Targeted Proteomic Quantitation of NRF2 Signaling and Predictive Biomarkers in HNSCC.

The NFE2L2 (NRF2) oncogene and transcription factor drives a gene expression program that promotes cancer progression, metabolic reprogramming, immune evasion, and chemoradiation resistance. Patient stratification by NRF2 activity may guide treatment decisions to improve outcome. Here, we developed a mass spectrometry-based targeted proteomics assay based on internal standard-triggered parallel reaction monitoring to quantify 69 NRF2 pathway components and targets, as well as 21 proteins of broad clinical significance in head and neck squamous cell carcinoma (HNSCC). We improved an existing internal standard-triggered parallel reaction monitoring acquisition algorithm, called SureQuant, to increase throughput, sensitivity, and precision. Testing the optimized platform on 27 lung and upper aerodigestive cancer cell models revealed 35 NRF2 responsive proteins. In formalin-fixed paraffin-embedded HNSCCs, NRF2 signaling intensity positively correlated with NRF2-activating mutations and with SOX2 protein expression. Protein markers of T-cell infiltration correlated positively with one another and with human papilloma virus infection status. CDKN2A (p16) protein expression positively correlated with the human papilloma virus oncogenic E7 protein and confirmed the presence of translationally active virus. This work establishes a clinically actionable HNSCC protein biomarker assay capable of quantifying over 600 peptides from frozen or formalin-fixed paraffin-embedded archived tissues in under 90 min.

Recurrent laryngeal motor nerve conduction studies in a rat model: Establishing an objective measure for investigating laryngeal innervation.

INTRODUCTION/AIMS: Disease or injury can cause neuromuscular changes to the larynx that can affect voice, breathing, and swallowing. Motor nerve conduction studies have had limited use in the study of laryngeal neurophysiology, despite their importance in other anatomic sites. The aim of this study was to explore the feasibility of performing recurrent laryngeal motor nerve conduction studies (rlMNCS) in a rat model. METHODS: rlMNCS were performed in 15 rats under anesthesia. A bipolar stimulating electrode was placed on the recurrent laryngeal nerve (RLN) 5 mm below the cricoid cartilage. Via direct laryngoscopy, a recording electrode was placed transorally into the thyroarytenoid muscle. The RLN was maximally stimulated to determine the compound muscle action potential (CMAP). Three consecutive trials were averaged. RESULTS: The mean stimulating threshold to the RLN to achieve a CMAP from the thyroarytenoid was 1.7 ± 0.6 mA. RLN stimulation caused a visible adductor twitch of the vocal fold in all animals. The mean negative amplitude was 2.0 ± 0.8 mV, and the total area was 1.0 ± 0.4 mV ms. The CMAP latency and negative duration were 1.0 ± 0.1 ms and 0.9 ± 0.2 ms, respectively. DISCUSSION: rlMNCS are feasible and may be useful in understanding laryngeal neurophysiology with disease or injury. This work could provide a tractable animal model for studying and monitoring treatment of neuromuscular conditions affecting voice, breathing, and swallowing.

Factors associated with skin graft take in fibula and radial forearm free flap donor sites.

