 60 years (APC = 0.88%, P = 0.009). Mucoepidermoid carcinoma also exhibited an increasing trend among females (APC = 0.87%, P = 0.016). These findings indicate a significant increase in MSGC incidence in the US, with distinct demographic and clinicopathological patterns.

OBJECTIVE:Prior studies have demonstrated the feasibility of fluorescently labeled tilmanocept for sentinel lymph node biopsy (SLNB) in the oral cavity. We evaluated the added value of fluorescently labeled tilmanocept in sentinel lymph node biopsy (SLNB) of the oral cavity compared to gamma probe. METHODS:Healthy male New Zealand white rabbits received oral cavity injections of radioactive (Technetium 99m) and fluorescently (IRDye800) conjugated tilmanocept followed by either fluorescence guided (n = 14) or gamma probe guided (n = 14) SLNB performed 1 h, 48 h, or 5 days postinjection. Duration of the SLNB performed by two individuals was measured and compared using the two methods. RESULTS:Fluorescence guidance resulted in a 1.8-fold reduction in time of SLN removal (median 104 vs. 191 s, p = 0.05). For the fluorescence guided SLNB, 7.1% (1 of 14) had nonsentinel node tissue removed prior to the correct identification of the SLN, whereas gamma probe/radioactivity guided SLNB had 28.6% (4 of 14). When comparing operation time between attending and resident surgeon, time to identification of first SLN was not significantly different for fluorescence guided surgery (82 vs. 107 s, respectively) or with gamma probe (158 vs. 204 s); however, median times using the gamma probe were nearly double for both operators. We additionally show the durability of fluorescence signal up to 5 days and clear visualization of proposed tracer with commercially available intraoperative imagers. CONCLUSION:The use of fluorescent labeled tilmanocept decreases operative time needed for SLNB as well as reduces the amount of nonsentinel tissue removed. LEVEL OF EVIDENCE:N/A.

A 48-year old woman presented with episodic headaches, light sensitivity, and a painful, pulsating mass on her forehead. What is your diagnosis?

PIK3R1, a regulatory subunit of class IA phosphoinositide‐3‐kinase (PI3K), undergoes alternative splicing to generate multiple isoforms, primarily p85α and p55α. The canonical isoform p85α associates with the catalytic subunit p110α to form the active PI3K complex, which regulates key cellular functions such as growth, proliferation, survival, and metabolism. In this study, we performed a comprehensive pan‐cancer analysis integrating transcriptomic, proteomic, and genomic data to investigate the expression patterns of p85α and its splicing variant, p55α, and their associations with clinical outcomes. Our findings reveal that while p85α expression is significantly reduced, p55α is elevated in tumors as compared to normal samples. These alterations are linked to poor prognosis across multiple cancer types. Notably, we observed racial disparities in expression patterns, with African American patients exhibiting more pronounced downregulation of p85α and upregulation of p55α than European Americans, potentially contributing to differential clinical outcomes. This is the first study to systematically evaluate p85α and p55α expression across diverse cancers and populations, highlighting the role of alternative splicing in PI3K pathway dysregulation and its relevance to cancer progression and health disparities.

Human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HPV+ HNSCC) is one of the most rapidly increasing cancers in the United States. Although patients with HPV+ HNSCC exhibit better responses to chemoradiation compared with patients with HPV- HNSCC, a subset still develops recurrent/refractory disease. In this study, we investigated the role and mechanisms by which the HPV E5 oncogene promotes resistance to conventional chemotherapy and radiation. After treatment with chemotherapy and radiation, HNSCC cells expressing HPV E5 had decreased apoptosis, improved cell viability, enhanced DNA damage response, and increased tumor growth in murine xenograft models when compared with empty vector controls. Transcriptomic analysis showed an enrichment of signaling pathways associated with epithelial development and increased expression of stem cell markers, including SOX9 and SOX4, in HPV E5 cells compared with empty vector cells. Knockdown of SOX9 restored sensitivity to chemo- and/or radiotherapy in HPV E5-expressing cells. In patients with HNSCC, RNA sequencing analysis showed that high expression of HPV E5 was associated with enrichment of pathways involved in cellular senescence and stem cell pluripotency. Furthermore, an HPV E5-associated gene set was identified that correlates with poor patient prognosis and is significantly overexpressed in recurrent HPV+ HNSCC tumors. Taken together, these data identify HPV E5 as a viral mediator of resistance to standard-of-care therapies for HPV+ HNSCC by promoting a stem cell-like phenotype that enhances DNA damage responses and tumor cell survival. Thus, HPV E5 may contribute to chemotherapy resistance, tumor recurrence after radiation, and poor prognosis of patients with refractory or metastatic disease.Significance: HPV E5 upregulates SOX9 to induce stem cell-like characteristics in head and neck cancer that drive resistance to chemotherapy and radiation, providing rationale for developing therapeutic agents targeting HPV E5.

The NCCN Guidelines for Thyroid Carcinoma address the management of different types of thyroid carcinoma, including papillary, follicular, oncocytic, medullary, and anaplastic carcinoma. The NCCN Thyroid Carcinoma Panel meets at least annually to review comments from reviewers within their institutions, examine relevant new data from publications and abstracts, and reevaluate and update their recommendations. These NCCN Guidelines Insights focus on the panel's most recent recommendations regarding systemic therapies for thyroid carcinoma as well as the supporting clinical data.

