Objective:Salivary adenoid cystic carcinoma (ACC) is a rare head and neck malignancy with limited surveillance guidelines. ACC disease patterns are quite variable and unpredictable. Herein, we present an institutional retrospective review of salivary adenoid cystic carcinoma and discuss patterns and predictors of recurrence. Study Design:Retrospective cohort study. Setting:Single Quaternary Care Referral Center. Methods:We analyzed 102 adult patients with salivary gland adenoid cystic carcinoma between 1988 and 2019. Patient and tumor-related characteristics are summarized, and recurrence, survival, and metastatic site data are described. Results:Multivariable analysis showed that greater than 30% solid pathology was independently predictive of recurrence (HR 5.32, CI 2.23-12.66). Advanced stage disease (stages III and IV) was also predictive of recurrence (HR 3.77, CI 1.57-9.03). Nearly one-third of all patients experienced distant recurrence. Among patients experiencing distant recurrence, 29% were diagnosed with distant recurrence more than 5 years post-treatment. Conclusion:These data support long-term surveillance and clinical follow-up for patients with ACC.

OBJECTIVE:Treatment resistance remains a significant challenge in head and neck squamous cell carcinoma (HNSCC). Alterations in oxidative stress and hypoxia pathways predict poor therapeutic outcomes. The nuclear factor erythroid 2-related factor 2 pathway is the key regulator of oxidative stress response and an established mediator of treatment resistance. There is emerging evidence suggesting that its activation may influence hypoxia-inducible factor 1α (HIF-1α) signaling. However, this relationship remains poorly defined in HNSCC. STUDY DESIGN:Translational research. SETTING:University-based research laboratory. METHODS:This study utilized bioinformatic and experimental approaches to investigate the relationship between NRF2 and HIF-1α expression. Correlations among KEAP1, NFE2L2 (NRF2), and HIF1A (HIF-1α) mRNA were examined using The Cancer Genome Atlas (TCGA) and RNA-sequencing data from patient-derived HNSCC cell lines. Functional validation was performed using siRNA-mediated KEAP1 knockdown in vitro, followed by quantitative polymerase chain reaction (PCR) and Western blot analysis. RESULTS:TCGA analysis revealed that HIF1A mRNA positively correlated with NFE2L2 (NRF2), though the expression did not differ between NRF2 pathway-mutant and wild-type tumors. No significant correlation between HIF1A and NFE2L2 or KEAP1 mRNA was observed in HNSCC cell lines. KEAP1 knockdown achieved >90% transcript reduction and significantly increased the expression of the NRF2 target gene NQO1. Although NFE2L2 and HIF1A transcripts were unchanged, Western blot analysis demonstrated increased NRF2 and HIF-1α protein levels, suggesting a post-translational relationship. CONCLUSION:KEAP1 knockdown promotes NRF2 upregulation and HIF-1α protein accumulation in HNSCC independent of mRNA changes, suggesting protein-level or redox-mediated crosstalk between oxidative stress and hypoxia pathways.

Preclinical studies have evaluated murine double minue 2 (MDM2) inhibitors as a treatment for adenoid cystic carcinoma (ACC), but clinical trials are lacking. This phase I trial (NCT03781986) assesses the safety and antitumor activity of an oral MDM2 inhibitor, alrizomadlin (APG-115), +/- carboplatin in TP53 wild type unresectable recurrent/metastatic salivary gland cancers (R/M SGC) with a planned 1:1 randomization to carboplatin chemotherapy. The co-primary endpoints are determination of dose-limiting toxicity (DLT) and response rate (RR) for alrizomadlin monotherapy +/- carboplatin. Secondary endpoints include safety, survival, and RR by tumor histology. After enrollment of 4 patients to combination therapy, the trial was modified to a single arm study of alrizomadlin monotherapy due to excess toxicity. 1 DLT was seen in the combination arm, all patients had ≥ G3 treatment related adverse events (TRAE). 37 patients were enrolled to alrizomadlin monotherapy. 3 DLTs were encountered, 67% of patients had ≥ G3 TRAE. The RR was 15% with median progression free survival 10.5 months. These findings demonstrate encouraging tolerability of alrizomadlin monotherapy with antitumor activity in patients with TP53 wild type SGC, especially ACC.

