Hunter, Danielle E.

ABSTRACT Introduction Changes in peripheral eosinophils follow endoscopic sinus surgery (ESS) in chronic rhinosinusitis with nasal polyps (CRSwNP), with and without aspirin sensitivity. Cytometry by time‐of‐flight (CyTOF) identifies more immune cell subsets than conventional assays or clinical testing. We hypothesized that ESS‐associated changes in peripheral immune cell subsets differ between aspirin‐tolerant (AT)‐CRSwNP and aspirin‐exacerbated respiratory disease (AERD) as measured by CyTOF. Methods Pre‐ and post‐ESS peripheral blood from patients with CRSwNP ( n = 13) or with AERD ( n = 15) was analyzed for immune cell populations using a 37‐marker CyTOF panel. CITRUS analysis identified cell clusters most predictive of patient group or sample timepoint. Results In the combined cohort of 28 patients, circulating CD4 + naïve T cells and CD194 + neutrophils increased after ESS, while eosinophils decreased. When stratified by disease, CD4 + naïve T cells increased in both AERD and CRSwNP post‐ESS; however, eosinophils decreased after ESS only in CRSwNP. Neutrophil subsets showed a mixed response in both patient groups after ESS. Dendritic cells increased in CRSwNP but not in AERD after ESS. Individual timepoint analyses showed more CD141 + monocyte‐derived DCs (moDCs) in CRSwNP than in AERD pre‐ and post‐ESS. Pre‐ESS, both CD185 + and CD194 + neutrophils were higher in AERD compared to CRSwNP. Conclusion CD141 + moDCs were more prevalent in CRSwNP versus AERD, both before and after ESS. Naïve CD4 + T cells increased, and neutrophil subsets changed in all groups after ESS, but eosinophils decreased only in CRSwNP. Our study points to possible systemic immune effects of ESS and candidate immune signatures in CRSwNP and AERD.

Abstract Background: HPV16 (human papillomavirus) is a leading cause of oropharyngeal cancer (OPC) in the US. Antibodies to HPV16 early antigens are detectable in serum samples up to two decades before diagnosis and are potential biomarkers for early detection. In this study, we have developed and compared three custom HPV16 serology platforms for detection of OPC: MSD (Meso Scale Discovery) electrochemiluminescent detection, RAPID ELISA, and a novel semi-quantitative lateral flow assay (LFA) using a custom android application. Methods: For the MSD platform, recombinant HPV 16 antigens (E1, E2, E6, E7) were synthesized in mammalian cells and printed on multiplexed arrays. In RAPID ELISA, GST-tagged antigens (E1, E2, E6, E7) were expressed using mammalian in vitro transcription and translation and bound IgG detected. For the LFA, His-tagged recombinant proteins (HPV16 CE2 184-365aa and HPV16 E7) were synthesized, printed, and antibodies detected with protein G conjugated nanoparticles. Archived serum samples drawn from newly-diagnosed OPC patients (n = 60), with matching saliva samples (n=54, MSD assay only) were obtained from Mayo Clinic. Unmatched controls (n = 46) were obtained from AT Still. For the lateral flow assay, colorimetric image analysis was done using a titration curve of known concentration of monoclonal antibody. We optimized the biomarker panel (E2, E6, and E7 for MSD and ELISA, and for LFA E2, and E7) by using multiparametric analysis and plotting the receiver operating curves to the test performance, and assays were compared using the Cohen’s kappa statistic. Results: At 98% specificity, the MSD HPV16 serologic assay achieved 88% sensitivity, similar to RAPID ELISA, and the LFA assay at 85% sensitivity. The signal intensities for saliva samples were weaker at 24% sensitivity. The Cohen’s kappa value ranged from 0.74 to 0.78 for the comparison between all three assays. There was only one sample among all the cases that was negative by all three serology tests. Conclusion: All three assays have strong concordance for the detection of HPV16 early antigen serology. All the tests can facilitate large scale HPV serology research. Citation Format: Akshansh Kaushik, David Routman, Tej Patel, Joshua Eger, Padhmavathy Yuvaraj, Kathleen R. Bartemes, Marisa D. Griesel, Danielle E. Hunter, Katie Van Abel, Anu Mathew, Mingyue Wang, Leonid Dzantiev, Martin Stengelin, Jacob N. Wohlstadter, Ann Spolarich, Kristina R. Dahlstrom, Jennifer Blain Christen, Erich Sturgis, Karen Anderson. HPV serology for rapid detection of HPV positive oropharyngeal cancer [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 5099.

