INTRODUCTION:Eagle syndrome is an underrecognized condition caused by the elongation of the styloid process or calcification of the stylohyoid ligament, with an unknown true incidence. It is diagnosed clinically through symptoms related to compression of the surrounding neurovascular and musculoskeletal structures, along with radiographic confirmation of styloid elongation. Symptoms are variable and may include neck pain, dysphagia, tinnitus, dizziness, headaches, otalgia, and cognitive complaints. Surgical management via intraoral or transcervical approaches is the current standard of care. We propose a novel minimally invasive skull base approach designed to allow complete styloid removal with reduced tissue dissection. METHODS:A retrospective review was conducted in patients who underwent this minimally invasive skull base approach for Eagle syndrome between November 2020 and June 2024. Of 102 identified patients, 90 met the inclusion criteria. Demographics, operative time, intraoperative and postoperative complications, narcotic use, and symptom improvement were extracted from the electronic medical record. Data were analyzed using Excel. RESULTS:Patients were predominantly female (73.3%) and white (90%). The mean operative time for unilateral styloid removal was 53 min (±21.2). The most common postoperative complication was temporary numbness (42.2%), with resolution reported in 63.2% of affected patients. Overall, 86.7% (n = 78) of patients reported complete or partial symptom improvement by or before the 3-month follow-up. CONCLUSION:The minimally invasive skull base approach for Eagle syndrome demonstrates symptom improvement rates comparable to traditional intraoral and transcervical techniques while offering shorter operative times and limited tissue dissection. This approach appears to be an effective alternative surgical option with an acceptable complication profile.

Supplemental Figure 9. Marker genes by cluster using Leiden method.

Supplemental Figure 3. Boxplot of NF- κB values across the three cluster subtypes. By unsupervised consensus clustering and self-organizing map (SOM) approaches, we identified three distinct biological subtypes within HPV-positive HNSCC: Cl1 (immune-related), Cl2 (epithelial-mesenchymal transition [EMT]-related), and Cl3 (proliferation-related). This classification holds prognostic significance, with Cl1 exhibiting the most favorable survival outcomes, Cl2 associated with the poorest prognosis, and Cl3 displaying an intermediate survival rate [Locati, 2019]. In contrast to recent studies that classified HPV-related HNSCC into immune and keratinocyte subtypes, our analysis confirmed the immune-associated subtype while further resolving the keratinocyte group into two distinct clusters with differing biological profiles and prognostic implications. NF-κB signature is significantly activated in Cl1-immune related cluster compared to the other 2 clusters: A) Metanalyisis-HPV267 cohort (1); B) BD2-HPV286 cohort (2).

Supplemental Figure 8A. UMAP dimensional reduction plots with marker gene validating cell type assignments. Marker genes related to epithelial cells, endothelial cells, fibroblasts, mast cells, dendritic cells, and myeloid cells. Supplemental Figure 8B. UMAP dimensional reduction plots with marker gene validating cell type assignments. Marker genes related to natural killer, B, T and cycling cells.

Supplemental Table 2. Association of NF-κB high and low groups with clinical parameters in both Metanalysis-HPV267 (1) and BD2-HPV286 (2). P-value assessed by χ2 test, with the exception of age (t-test). Abbreviations: CHT, chemotherapy; RT, radiotherapy.