LEARNING OBJECTIVES: Identify factors associated with skin graft take in fibula free flaps (FFF) and radial forearm free flaps (RFFF) donor sites. STUDY OBJECTIVES: To determine which factors are associated with decreased skin graft take at the donor site in FFF and RFFF in head and neck patients. DESIGN: Retrospective Chart Review Case Series. SETTING: Multicenter Tertiary Care. METHODS: A multicenter retrospective review was performed at three institutions identifying patients who underwent free tissue transfer, specifically either FFF or RFFF, between 2007 and 2017. Patient demographics, medical history, and social history were examined including age, gender, BMI, smoking status, diabetes and preoperative anticoagulation use. Preoperative, intraoperative data, and postoperative data were also examined including tourniquet use, type of flap, area of skin graft, if the skin graft had a donor site or if it was taken from the flap, wound NPWT use, cast use, use of physical therapy, DVT prophylaxis, limb ischemia, heparin drip, and postoperative aspirin use. Statistical analysis was used to determine which factors were significantly associated with skin graft take. RESULTS: 1415 patients underwent a forearm or fibula flap and 938 patients underwent split-thickness skin graft. Of these, 592 patients had sufficient information and were included in the final analysis. There were 371 males and 220 females. The average age was 55.7. Complete skin graft take was seen in 480 patients (81.1%). On univariate analysis, patients with diabetes (p = .003), type of flap (fibula p < .001), skin graft area (p = .006), tourniquet use (p = .003), DVT prophylaxis (p = .008) and casting (p = .003) were significantly associated with decreased skin graft take rate. In a multivariate analysis, diabetes (OR 2.17 (95%CI 1.16-3.98)), fibula flaps (OR 2.86 (95%CI 1.79-4.76)), an increase in skin graft area (OR 1.01 (95%CI 1.01-1.01)), post-operative aspirin (OR 2.63 (95%CI 1.15-5.88), and casting (OR 2.94 (95%CI 1.22-7.14)) were associated with poor rates of skin graft take. CONCLUSION: Several factors affect skin graft take rate and should be considered when performing a skin graft for a donor site defect.

Tourniquet use and factors associated with hematoma formation in free tissue transfer.

PURPOSE: Our objective was to understand which variables are associated with hematoma formation at both the donor and recipient sites in head and neck free tissue transfer and if hematoma rates are affected by tourniquet use. METHODS: Patients were identified who underwent free tissue transfer at three institutions, specifically either a radial forearm free flap (RFFF) or a fibula free flap (FFF), between 2007 and 2017. Variables including use of tourniquet, anticoagulation, treatment factors, demographics, and post-operative factors were examined to see if they influenced hematoma formation at either the free tissue donor or recipient site. RESULTS: 1410 patients at three institutions were included in the analysis. There were 692 (49.1%) RFFF and 718 (50.9%) FFF. Tourniquets were used in 764 (54.1%) cases. There were 121 (8.5%) hematomas. Heparin drips (p < .001) and DVT prophylaxis (p = .03) were significantly associated with hematoma formation (OR 95% CI 12.23 (4.98-30.07), 3.46 (1.15-10.44) respectively) on multivariable analysis. CONCLUSIONS: Heparin Drips and DVT prophylaxis significantly increased hematoma rates in free flap patients while tourniquets did not affect rates of hematoma.

Zinc isotopic evidence for the origin of the Moon.

Volatile elements have a fundamental role in the evolution of planets. But how budgets of volatiles were set in planets, and the nature and extent of volatile-depletion of planetary bodies during the earliest stages of Solar System formation remain poorly understood. The Moon is considered to be volatile-depleted and so it has been predicted that volatile loss should have fractionated stable isotopes of moderately volatile elements. One such element, zinc, exhibits strong isotopic fractionation during volatilization in planetary rocks, but is hardly fractionated during terrestrial igneous processes, making it a powerful tracer of the volatile histories of planets. Here we present high-precision zinc isotopic and abundance data which show that lunar magmatic rocks are enriched in the heavy isotopes of zinc and have lower zinc concentrations than terrestrial or Martian igneous rocks. Conversely, Earth and Mars have broadly chondritic zinc isotopic compositions. We show that these variations represent large-scale evaporation of zinc, most probably in the aftermath of the Moon-forming event, rather than small-scale evaporation processes during volcanism. Our results therefore represent evidence for volatile depletion of the Moon through evaporation, and are consistent with a giant impact origin for the Earth and Moon.

Effect on laryngeal adductor function of vincristine block of posterior cricoarytenoid muscle 3 to 5 months after recurrent laryngeal nerve injury.