ABSTRACT Human papillomavirus (HPV) is comprised of >200 genotypes and has an ~8 kb, circular, double-stranded DNA genome. Transmission of HPV occurs through skin-to-skin contact and infection of squamous epithelial cells of cutaneous and mucosal surfaces. HPV genotypes are categorized as low- or high-risk (hrHPV) based on oncogenic potential. There are approximately 14 types of hrHPV that can cause several types of cancer, including HPV-associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC). Detection of HPV(+)OPSCC is traditionally accomplished using p16 immunohistochemistry (IHC) and HPV-specific testing, either DNA or RNA in situ hybridization (ISH) staining or DNA-based PCR of suspected tumor biopsy tissue. More recently, platelet-poor plasma (PPP) samples from patients with HPV(+)OPSCC have proven useful for detection and quantitation of fragments of HPV circulating tumor DNA (ctDNA). ctDNA has been shown to be useful in determining treatment response and monitoring for disease recurrence. In this study, a novel droplet digital PCR assay (ddPCR) was developed and validated for the detection and quantitation of ctDNA from 5 hrHPV genotypes in PPP. Analytical sensitivity ranged from 7.71 to 19.45 fragments of HPV ctDNA per milliliter of PPP across five hrHPV genotypes. In patients with confirmed primary or recurrent HPV(+)OPSCC or HPV(-)OPSCC, testing of corresponding PPP samples ( n = 32) by ddPCR demonstrated 90.63% (29/32) overall agreement with p16/HPV-ISH biopsy results. Compared with reference ddPCR assays performed at outside laboratories, our ddPCR assay yielded 90% (9/10) overall agreement. This assay may provide clinicians with a tool for monitoring HPV ctDNA prior to, during, and after treatment of an HPV-associated cancer. IMPORTANCE At least 14 genotypes of human papillomavirus (HPV) have been identified to have high oncogenic potential. While molecular diagnostic testing for HPV is widely available for liquid cytologic cervical samples, testing is limited for other sample types, including liquid biopsy samples, such as platelet-poor plasma (PPP). With the rising incidence of HPV-associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC), laboratory testing is an essential part of patient diagnosis, management, and surveillance. Here, we summarize the development and analytical performance validation of a multiplexed, droplet digital PCR (ddPCR) assay for the detection and quantitation of HPV circulating tumor DNA (ctDNA) in PPP. This assay may provide clinicians with a tool to address minimal residual disease for patients with an HPV-associated cancer.

6057 Background: The incidence rate of human papillomavirus associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC) has persistently increased over the past decade and is anticipated to rise further over the next decade. Cost effective, accurate, and less invasive screening options for HPV(+)OPSCC have remained stagnant leading to the interrogation of circulating tumor DNA in liquid biopsies such as plasma. We hypothesized that candidate methylated DNA markers (MDMs), previously described by our group, would be present in the plasma of patients with HPV(+)OPSCC and absent in controls. Methods: HPV(+)OPSCC cases were enrolled from a high-volume referral practice. Cancer-free control patients were identified from the surrounding 7-county catchment area and frequency matched to cases by age and alcohol use. cfDNA was extracted from 4 mL of plasma and bisulfite converted prior to Target Enrichment Long-probe Quantitative Amplified Signal assays for 15 MDMs. Strand counts for individual MDMs were normalized to a methylated genomic reference marker. An MDM score was derived via random forest modeling of the 15 MDMs. Area under the receiver operator characteristic curve (AUC) for discriminating cases from controls was evaluated for individual markers and for the MDM score. The sensitivity of the MDM score at 95% specificity was also evaluated. Results: The study consisted of 96 HPV(+)OPSCC and 100 cancer-free controls. The study population was predominately white (95%) with a median age of 61 yrs (IQR: 52-69 yrs). Eighty-nine percent of the cases were male with cancers affecting either the tonsil in 50 (52%) or base of the tongue in 46 (48%) patients; by design 50% of controls were male. Stage I:II:III:IV disease was present in 66%:22%:9%:3% of the cases. The median (IQR) AUC across the 15 MDMs was 0.83 (0.82-0.85) overall: 0.78 (0.77-0.83) for stage I disease and 0.92 (0.91-0.93) for stage II-IV disease. The MDM score for the combined panel of 15 MDMs achieved a cross-validated AUC of 0.93 (CI: 0.89-0.97) overall: 0.90 (CI: 0.84-0.96) for stage I disease and 0.98 (CI: 0.97-1.00) for stage II-IV disease. At 95% specificity, the cross-validated sensitivity of the MDM score for HPV(+)OPSCC was 80% (CI: 71-87%) overall: 73% (CI: 60-83%) for stage I disease and 94% (CI: 78-99%) for stage II-IV disease. Conclusions: We validated a set of previously discovered MDM markers in HPV(+)OPSCC in a robust case control cohort using plasma. The MDMs were detected in both early and late-stage disease. Additional prospective studies in larger intended use cohorts are needed to validate our results for clinical use.