6034 Background: HNSCC reirradiation with stereotactic body radiation therapy (SBRT) has emerged as a potential option for the management of locoregionally recurrent (R) or metastatic (M) HNSCC, however treatment toxicity remains a limiting factor. NBTXR3 is a novel intratumoral radioenhancer composed of functionalized hafnium oxide nanoparticles which locally amplifies radiation therapy (RT). In pre-clinical models, NBTXR3/RT has demonstrated an ability to generate cell killing effects at low RT doses as well as trigger local and systemic immune responses. Here we report outcomes in pts with R or R+M HNSCC who were treated with reirradiation with NBTXR3/SBRT followed by immune checkpoint inhibitors (ICIs). Methods: A phase I dose escalation/expansion trial [NCT03589339] evaluating NBTXR3/SBRT followed by ICI (nivolumab or pembrolizumab) included a subgroup with R or R+M HNSCC that were either naïve or resistant to prior ICI. Pts received an intra-tumoral NBTXR3 injection at dose of 22% or 33% of gross tumor volume (GTV), SBRT (35 Gy in 5 fractions), and ICI. Primary objective was safety and establishing RP2D of NBTXR3/SBRT/anti-PD-1 combination. The expansion part tested the RP2D (33% of GTV). Secondary objectives include efficacy. Results: From June 2019 to February 2025, 30 pts were treated: 16 ICI naïve, 14 ICI resistant with median age of 67 years, 90% ECOG 0-1, and 55% HPV negative. 20 pts (66.7%) had R disease, 10 (33.3%) pts had R+M disease. All injected lesions were in a previously irradiated H&N field. Median time from end of prior radiotherapy to NBTXR3 injection was 21.3 [5-229] months. Median GTV was 16.1 [3-110] mL. 4 pts (13.3%) experienced G≥3 injection-related AEs, 8 (26.7%) G≥3 RT-related AEs, and 6 (20%) G≥3 NBTXR3-related AEs. Carotid injury was not observed. Most frequent injection or NBTXR3-related AEs were injection site pain (10%), oropharyngeal pain (6.7%), tumor pain (6.7%), dysphagia (6.7%), and soft tissue necrosis (6.7%). 27 pts (90%) were evaluable for efficacy. For all disease (i.e. injected and non-injected), the objective response rate (ORR) was 48.1% (13/27) and disease control rate (DCR) was 77.8% (21/27). In R pts, ORR was 64.7% (11/17) and DCR was 88.2% (15/17). In R+M pts, ORR was 20% (2/10) and DCR was 60% (6/10). Survival outcomes in reirradiation pts with R and R+M HNSCC will be presented. Conclusions: Reirradiation with NBTXR3 with SBRT and anti-PD1 was feasible in R or R+M HNSCC with an adverse effect profile expected for this clinical setting. Encouraging preliminary efficacy outcomes have been observed, thus warranting further investigation. Clinical trial information: NCT03589339 .

Importance:Standard treatment for human papillomavirus (HPV)-associated oropharyngeal carcinoma (OPC) often includes 7 weeks of intensive chemoradiotherapy with substantial toxic effects. Lowering the radiation dose to gross disease may maintain cancer control with fewer toxic effects. Objective:To evaluate long-term oncologic outcomes among patients with HPV-associated OPC treated with definitive deintensified (chemo)radiotherapy. Design, Setting, and Participants:This cohort study evaluated consecutive patients treated with (chemo)radiotherapy between September 2014 and August 2022 at multiple institutions under one academic hospital system. Eligible patients had p16-positive OPC with a clinical stage from T0 to T3 and N0 to N2c (according to the American Joint Committee on Cancer [AJCC] Staging Manual, seventh edition) and favorable smoking history. Data were analyzed between August 2023 and October 2025. Interventions:Treatment was 60-Gy intensity-modulated radiotherapy with first-choice concurrent chemotherapy of weekly cisplatin, 30 mg/m2, or weekly cisplatin, 40 mg/m2. Patients with OPC with a clinical stage from T0 to T2 and N0 to N1 (according to AJCC, seventh edition) were recommended 60-Gy radiotherapy alone. Main Outcomes and Measures:Evaluation of locoregional recurrence was the primary outcome. Distant recurrence, progression-free survival (PFS), and overall survival (OS) were secondary outcomes. Results:A total of 240 patients received definitive deintensified (chemo)radiotherapy (207 receiving prospective protocol-adherent treatment and 33 receiving non-protocol-adherent treatment). Mean (SD) patient age was 60.3 (9.9) years (range, 33.0-84.0 years); 206 (85.8%) were male, and 34 (14.2%) were female. The cohort included 139 never smokers (57.9%) and 101 former smokers (42.1%) (median smoking history, 9.0 pack-years [range, 0.25-50.0 pack-years]). Concurrent chemotherapy was prescribed for 205 patients (85.4%), with 185 (77.1%) receiving cisplatin, and 165 (68.8%) receiving weekly cisplatin, 30 mg/m2. Median follow-up for surviving patients was 6.5 years (range, 0.44-11.0 years; 208 of 210 [99.0%] with minimum 2-year follow-up). Outcomes were as follows: 2-year OS was 97.9% (95% CI, 96.1%-99.7%), and 5-year OS, 92.4% (95% CI, 89.0%-96.0%); 2-year PFS, 94.1% (95% CI, 91.2%-97.2%), and 5-year PFS, 86.5% (95% CI, 82.1%-91.0%); 2-year locoregional recurrence, 1.3% (95% CI, -0.2% to 2.7%), and 5-year locoregional recurrence, 3.4% (95% CI, 1.1%-5.8%); 2-year distant recurrence, 4.6% (95% CI, 1.9%-7.3%), and 5-year distant recurrence, 7.3% (95% CI, 3.9%-10.7%). The median time to progression events was 1.9 years (range, 0.3-5.1 years); 12 of 26 recurrences (46.2%) occurred after 2 years. Thirty-three patients with low-risk disease (clinical stage T0-T2, N0-N1, according to AJCC, seventh edition) received radiotherapy alone, which resulted in a 5-year PFS of 93.8%, with no patients experiencing a locoregional recurrence. Conclusions and Relevance:In this cohort study, modest dose deintensification of 60-Gy radiotherapy in patients with HPV-associated OPC shows favorable rates of locoregional control. These findings provide support for ongoing investigations of deintensified radiotherapy.