Tumor genome sequencing can identify genetic mutations that, when translated, lead to mutant peptides. Such mutant peptides, when shown to be immunogenic, may be designated as neoantigens. We identified three mutant peptides (PLVAP, MBOAT7, and Neurofascin) from lung cancer patients and characterized their antigenicity in this study. T cells and B cells were isolated from peripheral blood mononuclear cells (PBMC) and tumor-infiltrating lymphocytes (TILs); dendritic cells (DCs) generated from PBMC were pulsed with the mutant peptides or their corresponding wild type (WT) peptides, in order to assess peptide anti-tumor immunogenicity. DCs presenting mutant peptides conferred TILs, PBMC CD3 + T cells as well as CD19 + B cells to directly kill tumor cells, and enhanced proinflammatory cytokine (IFN-γ, TNF) secretion and antibody (IgG, IgM) production significantly more than DCs presenting WT peptides. Of note, we observed mutant epitope-induced B cell and antibody responses that signify the role of B cells in immune response to malignancy, which was largely overshadowed by our focus on T cells. In addition, we found that identified epitopes from one patient are immunogenic in 5 of the 6 patients tested in autologous settings, suggesting the existence of these mutant epitopes in multiple lung cancer patients as shared antigens. Together, the results encourage the generation of antitumor T cells and B cells via in vitro activation with the identified antigenic mutant epitopes for subsequent T cell and/or B cell-based immunotherapy in lung cancer patients.

Data Supplement -FDG Calculation Methods

PURPOSE:We conducted a phase II multicenter clinical trial to test the hypothesis that 18F-fluorodeoxyglucose positron emission tomography (FDG-PET)-based chemoradiation (CRT) dose deescalation would provide noninferior locoregional control compared with historical controls among patients with early-stage p16+ oropharyngeal cancer. We also hypothesized that human papillomavirus (HPV) circulating tumor DNA (ctDNA) changes during treatment predict locoregional recurrence (LRR). PATIENTS AND METHODS:Patients with stage I/II p16+ oropharyngeal squamous cell carcinoma were planned to receive radiation 70 Gy in 35 fractions with concurrent weekly carboplatin and paclitaxel. All patients underwent FDG-PET at baseline and at radiation therapy (RT) fraction 10. Patients with ≥50% decrease from baseline to mid-treatment metabolic tumor volume had treatment deescalated to 54 Gy in 27 fractions. The primary endpoint was LRR. Plasma HPV ctDNA was evaluated weekly and in surveillance. RESULTS:Of 84 evaluable patients, 43% met deescalation criteria. With a median follow-up of 37.8 months, 24-month LRR for the entire cohort was 7.8% [90% confidence interval (CI), 2.6%-12.6%], which was less than the 25% rate specified for assessing noninferiority, thereby meeting the primary endpoint. At 1 month after RT, the mean of multiple quality of life measures between the two groups was improved in the 54 Gy cohort, exceeding the minimal clinically important difference threshold. During CRT, week 1 percentage increase in ctDNA relative to baseline was significantly associated with worse loco-regional control (LRC; HR = 1.052 per 10 percentage point increase in ctDNA; 95% CI, 1.007-1.099; P = 0.023) and loco-regional progression-free survival (HR = 1.038; 95% CI, 1.002-1.076; P = 0.035). CONCLUSIONS:FDG-PET-based RT dose personalization resulted in promising LRR outcomes in early-stage oropharynx cancer with improved short-term patient-reported outcomes. Furthermore, HPV ctDNA changes early in treatment may predict LRC.

As rates of human papillomavirus-positive (HPV+) oropharynx cancer increase, there is increasing need for accurate biomarkers for diagnosis, treatment, and surveillance. The analytical performance of MyHPVscore, a droplet digital PCR laboratory-developed test, was characterized to detect circulating tumor DNA from multiple high-risk HPV (hrHPV) types (16, 18, 31, 33, 35, and 39) in plasma from patients with HPV+cancer. Using Clinical and Laboratory Standards Institute guidelines, MyHPVscore was developed and validated in a Clinical Laboratory Improvement Amendments-certified laboratory. Analytical controls and plasma samples from patients with HPV+oropharyngeal squamous cell carcinoma and noncancer controls were used to evaluate sensitivity, specificity, linearity, and analyte stability. MyHPVscore demonstrated high analytical performance for detecting multiple hrHPV types. Limits of blank were 2.7 (HPV16) and 2.6 (other hrHPV types) positive droplets/reaction; limits of detection were 15.2 and 20.8 positive droplets/reaction, respectively. The linear range was 15 to 62,500 targets/mL plasma (HPV16) and 12 to 100,000 targets/mL (other hrHPV types) with strong correlation (R2 > 0.99). Analytes were stable for 96 hours at -20°C and yielded consistent qualitative results after >400 days of storage. No cross-reactivity was observed between hrHPV types or low-risk types (HPV6 and HPV11). These results support the use of MyHPVscore as a laboratory-developed test for detecting HPV+oropharynx cancer. This method establishes an approach and standard controls that can benchmark other emerging circulating tumor DNA assays.