Importance Circulating tumor human papillomavirus DNA (ctHPVDNA) is an important biomarker for the presence of HPV-associated oropharyngeal squamous cell carcinoma (OPSCC), but little is known about early postoperative kinetics of ctHPVDNA clearance. Objective To investigate early postoperative kinetics of ctHPVDNA in patients with HPV-associated OPSCC. Design, Setting, and Participants This prospective cohort study was conducted at a single tertiary care center from January 4, 2020, to January 26, 2023. Patients with newly diagnosed HPV-associated OPSCC undergoing surgical management were enrolled. HPV status was defined as positive if findings of p16 immunohistochemistry and/or HPV DNA in situ hybridization and/or E6/E7 RNA in situ hybridization were positive. Exclusion criteria included history of prior head and neck cancer and metastatic disease at presentation. Data were analyzed from September 1, 2024, to April 25, 2025. Exposures Transoral robotic surgery with concurrent neck dissection. Main Outcomes and Measures Blood was drawn prior to surgery (pretreatment), 1 to 2 days after surgery (postoperative days 1 to 2), and approximately 2 weeks after surgery (postoperative week 2; range, 8 to 20 days). ctHPVDNA was quantified by a tumor tissue–modified viral (TTMV) HPV DNA test. Correlations were tested between the pretreatment and postoperative day 1 to 2 TTMV HPV DNA levels using Gaussian regression. Concordance between detectability at postoperative day 1 to 2 and postoperative week 2 was explored using negative predictive value and positive predictive value. Results Of 57 included patients with detectable pretreatment TTMV HPV DNA, 51 (89%) were male, and the median (IQR) age was 59 (54-66) years. A total of 35 patients (61%) had blood draws at all 3 time points; 16 (28%) had detectable TTMV HPV DNA on postoperative day 1 to 2. Pretreatment and postoperative day 1 to 2 TTMV HPV DNA levels had a medium positive linear correlation ( r = 0.31; 95% CI, 0.04-0.54). Undetectable TTMV HPV DNA on postoperative day 1 to 2 blood draw had a negative predictive value of 0.95 (95% CI, 0.74-1.00) for an undetectable level on postoperative week 2 blood draw, but a detectable level on postoperative day 1 to 2 blood draw only had a positive predictive value of 0.19 (95% CI, 0.04-0.46). Of the 16 patients with detectable TTMV HPV DNA pretreatment and at postoperative day 1 to 2, only 3 (19%) continued to have detectable TTMV HPV DNA at postoperative week 2. One patient had undetectable levels at postoperative day 1 to 2 and detectable levels at postoperative week 2. Conclusions and Relevance In this study, ctHPVDNA detectability early after surgery did not predict detectability at 2 weeks after surgery. ctHPVDNA clearance early after surgery could predict a negative test at 2 weeks. A negative blood draw finding on postoperative day 1 may be used to omit a postoperative blood draw at 2 weeks for minimal residual disease detection in future clinical trials.