Supplemental Figure 2. UMAP dimensional reduction plot with malignancy probabilities from scMalignantFinder. Low probability predictions (<0.5) were imputed with to a value of 0.5 for visual clarity.

Supplemental Figure 6. Survival Plot. Prognostic Value of NanoString NAC Classifier Measurements. In Figure 4, PFS was shown for (E) all patients and (F) de-escalation candidates in UNC and E1308 cohorts (non-smoker,

Supplemental Table 4. Association of NF-κB high and low groups with clinical parameters in combined UNC &E1308 cohorts (1) as pictured in Figure 4D-E. P-value assessed by χ2 test, except for age (assessed with t-test).

Supplemental Figure 4. Comprehensive comparison of all gene sets and potential summary metrics for the NF-κB signature using RNAseq from the UNC cohort. UNC Cohort originally presented in Schrank and colleagues PNAS 2023 (1). Correlation plot with scatter and Pearson correlation values. Nano str – the gene signature utilized in the nanostring assay including 232 NF-κB related genes, which are compared to 27 control genes, these genes were identified in our prior publications as outlined below and represent the combination of the PNAS and Onco large gene sets as defined below. Onco small – Top 50 NF-κB related genes identified in prior work using machine learning improved differential expression, prioritized by p-value in Schrank et at, Oncotarget 2022 (2), 49 of which are included (one excluded by probe design) in the NanoString panel. 29 of these genes are also identified as NF-κB related genes with an orthogonal approach in our prior work using WGCNA (1). Onco large – Top 75 NF-κB related genes identified in prior work using machine learning improved differential expression, prioritized by p-value in Schrank et at, Oncotarget 2022 (2), 74 of which are included (one excluded by probe design) in the NanoString panel 45 of these genes are also identified as NF-κB related genes with an orthogonal approach in our prior work using WGCNA (1). Pnas – 203 NF-κB related genes identified in our prior work using WGCNA, a network-based approach in Schrank and colleagues PNAS 2023 (3). Onco only – NF-κB genes identified by differential expression but not by WGCNA. Pnas only – NF-κB genes identified by WGCNA but not by differential expression. Mean – mean expression of signature genes. Median – median expression of signature genes. PCA – sample value of PC1, for PCA performed on signature genes only after expression normalization. ssGSEA – Signature sized normalized enrichment score (NES) calculated by the single sample gene set enrichment method.

Supplemental Figure 1. Correlation plots for the: A) Metanalyisis-HPV267 (1) cohort; B) BD2-HPV286 (2) cohorts, comparing various summary metrics (mean, ssGSEA enrichment score and for the NF-κB RNA signature in microarray gene expression data. Values represent Pearson correlation coefficients. *** p-value of Pearson correlation <0.0005.

Supplemental Table 1. Association of NF-κB pathway variants with patient clinical features. P-value assessed by χ2 test, except for age (assessed with t-test).

Supplemental Figure 5. NF-κB pathway mutations are found in cases with high NF-κB scores. A) NanoString NF-κB expression scores and NF-κB pathway mutational data from UNC cohort where both metrics were available. B) Non-linear least squares fit of mono-exponential function relating NanoString and RNAseq data. This fit was used to transpose the threshold defined by NanoString to the RNAseq data, where 3 more cases were available.

Supplemental Figure 7. IF staining was performed at UNC Pathology Services Core. Patients in the UNC RNAseq cohort were stratified by RNA NF-κB Signature score as previously published. (1) The following antibodies were used: NIK antibody: Cat#SC-8417 (Santa Cruz) and Pan CK: Leica NCL-L-AE1/AE3-601 (Leica). High resolution images of IF slides were obtained performed with the Aperio Versa 200 scanner (Leica Biosystems Inc.) at an apparent magnification of 10X. Images were uploaded to the eSlideManager database (Aperio) at the Pathology Services Core at UNC. ** p-value <0.00005, Wilcox test.