OBJECTIVES: It has been shown in a canine model that a single injection of vincristine into the posterior cricoarytenoid (PCA) muscle at the time of recurrent laryngeal nerve (RLN) injury effectively blocks its reinnervation and results in improved adductor strength. But clinically, such injuries are usually diagnosed weeks or months after onset. Vincristine injection does not affect a muscle that is already innervated; thus, there is a limited time frame following RLN injury during which a vincristine injection could effectively improve ultimate laryngeal adductor functional recovery. A series of delayed injections was performed in a canine model and results assessed. STUDY DESIGN: Animal (canine) experiment. METHODS: The RLN was transected and repaired, and vincristine (0.4 mg) was injected into the PCA muscle at the time of injury (n=12) or 3, 4, and 5 months later (n=8 each study group). Six months after RLN injury, laryngeal adductor function was measured. Results of vincristine injection without RLN injury (n=6) and longer-term (12 months) follow-up for time zero injections (n=4) are also reported. RESULTS: The animals injected at time zero had better adductor function than non-injected controls, as reported previously, and this result was further increased at 12 months. The 3-month delay gave results similar to the time zero group. The 5-month delay group showed no vincristine benefit, and the 4-month delay group gave an intermediate result. Vincristine to the PCA had no effect on adductor function when the RLN was left intact. Plasma levels showed 19% of injected vincristine reached systemic circulation, which was cleared within 69 hours. CONCLUSIONS: Vincristine injection of the PCA muscle after RLN injury, which blocks this antagonist muscle from synkinetic reinnervation, leads to improved laryngeal adductor functional recovery. The window of opportunity to apply this treatment closes by 4 months after RLN injury in the canine model. Human RLN recovery follows a similar time course and can reasonably be expected to have a similar therapeutic window.

Enhanced Abductor Function in Bilateral Vocal Fold Paralysis with Muscle Stem Cells.

INTRODUCTION: Patients with bilateral vocal fold paralysis (BVFP) experience airway obstruction because of loss of abductor function of posterior cricoarytenoid (PCA) muscles. We previously reported that implantation of autologous muscle progenitor (stem) cells into thyroarytenoid muscles during reinnervation resulted in improved adductor function. In this study, that same approach was applied to treating PCA muscles in a canine model of BVFP. DESIGN: Animal study. METHODS: Two canines underwent baseline measures of glottal resistance (GR), then complete transection and suture repair of both recurrent laryngeal nerves. Muscle stem cells were isolated from skeletal muscle and cultured. Two months later, GR was measured, and then 107 stem cells were implanted into one PCA muscle of each animal. After four more months, GR and glottal opening force (GOF) were measured and the muscles were harvested for histologic study. One control dog underwent the same procedures without stem cell implantation, for comparison. RESULTS: GR increased by 21%-25% over baseline at 2 months, but after stem cell implantation, improved to 10%-14% over baseline at 6 months. PCA muscle strength, as determined by GOF, was 61%-65% on control sides (no stem cells), and 78%-83% on treated sides (with stem cells). Histology confirmed survival of stem cells and a 50% higher rate of innervation of motor endplates in the stem cell treated sides. CONCLUSION: Autologous muscle progenitor (stem) cells show promise as a potential new therapy for patients with bilateral vocal fold paralysis. Additional studies are needed to determine the optimal number of cells, timing of implantation, and other variables before launching a clinical trial. LEVEL OF EVIDENCE: NA (animal study) Laryngoscope, 134:324-328, 2024.

How Do I Examine Laryngeal Dystonia?

Laryngeal dystonia is a potentially disabling task specific dystonia primarily affecting speech. The evaluation and diagnosis of laryngeal dystonia remain challenging, and often require a multi-disciplinary approach, involving collaboration among speech language pathologists, neurologists and laryngologists (1-5). It is crucial to correctly differentiate between the types of laryngeal dystonia due to the distinct therapeutic approaches and responses to botulinum toxin therapy or speech therapy. For educational purposes, we have divided laryngeal dystonia into two main types: adductor and abductor dystonia. In this article, we describe a series of examination techniques that can assist movement disorders neurologists diagnosing this condition, and appropriately differentiating the most common forms of laryngeal dystonia.

Poor Dental Health as a Risk Factor for Alveolar Ridge Malignancies.