AbstractObjectives/HypothesisCompare proteomic profiles of rabbit vocal folds (VFs) injected with micronized cross‐linked jellyfish collagen “collagen Type 0” (MX‐JC) against two clinical products for injection medialization laryngoplasty (IL).Study DesignAnimal model.MethodsLeft recurrent laryngeal nerve sectioning and IL were performed in New Zealand White rabbits (N = 6/group). Group 1 received (MX‐JC) and adipose‐derived stem cells (ADSCs), Group 2, MX‐JC alone; Group 3, cross‐linked hyaluronic acid; and Group 4, micronized acellular dermis. Animals were sacrificed at 4 and 12 weeks. Proteomic profiling of injected versus noninjected VFs by nano‐liquid chromatography, tandem mass spectrometry, and reactome gene ontology analysis was performed.ResultsOverall, 37–61 proteins were found to be upregulated and 60–284 downregulated in injected versus non‐injected VFs (>1.5 fold, false discovery rate‐adjusted p < .05). Over‐representation analysis (% of total) revealed top up‐regulated pathways at 4 and 12 weeks, respectively: Group 1, keratan sulfate metabolism (46%) and cellular processes (29%); Group 2, extracellular matrix (ECM)/collagen processes (33%) and beta oxidation (39%); Group 3, cellular processes (50%) and energy metabolism (100%); and Group 4, keratan sulfate metabolism (31%) and inflammation (50%). Top downregulated pathways were: Group 1, Inflammation (36%) and glucose/citric acid metabolism (42%); Group 2, cell signaling (38%) and glucose/citric acid metabolism (35%); Group 3, keratan sulfate metabolism (31%) and ECM/collagen processes (48%); and Group 4, glucose/citric acid metabolism (33%) and ECM/collagen processes (43%).ConclusionsMX‐JC “collagen Type 0” upregulates pathways related to ECM/collagen formation and downregulates pathways related to inflammation suggesting that it is promising biomaterial for IL.Level of EvidenceNA

Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37-gene set and overall genomic structure, ranging from 16,596 base pairs (bp) in the teleost zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most prevalent inherited diseases, affecting roughly 1 in every 5000 individuals. Currently, few effective treatments exist for those with mitochondrial ailments, representing a major unmet patient need. Mitochondrial dysfunction is also a common component of a wide variety of other human illnesses, ranging from neurodegenerative disorders such as Huntington’s disease and Parkinson’s disease to autoimmune illnesses such as multiple sclerosis and rheumatoid arthritis. The electron transport chain (ETC) component of mitochondria is critical for mitochondrial biology and defects can lead to many mitochondrial disease symptoms. Here, we present a publicly available collection of genetic mutants created in highly conserved, nuclear-encoded mitochondrial genes in Danio rerio. The zebrafish system represents a potentially powerful new opportunity for the study of mitochondrial biology and disease due to the large number of orthologous genes shared with humans and the many advanced features of this model system, from genetics to imaging. This collection includes 15 mutant lines in 13 different genes created through locus-specific gene editing to induce frameshift or splice acceptor mutations, leading to predicted protein truncation during translation. Additionally, included are 11 lines created by the random insertion of the gene-breaking transposon (GBT) protein trap cassette. All these targeted mutant alleles truncate conserved domains of genes critical to the proper function of the ETC or genes that have been implicated in human mitochondrial disease. This collection is designed to accelerate the use of zebrafish to study many different aspects of mitochondrial function to widen our understanding of their role in biology and human disease.

AbstractObjectivesTo examine the degree of agreement between MRI and histologically generated volumetric measurements of residual injection laryngoplasty material.MethodsFollowing left recurrent laryngeal nerve transection, rabbit vocal cords were injected with jellyfish collagen, Cymetra®, or Restylane®. Laryngeal tissue was harvested 4 or 12 weeks post injection followed by MRI imaging and histologic cross‐sectioning. Two raters estimated the volume of remaining injection material in specimens within MRI and histologic axial cross sections. Wilcoxon signed rank tests were employed to detect gross differences between inter‐rater measurements and between imaging modalities across time. Agreement between rater measurements and imaging (histology and MRI) was assessed using intra‐class correlation coefficients.ResultsData was available from 16 rabbits sacrificed at 4 weeks (n = 8) and 12 weeks (n = 8). Inter‐rater testing of MRI imaging revealed no significant differences (p > .05) between rater measurements across time points, and excellent agreement (0.93; 95% confidence interval 0.80–0.98) while histologically estimated volumes demonstrated a significant difference at 4 weeks (p < .05) and overall good agreement (0.89; 95% confidence interval 0.59–0.97). Comparison of MRI and histologically estimated volume measurements revealed significant differences at the 4‐week time point (p < .05) but not at 12 weeks (p > .05). Overall, there is only moderate agreement between MRI and histology estimates (0.72; 95% confidence interval 0.22–0.90).ConclusionsMRI imaging demonstrates good reliability and similar estimates of volume to histologically estimated measurements of residual injection laryngoplasty material at time points clinically relevant for future injection laryngoplasty experiments.Level of EvidenceNA.

