Single-Cell Transcriptomic Analysis of Primary and Metastatic Tumor Ecosystems in Head and Neck Cancer.

The diverse malignant, stromal, and immune cells in tumors affect growth, metastasis, and response to therapy. We profiled transcriptomes of ∼6,000 single cells from 18 head and neck squamous cell carcinoma (HNSCC) patients, including five matched pairs of primary tumors and lymph node metastases. Stromal and immune cells had consistent expression programs across patients. Conversely, malignant cells varied within and between tumors in their expression of signatures related to cell cycle, stress, hypoxia, epithelial differentiation, and partial epithelial-to-mesenchymal transition (p-EMT). Cells expressing the p-EMT program spatially localized to the leading edge of primary tumors. By integrating single-cell transcriptomes with bulk expression profiles for hundreds of tumors, we refined HNSCC subtypes by their malignant and stromal composition and established p-EMT as an independent predictor of nodal metastasis, grade, and adverse pathologic features. Our results provide insight into the HNSCC ecosystem and define stromal interactions and a p-EMT program associated with metastasis.

Stage Migration and Survival Trends in Laryngeal Cancer.

BACKGROUND: During the last two decades, significant advancements in the treatment of laryngeal cancer have occurred. Although survival of head and neck cancer patients has improved over time, the temporal trend of laryngeal cancer survival is an area of controversy. METHODS: From 2004 to 2016, 77,527 patients who had laryngeal cancer treated with curative intent in the United States were identified in the National Cancer Database. Relative and observed survival rates were assessed for temporal trends. Multinomial logistic regression investigated the relationship between American Joint Committee on Cancer (AJCC) stage and increasing calendar year. RESULTS: No significant improvement in 2- or 5-year observed survival (OS) or relative survival (RS) was observed. The 5-year RS ranged from 61.72 to 63.97%, and the 5-year OS ranged from 54.26 to 56.52%. With each increasing year, the proportion of stage 4 disease increased, with risk for stage 4 disease at the time of diagnosis increasing 2.2% annually (adjusted odds ratio [aOR], 1.022; 95% confidence interval [CI], 1.017-1.028; p < 0.001). This increase was driven by a 4.7% yearly increase in N2 disease (aOR, 1.047; 95% CI, 1.041-1.053; p < 0.001), with an annual 1.2% increase in T3 disease (aOR, 1.012; 95% CI, 1.007-1.018; p < 0.001) and a 1.2% increase in T4 disease (aOR, 1.012; 95% CI, 1.005-1.018; p < 0.001). CONCLUSION: Despite advances in the field, laryngeal cancer survival in the United States is not improving over time. This may be due to an increase in the proportion of stage 4 disease, driven primarily by increasing nodal disease. To achieve survival improvement commensurate with scientific and technologic advances, efforts should be made to diagnose and treat laryngeal cancer at earlier stages to prevent further stage migration.

Mucoepidermoid Carcinoma of the Parotid: Very Close Margins and Adjuvant Radiotherapy.

BACKGROUND/AIMS: The importance of adjuvant radiotherapy in patients with close margin resections for mucoepidermoid carcinoma of the parotid gland remains unclear. METHODS: Patients who underwent parotidectomy for mucoepidermoid carcinoma with or without adjuvant radiotherapy at a single academic tertiary care center from 2000 to 2014 were identified. Included patients had negative but close (≤2 mm) surgical margins without other high-risk histopathological factors including advanced T-stage, positive nodal disease, lymphovascular or perineural invasion, or high-grade histology. RESULTS: Nineteen patients were identified, of whom 15 (79%) were observed postoperatively, while 4 (21%) underwent adjuvant radiotherapy. There were no significant differences in extent of parotidectomy, elective neck dissection, T staging, or tumor size between patients who were observed and those undergoing adjuvant radiation. There were no locoregional or distant recurrences in any patients at a mean follow up 74.3 months. Patients undergoing adjuvant radiation, however, had significantly more intermediate-grade as compared to low-grade histology (75% vs. 13%, difference 62%, 95% CI 4% to 100%). CONCLUSIONS: Patients with negative but close (≤2 mm) surgical margins without other high-risk histopathological factors have excellent long-term locoregional control with surgery alone. The effects of adjuvant radiotherapy for those who have intermediate-grade disease remain uncertain.