OBJECTIVE: To determine the association between poor dental health and risk of oral cavity squamous cell cancer (OCSCC) at individual tumor subsites. STUDY DESIGN: Case-control and cross-sectional METHODS: A case-control study was performed using a population-based cohort in North Carolina (Carolina Head and Neck Cancer Epidemiology Study [CHANCE]). A secondary cross-sectional analysis was performed with an institutional cohort (WashU/Siteman). Cases were adults with primary OCSCC and an identifiable tumor subsite. In the CHANCE cohort, controls were adults without head and neck cancer. In the Washington University/Siteman cohort, patients with tongue cancer served as the comparator group. We used number of missing teeth (categorized 0-6, 7-24, 25-28) as a surrogate for poor dental health, which was self-reported in CHANCE and measured on a pretreatment computed tomography scan in the WashU/Siteman study. Adjusted odds ratios (aORs) for missing teeth were estimated for each tumor subsite using binomial logistic regression models. RESULTS: Near complete tooth loss (25-28 teeth) was associated with a 3.5-fold increased risk of alveolar ridge malignancy (aOR: 3.51; 95% confidence interval [CI]: 1.14-11.01, P = .03) in the CHANCE study. This association was confirmed in our cross-sectional analysis (WashU/Siteman study) where missing 25-28 teeth was associated with an increased risk of alveolar ridge compared to tongue cancer (aOR: 4.60; 95% CI: 1.97-11.10, P = .001). CONCLUSIONS: This study suggests an association between poor dental health and risk of alveolar ridge cancer independent of smoking, alcohol use, age, race, and sex. Future prospective and translational studies are needed to confirm this association and elucidate the mechanism of dental disease in alveolar ridge malignancies.

Endoscopic keel placement to treat and prevent anterior glottic webs.

We performed a retrospective chart review to examine and describe our clinical experience of use of the Lichtenberger technique to place silicone elastomer keels after lysis of existing webs or for prevention of webs following anterior commissure surgery in adults. Twenty-two patients were identified for inclusion, ranging in age from 24 to 80 years. For 18 patients with existing glottic webs,the surgical procedure involved laryngoscopy with complete lysis of the anterior glottic web by laser or sharp technique, followed by placement of a square of silicone elastomer that is sutured in place with the Lichtenberger needle holder and left in place for 3 to 5 weeks. The procedure was well tolerated, and successfully corrected the web in all but 2 cases. For 4 patients, the procedure was performed prophylactically at the time of anterior commissure surgery considered high-risk for web formation. The procedure does not require a tracheotomy, and patients can maintain a normal diet and have functional phonation while the keel is in place. This approach to treating anterior glottic webs offers several advantages over traditional open thyrotomy with keel placement and should be considered to treat or prevent anterior glottic webs.

Race and socioeconomic status interact with HPV to influence survival disparities in oropharyngeal squamous cell carcinoma.

BACKGROUND: HPV-related oropharyngeal squamous cell carcinoma (OPSCC) is associated with a favorable prognosis, yet patients of color and low socioeconomic status (SES) continue to experience inferior outcomes. We aim to understand how the emergence of HPV has impacted race and SES survival disparities in OPSCC. METHODS: A retrospective cohort of 18,362 OPSCC cases from 2010 to 2017 was assembled using the SEER (Surveillance, Epidemiology, and End Results) database. Cox proportional regression and Fine and Gray regression models were used to calculate hazard ratios (HRs) adjusting for race, SES, age, subsite, stage, and treatment. RESULTS: Black patients had lower overall survival than patients of other races in HPV-positive and HPV-negative OPSCC (HR 1.31, 95% CI 1.13-1.53 and HR 1.23, 95% CI 1.09-1.39, respectively). Higher SES was associated with improved survival in all patients. Race had a diminished association with survival among high SES patients. Low SES Black patients had considerably worse survival than low SES patients of other races. CONCLUSION: Race and SES interact variably across cohorts. High SES was protective of the negative effects of race, although there remains a disparity in outcomes among Black and non-Black patients, even in high SES populations. The persistence of survival disparities suggests that the HPV epidemic has not improved outcomes equally across all demographic groups.