Supplemental Table 3. NF-κB high and low patient samples with clinical parameters in combined scRNAseq data from Kurten and colleagues (1) No recurrence or progression events were noted in these 6 patients.

PURPOSE:Patients with human papillomavirus-associated head and neck squamous cell carcinoma (HPV+ HNSCC) have relatively favorable outcomes, but standard treatments like radiation or chemoradiation frequently result in long-term toxic side effects. Appropriate patient selection has been a barrier to effectively de-escalate therapy for HPV+ HNSCC, and the absence of accurate biomarkers likely contributed to failure of recent promising de-escalation trials that rely on histologic tumor characteristics, history of tobacco use, or tumor response to chemotherapy; This deficiency underlines the need to develop and validate an assay to accurately detect two subtypes of HPV+ HNSCC-one with good prognosis and one with poor prognosis. EXPERIMENTAL DESIGN:These two subtypes are distinguished by the activity of NF-κB in tumors. We first developed a DNA-based marker panel consisting of genes that when mutated would lead to NF-κB activation. Additionally, we developed a custom NanoString assay to determine the expression of NF-κB target genes. These assays were tested to determine their accuracy in detecting tumor subtype. RESULTS:We demonstrate that the NF-κB gene signature score, as determined using the NanoString assay, could more accurately classify HPV+ HNSCC as compared with the DNA-based marker panel. Patients with a high NF-κB gene signature score demonstrated significantly increased overall survival, indicating more sensitivity to (chemo)radiation treatment. CONCLUSIONS:The NF-κB gene signature score can accurately predict response to standard (chemo)radiation in HPV+ HNSCC. This molecular biomarker holds promise for clinical use in identifying patients who are likely to benefit from treatment de-escalation strategies, potentially reducing long-term side effects without compromising therapeutic efficacy.

Detection of circulating tumor DNA (ctDNA) has clinical value as a non-invasive method for therapeutic response monitoring and detection of minimal residual disease (MRD). In this study, we employed a tumor-informed approach for detecting ctDNA from plasma in a cohort of head and neck cancer (HNSCC) patients with matched sets of FFPE tissue (FFPET), peripheral blood, and plasma samples. Patients with locally advanced HNSCC were treated with induction chemoimmunotherapy, followed by surgical resection, and risk-adapted adjuvant therapy. Plasma was collected pre-treatment, post-induction (on-treatment), and after completion of all treatment (post-treatment). By leveraging whole genome sequencing (WGS), we can overcome the sparsity of ctDNA fragments in low tumor fraction (TF) settings via tracking of hundreds to thousands of single nucleotide variants (SNVs). Our approach identifies a patient’s unique compendium of tumor-related SNVs from their FFPET sample (after germline subtraction) and tracks this compendium of variants in their plasma samples to detect the presence of ctDNA. Three patients failed quality control criteria and were excluded from further analysis due to the presence of high proportions of C>T:G>A and C>A:G>T variants, known to be FFPE-related and oxidative damage-related artifacts, respectively. Three plasma samples from the post-induction timepoint were also excluded due to insufficient material for sequencing. Post-induction ctDNA was detected in 13 of 22 evaluable samples. Among the ctDNA positive (ctDNA+) post-induction patients, all of them (13/13) had measurable residual tumors on pathologic review, while (2/9) in the ctDNA negative (ctDNA-) group had measurable tumors. For patients with measurable tumors, mean tumor size in the ctDNA+ group (n=13) was 4 cm, while the mean in the ctDNA- group (n=2) was 2.6 cm. Post-treatment ctDNA was detected in 2 of 18 evaluable samples. Among the ctDNA+ post-treatment patients, 100% (2/2) had disease recurrence within 6 months, while disease recurrence was observed in 12.5% (2/16) of the ctDNA- group (occurring 7- and 32-months post-treatment completion). Lastly, we estimated plasma tumor fraction (TF) following Zivran et al (2020). ctDNA+ post-induction samples had a mean TF of 2.4 x 10-4 (n=13; ranging from 2.3 x 10-5 to 1.1 x 10-3) and ctDNA+ post-treatment samples had an order of magnitude lower mean TF of 5.8 x 10-5 (n=2; ranging from 5.7 x 10-5 to 5.8 x 10-5) indicating partial molecular response to treatment even in the presence of detectable residual ctDNA. In this albeit small clinical study, our tumor-informed ctDNA assay achieved clinical sensitivity of 87% and 50% at post-induction and post-treatment, respectively, and clinical specificity of 100% at both time points. Our findings underscore the utility of ctDNA as a promising biomarker for disease monitoring in head and neck cancer patients. Shetal A. Patel, Melanie Lou, Eddie Ho, Michelle Harwood, Seng Saelee, Dinesh Kumar, Steven Johnson, Siddarth Sheth, Jeffrey Blumberg, Catherine Lumley, Wendell Yarbrough, Michael K. Gibson, Jessica Bauman, Trevor Hackman, Jared Weiss, Sowmi Utiramerur. Whole genome sequencing of ctDNA enables disease monitoring and residual disease detection in head and neck cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3262.