A hybrid epithelial/mesenchymal state in head and neck cancer: A biomarker for survival with differential prognosis by self-reported race.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the 6th leading cause of cancer-related mortality, with racial disparities amplifying the challenges in treatment. Although the relationship between hybrid epithelial/mesenchymal (E/M) states and tumor progression is of interest, no studies have characterized the clinical relevance of hybrid E/M states in head and neck cancer outcomes among self-reported racial cohorts. METHODS: Given the overlap in gene expression between hybrid E/M malignant cells and cancer-associated fibroblasts, we utilized deconvolution of bulk RNA sequencing data from oral cavity and laryngeal squamous cell carcinoma tumors from The Cancer Genome Atlas. We utilized our previously collected single-cell profiles to generate inferred malignant profiles and then scored these for hybrid E/M. We then conducted a survival analysis on overall and disease-free survival among self-reported Black and White Americans. FINDINGS: The hybrid E/M state was differentially associated with head and neck cancer survival by self-reported race and ethnicity, with a stronger association in non-Hispanic Black patients. Black patients with a high hybrid E/M score had a higher risk of death or recurrence (hazard ratio [HR]: 4.18 [95% confidence interval (CI): 2.06, 8.49]) than White patients with a high hybrid E/M score (HR: 1.58 [95% CI: 1.11, 2.26]). CONCLUSION: Our results suggest a complex interplay of social structure, racism, and genetic diversity. We implore researchers to consider the social and biological context contributing to disparities. FUNDING: A.L.M. received support from the National Institute of Minority Health and Health Disparities (K01MD013897 [principal investigator (PI), A.L.M.]). S.V.P. received support from the National Institute of Dental and Craniofacial Research (R01DE032865 [PI, S.V.P.] and R01DE032371 [PI, S.V.P.]).

Modeling epithelial homeostasis and perturbation in three-dimensional human esophageal organoids.

Background: Esophageal organoids from a variety of pathologies including cancer are grown in Advanced Dulbecco's Modified Eagle Medium-Nutrient Mixture F12 (hereafter ADF). However, the currently available ADF-based formulations are suboptimal for normal human esophageal organoids, limiting the ability to compare normal esophageal organoids with those representing a given disease state. Methods: We have utilized immortalized normal human esophageal epithelial cell (keratinocyte) lines EPC1 and EPC2 and endoscopic normal esophageal biopsies to generate three-dimensional (3D) organoids. To optimize ADF-based medium, we evaluated the requirement of exogenous epidermal growth factor (EGF) and inhibition of transforming growth factor-(TGF)-ß receptor-mediated signaling, both key regulators of proliferation of human esophageal keratinocytes. We have modeled human esophageal epithelial pathology by stimulating esophageal 3D organoids with interleukin (IL)-13, an inflammatory cytokine, or UAB30, a novel pharmacological activator of retinoic acid signaling. Results: The formation of normal human esophageal 3D organoids was limited by excessive EGF and intrinsic TGFß receptor-mediated signaling. In optimized HOME0, normal human esophageal organoid formation was improved, whereas IL-13 and UAB30 induced epithelial changes reminiscent of basal cell hyperplasia, a common histopathologic feature in broad esophageal disease conditions including eosinophilic esophagitis. Conclusions: HOME0 allows modeling of the homeostatic differentiation gradient and perturbation of the human esophageal epithelium while permitting a comparison of organoids from mice and other organs grown in ADF-based media.

A 65-year-old woman diagnosed with PHACE syndrome.

PHACE syndrome is characterized by the association between infantile hemangioma and varied but characteristic systemic manifestations, including cerebrovascular and cardiac abnormalities. The disorder has primarily been diagnosed in children, with little information available regarding long-term outcomes in affected individuals. We report the oldest known individual with PHACE syndrome in the medical literature, a 65-year-old woman who was diagnosed after a transient ischemic attack.

Protocol for tumor dissociation and fluorescence-activated cell sorting of human head and neck cancers.

Tumors originating from the head and neck represent diverse histologies and are comprised of several cell types, including malignant cells, cancer-associated fibroblasts, endothelial cells, and immune cells. In this protocol, we describe a step-by-step approach for the dissociation of fresh human head and neck tumor specimens, followed by isolation of viable single cells using fluorescence-activated cell sorting. Our protocol facilitates the effective downstream use of techniques, including single-cell RNA sequencing and generation of three-dimensional patient-derived organoids. For complete details on the use and execution of this protocol, please refer to Puram et al. (2017)1 and Parikh et al. (2022).2.