Human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) is a growing subset of cancer cases distinct from HPV-negative by fewer genetic mutations and prevalent epigenetic dysregulation. We mapped H3K27ac-marked super-enhancers (SEs) via ChIP-seq in HPV+ patient-derived xenografts (PDXs) and normal oropharyngeal mucosa, identifying tumor-specific SE domains (T-SEDs) enriched for transcription factors (TFs) including TP63, FOSL1, and JUND. These SE-associated TFs regulate key oncogenic pathways and are downregulated by BRD4 inhibition with JQ1, highlighting sensitivity to epigenetic modulation. RNA-seq data revealed coordinated dysregulation of enhancer RNAs and mRNAs near T-SEDs, linked to upregulated pathways including epithelial-mesenchymal transition and E2F targets. JQ1 treatment significantly repressed these tumor-specific pathways, suggesting a therapeutic potential for targeting SE-driven transcription in HPV+ HNSCC. This study underscores the critical role of SEs in epigenetic and transcriptional dysregulation in HPV+ HNSCC, revealing therapeutic targets and providing a framework for future mechanistic studies in this area.

Differential gene expression and pathway analysis

Prognosis of HNSCC patients based on the status of E5 signature and expression distribution of E5 in single cell RNA-sequencing dataset

The discovery of tumor-derived neoantigens which elicit an immune response through major histocompatibility complex (MHC-I/II) binding has led to significant advancements in immunotherapy. While many neoantigens have been discovered through the identification of non-synonymous mutations, the rate of these is low in some cancers, including head and neck squamous cell carcinoma. Therefore, the identification of neoantigens through additional means, such as aberrant splicing, is necessary. To achieve this, we developed the splice isoform neoantigen evaluator (SINE) pipeline. Our tool documents peptides present on spliced or inserted genomic regions of interest using Patient Harmonic-mean Best Rank scores, calculating the MHC-I/II binding affinity across the complete human leukocyte antigen landscape. Here, we found 125 potentially immunogenic events and 9 principal binders in a cohort of head and neck cancer patients where the corresponding wild-type peptides display no MHC-I/II affinity. Further, in a melanoma cohort of patients treated with anti-PD1 therapy, the expression of immunogenic splicing events identified by SINE predicted response, potentially indicating the existence of immune editing in these tumors. Overall, we demonstrate SINE’s ability to identify clinically relevant immunogenic neojunctions, thus acting as a useful tool for researchers seeking to understand the neoantigen landscape from aberrant splicing in cancer.

Mechanisms of tumorigenesis in sinonasal squamous cell carcinoma (SNSCC) remain poorly understood due to its rarity. A subset of SNSCC is associated with human papillomavirus (HPV), but it is unclear whether HPV drives tumorigenesis or acts as a neutral bystander. Here, we show that HPV-associated SNSCC shares mutational patterns found in HPV-associated cervical and head and neck squamous cell carcinoma, including lack of TP53 mutations, hotspot mutations in PI3K and FGFR3, enrichment of APOBEC mutagenesis, viral integration at known hotspots, and frequent epigenetic regulator alterations. We identify HPV-associated SNSCC-specific recurrent mutations in KMT2C, UBXN11, AP3S1, MT-ND4, and MT-ND5, with KMT2D and FGFR3 mutations correlating with reduced overall survival. We establish an HPV-associated SNSCC cell line, showing that combinatorial small-molecule inhibition of YAP/TAZ and PI3K synergistically suppresses clonogenicity. Combining YAP/TAZ blockade with vertical PI3K inhibition may benefit HPV-associated SNSCC, whereas targeting MYC and horizontal inhibition of RAS/PI3K may suit HPV-independent SNSCC.

Background/Objectives: HPV+ head and neck squamous cell carcinoma has been shown to have a unique genomic background, requiring researchers to study it as its own distinct type of cancer. HPV+ tumors have been shown to exhibit fewer genetic mutations in cancer drivers as opposed to their HPV- counterparts. In this paper, we explored how targeting post-transcriptional changes, specifically alternative splicing events, could serve as a potential mechanism to treat HPV+ cancer. Methods: Using indisulam, a drug that targets alternative splicing through the degradation of RBM39, we treated various HPV+ and HPV- cell lines and assessed tumor cell viability. We also tested indisulam in vivo to evaluate its effect on tumor volume. Additionally, we analyzed gene expression differences between indisulam-treated subjects and their non-treated counterparts. Results: Indisulam treatment led to a reduction in tumor cell viability in both HPV+ and HPV- cell lines. In vivo experiments showed a reduction in tumor volume following indisulam treatment. Gene expression analysis revealed that indisulam induces consistent differential gene expression changes and highly enriches interferon pathways in treated HPV+ cell lines. Conclusions: These findings suggest that targeting alternative splicing via indisulam may be a promising therapeutic approach for HPV+ cancers. Further research is required to establish indisulam as a viable anti-cancer treatment in clinical settings.