Immune Cell Deconvolution Reveals Possible Association of γδ T Cells with Poor Survival in Head and Neck Squamous Cell Carcinoma.

(1) Background: The role of rare immune cell subtypes in many solid tumors, chief among them head and neck squamous cell carcinoma (HNSCC), has not been well defined. The objective of this study was to assess the association between proportions of common and rare immune cell subtypes and survival outcomes in HNSCC. (2) Methods: In this cohort study, we utilized a deconvolution approach based on the CIBERSORT algorithm and the LM22 signature matrix to infer proportions of immune cell subtypes from 517 patients with untreated HPV-negative HNSCC from The Cancer Genome Atlas. We performed univariate and multivariable survival analysis, integrating immune cell proportions with clinical, pathologic, and genomic data. (3) Results: We reliably deconvolved 22 immune cell subtypes in most patients and found that the most common immune cell types were M0 macrophages, M2 macrophages, and memory resting CD4 T cells. In the multivariable analysis, we identified advanced N stage and the presence of γδ T cells as independently predictive of poorer survival. (4) Conclusions: We uncovered that γδ T cells in the tumor microenvironment were a negative predictor of survival among patients with untreated HNSCC. Our findings underscore the need to better understand the role of γδ T cells in HNSCC, including potential pro-tumorigenic mechanisms, and whether their presence may predict the need for alternative therapy approaches.

Audiologic Follow-up in Patients With Head and Neck Cancer Treated With Cisplatin and Radiation.

OBJECTIVES: Evaluate factors associated with adherence to ototoxicity monitoring among patients with head and neck cancer treated with cisplatin and radiation therapy at a tertiary care center. METHODS: We performed a single-institution retrospective cohort study on adults with head and neck cancer treated with cisplatin and radiation therapy who participated in an ototoxicity monitoring program. The primary outcomes were rates of post-treatment audiograms at the following time points: one, three, six, 12, and greater than 12 months. Multivariable logistic regression was performed to identify risk factors associated with complete loss of follow-up after pre-treatment evaluation. RESULTS: Two hundred ninety-four head and neck cancer patients were analyzed. Overall, 220 (74.8%) patients had at least one post-treatment audiogram; 58 (20.0%) patients had more than one audiogram. The time point with the highest follow-up rate was at 3 months (n = 170, 57.8%); rates at the remaining times ranged from 7.1% to 14.3%. When controlling for covariates, patients without insurance and those with stage IV cancers were associated with complete loss of audiologic follow-up (aOR = 7.18, 95% CI = 2.75-19.90; aOR = 1.96, 95% CI = 1.02-3.77, respectively). Among 156 patients recommended for a hearing aid, only 39 (24.8%) patients received one. CONCLUSIONS: Head and neck cancer patients enrolled in an ototoxicity monitoring program demonstrate moderately high follow-up rates for at least one post-treatment audiogram. However, follow-up tapers dramatically after 6 months, and overall hearing aid utilization is low. Further research is needed to understand barriers to long-term audiologic follow-up and hearing aid utilization to decrease untreated hearing loss in cancer survivorship. LEVEL OF EVIDENCE: Level 3 Laryngoscope, 133:3161-3168, 2023.

Interim Results of a Phase 1/2 Open-Label Study of INO-3107 for HPV-6 and/or HPV-11-Associated Recurrent Respiratory Papillomatosis.