Experimental Modeling of Host-Bacterial Interactions in Head and Neck Squamous Cell Carcinoma.

The microscopic species colonizing the human body, collectively referred to as the microbiome, play a crucial role in the maintenance of tissue homeostasis, immunity, and the development of disease. There is evidence to suggest associations between alterations in the microbiome and the development of head and neck squamous cell carcinomas (HNSCC). The use of two-dimensional (2D) modeling systems has made significant strides in uncovering the role of microbes in carcinogenesis; however, direct mechanistic links remain in their infancy. Patient-derived three-dimensional (3D) HNSCC organoid and organotypic models have recently been described. Compared to 2D models, 3D organoid culture systems effectively capture the genetic and epigenetic features of parent tissue in a patient-specific manner and may offer a more nuanced understanding of the role of host-microbe responses in carcinogenesis. This review provides a topical literature review assessing the current state of the field investigating the role of the microbiome in HNSCC; including in vivo and in vitro modeling methods that may be used to characterize microbiome-epithelial interactions.

Prolonged operative time predicts postoperative deep venous thrombosis in head and neck cancer patients who undergo free flap reconstruction.

Objective: This study sought to quantify the deep venous thrombosis (DVT) incidence in head and neck cancer (HNC) patients undergoing free tissue transfer and to identify independent predictors of postoperative DVT. Materials and Methods: This is a cross-sectional study of the National Surgical Quality Improvement Program database from 2010 through 2020. The sample included all HNC surgical patients treated with free flap reconstruction. The study outcome was the presence of a DVT requiring treatment within 30 days of surgery. Univariate analyses were performed using chi-squared and independent t-tests. A multiple logistic regression model was created using all significant univariate predictors. Results: A total of 3954 patients were identified, of whom 53 (1.3%) experienced a postoperative DVT. The only medical comorbidity associated with DVT was COPD (RR = 2.7 [1.3, 5.4]; p < .01). Operative time longer than 9 hours (RR = 1.9 [1.0, 3.2]; p = .04) and length of stay longer than 10 days (RR = 1.9 [1.1, 3.2]; p = .02) were associated with greater DVT rates. In the multivariate analysis, only COPD (p < .01) and operative time (p = .02) were independently associated with DVT risk. The presence of a DVT was found to increase the relative risk of readmission (RR = 2.1 [1.2, 3.6]; p < .01) and non-home disposition (RR = 2.4 [1.7, 3.5]; p < .01). Conclusions: The incidence of DVT in HNC free flap patients was comparable to what has been reported in the general population of HNC surgery patients. Operative time >9 h and COPD history were independent risk factors for DVT in this subset of patients. Symptomatic DVTs necessitating treatment were accompanied by poorer post-hospitalization outcomes. Level of Evidence: Level 3.

Patient-derived three-dimensional culture techniques model tumor heterogeneity in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) outcomes remain stagnant, in part due to a poor understanding of HNSCC biology. The importance of tumor heterogeneity as an independent predictor of outcomes and treatment failure in HNSCC has recently come to light. With this understanding, 3D culture systems, including patient derived organoids (PDO) and organotypic culture (OTC), that capture this heterogeneity may allow for modeling and manipulation of critical subpopulations, such as p-EMT, as well as interactions between cancer cells and immune and stromal cells in the microenvironment. Here, we review work that has been done using PDO and OTC models of HNSCC, which demonstrates that these 3D culture models capture in vivo tumor heterogeneity and can be used to model tumor biology and treatment response in a way that faithfully recapitulates in vivo characteristics. As such, in vitro 3D culture models represent an important bridge between 2D monolayer culture and in vivo models such as patient derived xenografts.

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.

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.

Insurance Status Effect on Laryngeal Cancer Survival: A Population Based Study.