ABSTRACT Background The role of alternative splicing events (ASEs) in immune evasion and prognosis in head and neck squamous cell carcinoma (HNSC) is not well characterized. Methods Using The Cancer Genome Atlas data, we identified ASEs (using our novel algorithm OutSplice) and characterized associations between splice burden, immune infiltration (quantified by xCell) and prognosis with multivariable logistic regression and survival models. Results HSNC tumors with low splice burden and high immune infiltration had significantly better prognosis than tumors with high splice burden and low immune infiltration when controlling for age, pathologic stage, HPV status, and tumor mutational burden (hazard ratio = 0.61). High splice burden predicted decreased immune infiltration in HNSC, which was validated in five other cancer types and supported by murine models of oral squamous cell carcinoma. Conclusions High splicing burden, as defined by OutSplice, is a novel biomarker to predict poor both immune infiltration and prognosis in HNSC.

Expression of HPV E5, E6 and E7 in different HNSCC cells and normal keratinocytes (HaCaT)

Drug-tolerant persister cancer cells were first reported fifteen years ago as a quiescent, reversible cell state which tolerates unattenuated cytotoxic drug stress. It remains unknown whether a similar phenomenon contributes to immune evasion. Here we report a persister state which survives weeks of direct cytotoxic T lymphocyte (CTL) attack. In contrast to previously known immune evasion mechanisms that avoid immune attack, antigenic persister cells robustly activate CTLs which deliver Granzyme B, secrete IFNγ, and induce tryptophan starvation resulting in apoptosis initiation. Instead of dying, persister cells paradoxically leverage apoptotic caspase activity to avoid inflammatory death. Furthermore, persister cells acquire mutations and epigenetic changes which enable outgrowth of CTL-resistant cells. Persister cell features are enriched in inflamed tumors which regressed during immunotherapy in vivo and in surgically resected human melanoma tissue under immune stress ex vivo . These findings reveal a persister cell state which is a barrier to immune-mediated tumor clearance. Graphical abstract:

Background/Objectives: Immune checkpoint inhibitors (ICIs) are frontline treatment for advanced Merkel Cell Carcinoma (MCC), regardless of viral status. Frontline ICIs provide durable benefit to only half of patients, highlighting a need for alternative therapies. In this study, the objective is to leverage whole exome sequencing (WES) and transcriptome sequencing (WTS) to distinguish genomic alterations associated with ICI response. Investigate differential genomic alterations between virus-positive (VP) and virus-negative (VN)-MCC to identify novel therapeutic targets. Methods: A total of 95 MCC cases underwent WES and WTS. Utilizing computational pipelines applied to WES, we identified viral status and tumor mutational burden (TMB). RNA-seq data was used to characterize the immune microenvironment. Results: Of 95 MCC cases, 57 (60%) were VP-MCC and 38 (40%) were VN-MCC. Median TMB was higher in VN-MCC (27.5 vs. 1 Muts/Mb). Mutations in TP53, RB1, NOTCH1, KMTD2, KMT2C, and PIK3CA were primarily found in VN-MCC. MAPK Pathway Activity Score, NK cell infiltration, and the immune checkpoint gene CD276 in VN-MCC tumors were upregulated. No overall survival (OS) difference was identified between VP and VN-MCC, even after ICIs. Conclusions: MCC oncogenesis and treatment response transcend viral status. While mutational analysis confirms previous findings, assessment of the transcriptome and tumor microenvironment suggests alternate therapeutic targets.

shRNA mediated knockdown of SOX9 in HNSCC cells, measurement of apoptosis and DNA damage response in SOX9 silenced EV HNSCC cells

Background/Objectives: Non-melanoma skin cancer, which includes cutaneous squamous cell carcinoma (cSCC), ranks as the 5th most common cancer globally with high morbidity and more total deaths than melanoma despite having a lower mortality rate. While most cSCC cases can be treated with surgery, locally advanced, metastatic, and high-risk cSCC tumors are associated with a worse prognosis with higher rates of recurrence and require multimodality therapy. However, there is limited data on animal models of cutaneous squamous cell carcinoma for the use of combinatory immunotherapy and radiation. Methods: In this study, spontaneously generated tumors using DMBA/TPA were treated over three weeks with either IgG control, anti-PD1 antibody monotherapy, 8 Gy of localized radiation, or a combination of anti-PD1 and 8 Gy of radiation followed by anti-PD1 therapy. Results: We found that while anti-PD1 therapy showed a trend toward slowed tumor growth compared to controls, this difference was not statistically significant (p = 0.0775), with most mice showing continued tumor progression. Preliminary histological analysis suggested that anti-PD1 treatment increased CD8+ T cell infiltration, and the addition of radiation further enhanced CD8+ responses but added greater variability. A pathologic review revealed that irradiated tumors were associated with fibroblastic spindle-like cell morphology. Conclusions: This animal model represents a potential preclinical model for studying CSCC with limited responses to immunotherapy to understand potential mechanisms of resistance.

Measurement of apoptosis after knockdown of E5 in HNSCC cells and evaluation of DNA damage response in E5 expressed and E5 knockdown HNSCC cells