OBJECTIVE: To evaluate the safety, immunogenicity, and efficacy of INO-3107, a DNA immunotherapy designed to elicit targeted T-cell responses against human papillomavirus (HPV) types 6 and 11, in adult patients with recurrent respiratory papillomatosis (RRP; NCT04398433). METHODS: Eligible patients required ≥2 surgical interventions for RRP in the year preceding dosing. INO-3107 was administered by intramuscular (IM) injection followed by electroporation (EP) on weeks 0, 3, 6, and 9. Patients underwent surgical debulking within 14 days prior to first dose, with office laryngoscopy and staging at screening and weeks 6, 11, 26, and 52. Primary endpoint was safety and tolerability, as assessed by treatment-emergent adverse events (TEAEs). Secondary endpoints included frequency of surgical interventions post-INO-3107 and cellular immune responses. RESULTS: An initial cohort of 21 patients was enrolled between October 2020 and August 2021. Fifteen (71.4%) patients had ≥1 TEAE; 11 (52.4%) were Grade 1, and 3 (14.3%) were Grade 3 (none treatment related). The most frequently reported TEAE was injection site or procedural pain (n = 8; 38.1%). Sixteen (76.2%) patients had fewer surgical interventions in the year following INO-3107 administration, with a median decrease of 3 interventions versus the preceding year. The RRP severity score, modified by Pransky, showed improvement from baseline to week 52. INO-3107 induced durable cellular responses against HPV-6 and HPV-11, with an increase in activated CD4 and CD8 T cells and CD8 cells with lytic potential. CONCLUSION: The data suggest that INO-3107 administered by IM/EP is tolerable and immunogenic and provides clinical benefit to adults with RRP. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:3087-3093, 2023.

Cellular states are coupled to genomic and viral heterogeneity in HPV-related oropharyngeal carcinoma.

Head and neck squamous cell carcinoma (HNSCC) includes a subset of cancers driven by human papillomavirus (HPV). Here we use single-cell RNA-seq to profile both HPV-positive and HPV-negative oropharyngeal tumors, uncovering a high level of cellular diversity within and between tumors. First, we detect diverse chromosomal aberrations within individual tumors, suggesting genomic instability and enabling the identification of malignant cells even at pathologically negative margins. Second, we uncover diversity with respect to HNSCC subtypes and other cellular states such as the cell cycle, senescence and epithelial-mesenchymal transitions. Third, we find heterogeneity in viral gene expression within HPV-positive tumors. HPV expression is lost or repressed in a subset of cells, which are associated with a decrease in HPV-associated cell cycle phenotypes, decreased response to treatment, increased invasion and poor prognosis. These findings suggest that HPV expression diversity must be considered during diagnosis and treatment of HPV-positive tumors, with important prognostic ramifications.

Memory-like Differentiation, Tumor-Targeting mAbs, and Chimeric Antigen Receptors Enhance Natural Killer Cell Responses to Head and Neck Cancer.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is an aggressive tumor with low response rates to frontline PD-1 blockade. Natural killer (NK) cells are a promising cellular therapy for T cell therapy-refractory cancers, but are frequently dysfunctional in patients with HNSCC. Strategies are needed to enhance NK cell responses against HNSCC. We hypothesized that memory-like (ML) NK cell differentiation, tumor targeting with cetuximab, and engineering with an anti-EphA2 (Erythropoietin-producing hepatocellular receptor A2) chimeric antigen receptor (CAR) enhance NK cell responses against HNSCC. EXPERIMENTAL DESIGN: We generated ML NK and conventional (c)NK cells from healthy donors, then evaluated their ability to produce IFNγ, TNF, degranulate, and kill HNSCC cell lines and primary HNSCC cells, alone or in combination with cetuximab, in vitro and in vivo using xenograft models. ML and cNK cells were engineered to express anti-EphA2 CAR-CD8A-41BB-CD3z, and functional responses were assessed in vitro against HNSCC cell lines and primary HNSCC tumor cells. RESULTS: Human ML NK cells displayed enhanced IFNγ and TNF production and both short- and long-term killing of HNSCC cell lines and primary targets, compared with cNK cells. These enhanced responses were further improved by cetuximab. Compared with controls, ML NK cells expressing anti-EphA2 CAR had increased IFNγ and cytotoxicity in response to EphA2+ cell lines and primary HNSCC targets. CONCLUSIONS: These preclinical findings demonstrate that ML differentiation alone or coupled with either cetuximab-directed targeting or EphA2 CAR engineering were effective against HNSCCs and provide the rationale for investigating these combination approaches in early phase clinical trials for patients with HNSCC.