OBJECTIVE: To analyze insurance status effect on overall survival (OS) and disease-specific survival (DSS) in laryngeal cancer. STUDY DESIGN: Cross-sectional population analysis. SETTING: Surveillance, Epidemiology, and End Results (SEER) database. PARTICIPANTS: Laryngeal cancer patients from 2007 to 2016. MAIN OUTCOME MEASURES: Kaplan-Meier method with log-rank statistic analyzed OS and DSS by insurance status. Multivariable cox proportional hazard modeling generated survival prognostic factors. RESULTS: Of 19 667 laryngeal cancer cases, initial disease presentation was stage I: 7770 patients (39.5%), stage II: 3337 patients (17.0%), stage III: 3289 patients (16.7%), and stage IV: 5226 patients (26.6%). Patients had non-Medicaid insurance (15 523, 78.9%), had Medicaid (3306, 16.8%), or were uninsured (891, 4.5%). Mean and median OS for insured, Medicaid, and uninsured patients were 60.5, 49.6, and 56.6 and 74.0, 40.0, and 65.0 months, respectively. Following multivariable analysis, OS for insured, Medicaid, and uninsured patients was stage I: 87.9, 82.8, and 88.4 (P < .001), stage II: 79.1, 75.1, and 78.3 (P = .12), stage III: 68.7, 66.1, and 72.1 (P = .11), and stage IV: 57.1, 51.7, and 50.3 (P < .001) months. DSS mean survival times were 77.0, 65.8, and 67.7 months (P < .001) for insured, Medicaid, and uninsured patients. Age (HR: 1.02/year, P < .001) and black (HR: 1.15, P = .001) compared to white race predicted worse survival. Compared to insured status, Medicaid insurance carried a death hazard ratio of 1.40 (P < .001) and uninsured status had a death hazard ratio of 1.40 (P < .001). CONCLUSION: Insured laryngeal cancer patients had prolonged OS and DSS compared to Medicaid and uninsured patients. Medicaid patients had equivalent survival outcomes to uninsured patients. LEVEL OF EVIDENCE: 2c.

Adenoid Cystic Carcinoma of the Nasopharynx with Skull Base Involvement: Retrospective National Cohort Analysis of Treatment Paradigms, Outcomes, and Provider Density.

Objectives This article examines a national cohort of patients with nasopharyngeal adenoid cystic carcinoma (ACC) for incidence, skull base invasion, overall survival, and treatment paradigms. Design, Setting, and Participants Retrospective national population-based study using Surveillance, Epidemiology, and End Results program data of patients with ACC of the nasopharynx (NACC) and skull base between 2004 and 2016. Main Outcomes and Measures Primary outcomes included 5-year overall survival and odds of radiation treatment. Statistical analysis was performed using STATA 15.0 (STATACorp). p -Values < 0.05 were considered statistically significant. Results Of the 2,385 cases of ACC, 70 cases were classified as NACC. Twenty-one percent (15) involved invasion of the skull base or posterior pharyngeal wall, and 42% (30) were either stage 3 or stage 4. The 5-year overall survival for patients with NACC without skull base invasion was 67% which dropped to 40% with invasion into the skull base. Radiation was used as the primary form of therapy for 62% of NACC and 73% of NACC invading into skull base. Odds of receiving radiation therapy and 5-year survival were not affected by socioeconomic status or density of providers. Conclusion NACC is rare in incidence and was most commonly treated with radiation therapy when advanced in stage. Prognosis was dependent on invasion through posterior pharyngeal wall and skull base. Provider density and socioeconomic status did not affect odds of radiation or overall survival for NACC.

Predictors of survival following carotid blowout syndrome.

OBJECTIVES: Carotid blowout syndrome (CBS) is a rare, life-threatening complication for patients with head and neck cancer (HNC). The primary objective was to identify factors associated with survival following CBS. MATERIALS AND METHODS: A retrospective analysis of HNC patients treated at a single tertiary care hospital with CBS between 2016 and 2020 was performed. A multivariate Cox proportional-hazards model identified independent predictors of survival. A p value of <0.05 was considered significant. Kaplan-Meier survival analysis was performed. RESULTS: 45 patients were identified. The majority were male (80.0%) with a mean age of 64 years at time of blowout. Oropharynx was the most common primary site (48.9%) and 73.3% of patients had stage IV disease. 35 (77.7%) patients had active tumor at time of CBS. 93.3% of patients previously received RT with a mean total dose of 62.5 ± 14.8 Gy. Threatened/type I, impending/type II, and acute/type III CBS occurred in 6.7%, 62.2%, and 31.1% of cases, respectively. Patients underwent either embolization (80.0%) or endovascular stent placement (20.0%). The 30-day and 1-year OS rates were 70.1% and 32.0%, respectively. Primary oropharyngeal tumors (adjusted hazard ratio [aHR], 4.31 [1.30-15.15 95% confidence interval]), active tumor at time of CBS (aHR 8.21 [2.10-54.95]), ICA or CCA rupture (aHR 5.81 [1.63-21.50]), and acute/type III CBS (aHR 2.98 [1.08-7.98]) were independent predictors of survival. CONCLUSION: Primary oropharyngeal tumors, active tumor at time of CBS, ICA or CCA rupture, and acute/type III hemorrhage were independent predictors of survival. Multidisciplinary management and prompt, protocol-directed intervention may improve outcomes following CBS.