Abstract Head and Neck Squamous Cell Carcinoma (HNSCC), the 6th most common cancer with 800,000 new cases each year, is an extremely aggressive disease with poor overall survival that has remained unchanged for several decades. For patients that develop recurrent and metastatic (R/M) disease, treatment options are extremely limited, and despite the recent success of immune checkpoint inhibitor (ICI) therapies, overall response rates remain low. To overcome this gap in knowledge and improve survival, our team completed a Phase 2 clinical trial of the pan-VEGF inhibitor, axitinib, in R/M patients. Genetic analysis of tumors from this study revealed that 75% of patients with genomic aberrations in either PIK3CA or PTEN had a longer progression-free survival compared to 17% in those without a PI3K pathway alteration. Importantly, PI3K pathway aberrations are highly prevalent in HNSCC, accounting for 45-55% of all HNSCCs, suggesting the potential for a substantial clinical impact. In our genetically engineered mouse model (GEMM), axitinib significantly extended the overall survival of mice with K14:Pik3ca-H1047R, but not K14:control, tumors. Additionally, the use of in vitro cell proliferation and survival assays in HNSCC cell lines has shown that axitinib reduces cell numbers by 50%, increases γH2AX (DNA damage marker) protein levels as measured by western blots, and is more effective in PIK3CA mutant cell lines. Further, when testing the mechanism, we observed that axitinib, but not Lenvatinib (known VEGFR inhibitor), induced apoptosis in HNSCC cell lines. Deeper investigation showed that the HNSCC cell lines did not express any known axitinib targets, suggesting that axitinib may have an additional, unknown mechanism of action (MoA). To test for additional MoA, we used RNA sequencing of axitinib-treated HNSCC cell lines to compare the signal transduction pathways differentially regulated by axitinib compared to other VEGFR and PI3K inhibitors. Future studies will allow us to continue investigating axitinib’s mechanism of action and its therapeutic potential in PI3K aberrant HNSCC. Our findings support clinical advancement of axitinib and use of PI3K genetic status as a predictive marker for therapeutic response for a large subset of HNSCC patients with limited therapeutic options. Citation Format: Behirda Karaj, Jiayu Wang, Paul L. Swiecicki, An-Yun Teng, J. Chad Brenner. Evaluating the molecular mechanism of axitinib response in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5764.

Cells at the invasive front of oral squamous cell carcinoma (OSCC) occupy a partial epithelial-to-mesenchymal transition (p-EMT) state that drives invasion and therapeutic resistance yet remains poorly understood. To define the regulators of this metastable state, we developed an isogenic OSCC model that captures stable epithelial and p-EMT phenotypes and used it to perform a genome-wide CRISPR invasion screen. This unbiased strategy identified 17 modulators of invasion, most previously unrecognized in cancer and virtually unexplored in OSCC, that converge on cytoskeletal remodeling, adhesion, and epigenetic regulation. Within this network, EDIL3 emerged as a central driver of EMT, invasion, and chemoresistance in head and neck cancer, extending prior reports in other tumor types. Importantly, the invasion signature derived from our screen localized with striking specificity to the tumor invasive front of human OSCC, providing direct clinical validation. Together, these findings reveal a novel, targetable network of transient invasion drivers defining the OSCC tumor front.