Preexisting tumor-resident T cells with cytotoxic potential associate with response to neoadjuvant anti-PD-1 in head and neck cancer.

About 50% of patients with locally advanced head and neck squamous cell carcinoma (HNSCC) experience recurrences after definitive therapy. The presurgical administration of anti-programmed cell death protein 1 (PD-1) immunotherapy results in substantial pathologic tumor responses (pTR) within the tumor microenvironment (TME). However, the mechanisms underlying the dynamics of antitumor T cells upon neoadjuvant PD-1 blockade remain unresolved, and approaches to increase pathologic responses are lacking. In a phase 2 trial (NCT02296684), we observed that 45% of patients treated with two doses of neoadjuvant pembrolizumab experienced marked pTRs (≥50%). Single-cell analysis of 17,158 CD8+ T cells from 14 tumor biopsies, including 6 matched pre-post neoadjuvant treatment, revealed that responding tumors had clonally expanded putative tumor-specific exhausted CD8+ tumor-infiltrating lymphocytes (TILs) with a tissue-resident memory program, characterized by high cytotoxic potential (CTX+) and ZNF683 expression, within the baseline TME. Pathologic responses after 5 weeks of PD-1 blockade were consistent with activation of preexisting CTX+ZNF683+CD8+ TILs, paralleling loss of viable tumor and associated tumor antigens. Response was associated with high numbers of CD103+PD-1+CD8+ T cells infiltrating pretreatment lesions, whereas revival of nonexhausted persisting clones and clonal replacement were modest. By contrast, nonresponder baseline TME exhibited a relative absence of ZNF683+CTX+ TILs and subsequent accumulation of highly exhausted clones. In HNSCC, revival of preexisting ZNF683+CTX+ TILs is a major mechanism of response in the immediate postneoadjuvant setting.

Extracellular matrix deformations of the porcine recurrent laryngeal nerve in response to hydrostatic pressure.

Damage to the recurrent laryngeal nerve (RLN) caused by supraphysiological compression or tension imposed by adjacent tissue structures, such as the aorta, may contribute to onset of idiopathic unilateral vocal fold paralysis (iUVP) resulting in difficulty speaking, breathing, and swallowing. We previously demonstrated in adolescent pigs that the right RLN epineurium exhibits uniform composition of adipose tissue, with larger quantities along its length within the neck region in contrast to the left RLN that shows greater collagen composition in the thoracic region and greater quantities of adipose tissue in the neck region. In contrast, the epineurium in piglets was primarily composed of collagen tissue that remained uniform along the length of the left and right RLNs. Tensile testing of the left and right RLN in piglets and pigs showed associated differences in strain by RLN side and segment by age. The goal of this study was to investigate how external hydrostatic compression of the RLN affects the nerve's connective tissue and microstructure. RLN segments were harvested from the distal (cervical/neck) regions and proximal (subclavian for the right RLN, thoracic for the left RLN) regions from eight adolescent pigs and nine piglets. RLN segments were isolated and assessed under fluid compression to test hypotheses regarding epineurium composition and response to applied forces. Second harmonic generation (SHG) imaging of epineurial collagen was conducted at 0, 40, and 80 mmHg of compression. The cartesian strain tensor, principal strain (Eps1), and principal direction of the RLN collagen fibers were determined at each pressure step. Significantly larger values of the 1st principal strain occurred in the proximal segments of the pig left RLN when compared to the same segment in piglets (p = 0.001, pig = 0.0287 [IQR = 0.0161 - 0.0428], piglet = 0.0061 [IQR = 0.0033 - 0.0156]). Additionally, the median transverse strain Eyy) for the second pressure increment was larger in the right proximal segment of pigs compared to piglets (p < 0.001, pig = 0.0122 [IQR = 0.0033 - 0.0171], piglet = 0.0013 [IQR = 0.00001 - 0.0028]). Eyy values were significantly larger in the right proximal RLN versus the left proximal RLNs in pigs but not in piglets (p < 0.001). In contrast to piglets, histological analysis of pig RLN demonstrated increased axial alignment of epineurial and endoneurial collagen in response to compressive pressure. These findings support the hypothesis that the biomechanical response of the RLN to compressive pressure changed from being similar to being different between the right and left RLNs during development in the porcine model. Further investigation of these findings associated with age-related onset of idiopathic UVP may illuminate underlying etiologic mechanisms. STATEMENT OF SIGNIFICANCE: Damage to the recurrent laryngeal nerve (RLN) caused by compression imposed by the aorta may contribute to the onset of left-sided idiopathic unilateral vocal fold paralysis resulting in difficulty speaking, breathing, and swallowing. The goal of this study was to investigate how compression affects the connective tissue and microstructure of the RLN. We quantified the pressure induced deformation of the RLN using multiphoton imaging as a function of both location (proximal versus distal) and age (piglets, adolescent pigs). Our results demonstrate that the biomechanical response of the RLN to compression changes in the right versus left RLN throughout development, providing further evidence that the the left RLN is exposed to increasing dynamic loads with age.