Development and Validation of Machine Learning Models for Predicting Occult Nodal Metastasis in Early-Stage Oral Cavity Squamous Cell Carcinoma.

Importance: Given that early-stage oral cavity squamous cell carcinoma (OCSCC) has a high propensity for subclinical nodal metastasis, elective neck dissection has become standard practice for many patients with clinically negative nodes. Unfortunately, for most patients without regional metastasis, this risk-averse treatment paradigm results in unnecessary morbidity. Objectives: To develop and validate predictive models of occult nodal metastasis from clinicopathological variables that were available after surgical extirpation of the primary tumor and to compare predictive performance against depth of invasion (DOI), the currently accepted standard. Design, Setting, and Participants: This diagnostic modeling study collected clinicopathological variables retrospectively from 7 tertiary care academic medical centers across the US. Participants included adult patients with early-stage OCSCC without nodal involvement who underwent primary surgical extirpation with or without upfront elective neck dissection. These patients were initially evaluated between January 1, 2000, and December 31, 2019. Exposures: Largest tumor dimension, tumor thickness, DOI, margin status, lymphovascular invasion, perineural invasion, muscle invasion, submucosal invasion, dysplasia, histological grade, anatomical subsite, age, sex, smoking history, race and ethnicity, and body mass index (calculated as weight in kilograms divided by height in meters squared). Main Outcomes and Measures: Occult nodal metastasis identified either at the time of elective neck dissection or regional recurrence within 2 years of initial surgery. Results: Of the 634 included patients (mean [SD] age, 61.2 [13.6] years; 344 men [54.3%]), 114 (18.0%) had occult nodal metastasis. Patients with occult nodal metastasis had a higher frequency of lymphovascular invasion (26.3% vs 8.1%; P < .001), perineural invasion (40.4% vs 18.5%; P < .001), and margin involvement by invasive tumor (12.3% vs 6.3%; P = .046) compared with those without pathological lymph node metastasis. In addition, patients with vs those without occult nodal metastasis had a higher frequency of poorly differentiated primary tumor (20.2% vs 6.2%; P < .001) and greater DOI (7.0 vs 5.4 mm; P < .001). A predictive model that was built with XGBoost architecture outperformed the commonly used DOI threshold of 4 mm, achieving an area under the curve of 0.84 (95% CI, 0.80-0.88) vs 0.62 (95% CI, 0.57-0.67) with DOI. This model had a sensitivity of 91.7%, specificity of 72.6%, positive predictive value of 39.3%, and negative predictive value of 97.8%. Conclusions and Relevance: Results of this study showed that machine learning models that were developed from multi-institutional clinicopathological data have the potential to not only reduce the number of pathologically node-negative neck dissections but also accurately identify patients with early OCSCC who are at highest risk for nodal metastases.

Feeding Tube Placement Following Transoral Robotic Surgery for Oropharyngeal Squamous Cell Carcinoma.

OBJECTIVE: To identify factors that may predict the need for feeding tubes in patients undergoing transoral robotic surgery (TORS) in the perioperative setting. STUDY DESIGN: Retrospective chart review. SETTING: Academic tertiary center. METHODS: A retrospective series of patients undergoing TORS for oropharyngeal squamous cell carcinoma (OPSCC) was identified between October 2016 and November 2019 at a single tertiary academic center. Patient data were gathered, such as frailty information, tumor characteristics, and treatment, including need for adjuvant therapy. Multiple logistic regression was performed to identify factors associated with feeding tube placement following TORS. RESULTS: A total of 138 patients were included in the study. The mean age was 60.2 years (range, 37-88 years) and 81.9% were male. Overall 82.9% of patients had human papilloma virus-associated tumors, while 28.3% were current or former smokers with a smoking history ≥10 pack-years. Eleven patients (8.0%) had a nasogastric or gastrostomy tube placed at some point during their treatment. Five patients (3.6%) had feeding tubes placed perioperatively (<4 weeks after TORS), of which 3 were nasogastric tubes. Six patients (4.3%) had feeding tubes placed in the periadjuvant treatment setting for multifactorial reasons; 5 of which were gastrostomy tubes. Only 1 patient (0.7%) was gastrostomy dependent 1 year after surgery. Multiple logistic regression did not demonstrate any significant predictive variables affecting perioperative feeding tube placement following TORS for OPSCC. CONCLUSIONS: Feeding tubes are seldom required after TORS for early-stage OPSCC. With appropriate multidisciplinary planning and care, patients may reliably avoid the need for feeding tube placement following TORS for OPSCC.