OBJECTIVE:To evaluate the microbiologic characteristics, antibiotic resistance patterns, and clinical predictors of reconstruction in patients with osteoradionecrosis of the jaw (ORNJ). STUDY DESIGN:Retrospective cohort study. SETTING:Single tertiary care academic medical center. METHODS:Eighty-four adult patients treated for ORNJ between January 2023 and August 2024 were included. Clinical and microbiologic data were extracted from the electronic health record. Culture results, antibiotic resistance profiles, and reconstructive outcomes were analyzed. Logistic regression was used to identify predictors of autologous reconstruction, Gram-negative infection, and Actinomyces isolation. RESULTS:Among 84 patients (median age 63.3), 73.8% had cultures obtained, and 92.9% received antibiotics. Bacterial species were isolated in 58.2% of 134 cultures, with Actinomyces (23.9%) and Streptococcus anginosus (7.5%) most frequent. Gram-negative organisms were identified in 22.6% and independently predicted autologous reconstruction (odds ratio [OR] = 14.81; 95% confidence interval [CI]: 2.59-84.67; P = .0025). Fistula presence was significantly associated with Gram-negative infection (OR = 6.57; 95% CI: 1.76-24.47; P = .005). High resistance was observed to ampicillin (47.1%) and cefazolin (35.3%), while gentamicin showed the highest susceptibility (75.9%). Posterior mandibular involvement and pathologic fracture were associated with reduced odds of reconstruction. CONCLUSIONS:ORNJ exhibits a polymicrobial and antibiotic-resistant profile. Gram-negative infection is a key predictor of the need for autologous reconstruction and may reflect more severe disease. These findings support the use of culture-directed therapy and highlight the importance of integrating microbiologic and anatomic factors in ORNJ management. LEVEL OF EVIDENCE:IV.

Objective The use of perioperative antibiotics in sialendoscopy is nonstandardized and subject to surgeon preference. This study examines the association between perioperative antibiotic use in interventional submandibular gland sialendoscopy with stone removal and postoperative infection rates.Methods A multi-institutional retrospective cohort study of academic otolaryngology practices. Patients who underwent sialendoscopy of the submandibular gland for stone removal from January 1, 2015 to December 31, 2023 were included. Data were collected on patient demographics, comorbidities, disease factors, perioperative factors, and antibiotic use. The primary exposure was isolated postoperative antibiotic use. Secondary and tertiary exposures included any perioperative antibiotic use and isolated intraoperative antibiotic use. The primary outcome was postoperative infection.Results A total of 399 patients from four institutions were included, with an overall rate of infection of 5.01%. Perioperative antibiotics were used in 57.6% of cases, including intraoperatively in 44.9% and postoperatively in 31.1%. There was no statistically significant difference in infection rates between those who received postoperative antibiotics (RR: 1.48, 95% CI: 0.62-3.53), intraoperative antibiotics (RR: 1.10, 95% CI: 0.46-2.64), any perioperative antibiotics (RR: 1.01, 95% CI: 0.43-2.37), and no antibiotics. Additionally, postoperative antibiotic use was directly associated with age, history of prior sialendoscopy, stent placement, and systemic steroid use, and inversely associated with basket retrieval.Conclusions We found no association between the use of perioperative antibiotics and postoperative infection rates. While specific subgroups of patients with complex pathologies or high-risk intraoperative findings may benefit, this study suggests antibiotics are not indicated for routine interventional sialendoscopy procedures on the submandibular glands.Level of Evidence 3.