OBJECTIVE:We assessed correlations between tumor carbonic anhydrase IX (CAIX) staining, as a marker of tumor hypoxia, and CD8+ T-cell infiltration in a cohort of patients with advanced laryngeal squamous cell carcinoma undergoing a bioselection approach for definitive treatment. STUDY DESIGN:Retrospective cohort study. SETTING:Tertiary care hospital. METHODS:Patients with stage III to IV laryngeal squamous cell carcinoma treated under a bioselection paradigm were included. Immunohistochemistry for CD8+ T-cells and CAIX was performed. Nonparametric tests and Kaplan-Meier survival analyses were used to compare tumor CAIX status by clinicopathologic variables and CD8+ T-cell infiltration and to evaluate the role of CAIX and combination CAIX/tumor infiltrating lymphocytes (TIL) category on survival. RESULTS:Our cohort included 92 patients (n = 68 [73.9%] supraglottic). No difference in CAIX staining was seen by tumor subsite, stage, and response to induction chemotherapy (all P > .05). Thirteen (14.1%) tumors were CAIX-positive and showed significantly lower CD8+ T-cell infiltration than CAIX-negative tumors (18 [0-62] vs 32 [0-399], P = .028). Combination CAIX/TIL category was significantly associated with the likelihood of response (CAIX-/TIL[high] were less likely to respond) and in the group of responders, was predictive of a higher degree of tumor shrinkage (>80%). CONCLUSION:CAIX staining correlates with reduced CD8+ T-cell infiltration in patients with advanced laryngeal squamous cell carcinoma undergoing bioselection. The combination CAIX/TIL category is associated with the likelihood and degree of response to induction. The utility of CAIX status and other combination immune and hypoxia signatures as a biomarker of induction response and survival merits prospective evaluation.

Linear mixed model results for arm*time interaction EORTC QLQ30/HN35 PRO subscales.

Odds ratios (OR) and p values for multi-variable models for acute and late toxicity outcomes including covariates of p16+ OPSCC vs LAHNSCC, primary tumor volume (GTVp) and RT boost volume treated.

Merkel cell carcinoma (MCC) is an aggressive disease with poor survival outcomes and increasing incidence. There is a clear and present need for enhanced understanding of cellular mechanisms of tumorigenesis, validation of robust genetic signatures predictive of aggressive disease, and novel informatics tools to simplify analysis of Merkel cell polyomavirus (MCPyV)-host genome interactions. Genomic DNA was harvested from 54 MCC tumors for exome sequencing and in-depth genetic profiling of a 226-gene panel. We further developed a robust informatics package (MCPyViewer) optimized for MCPyV integration site analysis with graphical output to simplify usability for end users. Finally, we assessed the prognostic impact of specific genetic signatures on MCC-specific survival in our cohort. Our study included 54 patients (n = 44 MCPyV positive), 11 (20.4%) of whom had died of MCC at last follow-up. Human genes altered at high frequency included LRP1B (n = 10, 18.5%), FAT1 (n = 9, 16.7%), KMT2D (n = 9, 16.7%), and RB1 (n = 7, 13.0%). In 36 of 44 (81.8%) MCPyV-positive tumors, we identified viral integration into the human genome with a median of two events per tumor. In six tumors, MCPyV integrated into Catalogue of Somatic Mutations in Cancer tier 1 or tier 2 cancer-related human genes. IMPLICATIONS:A combined genomics score incorporating tumor mutational burden and copy-number variation was strongly prognostic of MCC-specific survival controlling for lymph node metastases and tumor MCPyV status; thus, our study adds critical understanding to prognostic markers and tumorigenic mechanisms in MCC.

OBJECTIVES:To determine if the assessment of CD8+ tumor-infiltrating lymphocytes (TILs) adds prognostic information to the PD-L1 combined positive score (CPS) in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC). METHODS:A retrospective case series was performed of 77 patients with R/M HNSCC between 2003 and 2019. From pre-treatment biopsies, CD8+ TILs and PD-L1 CPS were quantified on a tissue microarray. Associations of biomarkers with overall survival and immune checkpoint inhibition (ICI) response were assessed. RESULTS:Neither CD8+ TIL counts nor PD-L1 CPS alone were associated with overall survival; however, combined high TILs and CPS ≥ 1 were associated with improved survival compared to low TILS and CPS < 1 (18.5 vs. 10.0 months, p = 0.058). For patients treated with ICI (n = 28), PD-L1 CPS predicted ICI response more strongly than CD8+ TILs. CONCLUSIONS:In R/M HNSCC, the combination of PD-L1 CPS and CD8+ TILs is a stronger prognostic biomarker for overall survival compared with either biomarker alone.