Assessing Prognostic Value of Quantitative Neck Dissection Quality Measures in Patients With Clinically Node-Negative Oral Cavity Squamous Cell Carcinoma.

Importance: In clinically localized (T1-2) oral cavity squamous cell carcinoma (OCSCC), regional lymph node metastasis is associated with a poor prognosis. Given the high propensity of subclinical nodal disease in these patients, upfront elective neck dissections (END) for patients with clinically node-negative disease are common and associated with better outcomes. Unfortunately, even with this risk-adverse treatment paradigm, disease recurrence still occurs, and our understanding of the factors that modulate this risk and alter survival have yet to be fully elucidated. Objective: To investigate the prognostic value of lymph node yield (LNY), lymph node ratio (LNR), and weighted LNR (wLNR) in patients with clinically node-negative T1-2 OCSCC. Design, Setting, and Participants: In this cohort study, data were collected retrospectively from 7 tertiary care academic medical centers. Overall, 523 patients with cT1-2N0 OCSCC who underwent elective neck dissections after primary surgical extirpation were identified. Exposures: Lymph node yield was defined as the number of lymph nodes recovered from elective neck dissection. Lymph node ratio was defined as the ratio of positive nodes against total LNY. Weighted LNR incorporated information from both LNY and LNR into a single continuous metric. Main Outcomes and Measures: Locoregional control (LRC) and disease-free survival (DFS) were both evaluated using nonparametric Kaplan-Meier estimators and semiparametric Cox regression. Results: On multivariable analysis, LNY less than or equal to 18 lymph nodes was found to be significantly associated with decreased LRC (aHR, 1.53; 95% CI, 1.04-2.24) and DFS (aHR, 1.46; 95% CI, 1.12-1.92) in patients with pN0 disease, but not those with pN-positive disease. Importantly, patients with pN0 disease with LNY less than or equal to 18 and those with pN1 diseasehad nearly identical 5-year LRC (69.7% vs 71.4%) and DFS (58.2% vs 55.7%). For patients with pN-positive disease, LNR greater than 0.06 was significantly associated with decreased LRC (aHR, 2.66; 95% CI, 1.28-5.55) and DFS (aHR, 1.65; 95% CI, 1.07-2.53). Overall, wLNR was a robust prognostic variable across all patients with cN0 disease, regardless of pathologic nodal status. Risk stratification via wLNR thresholds demonstrated greater optimism-corrected concordance compared with American Joint Committee on Cancer (AJCC) 8th edition nodal staging for both LRC (0.61 vs 0.57) and DFS (0.61 vs 0.58). Conclusions and Relevance: Movement toward more robust metrics that incorporate quantitative measures of neck dissection quality and regional disease burden, such as wLNR, could greatly augment prognostication in cT1-2N0 OCSCC by providing more reliable and accurate risk estimations.

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids.

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.

Single-cell RNA sequencing identifies a paracrine interaction that may drive oncogenic notch signaling in human adenoid cystic carcinoma.

Salivary adenoid cystic carcinoma (ACC) is a rare, biologically unique biphasic tumor that consists of malignant myoepithelial and luminal cells. MYB and Notch signaling have been implicated in ACC pathophysiology, but in vivo descriptions of these two programs in human tumors and investigation into their active coordination remain incomplete. We utilize single-cell RNA sequencing to profile human head and neck ACC, including a comparison of primary ACC with a matched local recurrence. We define expression heterogeneity in these rare tumors, uncovering diversity in myoepithelial and luminal cell expression. We find differential expression of Notch ligands DLL1, JAG1, and JAG2 in myoepithelial cells, suggesting a paracrine interaction that may support oncogenic Notch signaling. We validate this selective expression in three published cohorts of patients with ACC. Our data provide a potential explanation for the biphasic nature of low- and intermediate-grade ACC and may help direct new therapeutic strategies against these tumors.