AbstractObjectives/HypothesisTest a new jellyfish collagen biomaterial aimed to increase duration of injection medialization laryngoplasty (IL) against two products in clinical practice.Study DesignAnimal model.MethodsLeft recurrent laryngeal nerve sectioning and IL were performed in New Zealand White rabbits (N = 6/group). Group 1 received micronized cross‐linked jellyfish collagen (MX‐JC) and adipose derived stem cells (ADSCs), Group 2, MX‐JC alone, Group 3, cross‐linked hyaluronic acid (X‐HA), and Group 4, micronized acellular dermis (MACD). Animals were sacrificed at 4 and 12 weeks. Major outcomes were MRI tissue volumes and histopathology.ResultsAfter 100 μL IL MRI volumes (means ± STD) at 4 and 12 weeks were: Group 1: 27.2 ± 15.6 and 13.1 ± 5.2 μL, Group 2: 60.8 ± 18 and 27.8 ± 2.47 μL, Group 3: 27.4 ± 12 and 10.6 ± 8 μL, and Group 4: 37.5 ± 11 and 9.85 ± 1 μL. Group 2 volumes were largest and Group 3 were smallest in all comparisons (P < .05). Histologically, low grade inflammatory responses were observed in Group 1, mild histiocytic infiltration in Group 2, widespread muscle fiber loss in Group 3, and plasmocytic infiltration in Group 4.ConclusionsMX‐JC showed the least resorption at 4 and 12 weeks among all groups. T cell inflammatory responses were observed with MX‐JC but were reduced by 12 weeks while B cell immune responses, indicative of antibody priming, were predominantly noted with MACD. MX‐JC + ADSC showed low grade immunity while the XHA showed greater myocyte loss compared to the other groups.Level of EvidenceNA Laryngoscope, 131:E2452–E2460, 2021

Craniofacial reconstruction of critical bone defects typically requires a bone graft. As graft availability may be restricted by disease or comorbidities, tissue engineering approaches are actively sought. The pericranium could provide new bone graft material. During development and repair, bone transitions through a chondrogenic phase. However, with tissue engineering, pluripotent cells can differentiate directly into bone cells. Does ability to recapitulate bone formation in vitro affect osteogenesis and vascularization of pericranium grafts? To answer this, we obtained tissue from nine patients with preplanned craniotomy surgery and studied three-dimensional osteogenesis and angiogenesis of pericranium-derived spheroids. First, we established growth and differentiation conditions on Matrigel. For each spheroid sample, we investigated (i) continuous osteogenic differentiation (COD) and (ii) osteogenic differentiation preceded by chondrogenesis (CD → OD). The effect of vascular endothelial growth factor (VEGF) was compared to VEGF supplemented with fibroblast growth factor, interleukin (IL)-1, IL-6, platelet-derived growth factor, and tumor necrosis factor-α, a growth factor mix (GFM) with possible synergistic effects. In this limited sample, we observed no age- or sex-related differences in cell expansion. Similarly, no statistically significant differences in osteogenic or angiogenic scores between COD or CD → OD spheroids were noted with regular media. In COD, however, VEGF statistically significantly increased angiogenesis compared to control media ( p = 0.007). Also, in COD, both VEGF and VEGF + GFM increased osteogenesis ( p = 0.047 and p = 0.038, respectively). By contrast, in CD → OD, neither VEGF nor VEGF + GFM yielded statistically significant angiogenesis or osteogenesis scores compared to control media. To understand these results, we characterized spheroid protein expression by nanoliquid chromatography coupled to tandem mass spectrometry. Nine angiogenic proteins were either uniquely expressed or upregulated in COD compared to CD → OD: (i) endothelial markers JUP, PTGIS, PTGS2, and TYMP, (ii) tissue remodeling factors CHI3L1 and MMP14, and (iii) metabolic pathways modulators ANGPTL4, ITGA5, and WNT5A. ANGPTL4, ITGA5, PTGIS, PTGS2, and WNT5A define a conserved angiogenic network and were >2-fold increased in VEGF compared to VEGF + GFM. Finally, we examined bone formation on printable poly-(propylene-fumarate) (PPF) scaffolds for individualized grafting. Under COD + VEGF conditions, PPF scaffolds loaded with pericranium-derived cells displayed hallmarks of spongiform-like bone formation. Thus, the human pericranium may be a potential repository for bone-generating cells with applications in craniofacial bone repair using tissue printing.