The c.-237_236GA>TT THAP1 sequence variant does not increase risk for primary dystonia.

BACKGROUND: Sequence variants in coding and noncoding regions of THAP1 have been associated with primary dystonia. METHODS: In this study, 1,446 Caucasian subjects with mainly adult-onset primary dystonia and 1,520 controls were genotyped for a variant located in the 5'-untranslated region of THAP1 (c.-237_236GA>TT). RESULTS: Minor allele frequencies were 62/2892 (2.14%) and 55/3040 (1.81%) in subjects with dystonia and controls, respectively (P=0.202). Subgroup analyses by gender and anatomical distribution also failed to attain statistical significance. In addition, there was no effect of the TT variant on expression levels of THAP1 transcript or protein. DISCUSSION: Our findings indicate that the c.-237_236GA>TT THAP1 sequence variant does not increase risk for adult-onset primary dystonia in Caucasians.

Development of In-Office Laryngeal Nerve Conduction Studies: Computed Tomography and Cadaveric Study.

OBJECTIVES/HYPOTHESIS: In-office recurrent laryngeal nerve conduction studies (NCSs) are a technique that can potentially provide information about laryngeal innervation. NCS is essential in the management of other neuropathies including carpal tunnel syndrome and spinal cord injury. We hypothesize that laryngeal NCS may have similar utility in managing patients with vocal fold paralysis, atrophy, and neurodegenerative disease. NCSs are technically challenging because they require transcervical stimulation of the recurrent laryngeal nerve (RLN). This study combines radiographic data with cadaveric dissection to describe the anatomic parameters for optimal RLN stimulation. STUDY DESIGN: Radiographic and Cadaveric Study. METHODS: Fifty computed tomography scans were reviewed to determine the dimensions for ideal needle electrode placement. These values were compared to measurements from 12 fresh human cadaveric neck dissections. Ultrasound imaging was utilized in select cases. The neck was dissected to assess the accuracy of electrode placement. RESULTS: Radiographically, the mean transcervical depth to the RLN was 33.2 mm ± 8.3 mm in males versus 29.4 mm ± 9.4 mm in females. The working space between the lateral trachea and carotid artery was 15.3 mm ± 3.6 mm on the right and 14.1 mm ± 2.9 mm on the left. After placement of stimulating electrodes into the cadaveric neck, the electrode tips were consistently within 8 mm of the RLN. Ultrasound guidance improved placement accuracy of the stimulating electrode. CONCLUSIONS: Laryngeal NCSs can provide detailed and objective information about laryngeal innervation that could dramatically improve the management of various neuropathies. In-office NCSs require technical precision, and this study describes anatomic factors that may affect the feasibility of performing this technique. LEVEL OF EVIDENCE: NA Laryngoscope, 131:1566-1569, 2021.